Screen The CRC Cell Line With Lentivirus-mediated Inducable SiRNA Specific For C-met

Colorectal carcinoma is one kind of common human malignantcarcinomas,the total survival rate was50-70%in five years. Lymph nodes andblood vessels transfer is one of the major biologic characteristics and also isthe indicator of the poor prognosis. Distant metastasis appeared in One third ofcolorectal cancer patients when they are diagnosed. Surgical treatment is stillthe effective treatments for the early and medium stage of colorectal cancer.Targeting therapy On the basis of radiotherapy and chemotherapy werenecessary for the middle and late stage of colorectal cancer. The targetedtherapy which uses VEGF and EGFR as the target has became the commonmethod to treat colorectal cancer, however, the primary or secondarydrug-resistance is one of the chief obstacle for the therapeutic effect. C-Met isone of the Tyrosine kinase receptors which over-expresses in colorectalcancer.The Tumor invasion and lymph node and liver metastasis-relatedmolecules (HGF) are ligands of C-Met. HGF/C-Met signal pathway canpromote proliferation invasion and metastasis of tumor cells, also inducetumor angiogenesis. Research indicates that the expression of HGF and itsligand c-Met in colorectal carcinoma were higher than in normal colorectalmuscosa. C-Met could be as a potential new therapeutic target for colorectalcarcinoma and targeting C-met with combination with be radiotherapy andchemotherapy will benefit for colorectal carcinoma treatment.We have already found that HGF could promote the formation andmigration of SW620colorectal carcinoma cells based on the earlier research,this experiment used Doxcycline to induce the inducible small interferingRNA expression to restraint the Tyrosine kinase receptor C-met expressionand discuss effects on the formation of SW620colorectal carcinoma cells andsensitivity of radiotherapy and chemotherapy by interfering c-Met expression. Objective: constructing the stable cell line using the virus with theplasmid expressing siRNA against human c-Met gene and discussing theinfluences of appropriate c-Met knockdown on SW620cells.Methods:1. Designed3pairs of siRNA to different sites of c-Met gene, then insertinto TA plasmid. Select the cells with best knockdown efficiency usingWestern Blot and RT-PCR after transiently transfecting the plasmid intoSW620cells. The scramble and empty SW620cells are conducted as negativecontrol and bank control.2. Co-transfect the pSD400Lentivirus vector with the siRNA of bestefficiency together with the auxiliary plasmid pRSV,pMDG and pMDL to293T cells to package the virus. Then collect the virus to infect SW620cells.24h later, use puromycin to select the cells stably expressing c-Met shRNA.3. Induce the stable cell line with Doxycycline to detect the expressionlevel of c-Met protein using Western Blot and RT-PCR and the influences ofc-Met knockdown on SW620cells multiplication.Results:1. TA vector was constructed successfully, test the righteous of plasmidthrough restriction and sequencing, the result of realtime–PCR show thatTA-c-Met-shRNA1plasmid lead expressing30%of c-Met after transientlytransfection; the result of western blot show that TA-c-Met-shRNA1(shRNA1)lead to40%of c-met expressing which is lower than TA-c-Met-shRNA2(shRNA2)(63%,64%),TA-c-Met-shRNA3(shRNA3)(54%,88%). The knock-down potency of the shRNA1is the best.2. The virus was packaged. After one-week selection, we got somemonoclones and then cultured them.3. Inducing the pSD400-c-Met-shRNA-SW620by DOX of differentconcentration, the expression of c-met in stable cell line shows a tendency ofdecrease with the DOX concentration increase, testing by Western-blot andRT-PCR. Stable cell line was constructed successfully.4. The suppression of cell reproduction emergy in pSD400-c-Met-shRNA -SW620cell line induced by doxycycline in the third day because the c-metwas appropriately knock-down showing statistically significant in cellreproduction of pSD400-scramble-SW620,SW620,no matter inducing bydoxycycline The he cell reproduction of SW620cell, in which the c-met isappropriately knocked down, is obviously lower than that of control group.Conclusion:1. The knockdown efficiency of siRNA to different sites of c-Met genedifferent.2. We can get the stable cell line which can inducibly express siRNAagainst c-Met.