Inhibitory Effect Of Bcl-xL And B-raf Double Target RNAi On Proliferation Of SW480 Cell Strain

As a kind of common malignant tumor, the incidence of cancer of colon increased rapidly in recent years. It is the second women malignant tumor in European and America nations. The incidence of colon cancer increases by 4 percent every year in China. Its mortality is from fourth to sixth in total malignant tumors, with a so high mortality, it seriously endangers the life and health of human. Traditional therapeutics of operation excision combined with radiotherapy and chemotherapy have some effects. To explore safe and effective gene therapy method, this research work constructed RNAi plasmids targeting to Bcl-xL, B-raf and both of which respectively to study their inhibition effect on the proliferation of the cancer cell strain SW480 of colon by silencing the expression of Bcl-xL and B-raf. Our purpose is to study the possibility to cure the cancer of colon by silencing the protooncogene related with cancer of colon with RNAi technology and get some basic knowledge for further clinical application. The selected and designed target sequence in this study is not reported by others. The mechanism of tumor formation is not very clear. Abnormal activation and over expression of proto-oncogene is very important factor. So the protooncogene is potential target molecular for tumor therapy. Bcl-xL, B-raf and other anti-apoptosis is usually expressed in the cancer cells of colon, so special siRNA targeting to these gene can inhibit the growth of tumor cells and induce apoptosis. The gene silencing function of RNAi is powerful, and only small space in vector is needed for promoter and target sequence. One vector can carry various promoters and target sites and interferen many genes simultaneously so as to increase the effect of anti-tumor gene therapy. So far research work indicates that the effect of RNAi in mammal is induced by siRNA, which is from 13 to 23 bp with from 2 to 3 nt protrudent nucleic acid at the 3′. The siRNA can combine with RISC in cells, and then recognize the completely complementary homogenous mRNA compared with the siRNA, the mRNA would be degraded at last. The methods to get siRNA include: (1) Chemistry synthesis; (2) In vitro transcription. (3) Transcripted in cells by siRNA expression vector. Chemistry synthesized and in vitro transcripted siRNA has short half life, transient inhibition effect and high cost. In vivo shRNA expression vector has stable RNAi effect, so it is convenient for long term gene function study. The shRNA expression vector include virus vector and nonvirus vector, shRNA virus expression vector has high transfection efficiency but low safety, so it is needed to study nonvirus vector that has high safety.The theory basis of using RNAi to cure the cancer of colon is to terminate the expression of protooncogene with siRNA according to the base complement principle and make the cancer cells of colon differentiate to maturity or induce apoptosis.This research work take the method of in vitro synthesizing siRNA. Firstly design oligonucleotide sequence, construct siRNA expression vector targeting to Bcl-xl, B-raf and both of which respectively in vitro, these siRNA can be transcripted into single strand RNA, and then form special hair structure. The key to shut down the expression of Bcl-xL and B-raf gene is the selection and designation of oligonucleotide sequence, the selection of oligonucleotide sequence is very strict. This study consulted the principles to construct RNAi vector made by Elbashir, Tuschl and Ginix, designed the oligonucleotide, which can transcript under the effect of HI promoter in the vector, and the transcripted RNA can form hair pin structure. It has been confirmed that the dsRNA with hair pin structure can keep long time for which the specific gene expression being shut down. The 19 nt oligonucleotide sequence should be chosen in the coding region with AA start in the two genes, the 5'and 3' nontranslationed region and region around translation start code, where the regulation protein biding sites are rich, should be avoid, for that may interfere the formation of hair pin structure. In this study, the GC content of Bcl-xL, B-raf are 33.3 percent and 42.9 percent respectively, which are in the span of from 30 percent to 55 percent, and the 19 nt oligonucleotide can not form dimmer by themselves. The H1 promoter is powerful to transcript short strand RNA and prone to transcript with start of purine bases. The termination sequence of transcription is poly (T), so the first base should be designed as purine G, and poly (T) should be added to the downstream of the sequence as termination signal of transcription, 9nt nucleic acid should be added between the 19bp positive and negative sequence to compose round structure when forming hair pin structure. To guarantee the specificity of this sequence, homogenous sequence is not found by genome data search through NCBI.The principle to select high effective silencing siRNA is concluded in this study and applied to select interfering sequence targeting to Bcl-xL and B-raf. Interfering vectors pSiBcl-xL and pSiB-raf targeting to Bcl-xL and B-raf respectively are constructed, which is confirmed by enzyme digest. This study provided a technology platform for the construction of interfering vector with double targets and explored the optimum condition for the transfection of interfering vector. The results of RT-PCR and Western-blot indicated that: pSiBcl-xL, PSiB-raf and pSiBcl-xL/B-raf can inhibit the expression of Bcl-xL and B-raf mRNA and protein. The inhibition efficiency of pSiBcl-xL/B-raf is higher than the interfering vector with single target. It is confirmed by jj and MTT that: the interfering effects appears after the cells are transfected 24h, the proliferation of SW480 is mostly inhibited after being transfected 48h, the inhibition rate to the proliferation of the cells of pSiBcl-xL/B-raf is 20 percent higher than pSiBcl-xL and PSiB-raf. Our results indicated that the siRNA targeting to Bcl-xL and B-raf can specially down regulate the level of Bcl-xL and B-raf mRNA and inhibit the expression of Bcl-xL and B-raf protein but can not inhibit the expression of GAPDH, the siRNA targeting to GAPDH can only specially inhibit the expression of GAPDH mRNA and protein, but can not influence the expression of Bcl-xL and B-raf protein. It can be concluded from this result that the siRNA targeting to Bcl-xL and B-raf appears targeted gene silencing effects. The research results also indicated that double targets interfering vector has higher inhibition effect to the proliferation of the cancer cell SW480 of colon than single target interfering vector, the former one may be developed to a therapeutic drug to the cancer of colon. The results of this study provided new clue and method for the gene therapy of cancer of colon that took Bcl-xL and B-raf as targets.The designation and selection on siRNA for mammal cells have no general principles for the mechanism of RNAi is not absolutely clear, and construction of highly stable and effective expression vector is under exploring, a lot of work needs to be done. But the high specificity of RNAi and easy process of technology, short cycle time, stable expression in cells and high efficiency make the RNAi appears promising prospect in gene therapy of tumor. As theory and technology of RNAi developes, RNAi should have broader prospect in the research for gene therapy of cancer of colon.