Detection Of Inhibition Of Survivin On Induced Apoptosis Of Lung Cancercell Lines A549 Cell By RNA Interference

At present, lung cancer is one of the malignant tumors in the world,because its incidence rate and death rate are maximal.Though we can deploy the combined therapy ,such as operation,radiotherapy,chems and so on,the curative effect is not good.Anti-apoptosis related gene expression of the tumor tissue up-regulation,apoptotic cancer cell decrease and the ability of cancer cell's drug fast increase is a most important reason.Many studies indicated that there is very important meaning for cleaning the tumor cell efficiently by using antisensenucleic acids technolagy to stop the anti-apoptosis gene expression of tumor and induce apoptosis selectly,which is a significant strategy for gene threapy tumor. However,the target difference and specificity for inducing apoptosis are two important factors ,which restrict its curative effect.The key point for inducing tumor apoptosis targetably is to select the specific expressional gene of tumor cell and restrain its expression specificly. RNAi is a new way for depressing gene expression ,it has many feature, such as high efficiency, high specificity, fast excellence ,transmiting to future generations and so on.It may be a new method for gene therapy of tumor in one day. Survivin is a new member of IAPS and be found recently.It is the minimal proteinum in the IAPs family up to now,and it is the most fortis survivin at present.It is the ideal target gene for treating lung cancer, because of its expressed specificity(only be expressed in homo-sapiecs embryonic tissue and tumor tissue)and the effects to cell multiplication and apoptosis.Therefore,there is important meaning for guidancing the gene therapy of lung tumor by using RNAi technology to repress the expression of survivin gene ,observe and analyze the effect to lung asenocarcinoma cell A549 multiplication and apoptosis,induce the relation between survivin gene and the occurrence,development,therapy and prognosis.Objective ; Study the relation between survivin gene and its occurrence,development,therapy and prognosis by using RNAi technology to repress the expression of survivin gene ,observe and analyze the effect to lung adenocarcinoma cell A549 multiplication and apoptosis.Obtain the best siRNA target sequence ,which is to aim directly at Survivin mRNA and the expressed proteinum of adenocarcinoma of lung by comparing the apoptotic effect of few specific small siRNA.Contents and methedsContents :Observe the expressed efficacy of protein and survivin mRNA to select effective and specific siRNA by using different siRNA to restrain the apoptotic and proliferative of A549 cell.Metheds:Design and synthesis three pair of siRNA frag which is releted to Survivin gene and a pair of negative conreol siRNA frag which was labeled by FAM according to Survivin gene codogenic casing through bioingormatics,the siRNA frag be inquested by BLAST and sure it is the survivin specific squence.We can transfect A549 cell through hangosome infection protocol and then observe the corpuscular transfection efficiency by inverted microscope, detect cell survival rate by MTT colorimereic analysis antigenic,detect cell cycle and apoptotic index by flow cytometry(FCM), we can gain the specific and effective siRNA by all these management.At last ,we observe the restraining efficiency of the protein-expression and survivin mRNA by RT-PCR and western blot.Result; The transfection efficiency of A549 cell which be trasfected through hangosome infection protocol is about 30%.The negative control cell which be labeled by FAM is green and transparent under the fluor inverted microscope,and the cell debris increased.The method of MTT show that siRNA has inhibitory action to cell mltiplication of A549 and the inhibition ratio is higher than other group.We also find that the siRNA transfection cell is stopped at the time of G2/M and the apoptotic rate is higher than other group if we collect the cell and detect its cell cycle and apoptosis after be transfection 48h. The method of MTT indicate that siRNA164 is more specific and effective than others, so we choose siRNA164 to undertake the experiment of western blot and RT-PCR.The methed of western blot indicates that the expression of survivin protein which be trasfected decreased and the gray scale of proteinic strap is lower than other group,There is signifficance meaning in Statistic. The experiment of RT-PCR indicates that the exprission of survivin mRNA decrease obviously after 48h by the effect of siRNA164 and the expressed strap brightness of Survivin mRNA is lower than others,but the level of the blank group ,hangosome group and negative control group has no marked change.Conclusion;Three pairs of siRNA squence aimed to survivin are be constructed successed in this experiment. The methods of MTT show that siRNA has inhibitory action to cell mltiplication of A549 and the inhibition ratio is higher than other group.The method of FCM indicates that the siRNA 164 has more depressive effect to survivin gene than others,so we choose it as the best sequence .The method of RT-PCR and western blot indicates that the expression of survivin protein which be trasfected decreased,it make us sure that the siRNA can close the expression of survivin gene.The effects of siRNA for restraining the A459 cell multiplication and apoptosis of make us sure that siRNA can be used to block up the expression of survivin specificnessly.So the RNAi technolagy which be on account of survivin may be a new diagnosis gene and a therapy target in many maligant tumor area (include lung cancer) one day.