| Objective: Human arrest defective 1 (hARD1) is a human homologous gene of yeast N-acetyltransferase ARD1, which binds to the NAT1 gene to produce a Nat A complex that catalyzes the acetylation of multiple proteins in cells. The occurrence and development of colorectal cancer is closely related to the expression of hARD1 gene and apoptosis. However,whether hARD1 gene expression can affect the occurrence and deveploment of colorectal cancer by influencing apoptosis pathway and the specific molecular mechanism is not clear.In this study, the colorectal cancer cell line SW620 as the research object to study the relationship between hARD1 and the expression level of apoptosis-related factors, explore the effect of hARD1 gene silencing on apoptotic signaling pathway proteins, and then reveal the specific molecular mechanism of hARD1 gene regulating apoptosis of colorectal cancer cells, hoping to provide theoretical basis for early diagnosis and treatment of colorectal cancer.Methods:1. Resuscitation, culture, passage and cryopreservation of human colorectal cancer cell line SW620.2. The experimental group was transfected into cell lines by Lipofectamin 2000 transfection reagent with ARD1 siRNA. The negative control group was transfected with NC siRNA. The blank control group were cultured without special treatment.3. The silencing efficiency of hARD1 in each group was detected by RT-PCR.4. Flow cytometry and PI staining were used to detect the changes of colorectal cancer cell cycles in each group.5. Detection of apoptosis of colorectal cancer cells in each group by flow cytometry and AnnexinV+PI staining.6. The proliferation of colorectal cancer cells was detected by EdU method.7. The content of ROS in colorectal cancer cells was detected by fluorescence probe method.8. The mRNA expression levels of apoptotic pathways related factors (TNF-?, Fas, caspase8,Apaf-1, caspase9, Bcl-2, Bad) were detected by fluorescence quantitative PCR in colorectal cancer cells.9. Western blot was used to detect the protein expression levels of apoptotic pathways related factors (TNF-?, Fas, caspase8, Apaf-1, caspase9, Bcl-2, Bad) in colorectal cancer cells.Results:1. The human colorectal cancer cell line SW620 were transfected by ARD1 siRNA and NC siRNA, the silence efficiency of hARD1 in each group was detected by RT-PCR: The silence efficiency of the negative control group was 0%, the silence efficiency of the experimental group was 62%.2. Flow cytometry and AnnexinV+PI staining were used to detect the cycle and apoptosis of colorectal cancer cells. The results showed that the cycle of the three groups did not change significantly after the ARD1 gene was silenced. The apoptosis level of the experimental group increased markedly, whereas the negative control group and the blank control group were not significantly different.3. EdU detection of cell proliferation tips: The experimental group compared with the blank control group and the negative control group, the cell proliferation ratio decreased markedly.There was no significant difference in the cell proliferation ratio between the negative control group and the blank control group.4. Fluorescence probe method for detecting ROS content tips: The experimental group compared with the blank control group, the ROS content increased markedly, the negative control group and the blank control group compared the ROS content with no obvious difference.5. Fluorescence quantitative PCR detection of mRNA expression levels in each apoptosis factor suggest: The mRNA expression level of apoptotic factor (caspase 9) in the experimental group was significantly higher than that in the blank control group and the negative control group, while the mRNA expression level of apoptosis pathway related factors( TNF-?,caspase8,Fas,Apaf-1,Bcl-2,Bad ) in three groups had no significant change.6. Western Blot was used to detect the protein expression level of each apoptotsis factor. The results showed that the protein expression level of apoptosis pathway related factors(caspase8, caspase9, Fas, Apaf-1 and Bcl-2) in the experimental group was significantly higher than that in the blank control group and the negative control group, while the protein expression level of the apoptosis pathway related factors (TNF-a, Bad) in the three groups had no significant change.Conclusions:1. The hARD1 gene silencing had no significant effect on the cycle of SW620 cells,but could obviously promote cell apoptosis and inhibit cell proliferation.2. This study confirmed that hARDl gene silencing can promote cell apoptosis by modulating the transcription level and translation level of some factors in the pathway of apoptosis.3. hARD1 gene may serve as a potential target for the development of colorectal cancer and provide a theoretical basis for knockout hARD1 gene therapy for colorectal cancer. |
PDF Full Text Request