| OBJECTIVE:Human arrest defective 1(hARD1)is an N-acetyltransferase(NAT)that catalyzes the acetylation of multiple proteins in vivo.According to the research,hARD1 is closely related to a variety of tumors,including colorectal cancer,and has been one of the new ideas and research points for cancer diagnosis in recent years.In this study,the recombinant plasmid pCMV6-Entry-ARD1 was transfected into colorectal adenocarcinoma cell line SW620 to establish a stable colorectal adenocarcinoma cell line with hARD1 expression.To observe the proliferation and apoptosis of colorectal adenocarcinoma cells.And the mRNA expression level and protein expression level of apoptosis-related factors were measured.The effects of hARD1 overexpression on the expression of apoptotic factors were compared.METHODS:1.Recovery and culture of colorectal cancer SW620 cell line;2.The recombinant plasmid pCMV6-Entry-ARD1 was transfected into SW620 colorectal cancer cell line by liposome-mediated transfection technique and G418 cell screening method.The transfected cells were detected by QPCR and Western blot.The empty plasmid was introduced into SW620 colorectal cancer cell line to construct negative control group.3.The cell cycle was tested by flow cytometry in the experimental group,the blank control group and the negative control group.4.Annexin/PI staining was used to detect the apoptosis of three groups.5.EDU method was used to detect the proliferation of the experimental group,the blank control group and the negative control group.6.The expressions of TNF-a,Fas,caspase-8 and BcL-2 in mRNA and protein were detected by Q-PCR and Western blot.The expression levels of Fas,caspase-8 and BCL-2 in the three groups were not different(P>0.05).RESULTS:1.The SW620 colorectal cancer cell line transfected with recombinant plasmid pCMV6-Entry-ARD1 was able to achieve high expression of hARD1 successfully.2.The colorectal cancer cell line with hARD1 stable expression was successfully screened by G418 reagent.The relative expression of ARD1 in the experimental group,blank control group and negative control group were 4.23±0.11,0.82±0.04 and 1±0.04 respectively by Q-PCR.The expression of ARD1 in the experimental group was significantly higher than that in the negative control group(P<0.05).There was no significant difference between the negative control group and the blank control group(P>0.05).There was no significant difference between the control group and the blank control group(P>0.05).The expression level of ARD1 protein in the experimental group was 1.60± 0.02,0.81±0.02 in the negative control group and 1±0.02 in the blank control group.The data were analyzed by variance analysis group(P<0.05).The expression of ARD1 protein in the experimental group was significantly higher than that in the control group(P<0.05).There was no significant difference between the negative control group and the blank control group(P>0.05),indicating that the ARD1 protein level in the experimental group was significantly higher than that in the control group(P<0.05),and there was no significant difference between the negative control group and the blank control group The expression of hARD1 overexpression was confirmed by the above assay.3.Cell cycle test showed that GO/G1 phase cells accounted for 53.0%of the blank control group,51.8%in the experimental group and 52.4%in the negative control group.S phase cells accounted for 17.3%of the blank control group and 13.0%in the experimental group.Group accounted for 16.4%;G2/M phase cells:blank control group accounted for 18.0%,experimental group accounted for 20.1%,negative control group accounted for 18.9%;three groups of cells cell cycle by chi-square test difference was not statistically significant(P>0.05).4.Apoptosis detection showed that the level of apoptosis was 1.9%in the experimental group,8.8%in the blank control group and 8.6%in the negative control group.There was no significant difference between the blank group and the negative control group(P<0.05),which indicated that the apoptosis rate of the experimental group was significant.5.EDU test showed that the cell proliferation level was 41%in the experimental group,28.3%in the blank control group and 25%in the negative control group.The proliferation of the three groups was analyzed,and the proliferation and negative The difference between the control group and the blank group was statistically significant(P<0.05).There was no significant difference between the negative control group and the blank group(P<0.05).6.(P<0.05).There was no significant difference between the blank control group and the negative control group(P>0.05).There was no significant difference between the blank control group and the negative control group(P<0.05),and there was no significant difference between the blank control group and the negative control group(P>0.05).There was no significant difference in the expression level of Fas,caspase-8 and BCL-2 between the three groups(P>0.05).The expression of TNF-a and caspase-8 in the experimental group was significantly lower than that in the control group and the negative group(P>0.05).There was no significant difference in the expression level of Fas between the three groups(P<0.05).There was no significant difference between the blank control group and the negative control group(P>0.05).The expression of BCL-2 was up-regulated and the difference between the experimental group and the blank control group and the negative group was statistically significant(P<0.05).There was no significant difference between the blank control group and the negative control group(P>0.05).CONCLUSION:1.The experiment shows that the high expression of hARD1 in colorectal cancer cells resulted in decreased cell apoptosis,cell proliferation rate increased,but the high expression of hARD1 after the cell cycle had no obvious change;Indicating that hARD1 can reduce the apoptosis of colorectal cancer cells and promote the proliferation of colorectal cancer cells.2.The expression of apoptotic factor TNF-a was down-regulated at the mRNA level and protein level,and the expression of caspase-8 was down-regulated at the protein level,and the expression of BcL-2 was up-regulated at the protein level.Indicating that hARD1 may inhibit the apoptosis of colorectal cancer cells by promoting or inhibiting the expression of multiple apoptosis factors. |
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