Over-expression Of Tumor Suppressor Gene DLC-1and Its Effect On The Biological Behaviour And Wnt Signaling Pathway In Colorectal Carcinoma Cell Lines

BackgroundColorectal cancer （colorectal cancer, CRC） is one of the most commonmalignant tumor and its metastasis and recurrence is still the mainchallenges in clinical work. The current study suggests that Wnt signalingpathway plays a very important role in the development of colorectalcancer. Sevral components of the Wnt signaling pathway are closelyrelated to changes in the incidence of colorectal cancer, but the mechanismof which is not yet clear. Tumor suppressor gene DLC-1impacts series ofintracellular signal transduction pathways. Its low expression is related tothe development of colonrectal cancer as well as its invasion andmetastasis. Present studies suggests that crosstalk of the signaltransduction pathways exists in CRC. DLC-1maybe is related to Wntsignaling pathway in CRC. But few studies on that are reported. ObjectiveOur study is aim to generate recombinant Lentiviral vectors which canstably over-express DLC-1gene in SW480cells, and to detect the effect ofover-expression of DLC-1on the biological behaviour and expressionlevels of Wnt signaling pathway related genes in colorectal carcinoma celllines.Method1. The target sequence of DLC-1gene is determined according to theGenebank database （gene number NM₀06094.4）.2. Target sequence of DLC-1was connected with plasmid pcDNA3.1（+）-GFP with restriction enzymes and DNA ligase, constructingrecombinant plasmid pcDNA3.1（+）-GFP-DLC-1.3. The integrity of positive clones of recombinant plasmids were detectedthrough double digested gel electrophoresis and gene sequencing.4. The recombinant plasmid pcDNA3.1（+）-GFP-DLC-1and lentiviralpackaging plasmid pHelper1.0and pHelper2.0were co-transfectedinto293T cells mediated by LipofectamineTM2000, packaging intolentiviral particles. 5. The expression of green fluorescent protein （GFP） in293T cells wasobserved with a fluorescence microscope and the titer of therecombinant lentivirus was calculated.6. Colorectal cancer SW480cells were infected with the recombinantlentiviral vectors and the infection efficiency was detected with flowcytometry.7. Real-time PCR and Western blot were used to detect the the expressionlevels of DLC-1mRNA and protein in the transfected SW480cells.8. The cell proliferation was analyzed by MTT; colony-forming wasanalyzed by plate colony-forming assay; cell cycle and apoptosis wasanalyzed by Flow cytometry; migration and invasion ability wasanalyzed by tanswell assay, respectively.9. The SW480cells transfected with DLC-1gene were sub-cultured toestablish stable transfected cell lines.10. Real-time PCR and Western blot were used to analysis the expressionlevels of DLC-1and Wnt signaling pathway gene GSK-3β, c-mycand β-catenin in the SW480cells transfected with DLC-1gene. Result1. Double-enzyme-digestion and gene sequencing confirmed that therecombinant plasmid pcDNA3.1（+）-GFP-DLC-1was constructedcorrectly.2. Green fluorescence protein was observed under a fluorescencemicroscope in293T cells; The virus titer was1x109UT/ml; the transfectionefficiency was90%which was very high.3. Real-time PCR revealed that expression level of DLC-1gene in thetransfected SW480cells was significantly higher than that of negativecontrol group （P＜0.0001）, While the difference between blank controlgroup and negative control group was not significant （P>0.05）.4. Western blot got the same result that expression level of DLC-1genein the transfected SW480cells was significantly higher than that ofnegative control group （P＜0.0001）, While the difference between blankcontrol group and negative control group was not significant （P>0.05）.5. The proliferation activity of transfected SW480cells was decreased（P<0.01）; the colony formation was decreased （P<0.01）; cell cyclearrest in G1phase （P <0.01）; apoptotic cells were significantly increased（P <0.0001）; the migration ability of SW480cells was inhibited （P <0.01） as well as the invasion ability （P <0.001）.6. The expression levels of Wnt pathway gene c-myc and β-catenin weresignificantly decreased while the expression level of GSK-3β wassignificantly increased.ConclusionRecombinant lentiviral vectors expressing DLC-1were successfullyconstructed, and the DLC-1gene carried by which was over-expressed incolorectal SW480cells, which can suppress the proliferation, cloning,migration and invasion of colorectal cancer SW480cells and can influencethe cell cycle, promote apoptosis, and expression levels of Wnt signalingpathway gene was inhibited. The research laid the theoretical andexperimental basis for further research into the molecular mechanisms ofDLC-1inhibiting Wnt signaling pathway, thus provides a new idea oftargeted therapy for colorectal cancer.