MiR-1/G6PD Promoting Malignant Progression In High-risk Human Papillomavirus Positive(HPV16/18+)cervical Cancer

Objective:G6PD is overexpressed in a variety of tumors and is involved in the pathogenic process of tumors.In HPV16/18-infected human cervical cancer cells,the expression of G6PD was positively correlated with the malignancy of cervical cancer cells.Previous studies by our group have found that miR-1 inhibits the proliferation of cervical cancer cells and promotes apoptosis by binding to the specific base sequence of the 3 ’UTR of the G6PD gene and reducing the expression of endogenous G6PD,but its specific molecular mechanism is not clear.Therefore,in this study,cervical cancer cell lines with miR-1 overexpression vector,miR-1 expression inhibitory vector,G6PD overexpression and G6PD expression inhibitory vector were transfected into HPV-(C33A),HPV16+(Siha)and HPV18+(Hela)cervical cancer cells using miR-1 overexpression vector,miR-1 expression inhibitory vector,G6PD overexpression and G6PD expression inhibitory vector to obtain cervical cancer cell lines with miR-1/G6PD overexpression or defective expression.Cell proliferation activity,cell cycle-related protein and apoptosis-related protein were studied in each group of cells to further investigate the molecular mechanism of miR-1/G6PD in the pathogenic process of high-risk HPV16/18 infected cervical cancer.Methods:Lentivirus-transfected cell lines C33A,Siha and Hela were named plenti-miR-1(miR-1 overexpression treatment group);lemiR(miR-1 overexpression blank control treatment group);miR-1-sponge(miR-1 expression inhibition treatment group);CX-control(miR-1 expression inhibition blank control treatment group);plenti-G6PD(G6PD overexpression treatment group);G6PD control(G6PD overexpression blank control treatment group);G6PD-siRNA(G6PD expression inhibition treatment group)and Empty-siRNA(G6PD expression inhibition blank control treatment group).After successful transfection,the expression levels of miR-1(real-time PCR,RT-PCR)and G6PD(RT-PCR,Western blot)in each cell line were detected after miR-1 overexpression and expression silencing,the cell growth of each group was detected by CCK-8 assay,and the expression levels of cell cycle-related proteins(cyclin D1,cyclin D2,cyclin E)and apoptosis-related proteins(Bcl-2,Bcl-xL and P53)in each group were detected by Western blot.Results:Overexpression of miR-1 significantly inhibited G6PD expression in Siha/Hela cells,while inhibition of miR-1 significantly up-regulated endogenous G6PD expression in Siha/Hela cells.Excessive administration of G6PD partially reversed the inhibitory effect of miR-1 on G6PD expression;G6PD-siRNA treatment also partially reversed the effect on G6PD expression after miR-1 expression inhibition.In the cell viability assay,miR-1 overexpression significantly inhibited the proliferation ability of high-risk HPV 16/18+human cervical cancer cells;on this basis,G6PD overexpression could increase the viability of miR-1 overexpressing Siha/Hela cells;while the proliferation ability of Siha/Hela cells was significantly increased after miR-1 expression intervention,and intervention of G6PD expression in miR-1 expression inhibited Siha/Hela cells could partially counteract the increased viability of cervical cancer cell lines induced by miR-1 expression intervention.After miR-1 overexpression,Cyclin D1,Cyclin D2,and Cyclin E proteins were significantly down-regulated in Siha/Hela cells compared with miR-1 overexpression blank control;however,Cyclin D1,Cyclin D2,and Cyclin E proteins were significantly up-regulated in Siha/Hela cells after miR-1 expression was down-regulated.MiR-1 overexpression significantly down-regulated the protein levels of Bcl-2,Bcl-xL and P53 in Siha/Hela cells;while miR-1 expression inhibition significantly increased the protein levels of Bcl-2,Bcl-xL and P53 in Siha/Hela cells.Conclusion:1.miR-1 can regulate the expression of G6PD in cervical carcinoma cells.2.miR-1 affects the proliferation of cervical cancer cells through the regulation of G6PD.The high expression of G6PD protein after miR-1 expression inhibition may promote the growth and proliferation of HPV16/18+human cervical cancer cells by up-regulating cyclin D1,CyclinD2 and CyclinE.3.miR-1 affects apoptosis by regulating G6PD,and low G6PD expression after miR-1 overexpression may promote apoptosis in HPV16/18+human cervical cancer cells by down-regulating Bcl-2/Bcl-xL protein expression and inhibiting P53 protein expression.