Establishment Of MiRNA Expression Proming Of Oral Submucous Fibrosis And Preliminary Investigation Of Its Function

Background and Objective:Oral submucous fibrosis (OSF) is a chronic pre-cancerous disease of insidious onset characterized by the deposition of fibrous tissues in the oral submucosa, with about7.6%malignant transformation rate. Recent epidemiological studies show an increase in the high incidence area, incidence rate and malignant transformation of OSF. many factors such as chewing betel nut, disorders of collagen metabolism, immune, genetic, microcirculation and hemorheology play important roles in the pathogenesis of OSF. We have previous found that Salvia in combination with low-dose prednisolone was effective in the treatment of OSF. However, the exact pathogenesis of OSF is not yet known, as well as the treatment.MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs that post-transcriptionally regulate gene expression, involved in regulation of various physiological and pathological processes such as growth and development, organogenesis, hematopoiesis, cell proliferation, differentiation and apoptosis. Many recent reports have demonstrated the role of miRNAs in myocardial fibrosis, kidney fibrosis and liver fibrosis. However, the relationship between miRNA and OSF is not clearly. To provide new clues to the pathogenesis and treatment of OSF, our study investigates the function and mechanism of miRNA in the OSF.Methods:(1) select three case with typical clinical pathological features of OSF and its paired normal tissue (internal control). The miRNA expression profiles between the OSF and its paired control were compared by using the Affymetrix analysis. After extraction miRNA from the mid-late OSF tissue specimens and the control normal tissue, some of the differentially expressed miRNAs were validated by real-time quantitative PCR.(2) Choose the computational prediction of miRNA targets from the TargetScan sites (www.targetscan.org,pictar.mdc-berlin.de/cgi-bin/PicTar vertebrate.cg,www.mirbase.org).(3) Treatment the primary normal oral mucoses cells by Arecoline, we detected the interesting miRNA expression at72h. Following, the primary cells OSF-Fb were treated with a combination of Salvia and low-dose prednisolone, then detection the different miRNAs at72h.(4) Using a variety of bioinformatics software tools, it realved that COL4A4was the putative target of miR-203. Examine the expreesion of COL4A4in the indcated cells by Western blot analysis.(5) we integrated a fragment of the COL4A43'UTR containing the target sequence into the pMIR-REPORT luciferase vector. Then, the COL4A43'UTR-luciferase construct and miR-203were contransfected into293T cells. Beta-gal was used as a control to monitor transfection efficiency.24h after transfection, cells were harvested and luciferase activity was measured.Results:(1) The miRNA microarray chip analysis identified19miRNAs differentially expressed with1.5-fold or greater change. Of these,13microRNAs were upregulated and6were downregulated. The results of expression data obtained by microarray analysis were in good agreement with those obtained by real-time quantitative PCR analyses.(2) The expression of miRNAs from the samples of middle-and late-stage OSF were consistent with the miRNA expression profiles observed from the microarray data. (3) Microarray-Bioinformatics analysis revealed that the miR-203, miR-23b, miR-200c of Oral Submucous Fibrosis play important roles in the development of OSF and subsequently malignant transformation.(4) Arecoline exposure induced changes in the expression of miRNAs in normal mucosal cells. Salvia in combination with low doses of prednisolone could reverse the miRNA expression.(5)Bioinformatics analysis revealed that the COL4A43'UTR has a conserved site (site1) and3non-conserved sites (site2, site3, site4) complementary to the miR-203.(6) The expression of COL4A4in OSF was significantly higher than normal control. Arecoline induced the expression of COL4A4in normal mucosal cells. Salvia in combination with low-dose prednisolone downregulated the expression of COL4A4in OSF cells.(7) miR-203could significantly inhibit site1-luciferase reporter activity, partially inhibited site3-and site4-luciferase reporter activity, while no significant effect on site3-luciferase reporter activity.Conclusion:(1) construction of miRNA expression profiling of the OSF.(2) The clinical evidences proved that miRNAs play essential role in the OSF.(3) In vitro, Arecoline changed the expression of miRNAs in normal oral mucosal cells, while the Salvia in combination with prednisolone could reversed it.(4) miR-203was negatively correlated with the expression of COL4A4protein in OSF. Moreover, miR-203can directly repress COL4A4mRNA.