| Objective: Cervical cancer(CC)is currently the fourth most common cancer and the fourth most common cause of cancer death among women in the world.It is necessary to explore the pathogenesis of cervical cancer,potential therapeutic targets and early diagnosis markers.Cervical cancer is mainly caused by human papillomavirus(HPV)infection,of which HPV16 is the most frequently detected genotype.HPV E6 has transforming properties,and it promotes the malignant progression of cervical cancer by integrating into the host genome and inducing functional inhibition of the tumor suppressor gene p53.Micro RNAs(miRNAs,miRNAs)are important tumor regulators that can act as oncogenes or tumor suppressor genes to affect cellular biological functions,thus playing a role in the occurrence and development of cervical cancer.miRNAs are involved in various epigenetic changes and DNA methylation processes present in HPV infection and cervical cancer.The abnormal expression of miRNA in cervical cancer is closely related to HPV16 infection,and the targeted interference of HPV16 E6 expression can affect the expression of various miRNAs.Studies have shown that miR-320 a plays a tumor suppressor role in cervical cancer,which inhibits cervical cancer cell proliferation,migration,invasion and EMT and induces apoptosis by targeting FOXM1 expression.Whether miR-320 a expression is regulated by HPV16 E6 is currently unclear.miRNAs down-regulate the expression of their target genes at the post-transcriptional level by binding to the 3’-untranslated region(UTR)of mRNA,resulting in translational repression or degradation of their target genes.Topoisomerase IIα(TOP2A)can relax positive/negative DNA supercoils to regulate DNA topology,resolve chromatid segregation and chromosome condensation during DNA transcription and replication,and is necessary for DNA replication and chromosome segregation.Its expression is significantly elevated in proliferating cells and cancer cells.Studies have shown that infection with HPV increases the expression of TOP2 A in cervical cells,and whether its expression is regulated by HPV16 E6 and its mediated miRNA is still unclear.In conclusion,this study screened differentially expressed miRNAs in cervical cells from HPV16-infected and uninfected cervical cancer patients by analyzing the gene expression omnibus database(GEO)GSE81137 data set,and screened out the miRNAs that play a tumor suppressor role and are related to cervical cancer.The effect of miR-320 a and HPV16 E6 with good prognosis in cervical cancer on the expression of miR-320 a in cervical cancer,and to verify whether HPV16 E6 regulates the expression of its downstream target gene TOP2 A through miR-320 a and affects the proliferation and apoptosis of cervical cancer cells in vitro and in vivo,migration,invasion and EMT,to provide a theoretical basis for the early diagnosis of cervical cancer and the development of therapeutic drugs.Methods: The first part of this study: By analyzing the GSE81137 dataset,the differentially expressed miRNAs in cervical cells of HPV16 infected and uninfected cervical cancer patients were screened,and the expression of miRNAs in cervical cancer tissues and the relationship with the prognosis of cervical cancer patients were analyzed by bioinformatics.HPV16-positive and negative cervical cancer tissues and cells were collected,and the expression of miR-320 a was detected by q RT-PCR.The miR-320 a inhibitor was transfected into Si Ha cells knocking down HPV16 E6 or the miR-320 a mimic was transfected into C33 a cells overexpressing HPV16 E6,and the expression of miR-320 a in cells was detected by q RT-PCR;the E6 protein in cells was detected by Western blot and apoptosis-related protein expression;cell proliferation was detected by CCK-8,Edu and plate clone formation assay;cell apoptosis rate was detected by flow cytometry;cell migration and invasion ability were detected by Transwell assay;Western blot and immunofluorescence were used The expression of epithelial-mesenchymal transition(EMT)marker proteins was detected.HPV16 E6 knockdown Si Ha cells or HPV16 E6 overexpressing C33 a cells were subcutaneously injected into nude mice,and administered with antagomiR-320 a or agomiR-320 a to detect tumor volume and tumor weight;Western blot was used to detect HPV16 E6 protein expression in transplanted tumors;The expression of miR-320 a in the transplanted tumor was detected by q RT-PCR;the expression of Ki-67 in the transplanted tumor was detected by immunohistochemistry;the apoptosis of the transplanted tumor was detected by TUNEL;the expression of EMT marker protein in the transplanted tumor was detected by Western blot.The downstream target genes of miR-320 a were analyzed by bioinformatics,and the up-regulated genes in cervical cancer tissues were screened from the GSE6791 and GSE9750 data sets.The shared genes of the three were screened by Wayne analysis,and the PPI of the shared genes was drawn through the STRING online analysis website.The core genes were screened by Cytoscape software,and the expression of core genes in cervical cancer tissue was verified by bioinformatics.The second part of this study: HPV16-positive and negative cervical cancer tissues and cells were collected,and the expression of TOP2 A was detected by q RT-PCR and Western blot.The potential binding sites of TOP2 A mRNA and miR-320 a were predicted by the online bioinformatics database,and the binding of TOP2 A mRNA to miR-320 a was verified by dual-luciferase reporter gene assay and AGO2-RIP.Si Ha cells knocked down HPV16 E6 or C33 a cells overexpressed HPV16 E6,and the expression of TOP2 A was detected by q RT-PCR and Western blot.Si Ha cells and C33 a cells were transfected with miR-320 a mimic and miR-320 a inhibitor,and the expression of TOP2 A was detected by Western blot.Si Ha cells with knockdown of HPV16 E6 were transfected with miR-320 a inhibitor or C33 a cells overexpressed with HPV16 E6 were transfected with miR-320 a mimic,and the expression of TOP2 A was detected by q RT-PCR and Western blot.The Si Ha cells with knockdown of HPV16 E6 overexpressed TOP2 A or the C33 a cells overexpressed with HPV16 E6 were knocked down with TOP2 A.The mRNA expressions of miR-320 a and TOP2 A were detected by q RT-PCR;the expressions of TOP2 A protein and apoptosis-related proteins were detected by Western blot.;Cell proliferation was detected by CCK-8,Edu and plate colony formation assay;cell apoptosis rate was detected by flow cytometry;cell migration and invasion ability were detected by Transwell assay;epithelial-mesenchymal transition was detected by Western blot and immunofluorescence(epithelial-mesenchymal transition,EMT)marker protein expression.Nude mice were injected subcutaneously into Si Ha cells with knockdown of HPV16 E6 and overexpression of TOP2 A or C33 a cells overexpressed with HPV16 E6 and knockdown of TOP2 A,and the tumor volume and tumor weight were detected;Western blot was used to detect the protein expressions of HPV16 E6 and TOP2 A in the transplanted tumors;q RT-PCR was used to detect the mRNA expression of miR-320 a and TOP2 A in the transplanted tumor;the expression of Ki-67 in the transplanted tumor was detected by immunohistochemistry;the apoptosis of the transplanted tumor was detected by TUNEL;the expression of EMT marker protein in the transplanted tumor was detected by Western blot.Results: The first part of this study: The expression of miR-320 family is down-regulated in cervical cells of patients infected with HPV16 cervical cancer,and the expression of miR-320 a is down-regulated in cervical cancer tissues and the survival rate of patients with high expression is high.The expression of miR-320 a was down-regulated in HPV16-positive cervical cancer tissues and cells.Inhibiting miR-320 a down-regulated the expression of miR-320 a in Si Ha cells that knocked down HPV16 E6,increased the protein expressions of Bcl-2,N-cadherin and Vimentin,decreased the protein expressions of Bax,cleaved caspase3 and E-cadherin,and promoted cell proliferation,migration,invasion and EMT,inhibit cell apoptosis,promote the growth of transplanted tumors,increase the expression of Ki-67 in the transplanted tumor,reduce the positive rate of TUNEL in the transplanted tumor,down-regulate the expression of E-cadherin protein in the transplanted tumor,and up-regulate the protein expression of N-cadherin and Vimentin in the transplanted tumor;Overexpression miR-320 a up-regulated the expression of miR-320 a in C33 a cells overexpressing HPV16 E6,decreased the expression of Bcl-2,N-cadherin and Vimentin proteins,increased the expressions of Bax,cleaved caspase3 and E-cadherin,and inhibited cell proliferation,migration,invasion and EMT,promote cell apoptosis,inhibit the growth of xenograft tumor,reduce the expression of Ki-67 in xenograft tumor,increase the positive rate of TUNEL in xenograft tumor,up-regulate the expression of E-cadherin protein in xenograft tumor,downregulate the protein expression of N-cadherin and Vimentin in xenograft tumor.The core genes of miR-320 a target genes upregulated in cervical cancer include cell cyclin A2(CCNA2),topoisomerase IIα(TOP2A),kinesin family member 20A(kinesin family member 20 A,KIF20A),centromere protein F(CENPF),RAD51-associated protein 1(RAD51AP1)and kinesin family member 20A(KIF23),which are in cervical cancer tissue are highly expressed.The second part of this study: TOP2 A was up-regulated in HPV16 positive cervical cancer tissues and cells.A potential binding site for miR-320 a exists on TOP2 A mRNA,and TOP2 A mRNA binds to miR-320 a.Knockdown of HPV16 E6 decreased TOP2 A expression in Si Ha cells,and overexpression of HPV16 E6 increased TOP2 A expression in C33 a cells.Inhibition of miR-320 a up-regulated the expression of TOP2 A in Si Ha and C33 a cells,while overexpression of miR-320 a down-regulated the expression of TOP2 A in Si Ha and C33 a cells.Inhibition of miR-320 a up-regulated the expression of TOP2 A in Si Ha cells with knockdown of HPV16 E6,while overexpression of miR-320 a downregulated the expression of TOP2 A in C33 a cells overexpressed with HPV16 E6.Overexpression of TOP2 A increases the protein expressions of TOP2 A,Bcl-2,Ncadherin and Vimentin in Si Ha cells with knockdown of HPV16 E6,reduces the protein expressions of Bax,cleaved caspase3 and E-cadherin,promotes cell proliferation,migration,invasion and EMT,and inhibits cell apoptosis,promote the growth of xenograft tumors,increase the expression of Ki-67 in xenograft tumors,reduce the positive rate of TUNEL in xenograft tumors,down-regulate the expression of E-cadherin protein in xenograft tumors,and up-regulate the protein expressions of N-cadherin and Vimentin in xenograft tumors;knockdown of TOP2 A reduces the overexpression of HPV16 E6 C33 a cells express TOP2 A,Bcl-2,N-cadherin and Vimentin proteins,increase the expression of Bax,cleaved caspase3 and E-cadherin proteins,inhibit cell proliferation,migration invasion and EMT,promote cell apoptosis,and inhibit cell xenograft tumors Growth,decreased the expression of Ki-67 in the transplanted tumor,increased the positive rate of TUNEL in the transplanted tumor,up-regulated the expression of E-cadherin protein in the transplanted tumor,and down-regulated the protein expression of N-cadherin and Vimentin in the transplanted tumor.Conclusion: HPV16 E6 promotes cervical cancer cell proliferation,migration,invasion and EMT and inhibits apoptosis in vivo and in vitro by downregulating the expression of miR-320 a and increasing the expression of its downstream target gene TOP2 A. |
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