The MRNA Alternative Splicing Changes Of Seven Genes In BCL-2 Family In Several Tumor Cell Lines Under The Cisplatin Treatment

The tumor is formed by the abnormal proliferation of tissue cells,and its unrestricted growth is the result of the inhibition of apoptosis.The occurrence and development of tumor can not be separated from the abnormal apoptosis.Inhibition of apoptosis can promote tumor,but it can also become a target of tumor treatment.Inducing apoptosis of cancer cells is a important mechanism in which chemotherapy drugs kill cancer cells.The most of the genes involved in apoptosis have mRNA alternative splicing isoforms,some of which translate into proteins that can affect apoptosis.Some of these alternative splicing isoforms can promote apoptosis,but some of them can play an inhibitory effect on apoptosis.It has been shown in some reports that the mRNA alternative splicing of some genes related with cell cycle or apoptosis changed in cancer cells after chemotherapy treatment.Many chemotherapy drugs mainly cause DNA damage inducing apoptosis mediated by mitochondria which leads to cell death.One interesting question is how mRNA splicing of genes associated with mitochondrial apoptotic pathway will change in cancer cells after DNA damage drug treatment.In this study,some genes of the Bcl-2 family were selected to check the change of mRNA alternative splicing in some cancer cell lines after cisplatin treatment.Objective:To investigate and analyze the mRNA alternative splicing changes of Bcl-2 family genes(Bcl2Al,Bad,Bak,Bcl2L12,Bcl-X,Bok,Bim)in multiple cancer cells under the treatment of cisplatin,a chemotherapeutic drug.Method:Lung cancer(A549 and H1299)and cervical cancer(SiHa,CaSki and C33A)cell lines were treated by gradient dose of cisplatin-Total RNAs of the control cells and the treated cells were abstracted 24h afterthe treatment.The mRNA isoforms were detected by semiquantitative RT-PCR.The PCR products were resolved in agarose gel Gel images were taken and the ratios of found mRNA isoforms were analyzed by gel image software and statistical analysis.Result(1)Bcl2Al(anti-apoptotic):Two isoforms were detected,a isoform 137bp and b isoform 81bp,respectively.In A549 cells,two isoform ratios did not change significantly,but their mRNA level increased when the dose of cisplatin went up.But the ratio and mRNA level both didn't change significantly in H1299 cells.(2)Bad(pro-apoptotic):only one isoform was detected,and there is no obvious change in its mRNA level.(3)Bak(pro-apoptotic):only one isoform was detected,and there is no obvious change in its mRNA level.(4)Bcl2L12(anti-apoptotic):Two isoforms were detected,a isoform 426bp and b isoform 153bp,respectively.In A549 cells,the ratio and level of these isoforms did not change significantly.In H1299 cells,the ratio and mRNA level of a isoform increased,but those of b isoform decreased,and total mRNA of BCL2L12 decreased when the dose of cisplatin went up.(5)Bcl2-X:Both in A549 and H1299 cells,two isoforms were detected,respectively,anti-apoptotic Bg12-XL(351bp)and pro-apoptotic Bcl2-XS(162bp);and the Bcl2-XL ratio decreased,but the Bcl2-XS ratio increased;whereas the mRNA level of these two isoforms did not change significantly.(6)Bok(pro-apoptotic):Only one isoform was detected.There is no obvious change in its mRNA level in A549 cells,but in H1299,the mRNA level of this isoform decreased when the dose of cisplatin went up.(7)Bim(pro-apoptotic):BimEL,BimL,BimS,Bim,Bim alpha 2,alpha 4 and beta 7 were detected in 5 cancer cell lines.The ratio and mRNA relative level of these isoforms changed with the increase of the concentration of cisplatin.The major changes were resulted from the ratio and mRNA level of BimL isoform in these cell lines.ConclusionUnder the treatment of cisplatin,the transcription and mRNA alternative splicing of some genes related with cell apoptosis in Bcl-2 family changed with the increase of the concentration of cisplatin accordingly to promote cell apoptosis.Bim,one of the important pro-apoptotic genes through mRNA transcription and mRNA alternative splicing of two kinds of mechanism changed the RNA level and isoforms ratio of 6 kinds of isoforms,to promote apoptosis in 6 cancer cell lines.BimL is the major isoform up-regulated.