The Mechanism Of HPV Promoting Invasion And Metastasis Of Cervical Cancer Cells Through Upregulating ICAT

Objective: This study aims to explore the potential molecular mechanism of HPV16 promoting the proliferation,invasion,metastasis,and the EMT progression of cervical cancer through regulating the expression of ICAT.Methods:(1)Exogenous knockdown of HPV16-positive cervical cancer cell lines SiHa and CasKi HPV16 E6 and E7 by small fragments of interfering RNA was used to detect the expression levels of miR-23b-3p and ICAT in SiHa and CasKi cells.(2)The direct binding sites of miR-23b-3p on ICAT was predicted by bioinformatics website: Target Scan(http://www.targetscan.org),Star Basev3.0(http://starbase.sysu.edu.cn/)and miRBase database(http://www.mirbase.org/),and dual luciferase report experimental was performed to verify that miR-23b-3p could directly bind to the 3′UTR of ICAT mRNA.(3)miR-23b-3p mimic or miR-23b-3p inhibitor was transfected into SiHa and CasKi cells respectively to increase or reduce the expression of miR-23b-3p.CCK-8 experiment,wound healing experiment,Transwell experiment and Western blot were used to detect cell proliferation,lateral migration,longitudinal migration,invasion ability and EMT pathway related protein marker of above recombinated cells.(4)miR-23b-3p mimic and Ad-ICAT were co-transfected into SiHa and CasKi for recovery test.CCK-8,wound healing experiment,Transwell experiment and Western blot experiment were used to analyze whether the over-expression of ICAT could reverse the inhibiting effects of miR-23b-3p mimic on the proliferation,transverse migration,longitudinal migration invasion ability and EMT of cervical cancer cells.(5)HPV16 E6,E7 siRNA and miR-23b-3p inhibitor were co-transfected into SiHa and CasKi,respectively.CCK-8,wound healing experiment,Transwell experiment and Western blot experiment were used to determine whether inhibition of miR-23b-3p expression could reverse the inhibiting effects of HPV16 E6,E7 siRNA on proliferation,lateral migration,longitudinal migration,invasion,EMT ability and ICAT expression of cervical cancer cells.Results: After exogenous knockdown of E6,E7 expression,the mRNA and protein expression levels of ICAT decreased significantly,while the expression level of miR-23b-3p significantly increased in SiHa and CasKi cells.Dual luciferase report assay confirmed that miR-23b-3p could directly bind to the 3′UTR of ICAT mRNA.After overexpression of miR-23b-3p,the ICAT expression level was down-regulated in SiHa and CasKi.Inverse results were obversed while inhibition of miR-23b-3p.CCK-8,Wound-Healing and Transwell experiments showed that the overexpression of miR-23b-3p in SiHa and CasKi cells could inhibit the proliferation,migration and invasion of cervical cancer cells.The expression of proliferation-related protein markers and EMT protein markers were detected by Western blot.The expression levels of PCAN and Cyclin D1 were up-regulated.In the rescue experiment,the overexpression of ICAT in SiHa and CasKi cells could partially reverse the inhibition of proliferation,migration,invasion and EMT process of cervical cancer cells by co-transfection of miR-23b-3p mimic.Knockdown of E6,E7 expression in SiHa and CasKi cells and subsequently inhibiting the expression of miR-23b-3p can partially reverse the inhibiting effect of HPV16 E6 and E7 siRNA on the proliferation,migration,invasion and EMT process of SiHa and CasKi cells.Conclusion: HPV16 E6 and E7 can upregulate ICAT expression through miR-23b-3p,thus promoting the proliferation,migration,invasion and EMT of cervical cancer cells.