An Experimental Study On Bcl-xl Gene Expression And RNAi In Bladder Carcinoma

Bladder cancer is the most common tumors of genitourinary tract. The most important clinical character of it is high incidence rate, high recurrence rate, and low malignancy. TCCB has been regarded as genie disease and regulationed by multipathways and multitargets, but the exact mechanisms of TCCB is unclear. In recent years,molecule mechanisms about TCCB is always the hot point,such as gene mutation, protooncogene activation, antioncogene inactivation, apoptosis disequilibrium and so on.Apoptosis is programmed cell death. When the machinery of apoptosis is improperly regulated, it will result in tumorigenesis and tumor progression. In the case of cancer, an abnormally increased cellular lifespan as a consequence of reduced apoptosis is ideal to favor the insurgence of genetic mutations, as well as to shield transformed cells from death induced by chemotherapy or radiotherapy, and to promote their survival at distant sites. Therefore, it is not surprising that manipulation of apoptosis has emerged as a new therapeutic strategy to help eliminate cancer cells.As protein gene of antiapoptosis, Bcl-xl can protect cells which its were a-voided apoptosis by virus, oxidant stimulated and induced . The high expression of Bcl-xl is one of important reasons that it lead to occurrence of tumor and development of tolerance . Therefore, the objective that utilization of RNA technique and expression of anti - Bcl-xl gene achieves and accelerates apoptosis of tumor cells is a kind of new pathway investigated actively at present. Overexpres-sion of Bcl-xl, an anti - apoptotic member of bcl - 2 family, contributes to the apoptosis resistance of cancer cells.RNAi is a sequence - specific, post - transcriptional gene silencing mechanism , which is triggered by double - stranded RNA (dsRNA) and causes the degradation of mRNA homologous in sequence to the dsRNA. These siRNA were shown to avoid the well - documented nonspecific effects triggered by longer double - stranded RNAs in mammalian cells.Here We design and chemically synthesize 21 nucleotides ( nt) siRNAs targeting against Bcl-xl and study its effects of on gene silencing and biological behavior of human T24 cells. We hope the results will provide rationale for the therapy used RNA - based drugs for the treatment of TCCB.Objective1 . To detect the expression of the inhibitor of apoptosis gene,Bcl-xl,and its correlation gene Bak in bladder carcinoma, and study the relationship of theses factors on malignant biological behaviour of TCCB.2 . To provide an easy , quick and economical method to synthesize of siRNA, and the reactive condition is further optimized.3. To investigate the expression of Bcl-xl in TCCB and the specific inhibition of gene expression in T24 cells by small interfering RNA.4. To study the expression of Bcl-xl on chemotherapeutics siRNA blocking the gene, inducing apoptotic effect, and its synergistic effect DDP, in human bladder cell line in vitro.Methods1. fourty - nine cases of the clinic pathologic information of TCCB patiat was studied retrospectively. By polyclonal antibody were investigated by staining respectively. The above dates were analyzed statistically together.2. according to the principles on the web site www. oligoengine. com, we designed 3 siRNAs sequences. After synthesis of DNA templates, single stranded RNAs were transcribed in vitro from the templates using T7 promoter. High quality siRNAs were obtained after annealing, digestion and purification.3. siRNAs were transfected into T24 by using LipfectamineTM 2000 reagent. Expression of Bcl-xl protein was study by mianyizuhua . Down - regulation of Bcl-xl was detected by RT - PCR and Western - blot, and the most effctive siRNA was selected. 4 . Spontaneous apoptosis of cells was detected by acridine orange (AO) , plate colony assays were used to detect the cellular proliferation after trans - fection. Apoptosis and morphologic changes of tumor cells were analyzed by flow cytometry .5. Statistical analysis: Biostatistic analyses were done by SPSS 11.0 software package. Data from the experiment was analyzed by ANOVA.Results1. In TCCB Bcl-xl and Bak are positive with different level. The difference is significant. The contents of Bcl-xl, Bak pro were significantly increased with stage and grade, respectively;It was showed that there were significantly correlation between Bcl-xl or Bak and stage or grade of TCCB.2 . siRNA were synthesized successfully in vitro and their lengths were 21bp.3. Compared with blank controls, the expression of Bcl-xl at mRNA level was markedly down - regulated in siRNA - transfected T24 .4. Western - blot analysis show expression of Bcl-xl protein after transfec-tion in T24 cell is higher than in the control group.5. MTT results-. The inhibition rates of siRNA3 group after transfection were remarkably higher than the control (p < 0. 01). The results indicated that cancer cells growth could be inhibited by the siRNA3. The inhibition rates of siRNA3 groups increased with time, which revealed that inhibition could keep some time.6. Plate cloning assays results;The clone of A^BNC on the 7th day after transfection were237 ±8^ 202 ± 10^ 87 ± 11 ,the values of the control CI were obviously lower (P < 0.05). The experiment results indicated that when Bcl-xl expression was inhibited, the proliferation of cancer cells was suppressed too.7. Flow cytometrv detection, showed, apoptotzc peak was seen in siRNA3groups 24h after transfection, which get obviously as transfection time prolonged, apoptosis index (AI) of siRNA3 groups was significantly higher than that of the normal control transfection;AO fluorescent staining showed the characteristic of typical apoptotic changes, including cell atrophy, condensed chromatin, condensed and heavily stained nuclei.Conclusions1. The expression of Bcl-xl in Bladder carcinoma were significantly higher than those in nomal bladder tissue, which could also be tested in bladder carcinoma cell lines. It suggested that Bcl-xl is involved in tumorgenesis and tumor progression, and Bcl-xl may be a good biomarker in early diagnosis and super-viseing the development of Bladder carcinoma.2. The synthesized siRNAs in vitro was able to down - regulate the expression of Bcl-xl. There were different capabilities of the specific siRNAs down -regulation. Although Bcl-xl siRNA could efectively inhibit the Bcl-xl expression, it could not completely silence the target genes. The transient transfected Bcl-xl siRNA3 could effectively inhibit the growth of the cancer cells and induce theirs apoptosis.3. The transient transfected Bcl-xl siRNA3 could effectively inhibit the growth of the cancer cells and induce theirs apoptosis. These findings identified surviving pathways in cancer, as a new target for disrupting cell viability.