The GTPBP4 Gene Silenced By Lentivirus-mediated RNA Interference To Explore The Effects On The Biological Behavior Of Human Colon Cancer HT29 Cells

Objective: Silencing GTPBP4 gene by lentivirus-mediated RNA interference to investigate the effects of GTPBP4 gene on the biological behavior of human colon cancer HT29 cells.Methods: 1.The selection of lentivirus for multiplicity of infection(MOI)and transfectionThe lentivirus GTPBP4 RNAi vectors and the negative control lentivirus vectors were used in this study.They were constructed by Shanghai Genechem Company.Firstly,we screened out the optimal multiplicity of infection(MOI)for HT29 cells by a pre-experiment.Next,the HT29 cells in logarithmic growth phase were divided into experimental and negative control groups.Then they were plated in 6-well plates on the day before transfection.After 72 hours of transfection,the fluorescence intensity of GFP marker was observed under a fluorescence microscope.Based on this,the efficiency of transfection was simply acquired.2.Real-time PCR and Western blot were used to detect the silence efficiency of GTPBP4 gene after transfection(1)Post-transfection 72 h,the total RNA was extracted from the HT29 cells of experimental and negative control groups respectively.Then,cDNA was synthesized according to Takara’s reverse transcription kit instructions.Finally,mRNA level of GTPBP4 gene was detected by Real-time PCR to determine the silencing efficiency;(2)Post-transfection 72 h,the total proteins were extracted from the HT29 cells of experimental and negative control groups respectively.The inactive proteins were detected by Western blot.Finally,the protein bands were obtained and the silencing efficiency of GTPBP4 at the protein level was analyzed.3.After silencing GTPBP4 gene by lentivirus,the effects on the biological behavior of HT29 cells(1)After GTPBP4 gene silenced,the proliferation of HT29 cells in vitro was detected by CCK-8.(2)Plate clone formation assay was used to detect the change of tumorigenesis ability of single cell in vitro.(3)Scratch assay was used to detect the ability of migration of HT29 cells in vitro.(4)Transwell assay was used to detect the invasive ability of HT29 cells in vitro after GTPBP4 gene silenced.(5)The PI-FACS method was used to detect the change of cell cycle distribution of HT29 cells after GTPBP4 gene silenced.(6)The apoptosis rate was detected by the Annexin V-APC single staining assay.4.The expression and location of p53 and Survivin protein were detected.Western blot and immunofluorescence assays were used to detect the expression and location of p53 and Survivin protein after GTPBP4 gene silenced by lentivirus.Results: 1.The screen of lentivirus for MOI and transfection.The optimal MOI of GTPBP4 RNAi transfected into HT29 cells was 10.2.mRNA and protein levels of GTPBP4 gene in experimental group were significantly decreased after GTPBP4 gene silence.Real-time PCR and Western blot were used to detect the efficiency of transfection.In the experimental group,the results of PCR and Western blot assays showed that the mRNA and protein levels of GTPBP4 were significantly decreased,compared with negative control group.GTPBP4 RNAi effectively inhibit the mRNA and protein expression of GTPBP4.3.The effects of lentivirus silencing GTPBP4 gene on the biological behavior of HT29 cells.(1)The results of CCK-8 showed that the mean of OD values of experimental group were 0.77±0.01,1.06±0.02,1.64±0.02,2.01±0.06 respectively at 24 h,48h,72 h,96h after transfection,and the mean of OD values of negative control group were 0.92±0.03,1.34±0.04,2.27±0.02,3.17±0.09.The experimental group OD values were less than the control group,and the differences between the two groups were statistically significant(P <0.05).(2)The results of plate clone formation showed that the number of colon formation in the experimental group was significantly smaller than that in the control group(67.00±3.61 vs.115.70±10.02),and the difference between this both groups was statistically significant(P <0.05).(3)The results of scratch experiment showed that the migration distance of the experimental group was shorter than that of the control group(500.01±1.82 PX vs.781.23±10.51 PX).(4)The results of Transwell showed that the mean of number of cells passing through the Matrigel in the experimental group was less than that of the control group(91.40±4.39 vs.263.80±11.90),and the difference was statistically significant(P <0.05).(5)The results of PI-FACS showed that the number of cells at G1 phase increased in experimental group,while the number of cells in S phase decreased significantly,compared with control group.(6)The results of Annexin V-APC showed that the proportion of HT29 cell apoptosis in the experimental group was significantly higher than that in the negative control group.4.The expression and location of p53 and Survivin protein of HT29 cells.When the GTPBP4 gene of HT29 cells silenced,the expression of p53 protein was significantly up-regulated and the expression of Survivin protein was down-regulated.While the results of immunofluorescence showed that there was no difference of the location of p53 and Survivin between the experimental group and the negative control group.Conclusion:1.Lentivirus-mediated GTPBP4 RNAi could infect human colon cancer HT29 cells,and could silence the GTPBP4 gene effectively.2.Inhibition of GTPBP4 gene of HT29 cells could inhibit the proliferative activity,clonality,invasiveness and migration capability in vitro,also could change the cell cycle distribution of HT29 cells to induce apoptosis.3.GTPBP4 gene may act as oncogene in the development of colorectal cancer.4.There may be a relationship between GTPBP4 gene and p53,Survivin.And this close association was possible to play a crucial part in the development of colorectal cancer.