| Now, the malignant tumor has become one of the main cutthroat of the health of human life. Although there are many new techniques coming out, it's impossible to keep cancer from relapse or metastasis radically. Therefore, it is still a puzzle to cure cancer. This also forces researchers and doctors to keep on looking for a new breakthrough, and try to overcome cancer.It's known that reduced apoptosis correlates with the occurrence and the progress of cancers, as well as the tolerance of cancer cells to chemotherapy or radiotherapy. So it is not surprising that induction of apoptosis has emerged as a new therapeutic strategy to help eliminate cancer cells. Survivin is a member of IAP (inhibitor of apoptosis protein) family, which has been discovered in 1997. It is widely expressed in almost all kinds of cancer tissues, but scarcely in normal adult tissues. Survivin is a multifunctional molecule. It can inhibit the functions of effect caspases, caspase3 and 7, by binding them to keep tumor cells from apoptosis. Accordingly, survivin prevents apoptosis induced by Fas, Bax and anticancer drugs. Survivin is excessively expressed in G2/M phase of the cell cycle and associates withmicrotubules of the mitotic spindle in a specific and saturable reaction that is regulated by microtubule dynamics. Recently, survivin has emerged as a predictive and prognostic marker in some kinds of clinical cancer research. Gene therapy and immunotherapy that aim at survivin gene have also been developed gradually, which makes survivin a new therapeutic target in cancer treatment.Objective:To inhibit the expression of survivin gene by RNA interference in human breast cancer SKBr-3 cells and cervix cancer HeLa cells. And then to investigate the proliferation ability of the survivin-knockdown cancer cells in vitro and in vivo, and the sensitivity of these cells to anti-cancer drugs or irradiation.Methods:1. Oligonucleotides were desiged and synthesized, and then were put into pSUPER vector to construct two eukaryotic expression vectors for RNA interference of human survivin gene. Cancer cells which have high expression of endogenous survivin mRNA was co-transfected with the recombinant vector and pcDNA3 plasmid, and subjected to G418 selection. Single cell clones were collected and analyzed by RT-PCR to determine the level of survivin mRNA. The protein level of survivin was detected by Western blot, indirect immunofluoresence staining and immunocytochemical staining respectively.2. The transfectants cultured on cover slips were observed following HE staining. The proliferation of these cells was investigated through colony forming assay and MTT assay. The cell cycle phases were determined byflow cytometry (FCM).3. ApcDNA3 plasmid was generated by removing CMV promoter from pcDNA3. HI-SI DNA fragment was digested from pSUPER-Sl, and then was cloned into ApcDNA3 to get a reconstructed vector pcDNA3-H1-S1 which is resistant to G418. Cancer cells were transfected with this recombinant pcDNA3-H1-S1 vector, and were examined by Annexin-V staining, TUNEL and electron microscopy following induction with low doses of apoptosis stimuli. The sensitivity of these transfectants to chemical drugs or irradiation was examined by MTT assay or colony forming assay, respectively.4. Tumorigenicity assay was performed in nude mouse model to assess the malignant behavior of the survivin-knockdown cancer cells in vivo. The microvessel density was investigated by immunohistochemical staining of anti-CD34 antibody on the tumor tissue slices of nude mice models. Metastasis of tumor cells to other organs was measured by HE staining.Results:1. Two potent sequences encoding survivin-target siRNA were deviced and synthesized, and the RNAi eukaryotic expression vectors, pSUPER-Sl and pSUPER-S2, were subsequently constructed. The SKBr-3 cells which possess high expression of survivin gene were co-transfected with pSUPER-S1 and pcDNA3 at the ratio of 10:1. Both the mRNA levels and protein expression of suvivin gene in different stably transfected of SKBr-3 cell clones were reduced significantly, as detected by RT-PCR, western blot, indirect immunofluoresence staining and immunocytochemical staining. As a control, the expression of XIAP was unchanged, suggesting the specific gene silencing of pSUPER-S1 vector.2. As determined by HE staining, pSUPER-S1-transfected SKBr-3 cells became big, flattened and irregular, whereas the control cells showed a normal morphology. FCM analysis revealed that most of pSUPER-S1-transfected SKBr-3 cells were blocked in G1 phase of cell cycle (74.03+8.91%). The results of MTT assay and colony forming assay are consistent: the proliferation of these treated SKBr-3 cells was slowed down because of the deletion of survivin gene expression.3. A new recombinant RNAi vector pcDNA3-H1-S1 (S1&P) and its negative control pcDNA3-Hl (H1&P) both of which harbored a G418 resistant neo gene were constructed successfully, and were confirmed by DNA sequencing. H1&P and S1&P constructs were transiently introduced into SKBr-3 cells, which were exposed to low doses of TNFcu DOX or 60Co y-irradiation 48h later. TUNEL assay showed that knock-down of survivin gene resensitized SKBr-3 cells to apoptosis in different degrees. Annexin-V test indicated that the stably transfected SKBr-3 cells (S1-1) had a more sensitive response to the above 3 kinds of stimuli than transiently transfected SKBr-3 cells.4. SKBr-3 cells and HeLa cells were stably transfected with Sl&P, respectively. Cells were collected for discrepancy microscope observation, sensitivity test to chemical drugs or irradiation. Spontaneous apoptosis were observed both in S1&P transfected SKBr-3 cells and HeLa cells. Cells were harvested after exposed to 0.5ug/ml DOX for 12h. In electron microscopy analysis, Sl&P transfected cells displayed typical apoptotic characters, such as shrinkage nuclei, chromatin condensation, plasma membrane blebbing, chromatin breakage fragments, etc. The data obtained from sensitivity test impressed us that HeLa-S1&P cells were more sensitive to the apoptotic...
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