| Objective: High-risk human papillomavirus (HPV) is the cause of90%cervical cancer. HPV16and18account for about70%. High-risk HPV infectsthe host cells persistently, then E6/E7oncogenes are integrated into the hostcells, which makes the cells immortalized and have the ability of invasion andmetastasis. That is the main pathogenesis of cervical cancer.The process of epithelial cells changing to mesenchymal charactericsticsis called epithelial-mesenchymal transformation (EMT). EMT makesepithelial cells lose their adhesion and obtain mesenchymal morphology andcharacteristics. A typical feature of EMT is that the E-cadherin converts to theN-cadherin. EMT is closely related to the mechanism of invasion andmetastasis of tumors. Therefore, it is also the focus of a variety of cancerresearch. However, related study on cervical cancer is far from enough.Whether the EMT process of cervical cancer cells is associated with thehigh-risk HPV still needs more study urgently.A variety of signaling pathways associated with the occurrence of EMT,among which Wnt pathway is very important. Aberrant activation of the Wntpathway induced EMT was confirmed in the study of a variety of tumors. Butit is unclear what role of the Wnt pathway in the EMT of cervical cancer.In recent years the RNA interference (RNAi) has become a new targetgene silencing technology which plays a role at the post-transcriptional level.siRNA, shRNA and miRNA is powerful tools of the RNAi technology.Although techniques of transfection varied in vivo and in vitro, RNAitechnology were very efficiency and specificity.In this study, HPV16E6/E7genes was inhibited by using RNAitechnology in the xenografts of human cervical carcinoma SiHa cells in nude mice, and the inhibitory effects on the xenografts by silencing HPV16E6/E7genes were observed. We detected the expression levels of mRNA and proteinof E-cadherin, N-cadherin, Wnt and β-catenin by Real-Time PCR andimmunohistochemistry, to confirme the role of HPV16E6/E7in EMT ofcervical cancer and Wnt pathway.Methods:1Culturing HPV16-positive SiHa cells which belong the cells of humancervical squamous cancer.2Plasmids containing miRNA or not was amplified and extracted.HPV16E6-miRNA and HPV16E7-miRNA were interfering plasmids.Neg-miRNA was used as the negative control.3The SiHa cells were inoculated into subcutaneous of the armpit near theoutside of the right upper limb in nude mice. Observing the situation and ofxenografts and then the nude mice were randomly divided into four groupsincluding two control groups and two treatment groups. Saline control groupand the empty plasmid group (Neg-miRNA group) were control groups.HPV16E6gene silencing group (HPV16E6-miRNA group) and HPV16E7gene silencing group (HPV16E7-miRNA group) were treatment groups.4Xenografts of four groups in nude mice were respectively injected withsaline, Neg-miRNA, HPV16E6-miRNA, HPV16E7-miRNA. The injectionwas multi-point intratumoral. We observed xenografts growth and measuredits volume before each injection.5We removed the tumors from nude mice, and measured its volume andweight. Then the mRNA and protein levels of the tumor specimens weredetected by Real-Time PCR and immunohistochemistry. Detection indicatorsinclude HPV16E6, HPV16E7, E-cadherin, N-cadherin, Wnt1and β-catenin.Then we analyzed the role of E6/E7genes silencing on tumor growth, EMTand Wnt pathway.Results:1Establishment of xenografts of nude mice.1.1Tumor-bearing mice all survived. Obviously visible xenografts could be seen after nearly20days after subcutaneous inoculation when volume ofxenografts reached about80-120mm3.Tumor formation rate was100%. Theshape of visually xenografts was irregular. Ulceration and bleeding can occurin some xenografts. Mice of four groups were not significantly listlessness,weight loss and so on. During the experiment, the weight of mice did notchange significantly.1.2General observation of xenografts: tumor growth is non-invasive. Tumorswith clear boundaries were easily separated. Gray section of tumor showsnecrosis, and there are no obvious blood vessels.2Average volume of xenografts of two treatment groups with miRNA were allless than two control groups (p<0.05). Similarly, average weight of xenograftswas significantly reduced (p<0.05).3We detected the expression of mRNA of HPV16E6/E7in each group byReal-Time PCR.The expression of HPV16E6mRNA in HPV16E6genesilencing group, HPV16E7gene silencing group and empty plasmid groupwere all compared with the saline control group,and only the expression ofHPV16E6mRNA in HPV16E6gene silencing group was downregulated(p<0.05). The expression of HPV16E7mRNA in HPV16E6gene silencinggroup, HPV16E7gene silencing group and empty plasmid group were allcompared with the saline control group, and only the expression of HPV16E7mRNA in HPV16E7gene silencing group was downregulated(p<0.05). Thisset of data shows that HPV16E6/E7genes were successfully silenced byintratumoral injection of cervical cancer xenografts with HPV16E6-miRNAor HPV16E7-miRNA.4We detected the expression of mRNA and protein of some indicators aboutEMT by Real-Time PCR and immunohistochemistry. The expression ofE-cadherin mRNA and protein in HPV16E6gene silencing group and HPV16E7gene silencing group were all upregulated comparing with the salinecontrol group (p<0.05).And the expression of N-cadherin mRNA and proteinin HPV16E6gene silencing group and HPV16E7gene silencing group wereall downregulated comparing with the saline control group (p<0.05). The expression of two indicators about EMT in the empty plasmid group had nodifference with the saline control group (p>0.05). This experimentillustratested that “transformation of cadherins”occurred after HPV16E6andE7gene silencing in cervical cancer xenografts. N-cadherin converted toE-cadherin in two interfering groups.5We detected the expression of mRNA and protein of some indicators aboutWnt pathway by Real-Time PCR and immunohistochemistry. The expressionof Wnt1mRNA and protein in HPV16E6gene silencing group and HPV16E7gene silencing group were all downregulated comparing to the salinecontrol group (p<0.05). The expression of β-catenin mRNA and protein in thattwo groups were same to the Wnt1comparing with the saline control (p<0.05).The expression of β-catenin and Wnt1in mRNA and protein levels had nosignificantly difference comparing to the saline control group (p>0.05).Silencing of HPV16E6/E7genes in cervical cancer xenografts madeWnt1/β-catenin signaling pathway inhibited.Conclusion:1We established cervical cancer xenografts from SiHa cells in nude micesuccessfully.That can be used for in the study of cervical cancer in vivo.2HPV16E6/E7genes are silenced specifically and efficiently by HPV16E6-miRNA and HPV16E7-miRNA in vivo.3Silencing of HPV16E6/E7genes can inhibit the growth of cervicalcancer xenografts in nude mice.4Interfering HPV16E6/E7genes lead to that the expression ofN-cadherin mRNA and protein belong to mesenchymal were significantlydownregulated and the expression of E-cadherin mRNA and protein belong toepithelial were significantly upregulated in vivo.That was also epithelial-mesenchymal transition(MET). Thus we conclude that the expression ofHPV16E6/E7genes can promote the EMT of cervical cancer.5Interfering HPV16E6/E7genes lead to that the expression of Wnt1andβ-catenin were all downregulated wether at mRNA level or protein level invivo. Wnt1and β-catenin are the key regulatory molecules in Wnt pathway. Thus we conclude that the expression of HPV16E6/E7genes can promote theEMT of cervical cancer by abnormal activation of Wnt signaling pathway.Interfering HPV16E6/E7genes can lead to the EMT of cervical cancer by thesuppression of Wnt pathway. |
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