| Objective:To explore expression of CtBP2protein in BPH and prostate cancer tissue, and the relationship of clinicopathologic characteristics and patient outcome. We intend to silence genes CtBP2in prostate cancer cell by using short hairpin RNA(shRNA) interference technology, and to establish stable transfected cell lines. Then we propose to investigate the effect of CtBP2-shRNA on proliferation of prostate cancer cells by RNA interference targeting CtBP2gene silencing.Methods:1. Expression of CtBP2protein were detected by using immunohistochemistry in60cases of BPH and160cases of prostate cancer tissue, statistical analysis was performed with clinicopathologic parameters such as pathological Gleason scores, clinical stage and PSA. Kaplan-Meier survival analysis and multivariate Cox proportional hazard model analysis were used to study the relationship between survival time and CtBP2protein expression.2. qPCR and Western-blot were used to screen the prostate cancer cell strain expressing CtBP2highly. Three CtBP2-shRNA expression vectors, which targeted different nucleotides of CtBP2mRNA respectively, mediated by Liposomes were transfected into cells. We chose one plasmid that had the best silencing effect by RT-PCR and Western blotting.3. Transfected cells were selected by puromycin to become stably transfected cells by using the biotechnologies of cloning.The expression levels of CtBP2mRNA and protein were determined by qPCR and Western blotting.4. To assess the effect of CtBP2-shRNA on the cellular proliferation was examined by cell counting, colony formation assay and MTT assay.Results:1. The results of immunohistochemistry analysis showed that CtBP2was over-expressed in86%of prostate cancer tissues, decreased CtBP2expression was detected in benign prostate hyperplastic tissue, which was considered as normal expression(P<0.05); CtBP2over-expression was closely correlated with increased serum PSA level (P=0.018), advanced tumor stage (T3)(P=0.025), higher Gleason scores (P=0.019). Kaplan-Meier survival analysis revealed that the CtBP2expression level correlated significantly with the survival time of patients with PCa (P=0.027). Multivariate Cox proportional hazard model analysis showed that CtBP2expression is independent prognostic factor (P=0.043).2. The results of qPCR and Western blotting showed that CtBP2was highest expressed in PC3among three cell lines. CtBP2-shRNA expression vectors were transfected into PC3successfully. Compared with RNAi plasmid2and3, the RNAi plasmid1targeting the838-857nucleotides exerted more significant silencing effect on CtBP2expression(P<0.05). The RNAi plasmid targeting the838-857nucleotides of CtBP2mRNA was transfected into the PC3, and the PC3cells of stable transfection were obtained by puromycin screen. The expression level of CtBP2mRNA and protein of transfected PC3were significantly decreased(P<0.05).3.(1) Compared with groups including negtive control (Vector-PC3) and blank control (PC3cells),the proliferation of experimental group (CtBP2-shRNA-PC3) was decreased (P<0.05).(2) In the clone formation assay, the number of clones of the three groups were136±11,128±12and56±9respectively. The diameter of clones in the silence group was shorter than that in other groups, the suppression ratio was59.8%.Conclusions:OBP2was over-expressed in the prostate cancer tissue. Increased CtBP2expression was correlated with several malignant behaviors, e.g. increased serum PSA level, advanced tumor stage (T3), higher Gleason scores and poor patient outcome. The plasmid targeting838-857nucleotides of CtBP2mRNA was transfected into PC3cell efficiently, and repressed the expression of CtBP2in post-transcriptional level effectively. Also the shRNA targeting CtBP2gene could decrease the proliferation of PC3cells. These results suggested CtBP2was related to malignant behavior of prostate cancer, it could be a predictor of progress and poor patient outcome of prostate cancer. |
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