HPV Induced Cervical Cancer Cell Epithelial-mesenchymal Transition Through Wnt Pathway

Objective: To construct interference vectors of micro-RNA (miRNA)for HPV16E6, E7and HPV18E6, E7respectively, and transfect them intocervical cancer cell lines SiHa (HPV16+) and HeLa (HPV18+). Then observethe changes of gene expression related to Wnt/β-catenin signaling pathwayand epithelial-mesenchymal transition (EMT), including Wnt1, β-catenin,E-cadherin and N-cadherin after HPV16/18E6or E7gene was silenced. Thisprocess was mediated by miRNA that can suppress the expression of HPVE6or E7. Analyse the correlation between HPV and EMT process of cervicalcancer cells, so to clarify the process and mechanism that HPV lead cervicalcancer cells to EMT through by the Wnt signaling pathway. This study willthus provide theoretical basis for gene therapy of cervical cancer.Methods:1.Cultivate cervical cancer cell lines SiHa and HeLa whichare HPV18-positive.2.Construct microinterference ribonucleic acid (miRNA)and extrac plasmid: to study the mechanism which HPV lead to cervicalcancer, according to the genetic sequence of HPV16E6, E7and HPV18E6, E7,construct interference plasmid vectors for HPV16E6, HPV16E7, HPV18E6and HPV18E7respectively and the negative control plasmid. Extract plasmidfor transient transfection and stable transfection.3. Transient transfection andassay transfection efficiency: using liposome transfection technologyinstantaneous transfect cervical cancer cell lines SiHa and HeLa. Observe thecells and calculate the transfection efficiency through the invertedfluorescence microscope after forty-eight hours.4. Stable transfection: takedifferent concentrations of blasticidin effects on cervical cancer cells SiHa andHeLa to get the screening concentration and maintain concentration; thenobtain corresponding stably transfected cell clones.5. Using the Real-TimeQuantitative PCR to detect the interference efficiency of each target genes: using SiHa and HeLa as negative control, detect the expression of HPV16E6,HPV16E7, HPV18E6and HPV18E7mRNA after transfected by the relevantinterference plasmid and the negative control plasmid. Calculate eachsilencing effciency to screen the highest effective one for the follow-upexperiments.6. Detect the related gene of EMT: to further study themechanisms between HPV and the process of EMT in cervical cancer, weexamined the expression of Wnt1, β-catenin, E-cadherin and N-cadherinmRNA after interfering with the expression of HPVE6and E7generespectively through real-time quantitative PCR.Results:1.The cervical cancer cells SiHa and HeLa all grow strongly,and the morphology of cells are various observed by inverted fluorescencemicroscope.2.Fluorescence-positive cells accounted for70-80%among eachinterference plasmid and the negative control plasmid transfected cervicalcancer cells SiHa and HeLa after transfected by miRNA for48hours.3.Knockdown efficiency: in view of SiHa cells, using SiHa as the negativecontrol, by contrast of GAPDH, the expression of HPV16E6mRNA inHPV16E6-miRNA transfected SiHa cells, compared with that in the controlcells, decreased by77.8%through real-time quantitative RT-PCR assay; andthe expression of HPV16E7mRNA in HPV16E7-miRNA2andHPV16E7-miRNA4transfected SiHa cells, compared with that in the controlcells, decreased by67.3%and59.4%, suggesting that HPV16E7-miRNA2hadhigher silencing efficiency; the expression of HPV16E6and HPV16E7mRNAin Neg-miRNA transfected SiHa cells, compared with that in the control cells,didn’t have any change, P>0.05, suggesting that Neg-miRNA couldn’t affectthe expression of HPV16E6and HPV16E7mRNA. These suggested thatHPV16E6-miRNA and HPV16E7-miRNA2effectively transfected SiHa cellsand inhibited expression of HPV16E6and HPV16E7mRNA significantly. Inview of HeLa cells, using HeLa as the negative control, by contrast ofGAPDH, the expression of HPV18E6mRNA in HPV18E6-miRNAtransfected HeLa cells, compared with that in the control cells, decreased by87.3%through real-time quantitative RT-PCR assay; and the expression of HPV18E7mRNA in HPV18E7-miRNA1and HPV16E7-miRNA4transfectedHeLa cells, compared with that in the control cells, decreased by68%and45.5%, suggesting that HPV18E7-miRNA1had higher silencing efficiency;the expression of HPV18E6and HPV18E7mRNA in Neg-miRNA transfectedHeLa cells, compared with that in the control cells, didn’t have any change,P>0.05, suggesting that Neg-miRNA couldn’t effect the expression ofHPV18E6and HPV18E7mRNA. This suggestd that HPV18E6-miRNA andHPV18E7-miRNA1could effectively transfect HeLa cells and inhibit theexpression of HPV18E6and HPV18E7mRNA significantly. So we can useHPV16E6-miRNA, HPV16E7-miRNA2,HPV18E6-miRNA, HPV18E7-miRNA1and Neg-miRNA as the effective plasmid for subsequent stabletransfection.4.Take different concentrations of blasticidin effects on cervicalcancer cells SiHa and HeLa, the cells all died by≥9ug/ml of blasticididtreating10days. So the screening concentration of blasticidin to SiHa andHeLa was10ug/ml, the maintain concentration was5ug/ml.5. The expressionof EMT related genes: The expression of E-cadherin and N-cadherin: we cansee E-cadherin are low expression but N-cadherin are high expression in SiHaand HeLa cells through real-time quantitative RT-PCR assay. We detected theexpression of E-cadherin mRNA in HPVE6-miRNA and HPVE7-miRNAtransfected SiHa and HeLa cells, compared with that in the control cells,increased significantly, but the expression of N-cadherin decreasedsignificantly, P<0.05. And there didn’t have any change between the cellstransfectde by Neg-miRNA and the control. This suggeste that the knockingdown of HPVE6or E7gene in cervical cancer cells significantly promote theexpression of E-cadherin gene, at the same times, inhibite the expression ofN-cadherin gene. So HPV16/18E6and E7have a certain relation with theEMT process in cervical cancer cells. The expression of Wnt1and β-catenin:using SiHa as the negative control, by contrast of GAPDH, we detected theexpression of Wnt1and β-catenin mRNA in HPV16E6-miRNA andHPV16E7-miRNA2transfected SiHa cells, compared with that in the controlcells, decreased significantly, P<0.05, through real-time quantitative RT-PCR assay; the same tendency was found in HeLa cells that transfected withHPV18E6-miRNA and HPV18E7-miRNA1. But there didn’t find any changebetween the cells transfectde with Neg-miRNA and the control. These suggestthat the knocking down of HPVE6or E7gene in cervical cancer cellssignificantly inhibited the expression of Wnt1and β-catenin gene, therebyindicate that HPVE6or E7gene can activate the classical Wnt/β-cateninsignaling pathway which is one of the classic EMT signaling pathways topromote the occurrence of cervical cancer.Conclusion: miRNA interference vectors targeting HPVE6and E7weresuccessfully constructed, and the effective interference miRNA were identifiedwhich can interfere the expression of target gene, but the Neg-miRNA did nothave this function. We obtained the stably transfected cell clone, which maybe useful for the further research between HPVE6E7and cervical cancer. Theknocking down of targed genes can inhibit the expression of Wnt1andβ-catenin, suggesting in cervical cancer HPV can regulate the Wnt signalingpathway. As the expression of HPVE6and E7decreased, the expression ofE-cadherin that is one of epithelial markers increased, but the expression ofN-cadherin that is one of interstitial markers decreased. This indicates that inthe process of the occurrence of cervical cancer there exits EMT. EMT processis associated with the expression of HPVE6and E7, and E6/E7can promoteEMT through activating the classical Wnt/β-catenin signaling pathway.