| Objective:To investigate the effects and mechanisms of silencingEphrinB2gene by RNA interference on biological behavior of colorectal carcinoma in vitro,Provding theoretical and experimental basis forthe early diagnosis and gene therapy of colorectal carcinomaMethods:EphrinB2expressions of protein and mRNA in the cells of SW480,SW620,LOVO and HT29were detected by Western blotting and RT-PCR, respectively.Then the high expression of EphrinB2were determined for screening the right colorectal carcinoma cell line;Transfec plasmid(Psilencer2.1-E65,Psilencer2.1-E376,Psilencer2.1-E730)into293T cells(exogenous sieving target),the EphrinB2mRNA andprotein expression in the cells was detected by RT-PCR and westernblot for determining the bestone in terms of interference effect.Thentransfect it into human colorectal cancer cell that highexpression ofEphrinB2(endogenous authentication),There were three groupsin the experiment:①Control group(CON):untransfected plasmids②Negative control group(NC):transfectedwithNegative plasmid(pSilencer2.1)③RNA interference group (RNAi):Apoptosis were detected by flow cytometry,Screen and establishment of stable transfected cell lines.transfected with pSilencer2.1-EphrinB2-shRNA376.in the above three groups, The levelof expression of EphrinB2,VEGF,CD105and MMP9protein and mRNA were detected by Western blotting and RT-PCR,respectively.Cell cycle was detected by flow cytometry.Transwell assay was used to detect the ability of cell invasion. Scratch healing experiment was usedto measure the ability of cell migration.Cell proliferation was measured by MTT test.Results:Human colon carcinoma SW480cells EphrinB2expressionlevel is the highest;Plasmids containing Psilencer2.1-E376interferencesequence was the most effective to silence the EphrinB2gene,whichmade EphrinB2protein expression in the293T cells most significantly decreased.We successfully transfected pSilencer2.1-EphrinB2-shRNA376into Sw480cell. The apoptosis rate in control group (1.97±0.47)and negative control group(1.74±0.36) was significantly lower thaninRNAi group(26.69±0.66)(P<0.05). established the stably transfect celllines.The level of expression of EphrinB2,VEGF,CD105,MMP9protein and mRNA in RNAi group were Obviously reduced comparedtoControl group and negative control group(P<0.05),Then there havenosignificant difference between CON group and NC group.(P>0.05).Flow cytometry test the percentage of cells at S phase in RNAi group(18.32±0.38) was decreased significantly compared to control group(25.22±0.89) and negative control group (24.34±1.26)(P<0.05), G1phasein RNAi group (74.49±1.55) was increased significantly compared tocontrol group (55.98±1.66) and negative control group (57.78±2.22)(P<0.05).Transwell assay showed the cell number of Transmembranein RNAi group (37.33±5.51) was obviously reduced compared tocontrol group (109±4.36) and negative group (102±4.58)(P<0.05).Scratchhealing experiments showed the cell migration distance in RNAi grou p(18.33±0.71) was significantly reduced compared to control group(32.7±1.25) and negative group (30.57±1.36)(P<0.05).The results ofMTT experiment showed the cell proliferation rate in RNAi groupwasobviously lower than control group and negative group (P<0.05)Conclusion: Silenced EphrinB2gene expression by RNA interference。Silenced the EphrinB2gene in SW480cell could significantly reduced the protein and mRNA expression of EphrinB2,and obviouslyincrease apoptosis.Silencing of EphrinB2inhibited tumor angiogenesisand cell proliferation, invasion, migration of colon cancer SW480cells in vitro.EphrinB2may play a role in tumor angiogenesis and invasion,metastasis in colorectal cancer by regulating VEGF and MMP9... |
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