Construction Of Survivin ShRNA Vector And It's Effect On Colorectal Carcinoma Vivo And Invitro

Background and ObjectiveColorectal cancer (CRC), one of the most common human malignant tumors, is the second leading cause of cancer-related deaths in the United States. Currently, most CRC are treated by comprehensive therapy, such as surgical resection, chemotherapy and radiotherapy. However, the recurrence and liver metastasis are still major causes leading to therapy failure. Thus, it is important to predict and discover early recurrence and metastasis in order to treat promptly, which could elevate living quality and survi- val rate. With the development of molecular biology and gene engineering, gene therapy,including gene replacement, antisensenucleic acids, cytokine therapy and RNA interference,has been an important method of tumor prevention and cure. The key of gene therapy is to find suitable target gene and to make use of most advanced gene therapeutic tool.Survivin is a new member of the inhibitor-apoptosis gene family,owing special structure,overexpressed in many malignant tumor tissues. Recent studies indicate that the expression of Survivin was localized in the carcinoma cells and was not expressed in normal colorectal mucosas. Overexpression of Survivin in colorectal tumor cells has been correlated with descend of apoptotic index, shorten of overall survival rate,poor prognosis and increased recurrence. Many reports have shown that abrogation of Survivin is useful for tumor therapy. RNA interference is the most effective way to silence gene transcription. The process of RNAi is mediated by double-stranded RNAs that are homologous to the gene being suppressed. These dsRNAs are cleaved by Dicer into duplex 21-25nt in length,named small interfering RNA(siRNA).These fragments recognize and cleave homologous mRNA and lead to degradation of the gene. Short hairpin RNA(shRNA) can be automatically processed into siRNA in cell and is more stable and effective than siRNA. The aim of this study was to construct a shRNA expression vector against Survivin,explore its impact on colorectal cancer cells and construct nude mice bearing human colorectal carcinoma to see the vivo effect.Methods1.Eukaryon expression vector pGCsi-H1/Neo/GFP containing green fluorescence protein was used to construct shRNA plasmid directed to Survivin.2.Colorectal carcinoma cells SW480(high expression of Survivin) were cultured , Eukaryon expression plsmid shRNA-Survivin was transfected into SW480 by use of Fuge6.Transfection was monitored by fluorescence microscope;cell cycle and the apoptosis cells were examined using flow cytometry and transmission electron microscope; Survivin protein expression was examined by Western-blotting.3.Cell proliferation after transfection were analyzed by MTT.4.The change of cell migration was detected by transwell chamber.5.Construct nude mice bearing human colorectal carcinoma and observe the effect of shRNA on tumorigenesis in nude mice.6.The tumors were measured two-dimensionally in double blind ;Tumor volume and average volume change were calculated , so did tumor inhibitation rate.7.Survivin protein expression in nude mice were examined by immunohistochemical method.8.Tumor cell apoptosis were monitored by use of TUNEL and transmission electron microscope.9.The histological changes of liver, kidney and lung were observed with light microscope;peripheral blood hematological and biochemical parameter were detected.Results1 .Recombinant plasmid pGCsi-H1/Neo/GFP-Survivin was constructed successfully, and was confirmed by sequencing that shRNA encoding sequence was in coincidence with target nucleotide sequence of Survivin we engineered.2.eukaryotic expression plasmid containing green fluorescent protein(GFP) could be transiently transfected into SW480,and obtained stably transfected cells after G418 screening. Transfection efficiency was more than 90% observed with fluorescence microscope. FCM and transmission electron microscope could see cell cycle change and apoptosis. Survivin protein expression was downregulated after transfection and lasted quite a long tine.3.MTT showed that transfected cell proliferation was inhibited.4.Cell migration experiment (transwell chamber) showed cell migration capability decreased significantly after transfection.5.The tumorigenesis capability in nude mice injected with transfected cells was lower than that in nude mice injected with nontransfected cells. The tumor volume in transfected nude mice was smaller than in nontransfected nude mice,the inhibition rate was 67.86%.6.Survivin protein expression of xenograft tumor in transfected nude mice was inhibited by immunohistochemical method,and apoptosis was increased by TUNEL and transmission electron microscope.7.Plasmid security analysis revealed that the function and structure of important organs and hematological system in transfected nude mice did not appear abnormity.Conclusion1. Recombinant plasmid pGCsi-H1/Neo/GFP-Survivin was constructed successfuly.2.shRNA expression vector directed against Survivin could downregulate Survivin protein expression for a long time,inhibit SW480 cell proliferation,induce cell apoptosis and result in cell cycle changes.3.Colorectal tumor model could established in nude mice with SW480 cells.4.Cell migration capability after transfection by recombinant plasmid descended obviously,which indicates that Survivin might participate and influence tumorous transfer process besides inhibiting apoptosis and regulating cell cycle.Downregulation of Survivin protein might hamper tumor metastasis.5. The tumorigenesis capability in SW480 cells transfected by shRNA-Survivin reduced,the tumor growth was inhibited and apoptosis was induced.6. Recombinant plasmid had no significant toxicity and side effects.