| BackgroundEsophageal cancer is one of the most familiar malignant tumors of the human, the pathological type was squamous cell carcinoma and adenocarcinoma. Squamous cell carcinoma is the major pathology, accounting for more than90%.Carcinogenesis of malignant tumor is through a series of complex process, which is a pluralistic body reaction, its own course of development may involve a series of apoptosis pathway activation and inactivation of anti apoptotic pathway. So is the carcinogenesis of esophageal mucosa.With the improvement of local surgery, radiotherapy and chemotherapy, the effect of early esophageal cancer is getting better, but most patients have reached their middle and late period. So the treatment usually can not produce desired effects. The survival rate within five years is below20%. Esophageal cancer is harmful to people’s lives and health. At present,it is a hot spot in modern medicine that we use molecular biology technology from gene level to explore tumor occurrence, development and treatment.BIT1was found as a new gene when exploring the integrin-mediated regulation of Bcl-2expression. the protein was named as BIT1to denote its ability to regulate Bcl-2expression. Later results showed that it was evolutionarily conserved from bacteria to human. The BIT1transcript was prominent in human testis, prostate, skeletal muscle and liver, and was clearly detectable in human heart, spleen, placenta and colon. No significant BIT1mRNA sigal was seen in the thymus or in peripheral leukocytes. Some study showed that BIT1protein was localized in the mitochondrial intermembrane space in the physiological condition, when the BIT1protein was released into the cytoplasm or the BIT1protein was expressed in the cytoplasm, it can combine AES (amino-terminal enhancer of split), induce cell de-adhesion and Promote cell apoptosis. Here BIT1plays a promoting role in the process of cell apoptosis.So far The physiological function of BIT1normal is not clear. But the latest research suggests that BIT1protein localized in the Golgi, also the BIT1protein has the effect of inhibiting apoptosis. There is only a small amount of the literature about the relationship between BIT1and the tumor at home and abroad, besides he difference is very big among the result of researches. Currently, the relationship between BIT1and esophageal squamous cell carcinoma is rarely reported. Therefore, It is significance to discuss the BIT1mitochondrial protein for exploring the occurrence and regulation of apoptosis.ObjectiveThis study first uses the BIT1siRNA transfected with EC9706of esophageal squamous carcinoma cells,result in inhiBITling the expression of BIT1. The expression of BIT1protein was deceted by Western blot detection before and after interference. The proliferation activity of EC9706cell was deceted by MTT. Also the scratch test is used to measure scratch healing of EC9706cell. Finally to analyse the relationship between BIT1and esophageal carcinoma, and explore the its clinical significance in development and metastasis of esophageal carcinoma.Methods1. Transient transfection:Mix the plasmid vector of BIT1siRNA with lipo2000liposomes beforehand, then put the mixture into the esophageal squamous cell carcinoma EC9706cell, completed the transient transfection of BIT1.2. Western blot:Make the BIT1gene of EC9706cell silenced by siRNA, western blot technique was carried out to detect the change of the expression of BIT1protein in different time point.3. MTT:MTT technique was carried out to detect the change of the cell proliferation ability before and after transfection in different time point.4. The scratch test:After the BIT1gene was silenced by siRNA24h, The scratch test technique was carried out to detect the variation of the distance by cell migration in0h,12h,24h,36h.5. Statistical analysis:All the dates were analyzed by SPSS17.0statistical package. T detection was used to compare two sample mean. Each group were counted by single factor analysis of variance test. The level of significant difference was α=0.05.Results1Western blotThe gray value of untreated group was1.49±0.46;1.44±0.53in negative group,0.85±0.27in siRNA24h,0.53±0.06in48h,0.49±0.05in72h,0.77±0.19in96h,1.12±0.08in120h,1.43±0.30in144h,1.46±0.39in168h.The group of siRNA24h,48h,72h,96h,120h and untreated group or negative group,the difference was statistically significant.(P<0.05);the difference between untreated group and negative group was not statistically significant.the expression of BIT1was significantly reduced after siRNA24h. Especially, the interference efficiency was the highest in siRNA72h. The group of siRNA144h and168h was not associated with untreated group or negative group,age and the differentiation P>0.05)2MTTMTT show that the OD value of siRNA group was lower than the untreated group and the negative group.(P<0.05) the inhibition efficiency is the highest in72h, accounting for45.9%.From BIT1siRNA120h, the inhiBITlion efficiency of cell proliferation was weakened gradually. The difference was not statistically significan between siRNA168h and the negative group.(P>0.05)3The scratch testThe test show the scratches was healing obviously between the untreated group and the negative group from siRNA Oh to36h. The difference was statistically significant.But the scratches was not healing in siRNA group,(P>0.05) contracted to the untreated group and the negative group, the difference was statistically significant.(P<0.05)Conclusions1. BIT1plays an anti-apoptotic role’in esophageal squamous cell carcinoma.2. BIT1siRNA could effectively silence BIT1gene, the expression of BIT1protein is closely related to the time of transient transfection.3. BIT1is expected to become a reliable indicator for monitoring the tumor invasion and metastasis, iblocking the expression of BIT1become a new target for the future treatment of esophageal cancer. |
