Bcl-2 Small Interfering RNA in Inhibition of Human Colon Cancer Cells Apoptosis
|School||Dalian Medical University|
|Course||Biochemistry and Molecular Biology|
|Keywords||Bcl-2 siRNA Apoptosis Colorectal cancer|
Background and Purpose of Bcl-2 gene family according to the structure and function can be divided into two categories: antiapoptotic genes including Bcl-2, Bcl-x1, Bcl-w, mcl-1, etc.; pro-apoptotic gene Bcl- xs, Bax, Bad, Bak, Hrk and Bim. These two substances are combined with each other to suppress each other, often determined by its number of relatively how much apoptosis occurred or not. Bcl-2 proteins in the Bcl-2 family is the first to be found separated from the family members far more thorough study. It is located in the mitochondrial membrane, endoplasmic reticulum and nuclear membrane ion channels (ion channel) and dual function of the docked protein (docking protein)-mediated apoptosis pathway plays a key role. Bcl-2 protein through regulation of endoplasmic reticulum Ca2 participate in the nuclear internal and external material transport and membrane permeability transition (PT) to block cytochrome C release from the mitochondria, thereby preventing its Apaf-1, procaspase-9 interaction of ultimately inhibit Caspase-9, Caspase-3-induced apoptosis cascade process. In short, as an anti-apoptotic gene, Bcl-2 can protect cells from viruses, oxidants and other stimulus-induced apoptosis. Studies suggest that Bcl-2 gene and its associated protein is highly expressed in the inhibition of apoptosis is one of the important factors in tumorigenesis and drug phenomenon. Therefore the use of RNAi technology to achieve the purpose of promoting tumor cell apoptosis by inhibiting the expression of Bcl-2 gene is now actively exploring a new cancer treatment pathway. RNAi is a conserved from lower organisms to mammals have defense reaction. Double-stranded RNA in intracellular RNA enzyme Ⅲ (Ribonuclase Ⅲ, RNase Ⅲ) digested into approximately 21 ~ 23nt small fragment interference RNA (short interfering RNA, siRNA), thus the formation of siRNA-protein complex that RNA-induced silencing complex ( RNA-induced silencing complex, RISC). RISC degradation having homologous sequences by identifying messenger RNA (messenger ribonucleicacid, mRNA), and ultimately lead to the expression of specific genes the silencers (Gene silencing) . In this study, the inhibitor of apoptosis gene Bcl-2 siRNA transduced into colorectal cancer cell lines, combined with the study of tumor cell apoptosis, MTT assay, immunocytochemistry, Western blot, RT-PCR method, using in vitro cell culture experimental observations of the Bcl-2-siRNA on human colon cancer cell line, Caco-2 proliferation and apoptosis, and to explore the possible molecular mechanisms, to open up the Bcl-2-mediated RNA interference provided the experimental basis for the treatment of colorectal cancer . Method 1. Extraction of total RNA from human colorectal cancer cells, Bcl-2 gene-specific primers Bcl-2cDNA 861-1174bp fragment was amplified by RT-PCR, RT-PCR and Western blot analysis of the target protein in different colon cancer cell line expression. Liposomes Bcl-2-siRNA transfection in human colorectal cancer cells Caco-2, RT-PCR and Western blot analysis after transfection of target protein expression changes. 3.MTT assay Bcl-2-siRNA Caco-2 cell growth and proliferation, and apoptosis was observed using the TUNEL assay, Western blot was used to detect the apoptotic pathway downstream signaling molecules of Bcl-2 and apoptosis-related factors changes. 1 using RT-PCR, Western blot from human colorectal cancer cell lines were screened expression of Bcl-2-positive cell line Caco-2. 2. Use of liposomal transfection technology, the Bcl-2-siRNA transfected into human colon cancer cells Caco-2, Bcl-2 protein Bcl-2-siRNA caused downregulation and confirmed by RT-PCR and Western blot. With non-specific siRNA transfected cells (Caco-2/mock) and non-transfected cells Caco-2 compare, Caco-2/Bcl-2-siRNA cell proliferation did not change significantly (P gt; 0.05); TUNEL assay observed number of apoptotic cells was significantly increased (P lt; 0.01); increased expression levels of Bcl-2 downstream molecule Caspase-3 Western blot detection of apoptosis pathway, increased apoptosis; apoptosis regulation of survivin expression decline in positive correlation with Bcl-2. Conclusion inhibitor of apoptosis gene Bcl-2 siRNA transduced into colorectal cancer cell lines can make the Bcl-2 protein expression, and caused increased apoptosis; preliminary survivin, Caspase family detection, and discussed with the Bcl-2 interaction may be the molecular mechanism of Bcl-2 has opened survivin-mediated RNA interference combined treatment of colorectal cancer provided the experimental basis.