Arecoline Inhibits The Expression Of MiR-134-3p In Fibroblasts And Participates In The Formation Of ODF

Objective:The purpose of this study was to screen the mi RNA associated with the pathogenesis of Oral Submucosal fibrosis(OSF),and explore the mechanism of its influence on OSF development,so as to provide a theoretical basis for personalized treatment of OSF.Methods:1)RNA sequencing was used to screen for mi RNAs significantly differentially expressed in normal mucosal tissues and OSF tissues.Combined with literature review the screen with the onset of OSF micrornas associated;Real-time fluorescence quantitative PCR and in situ hybridization were used to further verify the results.2)In situ hybridization and immunohistochemistry were combined to detect and analyze the main effector cells of OSF pathogenicity associated mi RNA.3)The effects of OSF pathogenesis related mi RNA on the expression and secretion of COL1A2 and COL3A1 in fibroblasts were detected by transfection with mimic/inhibitor,enzyme-linked immunosorbent assay(ELISA)and real-time quantitative PCR.4)The effects of arecoline intervention(5 μg/m L)at different times(0Hr、 6Hr、12Hr、24Hr)on the expression of mi RNA,COL1A2 and COL3A1 related to OSF were detected by real-time quantitative PCR.The expression of COL1A2 and COL3A1 in fibroblasts was detected by realtime quantitative PCR after 24 hours of arecoline intervention after transfection with mimic.5)The expression level of OSF pathogenesis related mi RNA in 64 saliva samples was detected by real-time fluorescence quantitative PCR,and its possibility as a humoral detection molecule in personalized therapy was preliminarily analyzed.Results:1)RNA-seq screened 30 mi RNAs with significant differential expression;mi R-134-3p may be associated with the pathogenesis of OSF,and the verification found that mi R-134-3p was significantly downregulated in OSF tissues,which was consistent with the sequencing results.2)Combined with in situ hybridization and immunohistochemical analysis,it was found that mir-134-3p hybrid overlapped with fibroblasts stained with fibroblast specific protein-1.3)Real-time fluorescence quantitative detection of mi R-134-3p mimic/inhibitor fibroblasts showed that m RNA and protein levels of COL1A2 and COL3A1 in fibroblasts were significantly decreased when mi R-134-3p was overexpressed.When mi R-134-3p was silenced,the results were completely reversed.4)The expression of mi R-134-3p in fibroblasts decreased with the prolongation of arecoline intervention time.The expression of COL1A2 and COL3A1 was highest at 24 Hr and 6Hr respectively.Overexpression of mir-134-3p significantly reduced the expression of COL1A2 and COL3A1 in arecoline-induced fibroblasts within 24 Hr.5)The expression of mi R-134-3p in saliva of OSF patients was significantly lower than that of the normal population,which was consistent with the results of sequencing and tissue verification.Conclusions:Arecoline can inhibit the expression of mi R-134-3p in fibroblasts,promote collagen secretion in fibroblasts,and lead to collagen accumulation in local tissues,which may be involved in OSF formation.There are 9 figures,8 tables and 71 references...