HPV Induced Cervical Cancer Cell Epithelial-mesenchymal Transformation Through Transforming Growth Factor Pathway

Objective： Cervical cancer (CC) is one of the most frequentgynecological malignant tumor of female，which is the first of femalereproductive organs and is one of the major cancer of the world. Now thathuman papilloma virus namely HPVs infection, especially in persistentinfection with HR-HPV types, is the basic cause of cervical lesionprecancerous and cervical cancer.HPVs are small double link DNA virus with high host specificity andaffinity, which are Papovaviridae of subgroup A and composed by the core andthe capsid protein (L1and L2). Its genome contains eight open reading frameand one upstream regulatory region, E2、E6and E7are three open readingframe for cancer gene of the virus.More than120types of HPV subtypes havebeen found, molecular epidemiology found that more than two-thirds of thecervical cancer specimens were detected HPV16or18positive. The virus＇DNA can be randomly into the host genome and express the E6and E7oncogene and made the host cells immortalized. The E6and E7oncogene canexpress respectively the viral proteins E6and E7, which inactivate tumorsuppressor gene products p53and pRB, respectively, inhibiting the tumorsuppressor function and promoting the development of cancer.An epithelial-mesenchymal transition (EMT) is a biologic process thatallows a polarized epithelial cell to undergo multiple biochemical changes thatenable it to assume a mesenchymal cell phenotype, which can move freelybetween the cell matrix. It is a fundamental physiological process of embryodevelopment and tissue morphogenesis, especially the development of themesoderm, neural crest, and hupeh. But recent studies found that EMT hasclose correlation with the development、invasive and metastasis of cancer.Among the transcription factors and cytokines inducing EMT, transforming growth factor beta is proved to be the key factor of the induction of EMT.Now a large number of reports about TGF-β induce EMT have beenreported, whether the phenomenon existence in cervical cancer and itsrelationship with the occurrence and development process of HPV in cervicalcancer, remain to be research.Methods:①In vitro culture of HPV16positive squamous cellcarcinomas (SiHa) and HPV18positive carcinoma (HeLa) cell line. Design forHPV16E7MiRNA interference vectors respectively,12MR0018C-1、12MR0018C-2、12MR0018C-3and12MR0018C-4(hereinafter referred to as16E7-MiR-1、16E7-MiR-2、16E7-MiR-3and16E7-MiR-4) and for HPV18E7MiRNA interference vectors respectively,12MR0018D-1、12MR0018D-2、12MR0018D-3and12MR0018D-4(hereinafter referred to as18E7-MiR-1、18E7-MiR-2、18E7-MiR-3and18E7-MiR-4). The cells were transienttransfected with MicroRNA formulated into liposomes, transfectionefficiencies were established by Real-time PCR and its high efficiencyplasmid will be used for subsequent experiments.②Make stealth RNA ofHPV16E6and HPV18E6used by early team into Micrornas and build topcDNA6.2-GW/EmGFP-miR carrier, to build into plasimid respectivelyto aim directly at HPV16E6and HPV18E6(hereinafter referred to as16E6-MiR and18E6-MiR).③Construct stable cell lines on silencingHPV16and HPV18viral genome of E6and E7gene.④Real-time PCRdetect the differences about the genes EMT markers of E-cadherin、N-cadherin and beta-catenin expression befor and after transfection; Real-timePCR detect the differences about the genes TGF-β1、TGF-βRII（TβRII）andSmad4befor and after transfection.Results:1.Construction transfection recombinant plasmid successfully, weanalysed by using a quantitative Real-time PCR assay the relative messengerRNA(mRNA) levels of E6and E7in SiHa cells transfected by E6and E7MicroRNA (16E6-MiR、16E7-MiR-1、16E7-MiR-2、16E7-MiR-3and16E7-MiR-4). The inhibition effective rate was77%(P=0) about the gene of HPV16E6after transfect plasmid16E6-MiR；the inhibition effective rate was66%(P=0)and51%（P=0.003）about the gene of HPV16E7after transfectplasmid16E7-MiR-2and16E7-MiR-4, but there was no obvious inhibitoryeffect after transfect plasmid16E7-MiR-1and16E7-MiR-3.2.In HeLa cell line, Real-time PCR detect levels of E6and E7gene inHeLa cells transfected by E6and E7MicroRNA (18E6-MiR、18E7-MiR-1、18E7-MiR-2、18E7-MiR-3and18E7-MiR-4). The inhibition effective ratewas87%（P=0） about the gene of HPV18E6after transfect plasmid18E6-MiR；the inhibition effective rate was68%(P=0) and55%（P=0） aboutthe gene of HPV16E7after transfect plasmid18E7-MiR-1and18E7-MiR-4,but there was no obvious inhibitory effect after transfected plasmid18E7-MiR-2and18E7-MiR-3.3. For subsequent experiment we construct stable transfected cell lineswith the plasimid16E6-MiR and18E7-MiR-2in SiHa cell line. Real-timePCR detected that EMT markers of E-cadherin is increased (P <0.05) butmarkers of N-cadherin and β-catenin is decreased, when the genes of HPV16E6and E7were inhibited obviously（P＜0.05）.Meanwhile after the genes of E6and E7of HPV16were inhibited, weanalysed the level of gene TGF-β1、TβRII and Smad4were decreasedobviously（P＜0.05）.4. For subsequent experiment we construct stable transfected cell lineswith the plasimid18E6-MiR and18E7-MiR-1in HeLa cell line. Real-timePCR detected that EMT markers of E-cadherin is increased (P＜0.05) butmarkers of N-cadherin and β-catenin is decreased（P＜0.05) when the genesof HPV18E6and E7were inhibited (P＜0.05).Meanwhile after the genes of E6and E7of HPV18were inhibited,weanalysed the level of gene TGF-β1、TβRII and Smad4were decreasedobviously (P＜0.05).Conclusions：1. In cervical cancer cells, the HPV E6, E7gene canpromote epithelial-mesenchymal transition When the E6or E7gene isinhibited, the reverse process of mesenchymal-epithelial occured, thus offers a new way for the treatment of cervical cancer.2. The E6and E7genes of HPV16and HPV18virus may promote theprocess of EMT through the TGF-β/Smads signaling pathway, and thusimprove the ability of proliferation,invasion and metastasis in cervical cancercells.