Experimental Study On Silencing Bcl-2 Expression By Small Interfering RNA Inhances Radiosensitivity Of Esophageal Cancer EC9706 Cells

Background and AimEsophageal cancer (EC) is one of the most common malignant tumor in digestive tract. The fatality rate of EC is ninty percent, and the incidence of EC in malignant tumor stand the fourth. The formation of esophageal cancer is a complicated process. It's the result of the serious disorder of the regulation of cell growth and proliferation. Current research shows that the occurrence and development of esophageal cancer have some connection with the participation of various apoptosis related genes. Radiotherapy is a kind of important means of tumor clinical treatments. The radiation sensitivity of tumor cells have intimate relatation with the repairation of DNA damage, cell signal transduction, cell proliferation, apoptosis and cell cycle regulation. In recent years, some key molecule of signaling pathways in tumor cell can be blocked, imported and modified by using modern molecular biology technology. As a result, Increasing ionizing radiation induced tumor cell death and apoptosis and improving the radiation therapy effect is effective. This technology minimize the damage of normal tissue cell and become one of the hot spot of radiotherapy research for cancer.Bcl-2 family is an important apoptosis regulatory proteins in Mitochondria apoptosis way. It is divided into two main categories:one category resists apoptosis, including Bcl-2, Bcl-W, Bcl-XL, and mcl-1. They localize at the mitochondrial membrane, the endoplasmic reticulum and the nuclear membrane. The other category facilitates apoptosis, including Bcl-xs, Bax, Bak, Bad, Hrk, and Bim. They exist in the cytoplasm and cytoskeleton. 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 protein was one of the earliest genes of Bcl-2 family and it was studied comparatively thoroughly until now. Many results indicate that the Bcl-2 protein localizes at the the endoplasmic reticulum membrane, the outer mitochondrial membrane, and the nuclear envelope. It has double functions as ion channel and docking protein that facilite it is key role in regulation of apoptosis. Its mechanism of biology action in cell apoptosis may be concerned with a few factors such as Ca2+ dependent endonuclease, oxygen free radicals, control of cell signaling and so on. In the mitochondria apoptosis way, Bcl-2 protein can regulate the nuclear internal and external physical transshipment and permeability transition in order to stable the mitochondrial membrane and prevents the release of cytochrome-C from mitochondria. Through interaction with Apaf-1 and procaspase -9 protect cells from apoptosis that stimulated and induced by Caspase-9 and Caspase-3.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 most important reason that leads to tumorigenesis and drug tolerance development. There-fore 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 is a kind of specific gene silencing phenomenon caused by specific double-stranded RNA, the double-stranded causing of RNA interference was called siRNA. siRNA can combine with the target mRNA chain by identifying bases complementary pairing, and guiding the Nucleic acid enzyme cut and digest the mRNA chain. Eventually it leads to target genes silence after transcription. Recent research shows that the artificially siRNA synthesized in vitro transfecting into cells can lead to ultimately specific silence of gene expression.The main focus of this experiment was to observe the expression of Bcl-2 protein, the influence of cells apoptosis and radiosensitivity effect after RNA interference by transfecting Bcl-2-siRNA into human esophageal cancer EC9706 cells through utilizing the RNAi technique. The purpose of the research is to explore theoretical basis for new radiotherapy treament in esophageal cancer.Materials and methods1. siRNA for Bcl-2 gene targeting are designed and synthesized by Shanghai JiMa pharmaceutical technology limited company.2. The Bcl-2siRNAs were transfected into the EC9706 cell by Lipofectamine. At 72h after siRNAs transfeetion, Flow cytometric analysis technology were applied to detect the Bcl-2 protein expression level.3. Different experimental cell apoptosis rate were tested by AnnexinV-FITC/PI double dye apoptosis kit to research the influence of siRNA in cell apoptosis. 4. AnnexinV-FITC/PI double dye apoptosis kit was used to detect apoptosis of these cells interfered with X radiation combined with Bcl-2 siRNA.5. Clone forming assay was used to determine the effects of small intemring RNA (siRNA) specific to Bcl-2 gene on radiosensitivity of oesophageal cancer EC9706 cells.Results1. Bcl-2siRNAs can be efficiently transfected into human esophageal cancer cells EC9706 by using Lipofectamine.2. Flow cytometric analysis showed that the expression level of Bcl-2 protein in EC-9706 cells was obviously down-regulated in Bcl-2-siRNAs treatment group. There was no difference in Bcl-2 protein levels between negative-siRNA and untreated cells.3. The apoptotic ratio of transfected cells was significantly increased compared with negative-siRNA transfected and non-transfected cells. It shows that Bcl-2 siRNA can induce human esophageal cancer EC9706 cell apoptosis.4. Bel-2 siRNA interfering combined with X radiation can significantly increased the apoptosis rate of esophageal cancer EC9706 cell.5. After X radiation, Cell survival curves decline in Bcl-2-siRNA treatment group compared with negative-siRNA and untreated cells.ConclusionsBcl-2-siRNA was transfected into human esophageal cancer cells EC9706 can effectively down-regulated the expression of Bcl-2 protein and promote the cell apoptosis of human esophageal cancer cells. Bcl-2 gene siRNA could enhance the radiosensitivity of oesophageal cancer EC9706 cells and can be used as a powerful adjunct to conventional radio- therapy.