Bcl-2 Small Interfering RNA In Inhibition Of Human Colon Cancer Cells Apoptosis

Background and AimAccording to functional and structural criteria, the Bcl-2 gene family can be divided into two main categories. One category resists apoptosis, including bcl-2, bcl-xl, bcl-w, and mcl-1 etc. The other category facilitates apoptosis, including bax, bad,Bak,Hrk and Bim. Most members of the Bcl-2 family are able to form homodimers or heterodimers, they combine with each other and also restrain mutually. When the antiapoptotic proteins dimerize with proapoptoic proteins, they alter the ratios between the relative amounts of antiapoptotic and proapoptotic proteins that influence susceptibility to cell apoptosis.Bcl-2 was one of the earliest identified and separated cancer genes of Bcl-2 family proteins; otherwise it was studied comparatively thoroughly until now. It's mechanism of biology action in cell death may be concerned with a few factors such as Ca2+ dependent endonuclease, oxygen free radicals, control of cell signaling and so on. Many results indicate that the Bcl-2 protein localizes at the outer mitochondrial membrane, the endoplasmic reticulum membrane, and the nuclear envelope. It has double functions as ion channel and docking protein that facilite it's key role in regulation of apoptosis. Bcl-2 acts as an antioxidant and prevents the release of cytochrome-c from mitochondria by altering the mitochondrial permeability, which is associated with the apoptosis activating factor (Apaf-1) and procaspase-9 for subsequent activation of Caspase-9 and Caspase-3 who are the terminal effectors of cell apoptosis. Thus, as a protein gene of anti-apoptosis, Bcl-2 can protect cells from apoptosis that is stimulated and induced by virus and oxidants.At present, scientists in the biology fields have investigated extensively the important roles played by Bcl-2 in cell apoptosis, tumor occurrence and other serious diseases. Studies show that the high expression level of Bcl-2 gene and the associated proteins in human cells can inhibit cell apoptosis, which is considered as the mostimportant reason that leads to tumorigenesis and drug tolerance development. Therefore the objective that utilization of RNAi technique can down-regulate the Bcl-2 expression and eventually accelerates apoptosis of tumor cells, which is explored actively as a new way in tumor treatments.RNA interference (RNAi) is a kind of conservative response of defense from low organism to mammal existed, which has recently been used to silence gene expression in various species. Double-stranded RNA was cut about 21~23nt siRNA by endonuclease RNaseâ…¢in cell, and formatted RNA-induced silencing complex, RISC identified and degraded mRNA of homological rank, it leads to ultimately specific silence of gene expression.The main focus of this experiment was to observe the biology changes (especially the effects on inhibiting growth) after transfecting the Bcl-2-siRNA into human colon cancer cells Caco-2 in vitro through utilizing several techniques such as RT-PCR, Western blot, MTT and TUNEL assay. We also investigate some related molecules such as survivin and initially discussed its possible molecular mechanism in order to establish background for further study of gene function and clinical target therapeutic tool.Methods1. After extracting the total RNA from the Caco-2 cells of human colon cancer, the targeted Bcl-2 cDNA of 314bp was amplified by RT-PCR with the specific primers from 861 to 1174bp; applied Western blot to test the Bcl-2 protein expression level of different colon cancer cell lines.2. The Bcl-2-siRNAs were transfected into the Caco-2 cell by Lipofectamine 2000TM. At 24-96h after siRNAs transfection, RT-PCR and Western-blot were applied to detect the mRNA and protein expression level of Bcl-2 gene.3. The changes of growth and proliferation of transfected group and control group cells were analyzed by MTT method. The apoptotic ratio and survival condition were observed by TUNEL assay. In addition, the protein level of Caspase-3 in the cells was determined by Western blot in order to assess the occurrence of cell apoptosis. The apoptosis correlated factor such as survivin was detected.Results1. Positive Bcl-2 expression cell line Caco-2 was determined by utilizing RT-PCR and Western blot from the human colon cancer cell lines.2. Bcl-2-siRNAs can be efficiently transfected into Caco-2 cells by usingLipofectamine 2000TM. RT-PCR and Western blot assays showed that the expression level of Bc1-2 protein in Caco-2 cells was obviously down-regulated in Bc1-2-siRNAs treatment group. There was no difference in Bc1-2 protein levels between control-siRNAs and untreated cells.3. The proliferation of transfected cells had no apparent change compared with control-siRNAs transfected and non-transfected cells (P>0.05), whereas the apoptotic ratio was significantly higher in Bcl-2-siRNA treated group than those in control (P<0.01) detected by TUNEL assay. In addition Western blot indicated the increasing protein level of Caspase-3 after transfection as expected. The protein level of survivin decreased evidently after transfection by Western blot analysis, which is consisted with the change levels of Bcl-2.ConclusionsBcl-2-siRNA can effectively down-regulated the Bcl-2 protein and promote the cell apoptosis of human colon cancer cells,which might be a potential applicative way for colon cancer treatment.We initially detected the expression levels of survivin,Caspase-3 genes, and explained the possible molecular interacting mechanisms, thus prepared the experiment foundations to develop the combination RNAi treatment of Bcl-2 with survivin for human colon cancer.