Inhibiting Tumor Growth And Invation Of Colorectal Cancer By Blocking The Expression Of VEGFR-3 Using Interference Vector-based RNA Interference

Background:RNA interference(RNAi) technology is emerging as a very potent tool to generate a knockdown of a desired gene.Many reports believed that VEGFR-3 is associated with tumor metastasis.But whether VEGFR-3 can be link with proliferation of tumor has been unclear.The aim of this study was to investigate whether down-regulation vascular endothelial growth factor receptor-3 (VEGFR-3) by transcriptional gene silencing induced by short hairpin RNAs (shRNAs) on colorectal cancer could inhibit growth of colorectal tumor.Methods:According to the principle of RNAi design,we designed three pairs of 64bp interference fragment,which has a hairpin structure,both sticky ends. Three pSUPER-siRNA/VEGFR-3 expression vectors were constructed by double enzyme digestion and connected.Then the recombinant vectors were identified by double enzyme digestion and sequencing. The recombinant plasmids were transfected into human colorectal cancer LoVo cell lines with Lipofectamine 2000.Then characteristics of the cell lines were analyzed with growth curves by MTT.The expression levels of VEGFR-3 in the transfected LoVo cell lines were determined by real-time polymerase chain reaction and western blot.Then we selected the highest effect expressing vector and injected LoVo cells transfacted it and pSUPER vector into tumor xenograft model in nude mice.Tumor growth,microlymphatics and microvessels were compared for mice.Results:After enzyme digestion,the small interfering RNA targeting VEGFR-3 expression vector was successfully constructed.The recombinant vectors appear 281bp small fragments,while the empty vectors were 241bp.pSUPER -siRNA/ VEGFR-3 was transfected into LoVo cell.The mRNA expression of the VEGFR-3 gene were significantly downregulated(P＜0.05).After transfected the pSUPER-siRNA /VEGFR-3 and pSUPER into LoVo cell,the colon cancer tumor model in nude mice has been constructedr.The tumour growth of the forth was significantly slower than the latter.The tumor volume,4 weeks after administered,were significantly smaller in treatment group than in pSUPER control group((201.4±34.6) mm3 vs(439.6±31.9)mm~3).In pSUPER-siRNA/VEGFR-3 treatment group, the number of microlymphatics per microscopic field was(4.8±3.9) decreased compared with pSUPER control group(13.6±5.6).(p＜0.01).Conclusion:the present study showed that VEGFR-3-siRNA was an effective tool to inhibit VEGFR-3 expression in colon tumors cells;and reduced lymphangiogenesis in favour of enhancing survival.