A Pilot Study On The Effect Of Silencing Survivin Gene On The Sensitity Of HCT-8/VCR To Vincristine And Cell Apoptosis

BackgroundColorectal cancer is a common malignancy of the upper digestive tract .As the socio-economic development,lifestyle and eating habits change recently, the incidence of colorectal cancer in China has an increasing trend and is now in the 4th to 6th place only after gastric cancer, esophagus cancer and so on. Although the modern surgical methods have achieved some progress,it still lacks effective methods to prevent post-operative recurrence or metastasis in the treatment of colorectal cancer. In recent years, chemotherapy before and after perioperative has made significant effect in patients with colorectal cancer.However,chemotherapy against colorectal cancer usually fails because of the occurrence of drug resistance,which means the resistant characteristic of the tumor cells limits the effect.Drug resistance of tumors and tumor cell apoptosis are two hot spots in cancer research. The traditional concept of cancer treatment is chemotherapy drugs directly function on DNA, tubulin and other targets to kill tumor cells. With the gradual deepening of research on cell apoptosis,it is recognized that chemotherapy drugs can also kill tumor cells by inducing cell apoptosis and as a result,the inhibition against tumor cell apoptosis can lead to a variety of drug resistance against chemotherapy drugs.The mechanisms of drug resistance in colorectal cancer involve many aspects. So far, it is believed that the tolerance of tumor cells to drug-induced apoptosis, or the increased apoptosis threshold is one of the major mechanisms of drug resistance in colorectal cancer. It becomes a vital issue in research about drug resistance of tumors how to modulate the abnormal apoptosis pathway and restore the sensitivity of tumor to stimulating factor (such as chemotherapeutics)-induced apoptosis. Gene therapy is an important strategy that can reverse drug resistance in tumors.Survivin is a new member of IAP (apoptosis inhibitory protein) family, and it has three main features: 1.Survivin gene has been the intensest apoptosis inhibitory factor discovered so far.2. Survivin maintains the normal mitosis at the beginning of mitosis, and thereby promotes cell proliferation. 3. Survivin takes part in the formation of capillary network,in the inhibition of vascular endothelial apoptosis,and in promoting angiogenesis. Survivin mRNA and Survivin protein express overly in almost all studied tumor cells,such as lung cancer, stomach cancer,colon cancer,breast cancer, ovarian cancer, renal cancer and melanoma, which play an important role in the occurrence and development of malignant tumors as well as in drug resistance during chemotherapy, as Survivin is the strongest apoptosis inhibitory factor in IAPs. Thomberry and Lazebnik confirmed that the apoptosis pathway activated by caspase was closely related to the susceptibility of chemotherapeutics and the decrease of apoptosis reactivity caused by up-regulation of apoptosis-suppressing gene or down-regulation of apoptosis- promoting gene is one of the reasons of drug resistance of tumor cells. Another experiment showed that the reduced expression of Survivin can enhance the susceptibility of chemotherapy of non-small cell lung cancer cell H520 on cisplatin. Survivin mainly inhibits the activity of caspases through three different ways to function directly or indirectly against apoptosis: 1. Survivin combines stably with terminal effector Caspases-3, Caspases-7 in the form of homodimer and then inhibits the protein cutting effect of the two, blocking the apoptosis signals from the Fas / FasL pathway, and (or) the mitochondrial pathway and blocking tumor cell apoptosis.2. It combines with Caspases-9 induced by SMAC to inhibit its activity.3.It interacts with cell cycle protein kinase cdk4 and p34cdc2, so that p21 can be released from the complex of CDK4 and then combines with mitochondrial caspase-3.In the end, its activity and cell apoptosis are inhibited.Small double strands RNA(dsRNA) complement with target gene mRNA sequence is introduced into target cells, the dsRNA joint with the targent gene mRNA specificly and degrade it, then the cells express the phenotype that the target gene deleted, the whole process is called RNA interference(RNAi). Small interference RNA (siRNA) or shot hairpin RNA(shRNA) both are the medium of RNAi.RNAi has a character of specificity of sequence which can discriminate mutation gene sequence with normal gene sequence,finally only the mutation gene is knocked out. Otherwise RNAi has characters of high performance and magnification effective.shRNA is more stable than siRNA, and it can increase the time of RNAi.In conclusion, this study was conducted to investigate whether there were any effects on apoptosis of tumor cells after blocking Survivin gene and what the effects were by building colorectal cancer resistant cell line HCT-8/VCR and interfering targetedly with Survivin gene in colorectal cancer resistant cell lines by RNAi technology,then to determine whether there was a correlation beween Survivin gene and the apoptosis mechanism of resistance of tumor cells, further to demonstrate the role Survivin gene was playing in the occurrence and development of drug resistance and finally to provide some new clues for the treatment of resistant colorectal cancer.and tumor cell apoptosis mechanism of resistance in the fashion related? Survivin further found that the resistance gene and development of the role of resistance for the treatment of colorectal cancer provide new clues. At present, reports on this issue is still few, so there is need for us to discover something and verify something.Method1,HCT-8/VCR (colorectal cancer cell resistant to vincristine) was constructed.1.1,HCT-8/VCR was constructed by increased concentration-induced method when vincristine(VCR) was used as the revulsive and HCT-8 cell lines as the study subjects.1.2,50% inhibiting concentration (IC50) : IC50 was determined by the method of CCK-8 on HCT-8 and HCT-8/VCR which were in the logarithmic phase. Optical density (OD) of samples was detected by the application of Bio-TEK Microplate reader at the wavelength of 450nm and then the Cell Survival Rate was calculated. Inhibition rate=(1-OD_drug/OD_control)×100%. Dose-response curve was drawn on semi-logarithmic paper to determine IC50.1.3,The detection of Resistance Index: Resistance Index = IC50 of resistant cell /IC50 of normal cells.1.4,Determination of growth curve. HCT-8/VCR and HCT-8 that in the logarithmic growth phase were transported in centrifuge tube after cell dissociation, and then prepared for 10⁵/ml cell suspension. The 10⁵/ml cell suspension was inoculated in 6-well culture plate, 3ml each hole, and 3 holes were counted per 24h for 7days. After that, the averages were chosen to draw cell proliferation curve. Doubling time of cells in logarithmic proliferation period was determined by the formula of Td(h)=(Tlg2)/(lgN-lgN0).T represented the time of cells in logarithmic proliferation, No was the number of cells at the beginning of logarithmic proliferation,and N is the number of cells at the end of logarithmic proliferation.2,Survivin gene in HCT-8/VCR cells was silenced by RNAi technology ,and then gene expression, cell apoptosis and the susceptibility of cells to drug were observed. HCT-8/VCR cells were divided into four experimental groups as follows: Group A: RNA interference alone, Group B: RNA interference+drug (joint group), Group C: drug alone, Group D: negative control. Survivin-targeted siRNA was transfected into the HCT-8/VCR cells through the transfection technology of liposome. Each index was detected after culture for 48 hours: RT-PCR was used to detect the mRNA level of Survivin; The detection of Survivin protein: Western blot was used to detect the protein level of Survivin ; Hoechst 33258 staining was used to detect the karyomorphological diversify of apoptotic cells; Annexin V-FITC/PI double staining and flow cytometry analysis were used; Caspase-3 Detection Kits were used to detect the activity of Caspase-3 in each group; IC50 of HCT-8/VCR cells and HCT-8/VCR cells after RNA interference to VCR was detected by cck-8 in the experiment of improving the sensitivity of the drug resistant cells to drugs.Results1. HCT-8/VCR (colorectal cancer cell resistant to vincristine) was constructed.HCT-8 cell can grow and proliferate steadily in the culture medium containing 3.0μg/mL VCR after eight months'inductive culture.IC50 (HCT-8): 1.11±0.11μg/mL,IC50(HCT-8/VCR): 13.46±0.72μg/mL, p<0.05. Resistance index (RI) was 12.17.2.Survivin gene in HCT-8/VCR cells was silenced by RNAi technology ,and then gene expression, cell apoptosis and the susceptibility of cells to drug were observed.The result showed 48h after transfection, the expression of Survivin mRNA(F=958.837,P<0.05) and Protein decreased significantly (F=958.470,P<0.05) .HCT-8/VCR in the intervention group showed obvious morphologic features ofapoptosis like karyopyknosis,while there were few or no changes in the control group;The difference of the apoptosis ratio between the two doses of the Survivin siRNA group (F=2190.548 , P<0.05)and VCR group (F=1516.185 , P<0.05) was significant. The synergism between the Survivin siRNA group and VCR group was significant (F=467.371, P<0.05) ;The difference of the activity ratio of caspase-3 between the two doses of the Survivin siRNA group (F=68.175, P<0.05)and VCR group (F=193.624, P<0.05) was significant. The synergism between the Survivin siRNA group and VCR group was significant (F=10.107, P<0.05) . The sensitivity (IC50) of HCT-8/VCR cells to VCR before and after Survivin RNA interference detected by cck-8 were respectively 12.96±0.57μg/ml and 6.95±0.28μg/ml, which showed the sensitivity of drug resistant cells to drugs were increased (P<0.05) .Conclusion1 .HCT-8 cell can grow and proliferate steadily in the culture medium containing 3.0μg/mL VCR.2.Survivin siRNA induced cell apoptosis itself through down-regulating the expression of Survivin, reversed the tolerance of cells to cell apoptosis induced by VCR, with the apoptosis threshold value reduced, and thus finally increased the sensitivity of the colorectal cancer cells to VCR;there was a synergistic effect of cell apoptosis when siRNA was combined with middle or low-dose VCR.