Effect Of HARD1 Gene Acetylation On Growth And Apoptosis-related Factor Expression In Colorectal Cancer Xenografts In Nude Mice

Objective:The human Arrest Defective 1(hARD1)gene is a N-acetyltransferase(NAT).Its main function is to generate a NatA complex by binding to the N-acetytransferas 1(NAT1)gene and affect the function,stability,and interprotein molecules of the protein through acetylation.The interactions,in turn,play an important role in the regulation of the cell cycle.This study was to investigate the effect of hARD1 gene acetylation on growth and apoptosis-related factor expression in colorectal cancer xenografts in nude mice,and to provide new ideas for the diagnosis and treatment of colorectal cancer,and to provide new molecular biology theoretical basis for colorectal cancer genetic research.Methods:1.Resuscitation culture of S W620 human colon cancer cells;2.The SW620 cell line was divided into 5 groups:blank control group,hARDl overexpression group(PCDNA3.1-hARD1 overexpression plasmid),overexpression negative control group(empty PCDNA3.1 plasmid),hARD1 silencing group(hARD1 gene specific siRNA transfection)and silencing negative control group(siRNA-Ncontrol transfection);fluorescence staining technique was used to study the growth and morphological changes of colorectal cancer cells in each group.3.Twenty-five nude mice were randomly divided into 5 groups.Tumor cells were subcutaneously implanted in the ventral side of the nude mice by the above method.The nude mice were carefully cultured and observed for tumor growth,and the tumor growth curve was recorded.4.The tumors were measured every 3 days after the tumor formation in the above 5 groups of nude mice.The volume was calculated.After 3 weeks,the tumors were killed and removed by conventional methods.The tumor tissues were observed by morphological observation.The tumor cell apoptosis was detected by tunnel method.Happening.5.Fluorescent quantitative PCR,immunohistochemistry and Western blot were used to detect hARD1,NAT1 and apoptosis pathway-related factors(TNF-a,caspase8,Apaf-1,caspase9,Bcl-2.Bad)in tumor tissue.mRNA and protein expression levels.Results:1.SW620 cell line was successfully cultured in vitro.2.A total of 25 nude mice in the 5 groups grew well and 24 were successfully inoculated.The tumor formation rate was 96%,and the tumor growth curve was recorded.RESULTS:One-way analysis of variance(ANOVA)was performed on the volume of transplanted tumors in 5 nude mice and compared with each other.The results suggested that the expression of hARD1 had no significant effect on the growth of transplanted tumor in nude mice(P>0.05).3.The results of HE staining of transplanted tumors in nude mice showed no significant differences in the morphology of the transplanted tumors in 5 groups.The proportion of non-cancerous cells in the hARD1-siRNA group was higher(the ratio of non-cancer tissues was(>50%),necrotic tissue was more,and the distribution of cancer tissues was more scattered.Compared with the other groups,the cancer group accounted for a relatively high(>50%).4.TUNEL method was used to detect the apoptosis of tumor cells:the difference between group ARD1-siRNA and CK group and NC-siRNA group was statistically significant(x 2=4.985,P=0.0265,P<0.05),and there was no statistical difference between the CK group and the NC-siRNA group(? 2=0.232,P=0.63,P>0.05).After hARD1 gene silencing in nude mice,the level of apoptosis increased.5.The results of fluorescence quantitative PCR showed that the mRNA expression level of caspase9 and TNFa in the hARD1 overexpression group was significantly lower than that in the negative control group and the blank control group,but there was no significant difference in other groups.6.Western bolt detection results:the expression level of apoptosis pathway related factors(caspase9,caspase8,TNF a,bad)in the hARD1 overexpressed group was significantly lower than that in the negative control group and the blank control group,and there was no significant difference in the other groups.Conclusion:1.Overexpression of hARD1 inhibited the expression of some apoptosis genes and apoptotic pathway factors in human colon cancer SW620 xenograft tumor xenografts,and inhibited tumor apoptosis.2.The silencing of hARD1 gene promotes the expression of some apoptosis genes and apoptotic pathway factors in human colon cancer SW620 xenograft tumor cells and promotes tumor apoptosis.3.hARD1 gene silencing has no effect on the appearance of human colorectal cancer S W620 xenografts in nude nice,but promotes the apoptosis of nude mice xenografts.4.Through this experimental study,it is concluded that the acetylation of hARD1 has an effect on the expression of apoptotic genes and apoptosis pathway-related factors,thereby inhibiting apoptosis and promoting the growth of tumor cells.