The Study On Cell Proliferation And Apoptosis Effects Induced By Beclinl-siRNA In Pancreatic Cancer Cells

Objective: Autophagy, an evolutionary conserved process in which cell engulfscytoplasmic components such as impaired proteins and organelles withinautophagosome and delivers them to the lysosome for degradation, will contribute tomaintaining cellular homeostasis as a result of quality control of both proteins andorganelles. Emerging evidences have shown that autophagy has a complicatedcorrelation with many human diseases, including cancer.Beclin1is a phylogenetically conserved essential autophagy protein in manmal.Recently, more studies have demonstrated that it also plays an important role in thecancer development, which is also a haploinsufficient tumor suppressor. Current studiesreveal that down-regulation or monoallelic deletions of Beclin1are frequently found invarious cancers including human breast carcinoma, oophoroma, prostatic carcinoma andother cancers. Beclin1, also found participating in the course of anti-cancer drugstreatment, will become a novel approach in cancer treatment, although its molecularmechanism is unkown yet.Pancreatic cancer is a highly lethal disease usually diagnosed at an advanced stagefor which there is little or no effective therapy, including operation, radiotherapy andchemotherapy. Furthermore, molecule intervention, an important option in curing orcontrolling various cancers including pancreatic cancer, will offer both symptomaticand survival benefits and may act as a potent target for cancer therapy.The purpose of this study was to investigate the lower-expression of theautophagic gene Beclin1, which played a role in the regulation of tumorigenesis. Briefly,the down-regulation of expression of Beclin1gene was established by RNA interference,and the correlation between Beclinl inhibition and cell proliferation or apoptosis weredetected in Miapaca2cells by CCK-8, Western blot, real-time PCR and flow cytometry. Methods:1. Beclin1-siRNA was transfected into Miapaca2cells by using Lipofectamine2000, inhibition expression of Beclin1was analyzed via Western blot and Real-timePCR means.2. Cell viability was determined by cell counting Kit-8after transfection treatmentwith Beclin1-siRNA in Miapaca2cells.3. Cell cycle and apoptosis were analyzed by flow cytometry after transfectiontreatment with Beclin1-siRNA in Miapaca2cells.4. Cell cycle and apoptosis were analyzed by flow cytometry after Gemcitabinetreated Miapaca2cells of down-regulation of Beclin1transfected with Beclin1-siRNA.Results:1. Beclin1expression is down-regulated and stable inhibit after Beclin1-siRNAwas transfected into Miapaca2cells through using Lipofectamine2000, Western blotwas performed to analyze Beclin1protein changes, Beclin1mRNA was detected byreal-time operation with comparative Delta-delta Ct means method.2. There was not significant influence on the proliferation of Miapaca2cellsalthough inhibition expression of Beclin1induced by Beclin1-siRNA.3. Analysis via flow cytometry indicated that Beclin1inhibition induced by RNAihad a significant influence on the cell cycle arrest of Miapaca2cells. But there was notnotable influence on the apoptosis of Miapaca2cells.4. Analysis by flow cytometry indicated that there are significant influence on thecell cycle arrest and apoptosis after Gemcitabine treated Miapaca2cells ofdown-regulation of Beclin1transfected with Beclin1-siRNA.Conclusion: In summary, expression of the Beclin1was deregulated throughsmall interfering RNA technique. There was not significant influence on theproliferation of Miapaca2cells although Beclin1inhibition induced by RNAi. But therewere a significant change in the cell cycle arrest of Miapaca2cells. Miapaca2cells inthe G2phase and S phase were decreased. Beclin1-siRNA transfected group showed agreater antiapoptotic effect than the control group. Also apoptosis was significantlydecreased in Miapaca2cells treated with Gemcitabine plus Beclin1-siRNA compairedwith con cells. Down-regulation of Beclin1was disadvantageous to survival inhibitionof Miapaca2cells induced by chemotherapeutic anti-cancer drugs Gemcitabine.