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Overexpression Of Mist-1 In Mesenchymal Stem Cells Induces Amylase Expression

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Title:
Overexpression Of Mist-1 In Mesenchymal Stem Cells Induces Amylase Expression
Series Title:
19th Annual Undergraduate Research Symposium
Creator:
Daduya, Hannah
Language:
English
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Undetermined

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Center for Undergraduate Research
Center for Undergraduate Research
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Conference papers and proceedings
Poster

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Abstract:
Saliva produced by salivary acinar cells provides biological functions critical for oral health. Patients with Sjögren’s syndrome or radiation therapy for head and neck cancer suffer from extreme dryness of the mouth due to acinar cell loss. Mesenchymal stem cells (MSC) have provided a promising tool to regenerate damaged organ tissues. Previous research identified six key transcription factors expressed in differentiating mouse MSCs into salivary precursors in a co-culture system. The aim of this study was to examine the roles of Muscle, Intestine and Stomach Expression 1 (MIST-1), in the induction of salivary precursor cell markers in MSCs. cDNA of MIST-1 was amplified by PCR with primers that contain sequence for XhoI and HindIII restriction sites at 5' and 3' ends, respectively. MIST-1 cDNA and pcDNA3.1/BioID/Myc vector (Addgene) were digested with the restriction enzymes. The purified MIST-1 cDNA was cloned into the vector by ligation. Positive E. Coli colonies were selected after transformation. MSCs were transfected with the correctly ligated-plamid DNA. cMyc-tagged MIST-1 recombinant protein expression was detected in the nucleus by immunocytochemistry. Amylase, which serves as an acinar cell marker, was expressed in the cytoplasm of MIST-1 transfectants; however, the expression level was relatively low. In conclusion, MIST-1 was successfully overexpressed in MSCs, which resulted in the induction of amylase protein expression and will be further characterized to determine the critical roles of MIST-1 in driving MSCs into salivary epithelial precursors. ( en )
General Note:
Research authors: Hannah Daduya, Rahae Miller, Seunghee Cha - University of Florida
General Note:
University Scholars Program
General Note:
Faculty Mentor: Saliva produced by salivary acinar cells provides biological functions critical for oral health. Patients with Sjögren’s syndrome or radiation therapy for head and neck cancer suffer from extreme dryness of the mouth due to acinar cell loss. Mesenchymal stem cells (MSC) have provided a promising tool to regenerate damaged organ tissues. Previous research identified six key transcription factors expressed in differentiating mouse MSCs into salivary precursors in a co-culture system. The aim of this study was to examine the roles of Muscle, Intestine and Stomach Expression 1 (MIST-1), in the induction of salivary precursor cell markers in MSCs. cDNA of MIST-1 was amplified by PCR with primers that contain sequence for XhoI and HindIII restriction sites at 5' and 3' ends, respectively. MIST-1 cDNA and pcDNA3.1/BioID/Myc vector (Addgene) were digested with the restriction enzymes. The purified MIST-1 cDNA was cloned into the vector by ligation. Positive E. Coli colonies were selected after transformation. MSCs were transfected with the correctly ligated-plamid DNA. cMyc-tagged MIST-1 recombinant protein expression was detected in the nucleus by immunocytochemistry. Amylase, which serves as an acinar cell marker, was expressed in the cytoplasm of MIST-1 transfectants; however, the expression level was relatively low. In conclusion, MIST-1 was successfully overexpressed in MSCs, which resulted in the induction of amylase protein expression and will be further characterized to determine the critical roles of MIST-1 in driving MSCs into salivary epithelial precursors. - Center for Undergraduate Research, University Scholars Program

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University of Florida
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Copyright Hannah Daduya. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Overexpression of MIST 1 in Mesenchymal Stem Cells Induced Amylase Expression Hannah Daduya, Rehae Miller, Mahmoud Mona, Hui Li, K Lee and Seunghee Cha Oral and Maxillofacial Diagnostic Sciences, College of Dentistry, University of Florida, Gainesville, FL 32610 Materials & Methods Conclusions References Introduction Results Saliva is produced from salivary acinar cells. It provides various functions important for oral health, such as digesting food, cleansing food debris, lubricating oral mucosa, and buffering acidic pH to prevent caries; therefore, it is vital to everyday life. Patients with syndrome or radiation therapy for head and neck cancer suffer from extreme dryness of the mouth due to salivary acinar cell loss. Current treatments to aid dry mouth are usually palliative as the damage to the cells is irreversible. Bone marrow derived mouse mesenchymal stem cells ( mMSC ) may provide a tool to regenerate damaged organ tissues, specifically in this case, the salivary gland acinar cells for functional restoration. Our previous research identified six key transcription factors expressed in differentiating mouse mMSCs into salivary precursors. For my research, Muscle, Intestine and Stomach Expression 1 (MIST 1) was chosen, which belongs to the family of basic helix loop helix ( bHLH ) proteins, to understand its role in the induction of salivary gland marker proteins in the differentiating mMSCs MIST 1 is known to affect differentiation of serous exocrine cells including pancreas, salivary glands, gastric epithelium, and mammary gland alveolar cells. 1) YK Park, J Koh AE Gauna S Chen, S Cha. Identification of Regulatory Factors for Mesenchymal Stem Cell Derived Salivary Epithelial Cells in a Co Culture System. PLoS ONE 2014; 9(11): e112158. doi:10.1371/journal.pone.0112158 2) YJ Park, J Koh JT Kwon YS Park, L Yang, S Cha Uncovering stem cell differentiation factors for salivary gland regeneration by quantitative analysis of differential proteomes PLoS One 2017; 12(2): e0169677. cDNAs of MIST 1 were amplified by PCR with primers that contain sequence for XhoI and HindIII restriction sites at 5' and 3' ends. MIST 1 and the vector, pcDNA3.1/ BioID / Myc ( Addgene ), were digested with the restriction enzymes and purified the digestion products by gel elution. The purified MIST 1 cDNA was cloned into the vector by ligation, referred to as pcDNA3.1/MycMIST 1/ BioID The ligated products were introduced into E. Coli bacteria by transformation. Amplification of the constructs was carried out by selecting and growing positive clones that contained the gene. The gene was introduced into mMSCs by transfection. The mMSCs that express the gene of interest were verified and the effect of the overexpressed MIST 1 on the cells were examined. MIST 1 was successfully overexpressed in the nucleus of mMSCs which resulted in induction of amylase expression This construct will be utilized further to characterize the critical roles of MIST 1 in the transdifferentiation of mMSCs into salivary epithelial precursors. Figure 1. Vector B. Vector containing MIST 1 gene are confirmed through restriction digestion with XhoI and HindIII enzymes. MIST 1 pcDNA3.1/ BioID / Myc Figure 2. a d. Transfected cells. Immunochemistry was conducted to detect Amylase protein expression. a MIST 1 is expressed in the nucleus ( ). b. Amylase which serves as an acinar cell marker, is expressed in the cytoplasm ( ); although, the expression level was low. Nuclei are detected by DAPI staining (blue). e. Untransfected cells (x20) 3B. MIST 1 Induces Amylase Protein Expression RELATIVE INTENSITY Figure 3. a. MIST 1 transfected cells shows an increase Amylase gene expression. b. Western blotting shows and increase of Amylase protein expression in MIST 1 transfected cells. The h uman s alivary g land l ysate ( hSGL ) was compared alongside the untransfected and transfected cells. (* p<0.5 **p<0.06 by Student T test) MIST 1 Amylase Nucleus Composite MIST 1 induced and increased Amylase gene and protein in transfected cells. Amylase gene expression was measured by PCR. Amylase protein expression was detected by W estern blotting. AMY GADPH 55KDa 55KDa RELATIVE EXPRESSION 3A. MIST 1 Amylase Gene Expression cMyc tagged MIST 1 was cloned into XhoI / HindIII sites of pcDNA3.1 / Myc / BioID The construct ( pcDNA /MycMIST 1/ BioID ) was confirmed by restriction digestion. 6.4 Kb 0.594 Kb a. b. m MSCs were transfected with pcDNA /MycMIST 1/ BioID MIST 1 induced Amylase expression as detected by immunocytochemistry (ICC), PCR and Western blotting. a d c b e