The Effect Of MicroRNA-191-5p On Proliferation And Metastasis Of Oral Squamous Cell Carcinoma Cells Through Targeting TJP1

Purpose: The aim of this project is to explore the expression and biological function of miR-191-5p in oral squamous cell carcinoma through database analysis,and to investigate its role and molecular mechanism in OSCC proliferation and invasion,providing new ideas for clinical targeted therapy.The main points are as follows:(1)Study the biological behavior of miR-191-5p in the occurrence and development process of oral squamous cell carcinoma.(2)Preliminary exploration of the molecular mechanism of miR-191-5p regulating proliferation,migration and invasion of oral squamous cell carcinoma.Methods:(1)The expression of miR-191-5p in oral squamous cell carcinoma patient samples was analyzed by bioinformatics and the target of miR-191-5p was predicted,and the target genes were predicted and intersected by Targetscan,miRDB and miRmap databases,and then the ENCORI database was used for co-expression correlation analysis and screening;(2)In vitro experiments to explore the effect of miR-191-5p on the proliferation,migration and invasion ability of oral squamous cell carcinoma cells.The transfected overexpression group and knockdown group verified the transfection efficiency by qRT-PCR,and then the effects on cell proliferation were observed by CCK8,cloning experiment and EdU proliferation experiment,and the effects on the migration and invasion of squamous cell carcinoma cells were verified by scratch migration experiment and Transwell invasion experiment,respectively.(3)To explore the effect of miR-191-5p on epithelial-mesenchymal transition EMT: the protein expression content of epithelial markers E-cadherin and mesenchymal markers N-cadherin and Vimentin in the miR-191-5p overexpression group and knockdown group were detected by WB experiments;(4)Study on the mechanism of action of miR-191-5p on OSCC: the regulation of miR-191-5p on the predicted target gene TJP1 was verified by the diluciferase gene detection report,and then the m RNA and protein levels of miR-191-5p overexpression and knockdown were detected by qRT-PCR and WB,respectively;(5)Further explore the role of TJP1: si RNATJP1 was transfected into cells with low expression of miR-191-5p,si RNA-NC was transfected as a control,qRT-PCR verified the transfection efficiency,WB verified the expression difference of protein TJP1,and the effect of downregulation of TJP1 on the proliferation,migration and invasion ability of Cal-27 cells was observed.Results:(1)Through bioinformatics analysis,it was found that the expression of miR-191-5p in OSCC was significantly higher than that of normal oral tissue.The Targetscan,miRDB,and miRmap databases predicted the complementary target site TJP1 in the 3’ UTR of miR-191-5p,and TJP1 was significantly less expressed in OSCC tissues than in its paired noncancerous tissues.The correlation analysis of miR-191-5p and TJP1 was carried out through the ENCORI database,and the correlation was negative.(2)In vitro cell experimental results showed that overexpression of miR-191-5p could significantly promote the proliferation,migration and invasion of Cal-27 cells,while knocking out miR-191-5p could significantly inhibit the proliferation and migration invasion of Cal-27 cells.(3)The results of WB western blot showed that the expression of E-cadherin in the miR-191-5p inhibitor group was increased relative to the Control group and the negative control group,the expression of N-cadherin and Vimentin was lower,and the expression of E-cadherin in the miR-191-5p mimics group was lower than that of the Control group and the negative control group,and the expression of Ncadherin and Vimentin was higher.(4)TJP1 was validated as a direct target for miR-191-5p.Diluciferase reporter gene testing confirms the targeted relationship between miR-191-5p and TJP1.The PCR and WB results showed that the expression of TJP1 in the miR-191-5p inhibitor group was higher relative to the control group and the negative control group,and the expression of TJP1 in the miR-191-5p mimics group was lower relative to the control group and the negative control group,and it was seen that miR-191-5p and TJP1 were negatively correlated with the prediction results.(5)The proliferation,migration and invasion ability of Cal-27 cells was significantly enhanced after simultaneous downregulation of TJP1 in miR-191-5p inhibitor,indicating that downregulation of TJP1 could reverse the inhibition of proliferation and migration of Cal-27 cells by miR-191-5p to a certain extent.Conclusions: miR-191-5p is highly expressed in OSCC,and overexpressed miR-191-5p can promote the proliferation,migration and invasion of OSCC in vitro,and the downregulation of miR-191-5p may inhibit the proliferation and metastasis of oral squamous cell carcinoma by targeting TJP1,and increase tumor metastasis by regulating the EMT pathway.