The Effect Of HARD1 Gene Silence By ShRNA On The Apoptosis Of Colorectal Cancer Cell Induced By 5-Fu

Objective:The experiment used SW620 lines as study objects of Colorectal Cancer cell lines, Investigated the Effect of recombi nant plasmid hARD1-shRNA on the apoptosis of colorectal cancer cell induced by 5-Fu.So as to provide some experimental basis for molecular targeting therapy and molecular therapy combined with chemotherapy of colorectal cancer, and study the effects of chemotherapy sensitivity factor,and provide a basement for the further study of ARD1.Methods:1. Cultivation of SW620?shCON-SW620?hARD1-shRNA-SW620 cell strains.2. Detection the stability and transfection efficiency of recombinant plasmid pENTR /U6-hAR D1-shRNA-SW620. There were 3 groups: The experimental group:hARD1-shRNA transfect into SW620 cells. The negative control group:Empty plasmid transfect into SW620 cells. The control group:untransfected cells.3. MTT assay was be used to detect the effect of on the sensitivity of 5-Fu4. AnnexinV/PI Double staining was used to detect early apoptosis of colorectal cancer cells.5. PI staining was used to detect cycle of colorectal cancer cells.6. Western Blot was used to detect the expression of hARD1 protein in colorectal adenocarcinoma after the treatment by 5-Fu.7. Detect the change of ARD1 gene expression of colorectal cancer SW620 cell which was ransfected with hARD1- shRNA by RT PCRResults:1. SW620?shCON-SW620?hARDl - shRNA-SW620 were cultured successfully and good growth state in vitro.2. Detection the stability and transfection efficiency of recombinant plasmid pENTR/U6-hAR D1-shRNA-SW620:The inhibition rate of mRNA hARD1 after SW620 shRNA-ARD1 was 54.8%3. MTT assay was be used to detect the effect of on the sensitivity of 5-Fu: Different concentrations(0,1,,10,20,40,100,500, 1000?g/ml) was used into the every groups and to observe the cell growth in different time periods(0,12,24,48,72h), then record the OD to calculate the inhibition rate of tumor cells and IC50. Statistical analysis with SPSS20.0: The results show that the experimental group compared to the negative control group and the control group were statistically differences (P<0.05);the blank control group compared to the negative control group is no significant difference (P>0.05). The median inhibitory concentration (IC50) was significantly down regulated with results 344.953±28.539 ?g/ml in the experimental group,798.383±86.59 ?g/ml in the negative control group,and 735.338±36.530 ?g/ml in the blank control group.4. AnnexinV/PI Double staining was used to detect early apoptosis of colorectal cancer cells: Comparison of apoptosis in three groups without 5-Fu: The apoptosis rate in the experimental group was significantly higher than that in the blank group (P<0.05);With 5-Fu:The results show that the experimental group compared to the negative control group and the control group were statistically differences (P<0.05);the blank control group compared to the negative control group is no significant difference (P>0.05).5. PI staining was used to detect cycle of colorectal cancer cells: Without 5-Fu:The cell cycle was significantly arrested at G0/G1 in the experimental group. With 5-Fu:The proportion of G0/G1 phase cells in each group was increased after/ml 1000ug, and the proportion of G0/G1 phase cells was lower than that of the negative group (P<0.05).. Sub-G1 peak in high doses 5-FU (500ug/ml), G0/G1 phase cells in experimental group is higher than that of non drug group decreased (P?0.05) but the experimental group and negative control group and blank control group had no significant difference(P>0.05) and a lower proportion of the cell concentration and no drug group significantly increased (P<0.05) Sub-G1 peak appeared in the sub G1 peak, the proportion of cells in sub-G1 phase is smaller than that of non treated with 5-FU and low concentration of 5-FU treatment increased, the experimental group had a higher degree than those in negative control group and blank group high, the difference is statistically significant (P<0.05).6. Western Blot was used to detect the expression of hARDl protein in colorectal adenocarcinoma after the treatment by 5-Fu: T The hardl protein in three groups of cells relative expression levels of gray value analysis, results showed that: the experimental group and control set of pairwise differences were statistically significant (P< 0.05); compared to the blank control group and negative control group, the difference was not statistically significant(P>0.05).7. The result of RT-PCR:hARD1 mRNA expression level, the experimental group and the negative group, the experimental group and the blank group were statistically significant(P< 0.05), compared with the negative control group, the difference was not statistically significant(P>0.05).Conclusion:.1. ShRNA silencing ARD1 gene can improve the sensitivity of 5-Fu in colorectal adenocarcinoma SW620 cells, and has the effect of chemotherapy sensitization2. ShRNA silencing hARD1 combined with 5-Fu can promote the apoptosis of colorectal cancer SW620 cells caused by chemotherapeutic drugs 5-Fu.3. ShRNA combined with low concentrations of ARD1 gene 5-Fu SW620 cell cycle arrest in G0/G1 phase, combined with high concentrations of 5-Fu to promote apoptosis4. ShRNA ARD1 gene combined with 5-Fu can down regulate the expression of hARDl in colorectal adenocarcinoma cells.5. The experiment verified that shRNA-hARD1 down regulation ARD1mRNA in colorectal adenocarcinoma SW620.