| PART 1 Expression and clinical significance of Vasohibin-2 on epithelial ovarian cancerOBJECTIVE:To detect the expression level of Vasohibin-2(VASH2)in epithel ial ovarian cancer(EOC)and evaluate its clinical significance in EOC.METHODS:Quantitative real-time PCR(qRT-PCR)and immunohistochemistry(IHC)were used to detect the expression of VASH2 in EOC tissues,paraneoplastic tissues and normal ovarian tissues,and combined with clinical follow-up data.Kap lan-meier survival curve and Cox regression analysis were used to evaluate the pro gnosis of patients with EOC.RESULTS:The qRT-PCR method was used to detect the relative expression of VASH2 mRNA in fresh ovarian cancer tissues,paraneoplastic tissues and normal o varian tissues.The results showed that The VASH2 mRNA levels in the tissues of ovarian cancer stage ?-?,ovarian cancer stage ?-?,paraneoplastic tissues and n ormal ovary were 8.961 ± 0.723,3.136 ± 0.452,1.258 ± 0.214,and 1.362±0.537,respectively.The expression of VASH2 mRNA in stage ?-? EOC was significant ly higher than that in stage ?-?(P=0.012)and the average expression of VASH2 mRNA in EOC tissues was significantly higher than that in paraneoplastic tissues and normal ovarian tissues.(P=0.004).Immunohistochemical staining showed that th e positive rate of VASH2 protein expression in 83 cases of epithelial ovarian cance r was 95.18%(79/83),while the positive rate of paraneoplastic tissues and normal ovarian tissues were 2.41%(2/83)and 0%,respectively.(0/78),suggesting that the expression of VASH2 protein in epithelial ovarian cancer tissues is significantly hig her than that in paraneoplastie tissues and normal ovarian tissues(P=0.001).In addi tion,the results of clinically relevant data show that VASH2 protein expression in e pithelial ovarian cancer was correlated with the patient's FIGO stage,pathological g rade,serum CA125 and serum HE4(P<0.05),but not related to age,histological ty pe,or whether ascites found cancer cells.Cox regression analysis showed that high expression of VASH2 in epithelial ovarian cancer tissues(P=0.002)and late FIGO stage(P=0.001)were independent factors for poor prognosis of ovarian cancer.The Kaplan-Meier survival curve showed that the five-year survival rate of the VASH2 high expression group was significantly lower than that of the low/non-expression g roup.CONCLUSION:(1)Detected with VASH2 expression can be used as a biomarker to evaluate t he prognosis of patients with epithelial ovarian cancer.(2)High VASH2 expression predicts poor prognosis in patients with epithelial ovarian cancer.PART 2 Expression of Vasohibin-2 gene influence on biological behavior and epithelial-mesenchymal transition of epithelial ovarian cancer cells and its mechanism of chemotherapy resistanceOBJECTIVE:To investigate the expression of Vasohibin-2(VASH2)in ovarian cancer cell lines,and to investigate the effect of interference and overexpression of VASH2 on the biologieal behavior and epithelial-mesenchymal transition of ovarian cancer cell lines and its mechanism of chemotherapy resistance,in order to seek effective theoretical and experimental evidence for ovarian cancer treatment strategies.METHODS:1.The expression of VASH2 mRNA and protein in ovarian cancer cell lines HO-8910,A2780 and SK-OV3 was detected by qRT-PCR and Western Blot.The HO-8910 cell line with high expression of VASH2 was selected to construct shRNA vector.The HO-8910 cell line was transfected with shRNA-1,shRNA-2,shRNA-3,shRNA-4 and shRNA-NC by qRT-PCR and immunoblotting,and the change of VASH2 protein expression in the cells was observed.After transfection,the expression of VASH2 protein in ShRNA-3 group was the least,and the interference effect was the best'ShRNA-3 was selected for subsequent experiments.2.Subsequently,the HO-8910 cell line was divided into normal group,shRNA group,blank load transfection group,cisplatin group,shRNA+eisplatin group.MTT,Transwell experiment and eell seratch test was implemented.Effects of transfection was evaluated on tumor cell proliferation,survival,invasion and metastasis and observation of biological behavior of ovarian cancer cells combine with cisplatin.Immunoblotting was detected for apoptosis-related proteins,autophagy-related proteins and EMT related proteins;3.A2780 cell line with low expression of VASH2 was selected,VASH2 overexpression vector was constructed,and A2780 cell line was divided into normal group,blank load transfection group,VASH2 overexpression group,cisplatin group and VASH2 overexpression+cisplatin group,and the effects of VASH2 overexpression on tumor cell proliferation,survival,invasion and metastasis ability and the effect of cisplatin on ovarian cancer cell biology were detected by MTT assay,Transwell assay and cell scratch assay.Immunoblotting was used to detected with apoptosis-related proteins,autophagy-related proteins,and EMT-related proteins.Results:1.The expression of VASH2 mRNA and protein in ovarian cancer cell lines was detected by qRT-PCR and Western blotting.It was found that VASH2 was highly expressed in HO-8910 cell line and low expressed in A2780 cell line,the difference was statistically significant(P<0.05).2.Five shRNA vectors were transfected into VASH2 high expression cell line HO-8910,and the effect on VASH2 gene expression was detected.It was found that transfection of shRNA-3 inhibited the expression of VASH2 gene(P<0.05).3.VASH2 high expression cell line HO-8910 was transfected shRNA-3,MTT assay showed that the proliferation of tumor cells could be inhibited by interfering with VASH2 expression.The proliferation ability of shRNA group,eisplatin group and shRNA+cisplatin group tumor cells was significantly decreased.Moreover,the inhibition of tumor cell proliferation by shRNA combined with cisplatin was more obvious and time-dependent,the difference was statistically significant(P<0.05).Transwell cell invasion assay was compared and the results showed that:invasive ability of tumor cells in shRNA group,cisplatin group and shRNA+cisplatin group were significantly decreased,and the number of cells in the shRNA combined with cisplatin administration group was significantly lower than that in the other groups(P<0.05).In the cell scratch test,the scratch healing rate of shRNA+cisplatin cells were slow,indicating that shRNA-3 can effectively inhibit the migration ability of tumor cells(P<0.05).Western blot analysis showed that the expression of P53j Caspase-3 and PARP was up-regulated in HO-8910 cells interfering with shRNA-3,and the expression of AKt,LC3B and Vimentin protein was down-regulated(P<0.05).The expression of protein in shRNA+DDP group was changed more obvious(P<0.05).4.After transfected into VASH2 overexpression vector,A2780 cell line showed promoted proliferation ablitiy in MTT assay.The proliferation ability of VASH2 overexpression combined with cisplatin group was between the two groups.Compared with the normal group,cell invasion ability was decreased,however,compared with DDP group,cell proliferation ability was promoted(P<0.05).Transwell cell invasion assay showed that the invasive ability of A2780 cells transfected with VASH2 was increased,and the cisplatin-treated group inhibited the invasion ability of cells.The invasive ability of VASH2 overexpression combined with cisplatin group was between the two groups.Compared with the normal group,cell invasion ability was decreased,however,compared with DDP group,eell invasion ability was promoted(P<0.05).In the eell scratch test,the migration ability of A2780 cells transfected with VASH2 significantly increased,and the migration ability of A2780 cells after cisplatin administration was significaltly reduced.The migration ability of cells in combine with overexpression and DDP was between the two groups.Compared with normal group,cell migration was decreased,however,compared to the DDP group,cell migration was promoted.(P<0.05).After overexpression of VASH2,P53,Caspase-3 and PARP were decreased,and the expression of AKt,LC3B and Vimentin was increased.On the other hand,the expression of P53,Caspase-3 and PARP increased,and the expression of AKt,LC3B and Vimentin was decreased in DDP group.OE+DDP group showed a lower p53,Caspase-3 and LC3B expression level and higher expression level of AKt,PARP and Vimentin compared with DDP group.(P<0.05).CONCLUSION:(1)Interference of VASH2 can effectively inhibit the proliferation,invasion and migration of ovarian cancer cells.(2)Interference of VASH2 attnuates the malignant biological ability of ovarian cancer cells,increases apoptosis of ovarian cancer cells,reduces autophagy and EMT progression,and strengthen the anti-tumor effect of cisplatin.(3)Overexpression of VASH2 enhances the malignant biological ability of ovarian cancer cells,decreases apoptosis of tumor cell,and increases autophagy and EMT progression,moreover decreases the effect of cisplatin,increasing chemotherapy resistance.(4)VASH2 gene is an oncogene with good prognostic evaluation and targeted therapeutic value in epithelial ovarian cancer. |
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