Influence Of Survivin Gene Interfered By RNA Interference On The Biological Features Of Colorectal Carcinoma Lovo Cells

BackgroundColorectal carcinoma is one of malignant tumors terribly endanger human life worldwide.The death rate of colorectal carcinoma is very high in North America and Europe.Recent years the attack rate of colorectal carcinoma in our country is more and more higher than before.But now the clinical cure rate of colorectal carcinoma is very low,the main reasons are metaptosis and drug resistance of carcinoma cells. Following the developing of molecular biology,it brings a new hope from tumor gene therapy.The key of gene therapy is choosing target gene.Survivin gene is a new number of IAP,it is the most powerful apoptosis inhibiting gene.Because of the repression of apoptosis by Survivin gene carcinoma cells can keep on cleavaging and proliferating, induce carcinoma cells' metaptosis.And the patients or cell lines positive expressing Survivin resist chemotherapy and radiotherapy.Related researches find the positive rate of Survivin gene express in colorectal carcinoma is more than 60%,but it has even no express in normal tissue and colorectal benign tumor.Further more than half of the Survivin positive patients has been found metaptosis.Those prove Survivin gene has very important effect in the process of colorectal carcinoma cancerate.Small double strands RNA(dsRNA)complement with target gene mRNA sequence is introduced into target cells,the dsRNAjoint with the targent gene mRNA specificly and degrade it,then the cells express the phenotype that the target gene deleted,the whole process is called RNA interference(RNAi).Small interference RNA(siRNA)or shot hairpin RNA(shRNA)both are the medium of RNAi.RNAi has a character of specificity of sequence which can discriminate mutation gene sequence with normal gene sequence,finally only the mutation gene is knocked out.Otherwise RNAi has characters of high performance and magnification effective,shRNA is more stable than siRNA,and it can increase the time of RNAi.In our study,we transient transfect the Survivin-targeted siRNA into Lovo cells and detecting the expression of Survivin gene,and then base on the siRNA sequence we design Survivin-targeted shRNA sequence and synthesize the shRNA,and construct the shRNA plasmid expression vector,stable transfect into Lovo cells and sieve the stable expression cell line,detecting the cell growth cycle and cell proliferation.The objective of the study is provide the evidence to the feasibility of colorectal carcinoma gene therapy.MethodDesign Survivin-targeted siRNA,transient transfection into Lovo cells,and detecting the expression of Survivin gene.1.Design of siRNA First,9 siRNA sequences were designed by siRNA design software,and analyzed through BLAST,at last,we got 2 siRNA sequences only targeting Survivin gene(we called Survivin-siRNA-1 and Survivin-siRNA-2).2.Transient transfection Transfected the 2 Survivin-targeted siRNAs into Lovo cells through Lipofectamin 2000 and detected after 72hrs.3.Detecting Expression of Survivin mRNA was detected by real-time RT-PCR,expression of Survivin protein was identified by Western blot,cell apoptosis rate was evaluated by flow cytometry,and cell proliferation was detected by MTT assay.Construct the Survivin-targeted shRNA plasmid expression vector,stable transfection into Lovo cells and detecting the cell proliferation and growth cycle.1.Design of shRNA and construction of shRNA plasmid expression vector Based on the siRNA sequence,we designed shRNA sequence which has a loop.And cloned the shRNA into pGenesil-1 plasmid vector.The 2 pGenesil-Survivin-shRNAs were respectively transfection into Lovo cells by Lipofectamine 2000,and cultured the stable expression cell line by G418.2.Detecting The expression level of Survivin mRNA was detected by RT-PCR,cell growth cycle was evaluated by flow cytometry,and cell proliferation was detected by MTT assay.ResultsTransient transfection1.Expression of Survivin mRNA The result displayed that the expression of Survivin mRNA of the 2 Survivin-targeted siRNA groups' Lovo cells were both further lower than control Lovo cells'.2.Expression of Survivin protein The result displayed the 2 Survivin-targeted siRNA groups' were both lower than control Lovo cells group's.3.Apoptosis The results of 48hrs and 72hrs displayed the 2 Survivin-targeted siRNA groups' were both higher than normal Lovo cells group's.But the result of 96hrs the 2 act groups had no difference with control Lovo cell group.4.Cell morphology The two act groups could observed much green fluorescence(on cell envelope)and red fluorescence(inside cell),and the control Lovo cell group couldn't see anything.5.Cell proliferation The results of 48hrs and 72hrs displayed the ODa90nm of the 2 Survivin-targeted siRNA groups' were both lower than normal Lovo cells group's.But the result of 96hrs the 2 act groups had no difference with control Lovo cell group.6.Contrasting of the two siRNAs The effect of Survivin-siRNA- 1 was more powerful than Survivin-siRNA-2's.Stable transfection1.Expression of Survivin mRNA The result displayed that the 2 Survivin-targeted shRNA plasmids groups' were lower than control Lovo cells'.2.Cell growth cycle The result displayed the cells in G2 and M of the 2 shRNA plasmid groups' were both higher than control Lovo cells'.3.Cell proliferation The result displayed the speed of the 2 shRNA plasmid groups cells were both slower than control Lovo cells'.4.Contrasting of the two shRNA plasmid expression vectors The effect of pGenesil-Survivin-1 was more powerful than pGenesil-Survivin-2's.Conclusion1.The 3 steps method of design siRNA is a very frequent simple and effective process.2.pGenesil-1 is a plasmid vector which has EGFP,kanar resistance,Neor expression frame and U6 promotor.3.After the Survivin-targeted siRNA or shRNA plasmid expression vector were transfected into Lovo cells,the expression of Survivin gene was successfully depressed.The proliferation ability of the Lovo cell stable expressing Survivin gene deletion was much lower than normal Lovo cell.The results displayed that Survivin gene has the key function in colorectal carcinoma cells on proliferation and apoptosis regulation.4.The siRNAs contains 19 base pair,and GC rate of Survivin-siRNA-1(42%) and Survivin-siRNA-2(37%)are both between 35-50%.It's more powerful of Survivin-siRNA-1 than Survivin-siRNA-2,I think GC rate more near 50%and the effect are more better.