Mechamism Research On The Role Of Wntl In Ovarian Cancer Stem Cells

Ovarian cancer is one of the commonest malignancies of females’ reproductivesystem with high morbidity in the world. Although the diagnostic methods have beenimproved, the recurrence, metastasis and multi-drug resistance still lead to the low5-year survival rate of less than30%. Meanwhile, the pathogenesis of ovarian cancer isunclear which limited the advancement of the treatment and prevention. Therefore, toexplore the mechanisms of the occurrence, recurrence and metastasis of ovarian cancerand to figure out effective pathways to suppress the tumor formation would be of greatimportance in improving the survival rate of ovarian cancer patients.Stem cells might survive for a long time, which make it possible for the genemutation occurring during their proliferation or differentiation. More and more studieshave shown that the tumors originate from cancer stem cells. Tumor is thought to be akind of disease resulted from gene mutation during the process of the renewal of stemcells. Tumor cells share the similar characteristics with the stem cells: the capacity ofself renewing and proliferating infinitely, longer life, the activation of anti-apoptoticsignal paths and telomerase stimulation."Cancer stem cell" hypothesis deems: the smallamount of stem cells which act as high carcinogenesis subgroups play key roles in thetumor occurrence, recurrence, metastasis as well as the failure of treatments.Wnt signaling pathway is complicated with double functions as be responsible forthe regulation of animals’ development mode and play a crucial role in the regulation ofnormal growth and homeostasis of stem/progenitor cells. It is reported that manycarcinogenesis exist abnormal activation of this pathway. DSH is a key component ofthe membrane-associated Wnt1receptor complex. It is activated after combined withWnt and then inhibits the downstream protein complexes, including axin, GSK-3andAPC protein. The complex of axin/GSK-3/APC might promote the degradation ofβ-catenin, which are signaling molecules within the cell. When “β-catenin degradation complex” is inhibited, β-catenin in the cytoplasm stably exists. Parts of the β-cateninmight move into the nucleus and interact with the TCF/LEF transcription factor familyto initiate the expression of the genes.The exact mechanism of Wnt signaling pathway in the development of ovary andthe occurrence of ovarian cancer is still unclear, but the researches have proved that theactivation of Wnt signaling is closely related to drug resistance, maintenance of cancerstem cells and interactions with other signaling pathways. To explore the role of Wnt information and progression of ovarian cancer will provide a new target for the preventionand treatment of ovarian cancer.In this study, we first selected ovarian cancer cells marked by CD133+fromovarian cancer SKOV3cell lines and then detected the expression of molecules of Wnt1signal pathway in CD133+and CD133-cells. Secondly induced the Wnt1gene silencein SKOV3cell by eukaryotic vector pGPU6/GFP/Neo with Wnt1shRNA stablytransfection. The impacts of Wnt1gene down-regulation on proliferation, cell cycle andapoptosis of ovarian cancer cells were studied to explore the mechanism of drugresistance in ovarian cancer cells and its action in the carcinogenesis of ovarian cancer.Part I Expression and mechanism of Wnt1Signaling Molecule in ovariancancer SKOV3cellObjective: To explore the function of Wnt signal pathway in CD133+andCD133-ovarian cancer stem cells.Methods: CD133+and CD133-cells were selected by magnetic activated cellsorting system (MACS). Real-time RT-PCR to detect the expression of mRNA andprotein of Wnt1signal molecules in the two types of cells, respectively.Results: CD133+cells accounted for7.25%of the total ovarian cancer cells andCD133and Oct-4expressed. The expression of wnt1mRNA and protein in CD133+cells were significant higher compared with CD133-cells (p＜0.05). The expression ofβ-catenin mRNA and protein in CD133+cells were significant higher compared withCD133-cells(p＜0.05).Conclusions: Wnt1is activated in ovarian cancer CD133+cells and it may playroles in the maintenance of cellular morphology and function of CD133+cells. PartⅡ The Effect of RNAi applied to silent Wnt1on Carcinogenesis ofovarian CancerObjective: To explore the action of the expression of Wnt gene in carcinogenesisof ovarian cancer.Methods: The Wnt-shRNA probe was designed to interfere and knockdown thetarget gene expression in SKOV3cells. The RT-PCR and western blot were used todetect transcription level of mRNA and expression of protein of Wnt1in ovarian cancerSKOV3cells after LipofectamineTM2000transfection48hours.Results: The correct insertion was verified by partial nucleotide sequencing of the4constructed eukaryotic vectors expressing shRNA of Wnt1. In the Wnt1shRNA-1group, Wnt1shRNA-2group, Wnt1shRNA-3group, Wnt1shRNA-4group, blankgroup, negative control group and MOCK group of SKOV3cells, the expression ofWnt1mRNA compared to GAPDH was0.77±0.07,0.98±0.08,0.71±0.05,0.57±0.05,0.96±0.08,1.01±0.08,1.00±0.11, respectively. Contrast to MOCK group, the Wnt1mRNA expression in Wnt1shRNA-1group, Wnt1shRNA-3group and Wnt1shRNA-4group decreased (p=0.007,0.002,0.046), moreover the Wnt1mRNA expressioninWnt1shRNA-4group is significantly lower than the other3groups. The interferingand inhibiting efficiency of Wnt1-shRNA-4was comparatively ideal with the downregulation rate of64.3%. The expression of Wnt1protein was detected in SKOV3byWestern blot, and the protein of Wnt1-shRNA-4showed inhibited.Conclusions: construction is successful, and Wnt1shRNA significantly inhibitthe expression of Wnt1.. Part Ⅲ Wnt1siRNA inhibit the proliferation of ovarian cancer cells and thes ovarian cancer stem cellsObjective: Explore the impact on proliferation by Wnt1gene down-regulating inovarian cancer cells.Methods: Wnt1knockdown SKOV3cells were selected and colony formationexperiment and CCK8examination were adopted to check the effect of Wnt1deficiencyon proliferation of ovarian cancer cells. The flow cytometre examined cellular cycle andAV/PI double staining detect apoptosis. The proportion of CD133+stem cells wasdetect by flow cytometry, and Wnt1、β-catenin、CD133and Sox2protein expression were examined by western blot.Results: Knockdown Wnt1in ovarian cancer SKOV3cells induced significantinhibition on cell proliferation and increased proportion of apoptosis and the G2/Mphase cells. The SKOV3stem cells showed low capacity of self-renew and theproportion of cancer stem cells in ovarian cancer cells declined. The expression of Wnt1,Sox2and CD133protein decreased.Conclusion: Knockdown Wnt1inhibits the SKOV3cell proliferation, promotesthe apoptosis and maintains the cell in the G2/M stage. The Sox2may be downstreamgene of Wnt1signaling pathway and it can inhibit the proliferation of ovarian cancerstem cells after the transcript inhibition by the expression of Wnt1protein.