The Expression And Effect Of TGFBR2 In Cervical Cancer

Part I The expression and significance of TGFBR2 protein in human Cervical cancer tissuesObjective The transforming growth factor beta receptor type II gene (TGFBR2) is a cancer suppressor gene in the TGF-β/Smad pathway that plays an important role in cell growth arrest. Our previous study found that the TGFBR2 protein interacts with the hTERT protein by a yeast two-hybrid system. However, the association between survival and the expression of these two proteins and dual TGFBR2/hTERT tumor status has not been previously investigated.Methods The expressions of TGFBR2 and hTERT were detected by using SP immunohistochemical staining assay; Data between two groups were analyzed using chi-square test.Results Expression of TGFBR2 protein was significantly downregulated in cervical cancer tissues compared to the normal tissues, P< 0.05. In addition, it was also identified that the International Federation of Gynecology and Obstetrics (FIGO) stage, differentiation grade, pelvic lymph node metastasis, vaginal invasion, parametrial infiltration, adjuvant radiotherapy, recurrence, and TGFBR2 expression were significantly associated with overall survival (P< 0.05 for all). Furthermore, the multivariate analysis showed that TGFBR2 expression, lymph node metastasis, FIGO stage, recurrence, and differentiation grade were independent prognostic factors. More notably, a TGFBR2low/hTERThigh tumor status significantly predicted the worst overall survival (hazard ratio,2.23; 95% confidence interval,1.31-3.80).Conclusion In conclusion, our results suggest that a TGFBR2 expression is a predictor of poor prognosis and that the simultaneous use of small-molecule TGFBR2 analogues in cervical cancer patients may be an effective treatment strategy.Part II The effects of TGFBR2 knockdown on cell biological behaviors and telomerase activityObjective To investigate the role of TGFBR2 gene in mediating Hela cell proliferation and telomerase activity by RNA interference technology.Methods shRNA targeted TGFBR2 (shTGFBR2) was transfected into Hela cells using Lipofectamine 2000. Realtime PCR and western blot were performed to determine the effects of shTGFBR2 on TGFBR2 expression. Real-time PCR was then used to detect the mRNA level of TGFBR2. Then the recombinant plasmid of shNC and shTGFBR2 was transfected into Hela cell and western blot was used to detect the protein level of TGFBR2. Cells were detected by CCK8 assay on their proliferation.Results Compared with blank group and negetive control group, proliferation of Hela-shTGFBR2 cells showed a marked increase (P<0.05). In addtion, the telomerase activity were significantly increased in Hela-shTGFBR2 group.Conclusion By silencing TGFBR2 gene, the cell proliferation and telomerase activity of Hela cells were both enhanced.Part Ⅲ The influence of TGFBR2 on the sensitivity to cisplatin and its mechanismObjective To explore the influence of TGFBR2 on the sensitivity to cisplatin and its mechanism.Methods Plasmid of siRNA targeting on TGFBR2 and negative control were transfected into Hela cells with LipofectamineTM2000.After treated with cisplatin of different concentration, the viability of Hela cells was examined by CCK8 assay. Cell proliferation and cytotoxicity was measured by CCK8 assay. Cell cycle distribution and cell apoptosis was demonstrated by flow cytometry.Results CCK8 assay demonstrated that the viability of Hela cells was decreased along with the concentration of CDDP increasing. Flow cytometry assay proved that the apoptosis ratio of the shTGFBR2 group was decreased significantly compared with the control.Our results showed that TGFBR2 knockdown limited cell apotosis significantly(p<0.05). Furthermore, the suppression of TGFBR2 can decrease the chemosensitivity of cervical cancer cells to DDP significantly.Conclusions After the expression of TGFBR2 was suppressed by RNA interference with a eukaryotic expression plasmid, the sensitivity of Hela cells to cisplatin was decreased. TGFBR2 not only plays an essential role in the proliferation of Hela cells but also represents a potential approach to chemosensitization therapy in human cervical cancer.