Citation
Micrornas as Biomarkers in Gingival Crevicular Fluid

Material Information

Title:
Micrornas as Biomarkers in Gingival Crevicular Fluid
Creator:
Wahl, Kelsey Cronauer
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (54 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Dental Sciences
Dentistry
Committee Chair:
Holliday,Lexie Shannon
Committee Co-Chair:
Dolce,Calogero
Committee Members:
Caudle,Robert M
Graduation Date:
5/1/2020

Subjects

Subjects / Keywords:
biomarker -- gcf -- microrna -- orthodontic
Dentistry -- Dissertations, Academic -- UF
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Dental Sciences thesis, M.S.

Notes

Abstract:
In medicine and dentistry there is a significant focus on improving patient diagnosis and outcomes. With the identification of exosomes as methods of intercellular communication and genetic exchange, another avenue for biomarker discovery presented itself. Released from osteoclasts and inflammatory cells, extracellular vesicles carry genetic information, such as microRNAs (miRNAs), which can alter gene transcription in target cells and affect the inflammatory and bone remodeling processes. Gingival crevicular fluid (GCF) is a commonly used fluid for detection of biomarkers, including miRNAs. The objective of this study was to determine if miRNA146a could be sufficiently collected by GCF methods and which method was best for collecting this miRNA. This study compared three different methods which are the following: Periopaper strips, Durapore filter membrane, and micropipettes. Twelve adult volunteers were sampled and sampling took place over two visits. For each subject three teeth were sampled, each tooth with one of the proposed methods. A pattern was established so that collection method varied per tooth but repeated every fourth subject, for comparison. Collections were taken on the mesial and distal surfaces of each specified tooth. RNA isolation, cDNA synthesis, and real time polymerase chain reaction (qPCR) were performed on each sample to assess expression of miRNA146a. A reference gene, miRNA103,3p, was also used for assessment and normalization of data. Samples from seven subjects were analyzed and the results showed that there was no significant difference in method of collection for identifying and expressing miRNA146a. However, a secondary analysis of samples showed that there is a significant difference in expression of miRNA146a between subjects who were undergoing orthodontic treatment and those who were untreated. This study concluded that there is no statistical advantage to using one of the three collection methods over another for expression of miRNA146a. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2020.
Local:
Adviser: Holliday,Lexie Shannon.
Local:
Co-adviser: Dolce,Calogero.
Statement of Responsibility:
by Kelsey Cronauer Wahl.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
LD1780 2020 ( lcc )

Downloads

This item has the following downloads:


Full Text

PAGE 1

1 MICRORNAS AS BIOMARKERS IN GINGIVAL CREVICULAR FLUID By KELSEY CRONAUER WAHL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2020

PAGE 2

2 © 2020 Kelsey Cronauer Wahl

PAGE 3

3 To my husband and family for their never ending l ove, encouragement, and support

PAGE 4

4 ACKNOWLEDGMENTS I would like to thank m y mentor, Dr. L. Shannon Holliday, for his guidance and support with this project. I would like to thank Dr. Calogero Dolce and Dr. Robert Caudle for their assistance. I would like to thank all faculty, staff, and residents in the University of Florida Ort hodontic Department for their suppor t during residency. I would also like to thank my wonderful husband and my family for their love, support, and encouragement to pursue my passion.

PAGE 5

5 TABLE OF CONTENTS page ACKNO WLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ............................. 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 2 MATERIALS AND METHODS ................................ ................................ ................ 22 Participants and Eligibility ................................ ................................ ....................... 22 Study Design ................................ ................................ ................................ .......... 22 Sample Collection ................................ ................................ ................................ ... 23 PerioPaper ................................ ................................ ................................ ....... 23 Durapore Filter Membrane ................................ ................................ ............... 24 Microcapillary Tube ................................ ................................ .......................... 24 RNA Isolation ................................ ................................ ................................ .......... 25 cDNA Synthesis ................................ ................................ ................................ ...... 26 Real Time PCR ................................ ................................ ................................ ....... 26 Statistical Considerations ................................ ................................ ........................ 27 3 RESULTS ................................ ................................ ................................ ............... 31 4 DISCUSSION ................................ ................................ ................................ ......... 42 5 CONCLUSIONS ................................ ................................ ................................ ..... 47 LIST OF REFERENCES ................................ ................................ ............................... 48 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 54

PAGE 6

6 LIST OF TABLES Table page 2 1 Sample Collection Pattern for Subjects ................................ .............................. 27 2 2 Sample Identifiers ................................ ................................ ............................... 28 3 1 Ct Values for PerioPaper Samples ................................ ................................ ..... 39 3 2 Ct Values for Durapore Samples ................................ ................................ ........ 39 3 3 Ct Values for Microcapillary Tube Samples ................................ ........................ 39 3 4 ANOVA Descriptive Statistics for Full Data Set (Ten Samples) .......................... 40 3 5 ANOVA Descriptive Statistics for Subset of Data (Six Samples) ........................ 40 3 6 t test for Treated and Untreated Subjects ................................ ........................... 41

PAGE 7

7 LIST OF FIGURES Figure page 2 1 Microfug e® 18 Centrifuge ................................ ................................ ................... 28 2 2 27 20 Thermal Cycler ................................ ................................ .......................... 29 2 3 96 well Plate ................................ ................................ ................................ ....... 29 2 4 C 1000 Thermal Cycler ................................ ................................ ........................ 30 3 1 Amplification Plot of A1 and B1 Samples ................................ ........................... 35 3 2 Amplification Plot of C1 and D1 Samples ................................ ........................... 35 3 3 Amplification Plot of E1 and F1 Samples ................................ ............................ 36 3 4 Amplification Plot of A2, F2, M1 and M2 Samples ................................ .............. 36 3 5 Bar Graph Showing Full Dataset (Green) and Subset (Blue) of Mean Ct Values for miRNA 146a ................................ ................................ ...................... 37 3 6 Bar Graph Showing Full Dataset (Green) and Subset ( Blue) of Mean Ct Values for miRNA 103 3p ................................ ................................ ................... 37 3 7 Bar Graph Showing Mean Ct Values for miRNA 146a (Blue) and miRNA 103 3p (Green) and .......................... 38

PAGE 8

8 LIST OF ABBREVIATIONS ALP Alkaline phosphatase ANOVA Analysis of variance cDNA Complementary deoxyribonucleic acid Ct Cycle threshold DNA Deoxyribonucleic acid EV Extrac ellular vesicle GCF Gingival crevicular fluid HGF Human gingival fibroblast IL 1 Interleukin one beta IL 6 Interleukin six IL 8 Interleukin eight IRAK1 Interleukin one receptor associated kinase one miRNA Micro ribonucleic acid mRNA Messenger rib onucleic acid NF Nuclear factor kappa B PB Processing body PCR Polymerase chain reaction pre mRNA Precursor messenger ribonucleic acid pri miRNA Primary micro ribonucleic acid qPCR Quantitative polymerase chain reaction RANK Receptor activator nu clear kappa B RANKL Receptor activator nuclear kappa B ligand R T PCR Real time polymerase chain reaction

PAGE 9

9 RISC Ribonucleic acid induced silencing complex RNA Ribonucleic acid SLE Systemic lupus erythematosus TNF Tumor necrosis factor alpha TRAF6 T umor necrosis factor receptor associated factor six UTR Untranslated region Ct Delta cycle threshold Delta delta cycle threshold

PAGE 10

10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfi llment of the Requirements for the Degree of Master of Science MICRORNAS AS BIOMARKERS IN GINGIVAL CREVICULAR FLUID By Kelsey Cronauer Wahl May 2020 Chair: Lexie Shannon Holliday Major: Dental Sciences Orthodontics In medicine and dentistry there i s a significant focus on improving patient diagnosis and outcomes. With the identification of exosomes as methods of intercellular communication and genetic exchange, another avenue for biomarker discovery presented itself. Released from osteoclasts and in flammatory cells, extracellular vesicles carry genetic information, such as microRNAs (miRNAs), which can alter gene transcription in target cells and affect the inflammatory and bone remodeling processes. Gingival crevicular fluid (GCF) is a commonly used fluid for detection of biomarkers, including miRNAs. The objective of this study was to determine if miRNA 146a could be sufficiently collected by GCF methods and which method was best for collecting this miRNA. This study compared three different methods which are the following: Periopaper strips, Durapore filter membrane, and micropipettes. Twelve adult volunteers were sampled and sampling took place over two visits. For each subject three teeth were sampled, each tooth with one of the proposed methods. A pattern was established so that collection method varied per tooth but repeated every fourth subject, for comparison. Collections were taken on the mesial and distal surfaces of each specified tooth. RNA isolation, cDNA synthesis, and real time polymeras e chain

PAGE 11

11 reaction (RT PCR) were performed on each sample to assess expression of miRNA 146a. A reference gene, miRNA 103 3p, was also used for assessment and normalization of data. Samples from seven subjects were analyzed and the results showed that there was no significant difference in method of collection for identifying and expressing miRNA 146a. However, a secondary analysis of samples showed that there was a significant difference in expression of miRNA 146a between subjects who were undergoing orthodo ntic treatment and those who were untreated. This study conclu ded that there is no advantage to using one of the three collection methods over another for expression of miRNA 146a.

PAGE 12

12 CHAPTER 1 INTRODUCTION For many years, miRNAs have been stud ied to determine their roles in health and disease. 1 Compr ising 1 2% of all genes in mammals, nearly all biological processes are affected by miRNA control . 2 Structurally, mi RNA is a single stranded RNA, 21 25 nucleotides in length . 2 miRNA s ar e present in multiple organisms, inclu ding plants, worms, and humans, and ma ny are conserved across species. 3 Remarkably, components of miRNA machinery have been found in archaea and eubacteria, showing their lasting presence from ancient times to toda y. 4 Originally found to regulate developmental timing in worms, the function of miRNA was determined to be even greater , with the discovery of its involvement in networking and coordination of gene expression. 5 miRNAs have been shown to participate in cell cycle control, cardiac and skeletal muscle development, and neurogenesis . 6 Additional l y, they have been implicat e d in a number of diseases including cancers, heart disease, and neurologic al diseases. 7,8 Consequently, miRNAs are studi ed as markers of disease progression and for diagnostic purposes. 8 Production of miRNA takes places in the nucleus and cytoplasm of a cell . 9 In the nucleus, different areas of a gene are transcribed by RNA Polymerase II into long primary miRNAs (pri miRNA). 10 These piece s are either inserted into noncoding RNAs, fixed into introns of protein coding genes , or are clustered in polycistronic transcripts. 11 Processing of the pri mRNAs occurs by two R ibonucleas e III enzymes, Drosha and Dicer. 12 In the nucleus, the enzyme Drosha attaches to a pri mRNA and excises the next precursor, pre mRNA. 10 The pre mRNA is about seventy nucleotides long and can fold into a stem loop structure. 10 The pre mRNA is exported from the nucleus into the

PAGE 13

13 cytoplasm by Exportin 5. 13 Once in the cytoplasm , it is cleaved by the enzyme Dicer, producing a miRNA duplex intermediate that is abou t twenty base pairs in length. 14 The duplex conta ins one strand of mature miRNA, which becomes bound by a large Argonaute protein and becomes part of the RNA induced silencing complex (RISC). 15 The mechanism of action of miRNA is achieved th r ou gh the RISC and is attributed mainly to the Argonaute core . 15 The Argonaute protein not only binds the miRNA in forming the RISC, but it also joins with other Argonaute pr oteins to form the center of the RISC complex. 16 This Argonaute core is highly conserved amongst species and is responsible for the slicing activity that cleaves messenger RNA ( mRNA ) , as instructed by the miRNA . 17 The RISC binds to a target mRNA ( mRNA UTR ) , regulating the e xpression of the mRNA , and subsequently , the expression of genes. 18 Once bound, the RISC cleaves the target mRNA, inhibit ing protein synthesis and degrading the target mRNA. 19 If destined for degradation, the cleaved mRNA associates with cytoplasmic processing bodies (PBs). 20 The PBs localize to the core and contain enzymes needed for mRNA degradation, such as decapping enzymes and exonucleases. 20 Together , the PBs and RISC carry out the actions determ ined by their associated miRNA. 9 Furthermore , a single miRNA can regulate multiple targets, emphasizing the widespread effect of miRNAs throughout the body. 21 miRNAs have been studied as pote ntial biomarkers of health and disease. 1 Biomarkers are considered any structure or substance objectively measured in a sample to indicate health and disease . 22 Cytokines, such as interleukin 1 ( IL 1 ) and tumor necrosis factor alpha ( TNF ) have been studied as biomarkers in the medical

PAGE 14

14 and dental fields. 23 Studies of tooth movement have also observed interleukin 6 (IL 6) , interleukin 8 (IL 8) , dentine sialophosphoprotein and alkaline phosphatase (ALP) as biomarker candidates in orthodontic tooth movement . 24 26 Furthermore, receptor activator of nuclear factor kappa B (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) have been studied as biomarkers of bone resorption. 27 Studies show the presence of RANKL, a transmembrane protein, and RANK in extracellular vesicles (EVs) and suggest that EVs and their components can be detected in gingival crevicular fluid . 28,29 The term e was initially suggested to identify extracellular vesicles of endosomal origin. 30 On ce regarded as cellular trash, exosomes are now known as a form of intercellular communication that can alter functions of targ et cells. 31 m icroRNAs are found in exosomes and function by regulating gene exp ression post transcriptionally. 32 Some studies have observed the role of miRNAs in bone remodeling and inflammation. 33,34 One miRNA proposed as a key regulatory molecule of the inflammatory respons e is miRNA 146. 35 The miRNA 146 family consists of miRNA 146a and miRNA 146b. 35 Both can be induced by pro inflammatory cytokines, such as IL 1 a nd TNF . 35 It is also report ed that miRNA 146a negatively regulates the innate immune response by repressing IL 1 receptor associated kinase 1 (IRAK1) and tumor nec rosis factor receptor associated factor 6 (TRAF6). 36 IRAK1 and TRAF6 are significant because they are two crucial molecules in the nuclear factor kappa B (NF 36 IRAK1 and TRAF6 are significant because they are two crucial molecules in the nuclear factor kapp a B (NF pathway. 36 They act by increasing NF an increased expression

PAGE 15

15 of the pro inflam matory cytokines IL 6 and IL 8 , which are key mediators of inflammation. 36 In medicine, some chronic inflammatory disease s are shown to be related to miR NA 146a dysregulation. 35 Psoriasis, systemic lupus erythematosus (SLE), and rheumatoid arthritis are a few of the most significant. 37 39 In dentistry, studies have proposed miRNA 146a as an inflammatory biomarker by observing its expression in patients with periodontitis. 40,41 A study by Motedayyen et al. assessed miRNA 146a expression in subjects with healthy periodontium versus chronic periodontitis. 41 They found that subjects with c hronic periodontitis had significantly higher levels of miRNA 146a. 41 Furthermore, they found that elevated miRNA 146a was accompanied by a reduction in TNF 6, two pro inflammatory cytokines. 41 These results are similar to a nother study by Xie et al. which analyzed miRNA 146a expression in the inflammatory response in human gingival fibroblasts (HGFs). 41,42 In this study, they used polymerase chain reaction ( PCR ) to measure the expression of miRNA 146a after Porphyromonas gingivalis ( P. gingivalis ) lipopolysaccharide stimulation. 42 Their results showed a significant difference in miRNA 146a expression in the P. gingivalis stimulated HGFs, with a greater expression of miRNA 146a in these HGFs. 42 MicroRNA 146a expression in bone has also been of interest . 43 A study b y Nakasa et al. aimed to determine whether overexpression of miRNA 146a inhibits osteoclastogenesis. 44 In their animal study, they administered an intravenous injection of double stranded miRNA 146a into mice with a rthritis. 44 They observed that while not completely suppressed, the mice injected with miRNA 146a showed a reduction of pro -

PAGE 16

16 inflammatory cytokines TNF , IL , and IL 6. 44 Radiographically and histologically , those injected with the microRNA showed less destruction of cartilage and bone. 44 Further assessing the role of miRNA 146a in bone remodeling, a study by Holliday et al. looked at osteoclast exosomes, exosomal components and bone remodeling processes. 34 They discussed candidate molecules identified in exosome based regulation of bone remodeling. 34 Two of these candidates were miRNA 146a and miRNA 214. 34 Studies by Li et al. and Sun et al. detected miRNA 214 as enriched in osteoclast exosomes compared with exosomes from precursors. 33,45 The Holliday et al. st udy found similar results to Sun et al. regarding miRNA 214, which Sun et al. reported to be enriched 4 fold in osteoclasts. 33,34 Significantly, Holliday et a l . found that miRNA 146a was enriched over 80 fold in os teoclasts compared with precursors, making it a potential biomarker for bone remodeling and providing a rationale for further study of miRNA 146a as a candidate biomarker for orthodontic tooth movement. 34 A recent study of orthodontic tooth movement by Atsawasuwan et al. analyzed gingival crevicular fluid ( GCF ) samples of orthodontically treat ed and untreated subjects to observe the expression of microRNA 29 . 46 GCF samples were collected five t imes throughout treatment and the results showed the greatest expression of microRNA 29 in the subjects who underwent canine retraction . 46 Furthermore, they found that the highest concentration of their miRNA of interest was prese nt in the fraction of GCF containing EVs . 46 Their results showed an increased expression of miRNA 29 in the treated subjects , revealing a correlation between this miRNA , orthodontic tooth movement, and osteoclast function. 46 Furthermore, this study suggests that miRNAs in GCF can be candidate biomarkers for orthodontic tooth movement . 46

PAGE 17

17 Of the many miRNAs that impact ph ysiologic and disease processes there are miRNAs. 47 One such reference miRNA is miRNA 103 3p. 47 While found to promote human gastric cancer cell proliferation, it is an endogenous miRNA present in serum, plasma, urine, and cerebrospinal fluid. 48 A study by Song et al. identified suitable reference genes for quantitative PCR ( qPCR ) analysis of serum. 49 Of six candidate reference microRNAs, microRNA103 3p was found to be moder ately abundant in serum sa mples, suggesting it as a suitable miRNA for comparison. 49 For many years, GCF has been collected and analyzed for potential biomarkers. 50 Rather than collecting and examining saliva or other oral fluids, GCF is preferred due to its site specific natu re and better ability to express the metabolic status of localized tissue. 51 Its inclusion of bacterial and host cell molecules makes it ideal fo r evaluating cellular metabolism. 51 Considered a serum transudate or inflammatory exudate, GCF accumulates in the gingival crevice by leakage thr ough postcapillary venules in tissue. 51,52 When the lymphatic system is unable to remove the fluid, due to a greater filtrate volume, leakage occurs and fluid escapes into the gingival sulcus. 52 It is in the sulcus or at the gingival margin that GCF is collected. 53 Experimentally, this process is supported by an animal study by Del Fabbro et al. 54 They observed gingival fluid flow by measuring osmotic pressure at different points in the gingival crevice .* They also observed the thickness of epithelium to distinguish absorption or fluid releas e . 54 These methods of measurement are an improvement from past techniques, where quantities were pri marily based on estimates. 55 Collection of available GCF is a sensitive process because c ontamination of any kind can a ffect analysis and results. 53 If blood contaminates the sample it must be

PAGE 18

18 discarded , and plaque and saliva must be removed from the area. 56 Furthermore, collecting GCF samples is a challenge when fluid is sparse. 53 Enough fluid must be gathered for adequa te analysis , but increasing collection time can increase the risk for contamination. 53 It has been shown that GCF can be isolated from a heal thy sulcus, although only in small amounts. 51 It has been clinically demonstrated tha t GCF production greatly incre ases when gingival inflammatio n is present . 57 Damage to the epithelium leads to localized periodontal inflammation and increased vascular permeability, which increases the flow . 57 A healthy periodontium releases about 3 into the gingival sulcus, while sites with periodontal disease can release 58 Regardless of these limitations, many studies in health care have successfully used GCF collection techniques to obtain adequate fluid samples for a nalysis. 59,60 The gingival washing method, use of micropipettes, and use of filter paper strips are three main techniques for collecting GCF. 53 Each technique has i ts advantages and disadvantages, with the washing method best suited for sample s that need cells collected . 53 This technique involves injecting and aspirating a balanced salt solution of known amount into the gingival sulcus repeatedly , before collecting the final sample. 53 By mixing the fluids in this way it is difficult to determine the sample dilution and to control the final volume. 53 As described, the gingival washing method is not the most efficient system for gathering GCF but can be us eful if sample dilution is not of concern. 53 Using capillary tubes to collect GCF is more beneficial than gingival washing , if standardization of the GCF volume is desirable. 53 Because the micropipettes have a

PAGE 19

19 known diameter, GCF volume is easy to determine. 53 Tubes of varying diamet ers are available for GCF collection , and filling the tube fully with GCF would provide an accurate measurement of sample volume . 61 The tubes are placed in the gingival crevice and fluid flows into the micropipettes by capillary act ion. 53,61,62 The disadvantage of this method is that it may require more time to obtain an adequate volume of GCF. 53 Using absorbent paper strips is the favored technique for GCF collection because it is fast and least traumatic. 63 Many types and sizes of paper strips are available , such as Whatman chromatography paper and PerioP aper . 64 When using the strips the sample tooth is cleaned with a cotton pellet, isolated with cotton rolls, dried, and the n the paper strip is placed . 56 The strip is typically p laced into the gingival sulcus until slight resistance is felt , or at the coronal edge of the gingival sulcus. 53 Placement of the strip causes little to no irritation , and GCF is collected by fluid migration through the paper. 53 To observe miRNAs from GCF, RT PCR is often used . 65 PCR is a method for amplifying fragments of DNA. 65 This technique allows for segments of specific chromosome s to be amplified more than a million fold , and for testing of very small sequences of DNA , which could not otherwise be tested. 66 RT PCR is the ability to monitor the progress of the PCR as it occurs, since data is collected throughout the PCR process . 65 RT PCR records the point during cycling when the target is first detected and amplified, rather than reporting the am ount of target gathered after a fixed number of cycles, as in conventional PCR. 67 The real time PCR process consist s of twenty five to fifty cycles , and each cycle undergoes three phases at different temperatures. 65 The initial phase is denaturation,

PAGE 20

20 when the DNA helix is separated into two strands. 65 Denaturation occurs at a high temperature. 65 The second phase, annealing, occurs when the sample is cooled and the added 65 The third phase is amplification , which occurs at a high temperature and is when elongation of the primer occurs , creating a complementary DNA strand to the target. 65 Significantly, a fluorescent dye is used in q PCR and binds to double stranded DNA molecules by inserting between the base pairs. 68 The fluorescence is measured with each amplification cycle to determine relatively how much DNA has been ampl ifie d. 68 With the use of dye in PCR, the higher the starting amount of the target DNA , the sooner an increase in fluorescence is observed. 68 q PCR data is represented in an amplification plot , which shows each sample as a curve using cycle number and fluorescent signal. 69 At first glance, an amplification plot allows for basic interpretation of the data without numerical values . 69 In a plot, the curve farthest to the left signifies the highest concentration of the target and earliest signal amplification. 69 The curve farthest to the right signifies the lowest initial concentration of the target and subsequent latest signal amplification. 69 The data can be understood more specifically by observing key points in the amplification plot. 70 A horizontal line is present in each plot and denotes a threshold value, set by the machine. 70 The section of the curve below the threshold represents fluorescence that cannot be distinguished from background fluorescence. 70 The point at which fluorescence can b e sufficiently detected is when the curve just passes the threshold value . 70 This point is easily identified by the intersection between the curve

PAGE 21

21 and threshold line and is referred to as the cycle threshold, or Ct. 70 The ability to determine Ct values p rovides an easy method for comparison between samples. 7 0

PAGE 22

22 CHAPTER 2 MATERIALS AND METHODS Participants and Eligi bility The University of Florida Institutional Review Board for the Protection of Human Subjects approved this project (UF IRB 01, IRB201801700). Additional funding for this project was provided b y the Southern Association of Orthodontists (SAO). Gingival crevicular fluid samples were collected from twelve adult volunteers using the inclusion and exclusion criteria. The inclusion criteria consisted of the following: an age range of 18 to 40 years o ld, the presence of all six maxillary and mandibular anterior teeth, and the presence of all four maxillary premolars. Exclusion criteria included the presence of extremely poor oral hygiene or a history of smoking or tobacco use within the past year . The patient popu lation was representative of the patient population of the University of Florida College of Dentistr y Department of Orthodontics , with patients of all races, genders, and ethnicities included in the study. Study Design Collection s of all GCF samples were c arried out in two visits for each subject. The second visit was at least twenty four hours after the first visit. At the initial visit, all su bjects confirmed that they did not eat, drink, brush their teeth, or use mouthwash at least two hours prior to sam ple collection. Subjects confirmed this again at the second visit, prior to collection. At enrollment and prior to sample collection, all subjects provided written informed consent. Three methods of GCF collection were used and are the following: PerioPape r, Durapore filter membrane, and microcapillary tubes. Teeth numbers 5, 7, and 9, were used as collection sites. For each participant, the collection method varied per tooth, following the pattern listed in Table 2 1 .

PAGE 23

23 Sample Collection Each sample collect ion Eppendorf tube was labeled with a specific subject A hyphen then se followed by the tooth number that was collected. Table 2 2 shows the identifiers listed for the samples tha t were anal yzed . For each subject at each visit six total samples were collected, with two samples per technique. Plaque was removed from the buccal surface of each tooth along the gingival margin and samples were collected on the mesial and distal of each tooth. If any sample was visually contaminated with blood it was discarded and a new sample was taken. The specific collection techniques for each method are as follows: PerioPaper The tooth designated for sampling was cleaned of any plaque on the buccal surface using a cotton pellet. The tooth was then isolated with cotton rolls to prevent contamination by saliva and was air dried for five seconds. A PerioPaper® GCF strip (Oraflow Inc., Plainview, NY) was inserted about 2mm into the gingival sulcus, until mild resistance was felt, on the mesial aspect of the tooth. The strip was left in place for thirty seconds and was removed using a sterilized cotton plier. It was placed in a sterilized of phosphate buffered salien (PBS; pH 7.4, RNase free). The same process was repeated with a new PerioPaper strip on the distal aspect of the tooth . The samples were immediately placed in a freezer and kept at 80°C for further analysis.

PAGE 24

24 Durapore F ilter M e mbrane For each sample collection a Durapore filter membrane was cut to the same length and width as a PerioPaper strip, 2x8mm. The tooth designated for sampling was cleaned of any plaque on the buccal surface using a cotton pellet. The tooth was then iso lated with cotton rolls to prevent contamination by saliva an d was air dried for five Corp., Bedford, Mass., USA) was inserted about 2mm into the gingival sulcus, until mild resistance was felt, on the mesial aspect of the toot h. The strip was left in place for thirty seconds and was removed using a sterilized cotton plier. It was placed in a sterilized RNase free). The same process was repeated with a new Durapore filter membrane strip on the distal aspect of the tooth. The samples were immediately placed in a freezer and kept at 80°C for further analysis. Microcapillary T ube The tooth designated for sampling was cleaned of any plaque on the buc cal surface using a cotton pellet. The tooth was then isolated with cotton rolls to prevent (Drummond Scientific Co., Broomall, Pennsylvania, USA) was placed on the mesi al aspect of the tooth at the entrance to the gingival sulcus. The micropipette was held in place by the operator for five minutes. GCF was taken up into the micropipette by capillary action. A fter five minutes the GCF was eluted from the micropipette usin g a bulb provided by the company. The bulb is attached to one end of the micropipette and produces gentle air pressure, dispensing the GCF into a sterilized Eppendorf tube The same process was repeated with a

PAGE 25

25 new microcapillary tube on the distal aspect of the tooth. The samples were immediately placed in a freezer and kept at 80°C for further analysi s. RNA I solation A total of seven subjects and samples from ten visits were used for analysis, as shown in Table 2 2 . The QIAGEN kit protocol, miRNeasy Serum/Plasma Advanced Kit, for RNA isolation was used and began by thawing ea ch sample and placing them on ice containing thawed sample with PBS. Each tube was closed and vortexed for more than five seconds. The samples then rested at room temperature for three minutes. The samples were cen trifuged at 13,000 times gravity (×g) for three minutes at room temperature, to precipitate the pellet , as shown in Figure 2 1 . The supernatant from each was transferred to a new, labeled, reaction tube. 1 volume isopropanol was added and mixed by vortexin g. Each entire sample was transferred to an RNeasy UCP MinElute column, the lids were closed, and the columns were centrigured for fifteen seconds at greater than 8,000 ×g. The flow through was discarded. was pipetted into the Rneasy UCP MinElute spin column. The lids were closed and they were centrifuged for fifteen seconds at greater than 8 , 000 ×g. The flow through was spin column. The lids were closed and they were centrifuged for fifteen second at greater than 8,000 ×g. The flow UCP MinElute spin column. The lids were closed and they were centrifuged f or two minutes at greater than 8,000 ×g. The flow through and collection tubes were discarded. The Rneasy UCP MinElute spin columns were placed in new 2 mL collection tubes, supplied in the kit. The lids of the spin columns were kept open and they were

PAGE 26

26 cen trifuged at full speed for five minutes to dry the membrane. The flow through and collection tubes were discarded. The Rneasy UCP Min elute spin columns wer e placed free water was added diretly to the center of each spin column membrane. Incubation then occurred for one minute. The lids were closed and they were centrifuged for one minute at full speed. The RNA was then eluted. If ready for cDNA synthesis it was kept on ice for the next step, otherwise it was frozen at 80°C for further analysis. cDNA Synthesis cDNA synthesis was accomplished using the QIAGEN kit protocol, specifically the mi RCURY LNA Universal RT microRNA PCR protocol. If frozen, the RNA samples were thawed and kept on ice. From the kit, the 5x Reaction Buffer, Enzyme mix, and RNA spike were thawed in the fridge. A master mix was made by adding of the RNA spike to an Eppendorf tube. This tube was kept on ice. New, Eppendorf tubes were labeled with subject/sample identifiers and each RNA sample was added to the appropriately labele d tube. Master Mix was added to each tube and then each tube was ready for cDNA synthesis. The samples were incubated in a 2720 Thermal Cycler (Figure 2 2) for 65 minutes with the following specifications: 60 minutes at 42°C, 5 minutes at 95°C, at least 3 minutes at 4°C. Real T ime PCR To prepare for qPCR, a 96 well plate was used. A total of fou r 96 well plates were used and four runs of qPCR were completed. The first round of qPCR was for subjects A and B. The second round was for subjects C and D. The third round was for subjects E and F. The fourth round was for subjects A, F, and M. An example of a

PAGE 27

27 planned and labeled 96 well plate used for qPCR is shown in Figure 2 3. The QIAGEN kit protocol, miRCURY LNA Universal RT microRNA PCR protocol, was used for samp le preparation in the PCR plate and for setting the Thermal Cycler machine. In each labeled well the appropriate cDNA sample was added, along with nuclease free The plate was sealed, spun in a Micropla te Spinner for a few seconds and placed into the C1000 Touch Thermal Cycler (Figure 2 4) to undergo real time PCR. The settings were as follows: 39 amplification cycles at 95°C for 10 seconds, 60°C for 1 minute, and a ramp rate of 1/6 C/s^7 optical read. q PCR data was retrieved using the Bio Rad qPCR Analysis Software. From this software, raw data was retrieved as Ct values and amplification plots. Statistical Considerations One way ANOVA and student t test were performed to determine statistical significan ce, with statistical significance represented by a p value less than 0.05. Table 2 1. Sample Collection Pattern for Subjects Subject Tooth #5 Tooth #7 Tooth #9 A PerioPaper Durapore Micropipette B Durapore Micropipette PerioPaper C Micropipette Peri oPaper Durapore D PerioPaper Durapore Micropipette

PAGE 28

28 Table 2 2. Sample Identifiers Subject PerioPaper Durapore Micropipette A A1 P5 A1 P5 A1 D7 A1 D7 A1 M9 A1 M9 A2 P5 A2 P5 A2 D7 A2 D7 A2 M9 A2 M9 B B1 P9 B1 P9 B1 D5 B1 D5 B1 M7 B1 M7 C C1 P7 C1 P7 C1 D9 C1 D9 C1 M5 C1 M5 D D1 P5 D1 P5 D1 D7 D1 D7 D1 M9 D1 M9 E E1 P9 E1 P9 E1 D5 E1 D5 E1 M7 E1 M7 F F1 P7 F1 P7 F1 D9 F1 D9 F1 M5 F1 M5 F2 P7 F2 P7 F2 D9 F2 D9 F2 M9 F2 M9 M M1 P7 M1 P7 M1 D9 M1 D9 M1 M5 M1 M5 M2 P7 M2 P7 M2 D9 M2 D9 M2 M5 M2 M5 Figure 2 1. Microfuge® 18 Centrifuge. Courtesy of Dr. Kelsey Cronauer Wahl. Centrifugation of Samples for RNA I solation

PAGE 29

29 Figure 2 2. 2720 Thermal Cycler. Courtesy of Dr. Kelsey Cronauer Wahl. cDNA Synthesis Figure 2 3. 96 well P late Organized and L abeled. Courtesy of Dr. Kelsey Cronauer Wahl. Plate Prepared for Fourth R un of qPCR

PAGE 30

30 Figure 2 4. C1000 Thermal Cycler. Courtesy of Dr. Kelsey Cronauer Wahl. Plate L oaded and Machine P repared for qPCR

PAGE 31

31 CHAPTER 3 RESULTS Sa mples from seven of the twelve subjects were analyzed, and samples from only the first visit were analyzed for all seven subjects . For three of the seven subjects, their second visit samples were analyzed. Samples were collected using the three techniques of PerioPaper strips, Durapore filter membrane, and microcapillary tubes. qPCR was performed four times and amplification plots from all four runs were obtained using the Bio Rad qPCR An alysis Software and are shown from Figure 3 1 to Figure 3 4. Using thi s software , numerical raw data was also obtained as Ct values. Table 3 1 to Table 3 3 show s the Ct values obtained for each subject and organized based on GCF collection technique and miRNA. These tables also include the calculated Ct va lues for each subject , which uses miRNA 103 3p to normalize the data of miRNA 146a . Ct values were calculate by subtracting the Ct value of miRNA 1 46a from the Ct value of miRNA 103 3p. For each of the three collection techniques, the mean Ct value for miRNA 146a and miRNA 103 3p and the mean Ct value were calculated. A series of one way ANOVAs were conducted on the entire da taset of seven subjects (ten sample groupings ) and the descriptive statistics are presented in Table 3 4. This was done in or der to determine whether there are significant differences in the mean values of miR NA 146a, miR NA 1 03 3p, and Ct on the basis of condition, which consisted of Periopaper, Durapore, and microcapillary tubes. Significant mean differences were not found wit h regard to miR146a, F (2, 27) = 1.539, p = .233, miR103 3p, F (2, 27) = 1.103, p = .347, or Ct, F (2, 27) = 1.978, p = .158. All p values were above 0.05. As shown,

PAGE 32

32 means differed slightly by condition, while these mean differences were not significantly di fferent. A series of one way ANOVAs were conducted on a subset of the data, which exclu ded four sample groupings. Four sample groupings were excluded because the subjects were undergoing orthodontic treatment at the time of collection, as noted at the vis it . Six sample groupings were run for this one way ANOVA series and descriptive statistics are presented in Table 3 5. These analyses also failed to find significant mean differences on the basis of miR146a, F (2, 15) = 1.970, p = .174, miR103 3p, F (2, 15) = 1.347, p = .290, or Ct, F (2, 15) = .529, p = .600. All p values were above 0.05. Mean differences were again found here on the basis of condition, while these mean differences also failed to achieve statistical significance. Graphical representations of data from the fu ll dataset (ten sample) and subset (six sample ) are shown in Figure 3 5 and Figure 3 6 . Based on the statistical analysis we therefore fail to reject our null hypothesis and conclude that there is no significant difference in the recovery of miRNA 146a in GCF using Periopaper, Durapore filter membrane, or microcapillary tubes. An additional method of comparison was completed to determine the fold change in expression of miRNA 146a between the GCF collection methods. 71 The Livak Method was used to calculate the fold change of gene expression, referred to as 2^ ( 71 In this method, a reference is needed to compare conditions. Per ioPaper was used as the reference condition. Therefore the 2^ ( change expression of miRNA 146a for Durapore in relation to PerioPaper and for microcapillary tubes in relation to PerioPaper. For Durapore the 2^ (

PAGE 33

33 fold change decrease and the 2^ ( rocapillary tubes was a 3.12 fold change decrease. Since a few subjects were undergoing orthodontic treatment, t he data was a n alyzed further to assess whether there is a significant difference in miRNA 146a expression between two groups, defined a s treated and untreated. GCF collection technique was not a factor of interest in this analysis. The treated and untreated groups each consisted of five sampling groups. It is necessary to note that for one subject, the samples from the initial visit were included in the untreated group, while the samples from the second visit were included in the treated group. This was done since orthodontic treatment did not begin until the second visit for that subject. Independent sample t tests were conducted specific ally comparing the untreated subjects, with healthy periodontium, and treated subject s , with inflamed periodontium, on their mean values of miR NA 146a, miR NA 103 3p, and Ct. In these analyses , significant differences between these two groups were found wi th respect to miR146a , t (22.103) = 2.253, p < .05, and Ct, t (28) = 4.844, p < .001, though not with respect to miR103 3p, t (27.115) = .047, p = .963. As stated, p values were less than 0.05 for miRNA146a and the Table 3 6 presents the descriptive sta tistics associated with these analyses, which indicate small mea n differences on the basis of untreated subjects and subjects undergoing orthodontic treatment . In the two cases in which statistical significance was achieved, significantly higher m eans were found in the case of untreated subjects. This data is graphically shown in Figure 3 7 . Furthermore, the Livak Method was used to calculate the fold change of gene expression between the treated and untreated groups. 71 For this calculation -

PAGE 34

34 103 3p were used to calculate the fold change of gene expression of miRNA 146a between the two groups . The 2^ ( was an increase of 5.81 .

PAGE 35

35 Figure 3 1. Amplification P lot of A1 and B1 Samples Figure 3 2. Amplification Plot of C1 and D1 Samples

PAGE 36

36 Figure 3 3. Amplification Plot of E1 and F1 Samples Figure 3 4. Amplification Plot of A2, F2, M1 and M2 Samples

PAGE 37

37 Figure 3 5. Bar G raph S howing Full Dataset (G reen) an d Subset (B lue) of Mean Ct Values for miRNA 146a Based on GCF Collection Method Figure 3 6. Bar G raph Showing Full Dataset (Green) and Subset (Blue) of Mean Ct Values for miRNA 103 3p Based on GCF Collection Method

PAGE 38

38 Figure 3 7. Bar G raph Showing Mean Ct Values for miRNA 146a (Blue) and miRNA 103 3p (Green) and (Gold) in Treated and Untreated Subjects

PAGE 39

39 Table 3 1. Ct Values for PerioPaper Samples Subject microRNA 146a microRNA 103 3p A1 29.2545 27.3836 1.87092 A2 25.375 26.36 0 .985 B1 32.1364 28.8240 3.3124 C1 31.8422 27.8092 4.03296 D1 25.4571 23.2879 2.1692 E1 29.6349 24.7220 4.9128 F1 35.6996 31.2679 4.4317 F2 34.49 37.40 2.91 M1 28.935 28.45 0.485 M2 29.03 27.66 1.37 Table 3 2. Ct Values for Durapore Samples Subject microRNA 146a microRNA 103 3p A1 27.0819 25.4094 1.672 A2 26.685 25.12 1.565 B1 28.1666 24.7352 3.431 C1 34.0511 30.8830 3.168 D1 26.9159 23.8072 3.109 E1 29.4507 25.5267 3.924 F1 29.4714 24.7691 4.702 F2 28.765 26.9 1.865 M1 28.69 27.7 .990 M2 29.01 27.72 1.290 Table 3 3. Ct Values for Microcapillary Tube Samples Subject microRNA 146a microRNA 103 3p A1 30.639 27.5900 3.049 A2 30.555 28.06 2.495 B1 28.531 23.3338 5.197 C1 33.996 33.1189 .877 D1 37.628 31.9406 5.687 E1 29.951 25.3635 4.588 F1 37.303 32.0536 5.249 F2 28.925 25.86 3.065 M1 26.97 24.49 2.480 M2 28.59 26.18 2.410

PAGE 40

40 Table 3 4. ANOVA Descriptive Statistics for Full Data Set (Ten Samples ) Measure Condition Mean Standard Deviation Standa rd Error 95% Confidence Interval Lower Limit 95% Confidence Interval Upper Limit miRNA 146a PerioPaper 30.186 3.417 1.080 27.741 32.630 Durapore 28.829 2.105 .666 27.323 30.335 Micropipette 31.309 3.735 1.181 28.637 33.981 T otal 30.108 3.223 .589 28.904 21.311 miRNA 103 3p PerioPaper 28.316 3.874 1.225 25.545 31.088 Durapore 26.257 2.085 .659 24.766 27.748 Micropipette 27.799 3.446 1.090 25.334 30.264 Total 27.458 3.238 .591 26.249 28.667 PerioPaper 1. 869 2.491 .788 0.087 3.651 Durapore 2.572 1.257 .397 1.673 3.471 Micropipette 3.510 1.578 .499 2.381 4.639 Total 2.650 1.912 .349 1.936 3.364 Table 3 5. ANOVA Descriptive Statistics for Subset of Data (Six Samples) Me asure Condition Mean Standard Deviation Standard Error 95% Confidence Interval Lower Limit 95% Confidence Interval Upper Limit miRNA 146a PerioPaper 30.671 3.437 1.403 27.064 34.278 Durapore 29.190 2.625 1.071 26.435 31.944 M icropipette 33.008 3.893 1.403 28.923 37.093 Total 30.956 3.546 0.836 29.193 32.719 miRNA 103 3p PerioPaper 27.216 2.865 1.170 24.209 30.222 Durapore 25.855 2.538 1.036 23.191 28.519 Micropipette 28.900 4.054 1.170 24.645 33.155 Total 27.324 3.284 0.774 25.691 28.957 PerioPaper 3.455 1.233 0.503 2.162 4.749 Durapore 3.335 1.007 0.411 2.278 4.391 Micropipette 4.108 1.831 0.748 2.186 6.030 Total 3.632 1.362 0.321 2.955 4.310

PAGE 41

41 Table 3 6. t test for Treated and Untreated Subjects Mea sure Group Mean Standard Deviation Standard Error miRNA 146a Untreated 28.866 2.099 0.542 Treated 31.349 3.717 0.960 miRNA 103 3p Untreated 27.486 2.983 0.770 Treated 27.430 3.580 0.920 Untreated 1.381 1.574 0.406 Treated 3.919 1.282 0.3 31

PAGE 42

42 CHAPTER 4 DISCUSSION Candidate biomarker s have been studied in dentistry for many years, with a long term goal of identifying biomarkers to aid in early detection of disease or undesired outcomes. 72 Periodontal studies have assessed GCF for potential biomarkers to indicate periodontal infla mmation and periodontal disease. 73 Some studies have identified potential biomarkers including cytokines and miRNAs. 72 Significantly, miRNAs have been detected withi n extracellular vesicles, revealing their ability transfer genetic information between cells. 74 As shown in a study by Atsawasuwan et al., miRNA 29 was detected in GCF of orthodontically treated subjects and untreated subject. 46 Furthermore, the greatest amount of this miRNA 29 was detected in a portion of the sample containing EVs. 46 Other studies also identified miRNA in exosomes, with studies by Sun et al. and Li et al. showing the presence of miRNA 214 in exosomes of osteoclasts. 33,45 Holliday et al. showed that miRNA 146a was enric hed over 80 fold in osteoclasts, providing a foundation for further study of miRNA 146a as a candidate biomarker for bone remodeling processes and orthodontic tooth movement. 34 Identifying biomarkers of tooth movement can improve orthodontic diagnosis and treatment planning. Using a non invasive method to collect and identify cons tituents of a providing the best and most efficient treatment for the patient. This could be especially beneficial for patients at risk for adverse treatment outcomes, such as root resorption during orthodontic trea tment or ankylosis of impacted teeth. Furthermore, determining the most cost effective and reliable method for sample collection would be beneficial so that in the future these practices can be easily implemented in a clinical setting and available to prac titioners.

PAGE 43

43 GCF collection has been done for many years and shows local processes at the site of collection. 51 Studies have used GCF to identify m iRNA in EVs and proteins in EVs . 34,75 While many studies use PerioPaper for GCF collection , other te chniques have been suggested, with advantages and disadvantages to each. Th erefore, we performed our study to determine whether miRNA 146a could be adequately identified within GCF samples and whether a difference in miRNA 146a expression is noted between collection techniques. The results of our study show that there i s no statistically significant difference in miRNA 146 a expression between the PerioPaper, Durapore, or Microcapillary tube techniques. Using one way ANOVA we analyzed the data twice, first with the entire dataset (Table 3 4) and second with a subset of data (Table 3 5) . Statistical analysis of the full dataset and the subset of data did not yield p values less than 0.05 for expression of miRNA 146a and miRNA 103 3p between the three techniques. Furthermore, the p value for the normalized was not less than the significance value of 0.05 for the f ull dataset or subset of data. Two data sets were analyzed for significance because some subjects were noted to be in orthodontic treatment. For completion of data analysis, the subset of data (Table 3 5) did not include samples taken from subjects that we re noted at one or both of their collection visits to be in active orthodontic treatment. Analysis of both data sets suggests that there is no significant difference in the expression of miRNA 146a between the three collection methods. With studies often u sing multiple strips or micropipettes in one sulcus to obtain GCF, we designed our study as described to be more clinically applicable.

PAGE 44

44 Many studies using GCF for analysis use PerioPaper. These strips come cut, sterilized, packaged and are easy to insert and remove from the gingival sulcus. If interested in determining the volume of a sample, a Periotron 8000® (Oraflow, Plainview, NY, USA) machine can be purchased and used. Durapore filter membrane is less widely used and must be cut into strips to be used . While this requires more time for the operator, it is a more economic material. Both PerioPaper and Durapore are easy to use and comfortable for the subject but due to the nature of the materials, elution of the sample can lead to particles from both str ips entering the media. Contrasting, a microcapillary tube would collect and elute a more pure sample. Depending on the volume needed, microcapillary tubes can be purchased with different diameters. The microcapillary tube has a defined volume and should be a n efficient way to calculate volume. However, GCF volume is minimal and often the tube is not filled entirely , making it difficult to determine sample volume. Additionally, this technique is tiring for both the operator and the subject. Collection at each gingival sulcus occurred for five minutes, which is much longer than the thirty second collection time for PerioPaper and Durapore strips. The calculated f old change in gene expression of miRNA 146a was 1.63 for Durapore strips and 3.12 for microcapillary tubes. T hese values represent a decrease in fold change expression of miRNA 146a compared to Periopaper , with the microcapillary tubes showing the largest decrease in fold change of gene expression. While our results showed no significant difference in mi RNA 146a expression between the three techniques, the micropipettes a re more challenging to use, require more time for GCF collection, and are less clinically applicable than the PerioPaper and Durapore strips.

PAGE 45

45 With some subjects undergoing orthodontic trea tment, an additional analysis was performed to determine whether there was a difference in miRNA 146a expression between treated and untreated subjects. The data is shown in Table 3 6 as the average Ct values for each group for miRNA 146a, miRNA 103 3p and . I ndependent sample t tests were performed and the p values for miRNA 146a and are p < .05 and p <.001 , respectively. Both p values are less than the significance value of 0.05, meaning there is a significant difference in expression of miRNA 146a between treated and untreated subjects. These results show a significantly greater expression of miRNA 146a in the treated subjects. The p value for miRNA 103 3p is greater than 0.05 and, as seen in Table 3 6, the mean Ct values for both groups only differ by 0.056. With mean values so similar for miRNA 103 3p , it suggests that it is stable and a good choice for a reference gene. Furthermore, the calculated fold change in gene expression of miRNA 146a, 5.81, s ignifies a greater expression of miRNA 146a in s ubjects undergoing orthodontic treatment. This study agrees with a study by Holliday et al. , which detected an 80 fold chang e in miRNA 146a expression in osteoclasts . 34 The results of this study suggest miRNA 146a as a potential biomarker for orthodontic tooth movement and is promising for future studies. A limitation of our study is the small sample size. Increasing the number of subjects would be beneficial and provide more power to the study. While we aimed to be more practical in sample coll ection, the use of two collection strips or tubes per tooth is still not ideal for a clinical scenario. The use of one strip, or tube, would be more clinically applicable. RNA isolation, cDNA synthesis, and preparation of the PCR plate wells are technique sensitive and prone to error and contamination. Error in these

PAGE 46

46 processes can affect the quality and quantity of target miRNA expressed. Our study does include subjects having orthodontic treatment. In retrospect, making treatment an exclusion criteria woul d make the data set more homogenous for comparison of GCF collection techniques.

PAGE 47

47 CHAPTER 5 CONCLUSIONS GCF collection is non invasive and identification of biomarkers in GCF can improve treatment planning, assist in early identification of adverse outco mes, and may lead to therapeutic treatment approaches. The objective of this study was to determine if miRNA 146a could be sufficiently collected by GCF methods and which method was best for collecting this miRNA. Results from this study suggest there is n o difference in the PerioPaper, Durapore filter membrane, or microcapillary tube techniques for expression of miRNA 146a in GCF . Additional analysis of the data shows a significantly greater express ion of miRNA 146a in subjects undergoing orthodontic treatment compar ed with untreated subjects. These results suggest there is promise for identification of miRNA 146a as a candidate biomarker for orthodontic tooth movement.

PAGE 48

48 LIST OF REFERENCES 1. Velu VK, Ramesh R, Srinivasan AR. Circulating MicroRNA s as Biomarkers in Health and Disease. J Clin Diagn Res 2012;6:1791 1795. 2. Vidigal JA, Ventura A. The biological functions of miRNAs: lessons from in vivo studies. Trends Cell Biol 2015;25:137 147. 3. Padmashree D, Ramachandraswamy N. Identification and characterization of conserved miRNAs with its targets mRNA in Trichinella Spiralis. Bioinformation 2016;12:279 284. 4. Pillai RS. MicroRNA function: multiple mechanisms for a tiny RNA? RNA 2005;11:1753 1761. 5. Bhaskaran M, Mohan M. MicroRNAs: history, bio genesis, and their evolving role in animal development and disease. Vet Pathol 2014;51:759 774. 6. Ardekani AM, Naeini MM. The Role of MicroRNAs in Human Diseases. Avicenna J Med Biotechnol 2010;2:161 179. 7. Ha TY. MicroRNAs in Human Diseases: From Cancer to Cardiovascular Disease. Immune Netw 2011;11:135 154. 8. Kamal MA, Mushtaq G, Greig NH. Current Update on Synopsis of miRNA Dysregulation in Neurological Disorders. CNS Neurol Disord Drug Targets 2015;14:492 501. 9. Leung AKL. The Whereabouts of microRN A Actions: Cytoplasm and Beyond. Trends Cell Biol 2015;25:601 610. 10. Macfarlane LA, Murphy PR. MicroRNA: Biogenesis, Function and Role in Cancer. Curr Genomics 2010;11:537 561. 11. Dar D, Sorek R. Bacterial Noncoding RNAs Excised from within Protein Codi ng Transcripts. mBio 2018;9. 12. Johanson TM, Lew AM, Chong MM. MicroRNA independent roles of the RNase III enzymes Drosha and Dicer. Open Biol 2013;3:130144. 13. Wu K, He J, Pu W, Peng Y. The Role of Exportin 5 in MicroRNA Biogenesis and Cancer. Genomics Proteomics Bioinformatics 2018;16:120 126. 14. Sontheimer EJ. Assembly and function of RNA silencing complexes. Nat Rev Mol Cell Biol 2005;6:127 138.

PAGE 49

49 15. Wilson RC, Doudna JA. Molecular mechanisms of RNA interference. Annu Rev Biophys 2013;42:217 239. 16. Wei K, Wu L, Chen Y, Lin Y, Wang Y, Liu X et al. Argonaute protein as a linker to command center of physiological processes. Chin J Cancer Res 2013;25:430 441. 17. Ronemus M, Vaughn MW, Martienssen RA. MicroRNA targeted and small interfering RNA mediated m RNA degradation is regulated by argonaute, dicer, and RNA dependent RNA polymerase in Arabidopsis. Plant Cell 2006;18:1559 1574. 18. Pogue AI, Clement C, Hill JM, Lukiw WJ. Evolution of microRNA (miRNA) Structure and Function in Plants and Animals: Relevan ce to Aging and Disease. J Aging Sci 2014;2. 19. van den Berg A, Mols J, Han J. RISC target interaction: cleavage and translational suppression. Biochim Biophys Acta 2008;1779:668 677. 20. Aizer A, Shav Tal Y. Intracellular trafficking and dynamics of P bo dies. Prion 2008;2:131 134. 21. Shivdasani RA. MicroRNAs: regulators of gene expression and cell differentiation. Blood 2006;108:3646 3653. 22. Kinney JS, Ramseier CA, Giannobile WV. Oral fluid based biomarkers of alveolar bone loss in periodontitis. Ann N Y Acad Sci 2007;1098:230 251. 23. d'Apuzzo F, Cappabianca S, Ciavarella D, Monsurrò A, Silvestrini Biavati A, Perillo L. Biomarkers of periodontal tissue remodeling during orthodontic tooth movement in mice and men: overview and clinical relevance. Scient ificWorldJournal 2013;2013:105873. 24. Alhashimi N, Frithiof L, Brudvik P, Bakhiet M. Orthodontic tooth movement and de novo synthesis of proinflammatory cytokines. Am J Orthod Dentofacial Orthop 2001;119:307 312. 25. Kereshanan S, Stephenson P, Waddington R. Identification of dentine sialoprotein in gingival crevicular fluid during physiological root resorption and orthodontic tooth movement. Eur J Orthod 2008;30:307 314. 26. Farahani M, Safavi SM, Dianat O, Khoramian Tusi S, Younessian F. Acid and Alkalin e Phosphatase Levels in GCF during Orthodontic Tooth Movement. J Dent (Shiraz) 2015;16:237 245. 27. Lerner UH. Inflammation induced bone remodeling in periodontal disease and the influence of post menopausal osteoporosis. J Dent Res 2006;85:596 607.

PAGE 50

50 28. Hu ynh N, VonMoss L, Smith D, Rahman I, Felemban MF, Zuo J et al. Characterization of Regulatory Extracellular Vesicles from Osteoclasts. J Dent Res 2016;95:673 679. 29. Deng L, Wang Y, Peng Y, Wu Y, Ding Y, Jiang Y et al. Osteoblast derived microvesicles: A novel mechanism for communication between osteoblasts and osteoclasts. Bone 2015;79:37 42. 30. Harding CV, Heuser JE, Stahl PD. Exosomes: looking back three decades and into the future. J Cell Biol 2013;200:367 371. 31. H Rashed M, Bayraktar E, K Helal G, Abd Ellah MF, Amero P, Chavez Reyes A et al. Exosomes: From Garbage Bins to Promising Therapeutic Targets. Int J Mol Sci 2017;18. 32. Oliveto S, Mancino M, Manfrini N, Biffo S. Role of microRNAs in translation regulation and cancer. World J Biol Chem 2017; 8:45 56. 33. Sun W, Zhao C, Li Y, Wang L, Nie G, Peng J et al. Osteoclast derived microRNA containing exosomes selectively inhibit osteoblast activity. Cell Discov 2016;2:16015. 34. Holliday LS, McHugh KP, Zuo J, Aguirre JI, Neubert JK, Rody WJ. Exosomes: novel regulators of bone remodelling and potential therapeutic agents for orthodontics. Orthod Craniofac Res 2017;20 Suppl 1:95 99. 35. Tahamtan A, Teymoori Rad M, Nakstad B, Salimi V. Anti Inflammatory MicroRNAs and Their Potential for Inflammatory Diseas es Treatment. Front Immunol 2018;9:1377. 36. Saba R, Sorensen DL, Booth SA. MicroRNA 146a: A Dominant, Negative Regulator of the Innate Immune Response. Front Immunol 2014;5:578. 37. Shams K, Kurowska Stolarska M, Schütte F, Burden AD, McKimmie CS, Graham GJ. MicroRNA 146 and cell trauma down regulate expression of the psoriasis associated atypical chemokine receptor ACKR2. J Biol Chem 2018;293:3003 3012. 38. Tang Y, Luo X, Cui H, Ni X, Yuan M, Guo Y et al. MicroRNA 146A contributes to abnormal activation o f the type I interferon pathway in human lupus by targeting the key signaling proteins. Arthritis Rheum 2009;60:1065 1075. 39. Pauley KM, Cha S. miRNA 146a in rheumatoid arthritis: a new therapeutic strategy. Immunotherapy 2011;3:829 831. 40. Olsen I, Sing hrao SK, Osmundsen H. Periodontitis, pathogenesis and progression: miRNA mediated cellular responses to. J Oral Microbiol 2017;9:1333396.

PAGE 51

51 41. Motedayyen H, Ghotloo S, Saffari M, Sattari M, Amid R. Evaluation of MicroRNA 146a and Its Targets in Gingival Tis sues of Patients With Chronic Periodontitis. J Periodontol 2015;86:1380 1385. 42. Xie YF, Shu R, Jiang SY, Liu DL, Ni J, Zhang XL. MicroRNA 146 inhibits pro inflammatory cytokine secretion through IL 1 receptor associated kinase 1 in human gingival fibrobl asts. J Inflamm (Lond) 2013;10:20. 43. Kuang W, Zheng L, Xu X, Lin Y, Lin J, Wu J et al. Dysregulation of the miR 146a Smad4 axis impairs osteogenesis of bone mesenchymal stem cells under inflammation. Bone Res 2017;5:17037. 44. Nakasa T, Shibuya H, Nagata Y, Niimoto T, Ochi M. The inhibitory effect of microRNA 146a expression on bone destruction in collagen induced arthritis. Arthritis Rheum 2011;63:1582 1590. 45. Li D, Liu J, Guo B, Liang C, Dang L, Lu C et al. Osteoclast derived exosomal miR 214 3p inhib its osteoblastic bone formation. Nat Commun 2016;7:10872. 46. Atsawasuwan P, Lazari P, Chen Y, Zhou X, Viana G, Evans CA. Secretory microRNA 29 expression in gingival crevicular fluid during orthodontic tooth movement. PLoS One 2018;13:e0194238. 47. Chen J , Li K, Pang Q, Yang C, Zhang H, Wu F et al. Identification of suitable reference gene and biomarkers of serum miRNAs for osteoporosis. Sci Rep 2016;6:36347. 48. Yao Y, Suo AL, Li ZF, Liu LY, Tian T, Ni L et al. MicroRNA profiling of human gastric cancer. Mol Med Rep 2009;2:963 970. 49. Song J, Bai Z, Han W, Zhang J, Meng H, Bi J et al. Identification of suitable reference genes for qPCR analysis of serum microRNA in gastric cancer patients. Dig Dis Sci 2012;57:897 904. 50. Gupta S, Chhina S, Arora SA. A sy stematic review of biomarkers of gingival crevicular fluid: Their predictive role in diagnosis of periodontal disease status. J Oral Biol Craniofac Res 2018;8:98 104. 51. Barros SP, Williams R, Offenbacher S, Morelli T. Gingival crevicular fluid as a sourc e of biomarkers for periodontitis. Periodontol 2000 2016;70:53 64. 52. Lamster IB. Evaluation of components of gingival crevicular fluid as diagnostic tests. Ann Periodontol 1997;2:123 137. 53. Griffiths GS. Formation, collection and significance of gingiv al crevice fluid. Periodontol 2000 2003;31:32 42.

PAGE 52

52 54. Del Fabbro M, Galardi E, Weinstein R, Bulfamante G, Miserocchi G. Fluid dynamics of gingival tissues. J Periodontal Res 1998;33:328 334. 55. Pashley DH. A mechanistic analysis of gingival fluid producti on. J Periodontal Res 1976;11:121 134. 56. Rody WJ, Wijegunasinghe M, Holliday LS, McHugh KP, Wallet SM. Immunoassay analysis of proteins in gingival crevicular fluid samples from resorbing teeth. Angle Orthod 2016;86:187 192. 57. Bevilacqua L, Biasi MD, L orenzon MG, Frattini C, Angerame D. Volumetric Analysis of Gingival Crevicular Fluid and Peri Implant Sulcus Fluid in Healthy and Diseased Sites: A Cross Sectional Split Mouth Pilot Study. Open Dent J 2016;10:131 138. 58. Drummond S, Canavarro C, Perinetti G, Teles R, Capelli J. The monitoring of gingival crevicular fluid volume during orthodontic treatment: a longitudinal randomized split mouth study. Eur J Orthod 2012;34:109 113. 59. Rody WJ, Wijegunasinghe M, Wiltshire WA, Dufault B. Differences in the g ingival crevicular fluid composition between adults and adolescents undergoing orthodontic treatment. Angle Orthod 2014;84:120 126. 60. Khurshid Z, Mali M, Naseem M, Najeeb S, Zafar MS. Human Gingival Crevicular Fluids (GCF) Proteomics: An Overview. Dent J (Basel) 2017;5. 61. Subbarao KC, Nattuthurai GS, Sundararajan SK, Sujith I, Joseph J, Syedshah YP. Gingival Crevicular Fluid: An Overview. J Pharm Bioallied Sci 2019;11:S135 S139. 62. Sueda T, Bang J, Cimasoni G. Collection of gingival fluid for quantitat ive analysis. J Dent Res 1969;48:159. 63. Guentsch A, Kramesberger M, Sroka A, Pfister W, Potempa J, Eick S. Comparison of gingival crevicular fluid sampling methods in patients with severe chronic periodontitis. J Periodontol 2011;82:1051 1060. 64. Johnso n RB, Streckfus CF, Dai X, Tucci MA. Protein recovery from several paper types used to collect gingival crevicular fluid. J Periodontal Res 1999;34:283 289. 65. Garibyan L, Avashia N. Polymerase chain reaction. J Invest Dermatol 2013;133:1 4. 66. Goate AM. Molecular Biology. Alcohol Health Res World 1995;19:217 220. 67. Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA et al. Real time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 2006;19:165 256.

PAGE 53

53 68. Ruijter JM, Lorenz P, Tuomi JM, Hecker M, van den Hoff MJ. Fluorescent increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency. Mikrochim Acta 2014;181:1 689 1696. 69. Pabinger S, Rödiger S, Kriegner A, Vierlinger K, Weinhäusel A. A survey of tools for the analysis of quantitative PCR (qPCR) data. Biomol Detect Quantif 2014;1:23 33. 70. Karlen Y, McNair A, Perseguers S, Mazza C, Mermod N. Statistical signif icance of quantitative PCR. BMC Bioinformatics 2007;8:131. 71. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real time quantitative PCR and the 2( Delta Delta C(T)) Method. Methods 2001;25:402 408. 72. Branca F, Hanley AB, Pool Z obel B, Verhagen H. Biomarkers in disease and health. Br J Nutr 2001;86 Suppl 1:S55 92. 73. Jaedicke KM, Preshaw PM, Taylor JJ. Salivary cytokines as biomarkers of periodontal diseases. Periodontol 2000 2016;70:164 183. 74. Di Liegro CM, Schiera G, Di Lieg ro I. Extracellular Vesicle Associated RNA as a Carrier of Epigenetic Information. Genes (Basel) 2017;8. 75. Rody WJ, Chamberlain CA, Emory Carter AK, McHugh KP, Wallet SM, Spicer V et al. The proteome of extracellular vesicles released by clastic cells di ffers based on their substrate. PLoS One 2019;14:e0219602.

PAGE 54

54 BIOGRAPHICAL SKETCH Kelsey Cronauer Wahl was born and raised in South Florida by her parents Edward and Jul ie Cronauer. She completed her u ndergraduate education at Vanderbilt University where she majored in History of Art with a Pre Dental concentration. She pursued a career in dentistry and was accepted into the University of Florida College of Dentistry. Upon graduating, she began her specialty training in orthodontics at the University of Fl orida College of Dentistry, Department of Orthodontics. In April 2019 she married her wonderful husband, Tyler Wahl. Her husband, brother, Edward, and parents, Edward and Julie, have supported her every step of the way. Kelsey plans to practice orthodontic s in Florida after graduation in May 2020.