Study On MRNA Alternative Splicing Of Three Genes In The Caspase Family Under Cisplatin Treatment

Apoptosis is a controlled process for multicellular organisms to remove damaged or unnecessary cells during development or under certain physiological conditions,which plays an important role in the development and homeostasis of animals.The weakening of apoptosis can therefor lead to the resistance of tumor cells against treatment,and many anticancer therapies are to trigger enough DNA damage to break through these anti-apoptosis barriers and cause cell death.One of the pathways leading to apoptosis is mediated by the death receptor on the cell membrane(exogenous pathway),and the other is mediated by mitochondria(endogenous pathway).Generally speaking,the radiotherapy and chemotherapy for DNA damage are mainly caused by the endogenous pathway to induce apoptosis of tumor cells.In this pathway,many proteins from the two families of Bcl-2 and caspase are involved in the signal transduction.It is found that in the case of DNA damage,cells will rearrange quite a lot alternative splicing of pre-mRNA related to cell life genes,including the genes from caspase family.According to the NCBI database,there are many pre-mRNA splice isoforms in almost all the 14 members of the caspase family.Then how about the changes and rules of mRNA splicing of genes from caspase family in tumor cells when treated with cisplatin?Methods We chose representative samples of lung cancer cells(A549,H1229),cervical cancer cells(C33A,SiHa,CaSki)and human embryonic kidney cells(293FT)processed with cisplatin gradient concentration,then extracted the total RNA of the cells above,analyzed the composition of the 3 isoforms of mRNA gene and their changes of ratio and relative expression by semi quantitative RT-PCR,agarose gel electrophoresis and gel image analysis software.We have studied the 3 members from caspase family:caspase-2,caspase-9 and caspase-10,which belong to the initial apoptotic protease(initiator caspases).The 3 genes were tested in lung cancer cells and caspase-9 was tested in the other 4 cells as well.Results 1.In the study of caspase-2 gene,caspase-2sr isoform was not detected in lung cancer cells A549 and H1299 while caspase-2L(and s)isoforms were detected and their mRNA level increased with the increase of cisplatin concentration(P<0.05),but the proportion of caspase-2 L(and s)isoform was not changed with the increase of cisplatin concentration(P>0.05).This shows that the alternative splicing of mRNA is not the regulatory mechanism of the gene expression in the 2 cells under cisplatin treatment.2.In the study of caspase-10 gene,the design of primers were used to detect 2 kinds of different isoforms at the gene 3'end.In A549 the mRNA levels of caspase-lOL and s isoforms were increased following the cisplatin concentration increased,and the expression level of caspase-L was significantly higher than that of caspase-10s,showing that caspase-10 plays a role in apoptosis in A549 after cisplatin treatment.However,there was no change in the proportion of the 2 different isoforms at 3' end,suggesting that the alternative splicing mechanism of mRNA did not play a regulatory role at the 3' end of the gene.In H1299,caspase-10s isoform was not detected but caspase-10L isoform was detected.Its level of mRNA decreased following the cisplatin concentration increased and the expression level was significantly lower than that in A549,suggesting that it did not make any effect on apoptosis promoting.3.In the study of caspase-9,among all the 6 cells treated with concentration gradient,there 2 kinds of isoforms by CASP9f:r1 were detected:caspase-9a and c(NR-102732),but with increasing cisplatin concentration,there was no change in the ratio of these 2 isoforms in 6 kinds of cells(P>0.05);However,the isoforms of caspase-9a and b by CASP9f:r2 primers increased gradually in A549,H1299,SiHa,C33A and CaSKi,while the opposite trend was observed in 293FT cells.Compared with normal A549 cells,the proportion of caspase-9a/b in cisplatin-resistant A549 cells was significantly decreased after cisplatin treatment,which meant that this change was associated with the anti-apoptosis of cells.Conclusion In the 2 kinds of lung cancer cells,A549 and H1299,the alternative splicing mechanism of mRNA does not play a role in the regulation of the expression of both caspase-2 and caspase-10 genes;and in 5 kinds of tumor cells,caspase-9 takes part in cisplatin-induced apoptosis by regulating the ratio of caspase-9a/b mainly through the alternative splicing of mRNA,suggesting that regulation of caspase-9a/b the proportion may be one of the effective ways to improve cisplatin in the treatment of cancer.