Bcl-2Down-regulation By Small Interfering RNA Induces Beclin1-dependent Autophagy In Human SGC-7901Cells

Objective: Type Ⅰ programmed cell death (apoptosis) and typeⅡ(autophagy) are crucial physiological and pathological mechanisms thatregulate homeostasis, development, disease and elimination of malignant cells.Both of apoptosis and autophagy are a survival and a death process. Thefunction of autophagy in tumourigenesis is dual even now is controversial.Complex interrelationships are likely to exist between them. And now themolecular mechanism of apoptosis has been well studied. In the evolutionarilyconserved process of apoptosis, Bcl-2family proteins play important roles inits regulation. More recently, researches showed that Beclin1, described as theessential autophagy effector and haploinsufficent tumour suppressor, wasoriginally isolated as a Bcl-2interacting protein. In the latest decade studiesindicate that Beclin1interacts with Bcl-2through a BH3domain（Bcl-2-homology-3(BH3) amphipathic alpha-helix amino acids112–123）.Nevertheless, the function of the anti-apoptotic gene Bcl-2in autophagy hasnot yet well known. Gastric cancer is the second frequent cause of humancancer mortality in the world and Asia. Meanwhile it is the leading cause ofcancer mortality in China. Anti-apoptotic protein Bcl-2is overexpressed in themajority of human gastric cancers. Here, we study the role of anti-apoptoticprotein Bcl-2in autophagy through down regulating Bcl-2by small interferingRNA in human SGC-7901cells, and provide a new potential therapeuticstrategy in gastric cancer cells which overexpress anti-apoptotic protein Bcl-2and some evidence and experiment data for the treatment of siRNA in clinicalpatients.Methods:1Real Time RT-PCR and Western-Blot were used to detect thedegradation of anti-apoptotic protein Bcl-2by transfection of extrinsic Bcl-2special siRNA.2The essential autophagy effector Beclin1, which induced by down-regulation of anti-apoptotic protein Bcl-2, was detected by Real TimeRT-PCR and Western-Blot.3One-third of the DNA sample extracted from the nucleus chromosomalDNA was applied on2%and0.3%agarose gel, separated by horizontalelectrophoresis, and visualized by staining with ethidium bromide to evaluatethe molecular weights of the fragmented DNA of apoptosis.4Transmission electron microscopic examinations were carried out toobserve the formation of autophagosome which is the mark of autophagy.Results:1Treatment of SGC-7901cells with the three different siRNA (1,2,3) and the expression of the anti-apoptotic protein Bcl-2was significantreduction of about54%、63%、82%respectively as shown by Western Blot andthe inhibition of3#Bcl-2mRNA approximately98%as shown by Real TimeRT-PCR compared with the untreated cells.2Real Time RT-PCR and Western-Blot indicate that Beclin1induced bydown-regulation of anti-apoptotic protein Bcl-2was as much as70%,58%respectively.3The results of the nucleus chromosomal DNA applied on agarose gelelectrophoresis indicated that certain mechanisms other than apoptosis werecarried out in Bcl-2-down-regulated cells.4Transmission electron microscopy demonstrated that Bcl-2siRNAtreatment, but not control siRNA treatment, induced the formation ofautophagic vesicles with lysosomes and lysed cellular content in theautophagosomes.Conclusions:1Bcl-2special siRNA has emerged as a powerful tool for thesilencing of post-transcriptional gene expression.2Treatment of human gastric cancer SGC-7901cells with the Bcl-2special siRNA induces Beclin1-dependent autophagy.3Both of the results of the nucleus chromosomal DNA applied onagarose gel electrophoresis and the observation of transmission electronmicroscopic examinations clearly demonstrate that Bcl-2siRNA treatment, butnot control siRNA treatment, induced Beclin1-dependent autophagy other than apoptosis.