| Objective: according to epigenetics,DNA methylation plays an important role in the occurrence and development of colorectal cancer.Hypermethylation of Cp G islands in the promoter region of tumor suppressor genes leads to decreased expression or silencing of tumor suppressor genes is considered to be a common feature of tumorigenesis.Because DNA methylation is reversible,DNA methylation can be reversed by using DNA methyltransferase inhibitors to reactivate silent tumor suppressor genes,thus inhibiting tumor growth.Dexitabine has the strongest demethylation effect among several nucleoside DNA methyltransferase inhibitors,and has been used in the clinical treatment of malignant hematological diseases such as recurrent myelodysplastic syndrome,but its clinical application is limited because of its bone marrow toxicity and severe gastrointestinal reactions.Procaine hydrochloride(procainehydrochloride,PCA),as a non-nucleoside DNA methyltransferase inhibitor,inhibits the proliferation of human breast cancer MCF-7 cells,gastric cancer SGC-7901 and other tumor cells.However,there are few reports on the effect of PCA on the methylation degree of DNA and its biological function in colon cancer cells.Our group used human colon cancer HCT116 and SW480 cells as models to analyze the effect of PCA treatment on the methylation status and expression level of tumor suppressor gene TBX5,and to explore the molecular mechanism of PCA inhibiting tumor cell proliferation,so as to provide theoretical and experimental basis for the clinical treatment of colorectal cancer with PCA.Methods:(1)Human colon cancer HCT116 and SW480 cells were treated with different concentrations of PCA(0.5,1.0,2.0,4.0 and 8.0m M)for 48 hours,and the best concentration of inhibitory effect of PCA on the proliferation of two kinds of colon cancer cells was detected by CCK-8,and the best time of proliferation inhibition of HCT116 and SW480 cells treated with PCA for 24 hours,48 hours and 72 hours was detected by CCK-8.(2)the changes of proliferation of colon cancer HCT116 and SW480 cells before and after PCA treatment were studied by plate colony formation assay.(3)the effect of PCA on apoptosis of colon cancer HCT116 and SW480 cells was detected by flow cytometry.(4)Transwell assay was used to detect the effect of PCA on the invasive ability of human colon cancer HCT116 and SW480 cells.(5)the effect of PCA on the migration ability of human colon cancer HCT116 and SW480 cells was detected by cell scratch test.(6)High throughput sequencing of bisulfite amplifiers(BSAS)was used to detect the methylation status of TBX5 gene and the methylation status of TBX5 gene in human normal colon epithelial FHC cells before and after the treatment of PCA on colon cancer HCT116 and SW480 cells.(7)the expression level of TBX5 gene was detected by q RT-PCR before and after PCA treatment on colon cancer HCT116 and SW480 cells.Results:(1)PCA could inhibit the proliferation of colon cancer HCT116 and SW480 cells.By calculating the growth inhibition rate of PCA on the two kinds of cancer cells and the optimal concentration of IC50,the best concentration and the best working time were2 mmol /L and 48 h,respectively.(2)the results of plate colony formation assay showed that after PCA treatment of human colon cancer HCT116 and SW480 cells,the number of colonies formed in the experimental group was significantly lower than that in the control group,and the colony diameter in the experimental group was significantly lower than that in the control group.(3)the results of flow cytometry analysis showed that compared with the blank group,PCA treatment could significantly induce the apoptosis of HCT116 and SW480 cells,and the difference was statistically significant(P < 0.05).(4)the results of Transwell invasion assay showed that the number of transmembrane cells of HCT116 and SW480 human colon cancer cells treated with PCA was significantly lower than that of the blank group(P < 0.05).(5)the results of cell scratch test showed that the scratch healing rate of HCT116 and SW480 human colon cancer cells treated with PCA for 48 hours was significantly lower than that of the blank group(P < 0.05).(6)the methylation degree of Cp G island in the promoter region of TBX5 gene in HCT116 and SW480 cells was significantly higher than that in human normal colonic epithelial FHC cells by bisulfite expander high-throughput sequencing.The difference was statistically significant(P < 0.05).After HCT116 and SW480 cells were treated with PCA,the methylation degree of Cp G island in the promoter region of TBX5 gene decreased,and there was significant difference before and after treatment.(7)the results of q RT-PCR assay showed that the expression level of TBX5 gene in HCT116 and SW480 cells treated with PCA was significantly higher than that in the blank control group.Conclusion: Procaine hydrochloride may inhibit the malignant progression of colon cancer cells,such as proliferation,migration and invasion,by reversing the methylation of tumor suppressor gene TBX5 and increasing its expression. |
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