Stable Knockdown Of CDK8by Lentivirus-based RNAi Inhibits Colorectal Cancer Cells Growth

Objective:To investigate effect of stable knockdown of CDK8by lentivirus-based RNAi oncolorectal cancer cells growth in vitro and in vivo, and to illuminate the mechanisms.Methods:1. establish the colon cancer cell line stably transfected with the luciferase gene(SW480-luc).SW480cells were transfected with the PRC/CMV-luc plasmid and screen cellstably expressing luciferase reporter gene with G418. Resistant clones wereisolated and the luciferase activity were measured every5passages. The cloneswith low luciferase expression were discarded. SW480-luc cell clones weremaintained in the media supplemented with1400ug/mL G418for40passages.Compare the cell growth characteristics of the SW480-luc cells and the parentalSW480cells..2. Lentivirus production, infection, and isolation of cell clones with CDK8silenced.Lentivirus was harvested from293T cells co-transfected with optimized Lenti-PacHIV Expression Packaging Mix and the expression construct, SW480-luc cellswere transfected. The cells with CDK8knock-down was screened by puromycinselection, CDK8expression was detected using quantitative RT-PCR and Westernblotting.3. Investigate effect of stable knockdown of CDK8by lentivirus-based RNAi oncolorectal cancer cells growth in vitro and in vivo, and to illuminate the mechanisms.The cell proliferation was detected using MMT assay, and the c-Myc and MMP-7expression were detected using quantitative RT-PCR. shCDK8-1cells (the stablecell clone with CDK8knockdown) were subcutaneously inoculated into leftarmpits，the negative control mock-1were subcutaneously inoculated into rightarmpits，the tumor growth was detected using bioluminescent imaging system.Results:1. Establish4colon cancer cell lines stably expressing the luciferase gene, and thecell growth characteristics of the SW480-luc have no difference with the parentalSW480.2. Establish three cell clones with CDK8knockdown and three negative controlclones, the positive clones were denominated as shCDK8-1, shCDK8-2andshCDK8-3; the negative clones were denominated as mock-1、mock-2、mock-3.The CDK8expression of positive clones were respectively suppressed by74.7%，71.3%，68.5%at mRNA level. The inhibition rate at protein level was significant.the CDK8expression levels of negative control clones were respectively104%,108%，96%.3. MTT assay indicated strong inhibition in growth rate of shCDK8cells comparedto that of negative and blank control. quantitative RT-PCR results showed asignificant reduction in expression of β-catenin， c-Myc and MMP-7at themRNA level. The bioluminescent imaging results indicated that the tumor growthwas significantly suppressed in tumors with CDK8knockdown.4. Quantitative RT-PCR results showed a significant reduction in expression ofCDK8, β-catenin, c-Myc and MMP-7at the mRNA level in shCDK8-1tumortissue compared to mock-1tumor tissue, and immunohistology results showedthat the expression of CDK8reduced significantly at the protein level in tumorswith CDK8knockdown.Conclusion:1. The expression of CDK8can be significantly suppressed by the recombinant lentivirus.2. Silencing of CDK8gene can inhibit the proliferation in vitro, and β-catenin,c-Myc and MMP-7was down-regulated.3. tumor growth was significantly suppressed in tumors with CDK8knockdown.CDK8is expected to be the new target of gene therapy...