The Roles Of Survivin On Biological Behaviors Of Endometrial Cancer Cells And Its Signaling Pathway

Endometrial cancer is a common gynecological malignancy among women and adenocarcinoma is the most common type. The molecular mechanisms involved in the progression of endometrial cancer are unclear, which has hampered the development of an effective prevention.Survivin, a new member of inhibitor of apoptosis protein family, could regulate cell proliferation and cell apoptosis. It has been reported that survivin plays an important role in carcinogenesis, but little is known about the relationship between survivin and endometrial carcinogenesis, neither the roles of survivin on biological behaviors of endometrial cancer cells. Our experiments began with the investigation of the expressions of survivin in normal endometrium, atypical endometrium and endometrial cancer. RNA interfence technique was used, which could knock down survivin expression in endometrial cancer cells Ishikawa. Subsequently, RT-PCR, western blot, Brdu incorporation, flow cytometric assessment, MTT, wound healing assay and matrigel invasion assay were applied to explore the roles of survivin on proliferation, apoptosis, motility and invasion in Ishikawa cells. The results of our experiments suggest that abnormal expression of survivin is one of the important factors that affect biological behaviors of endometrial cancer cells.These experiments were divided into 4 parts, as following: â‘  Expression of survivin in endometrial cancer; â‘¡ The roles of survivin on proliferation and apoptosis in endometrial cancer cells; â‘¢ The roles of survivin on motility and invasion in endometrial cancer cells; â‘£ The regulation of EGFR/Ras/Raf/MAPK signaling pathway on survivin expression in endometrial cancer cells.Section I. Expression of survivin in endometrial cancer Objective To examine the expressions of survivin in normal endometrium,atypical endometriura, endometrial cancer tissues and cell lines and then explore the relationship between the aberrant expression of survivin and then the carcinogenesis of endometrial cancer.Methods RT-PCR, Western blot and immunochemistry were used to investigate the expressions of survivin mRNA and protein in normal endometrium, atypical endometrium, endometrial cancer tissues and Ishikawa, HEC-1-A cell lines.Results (1) Survivin mRNA was present in all samples of these three different types. The level of survivin mRNA was significantly higher in endometrial cancer than those in atypical and normal endometrium (P<0.05). The expression of survivin mRNA was strong in Ishikawa and HEC-1-A cells. (2) The level of survivin protein was significantly higher in endometrial cancer than those in atypical and normal endometrium (P<0.05 and P<0.01). The expression of survivin protein was strong in Ishikawa and HEC-l-A cells, consistently with survivin mRNA. The results were consistent with RT-PCR. The location of survivin protein was in the cytoplasm of endometrial cancer cell.Conclusions Overexpression of survivin might be relatd to the carcinogenesis of endometrial cancer.Section II. The roles of survivin on proliferation and apoptosis inendometrial cancer cellsObjective To investigate the roles and the related mechanisms of survivin on proliferation and apoptosis in endometrial cancer cells. Methods According the sequence of survivin mRNA, three survivin specific siRNAs and one non-target siRNA were designed, and cloned into pRNA plasmid vectors. The recombinant plasmids were transfected into Ishikawa cells. RNA interference was used to knockdown the survivin expression. Inhibition of survivin expression by RNAi was assessed by RT-PCR and western blot. Cell proliferation and cell apoptosis of Ishikawa cells before and after RNAi techniques were detected by the BrdU incorporation and flow cytometry, respectively. Moreover, in order to know the mechanisms of survivin regulating proliferation and apoptosis, wedetected the expressions of cyclin D1, cyclin E, p-RB, which were closely associated with proliferation, and the expressions of caspase-3, caspase-8, bcl-2, which were closely associated with apoptosis by western blot.Results (1) Three recombinant plasmids of survivn RNAi and one negative plasmid, which were named pRNAT-suv1,pRNAT-suv2,pRNAT-suv3 and pRNAT-neg, were constructed successfully. PRNAT-suv2 and pRNAT-suv3 could effectively inhibit the expressions of survivin. Compared with negative control groups, the levels of survivin mRNA in cells transfected with pRNAT-suv2, pRNAT-suv3 were inhibited (78. 97 ± 4. 13)% and (80. 23±5. 42)% ( P<0.01 ) , respectively. And the levels of survivin protein were down-regulated (81. 61 ± 3. 45)% and (82.63 ± 4.76)%(P<0.01), respectively. (2) Survivin RNAi also arrested of G₁ cell cycle and induced inhibition of cell proliferation. Cell proliferation rates of four groups were: control (52. 42 ± 3. 17 ) %, pRNAT-neg (49. 67±2. 52) %, PRNAT-suv2 (33 ± 2.65) % and pRNAT-suv3 (34 ±2.0) %, respectively. The difference of cell proliferation rates between RNAi groups and negative control groups was significant (P<0.01) . (3) The expressions of cyclin Dl and p-RB were down regulated after survivin RNAi, while the expression of cyclin E was unchanged. (3) Survivin RNAi also induced cell apoptosis. Cell apoptosis rates of four groups were: control (2.17±0. 05)%, pRNAT-neg (3. 99 ± 0. 17) %, pRNAT-suv2(38. 48±2.13)% and pRNAT-suv3(39. 39 ± 2. 55)%, respectively. The difference of cell apoptosis rates between RNAi groups and negative control groups was significant (P<0.01) . (5) Survivin RNAi also increased cleavage of caspase-3 and caspase-8, but it did not influence the level of bcl-2.Conclusions These results demonstrated that inhibition of survivin could decrease proliferation and induce apoptosis in Ishikawa cells. The mechanisms might be due to downregulation of cyclin Dl, p-RB and upregulation of cleaved caspase-3, cleaved caspase-8.Section III. The roles of survivin on motility and invasion in endometrial cancer cellsObjective To investigate the roles of survivin on motility and invasion in endometrial cancer cells.Methods The recombinant plasmids of pRNAT-suv2 and pRNAT-neg which have been constructed in section II were used to transfect Ishikawa cells. The cells were cultured in medium with G418 for 14 days and the clones stably and effectively expressing pRNAT-suv2 and pRNAT-neg were selected. Western blot was used to examine the effect of survivin RNAi. The motility, invasion and E-cadherin expression of cells transfected with pRNAT-suv2 and pRNAT-neg, or non-transfected cells were observed through wound healing assay, matrigel invasion assay and western blot. Results (1) Survivin expression was lower in cells stably expressing pRNAT-suv2 than those in non-transfected cells and cells stably expressing pRNAT-neg. (2) The motility and invasion in cells stably expressing pRNAT-suv2 were decreased comparing with non-transfected cells and cells stably expressing pRNAT-neg (p<0.01). (3) E-cadherin expression was higher in cells stably expressing pRNAT-suv2 than those in non-transfected cells and cells stably expressing pRNAT-neg (p<0. 05). Conclusions These results demonstrated that inhibition of survivin could decrease motility and invasion in Ishikawa cells, which might be associated with increased E-cadherin expression. The abnormal expression of survivin could promote invasion and metastasis in endometrial cancer.Section IV. The regulation of EGFR/Ras/Raf/MAPK signaling pathway on survivin expression in endometrial cancer cellsObjective To investigate the regulation of EGFR/Ras/Raf/MAPK signaling pathway on survivin expression in endometrial cancer cells. Methods EGF or TGF-α was added into the medium in which Ishikawa cells were cultured. The phosphorylation of ERK1/2, the key member of the MAPK family, and the expression of survivin protein were observed by Western blot when Ishikawa cells were treated with EGF or TGF-α at different time. U0126, the specific inhibitor of MAPK pathway, was also used to inhibit the phosphorylation of ERK1/2 and to study the expression of survivin protein.Results (1) EGF and TGF-α all induced the prompt phosphorylation of ERKl/2. The levels of survivin protein were increased to 8.35 and 9.42 times in the cells treated by EGF and TGF-α than those in the cells of control. (2) U0126, the inhibitor of MAPK pathway, completely inhibited the phosphorylation of ERKl/2 induced by EGF and TGF-α . Blocking the MAPK pathway with U0126 neutralized the upregulation of survivin protein expressions induced by EGF and TGF-α.Conclusions EGF and TGF-α could upregulate the expression of survivin protein through activating MAPK pathway in endometrial cancer. MAPK pathway was one of the upstream signaling pathways regulating survivin. In summury, the results demonstrated that survivin was over-expressed in endometrial cancer, and it could promote proliferation and invasion and inhibit apoptosis in endometrial cancer cells. MAPK pathway was one of the upstream signaling pathways regulating survivin. Our results provide a new strategy for further studying the carcinogenesis of endometrial cancer.