Construction Of Stable Silencing MiR-222-3p Colon Cancer HCT-116 Cell Line And Intervention Of Jianpi Qingre Huoxue Recipe

Objective:To construct a lentiviral vector-mediated stable silencing mi R-222-3p colon cancer HCT-116 cell line,and to explore the biological effects of Jianpi Qingre Huoxue recipe on autophagy regulation,cell proliferation,cycle,apoptosis and migration.Methods:TUD-hsa-mi R-222-3p Inhibitor lentiviral vector was constructed,then the lentivirus was packaged and infected with HCT-116 cells.The stable cell lines were screened by Puromycin,and the relative expression of mi R-222-3p was detected by RT-PCR.The drug-containing serum of Jianpi Qingre Huoxue recipe was prepared according to the method of serum pharmacology,and different doses of drug-containing serum were used to stably silence mi R-222-3p colon cancer HCT-116 cell line.The experiment was divided into blank control group(HCT-116 group),negative control group(NC group),mi R-222-3p I group and Jianpi Qingre Huoxue recipe low dose group,middle dose group and high dose group.The ultrastructural changes of cells were observed by transmission electron microscope.Cell proliferation and growth activity were detected by MTS,cell cycle were detected by flow cytometry,Apoptosis were detected by Annexin V-FITC,and cell migration was detected by Transwell chamber model.Results:(1)The lentiviral vector TUD-hsa-mi R-222-3p Inhibitor was successfully constructed by sequence comparison,and the virus titer was 1×10~8TU/ml determined by limited dilution method.Stable silencing of mi R-222-3p colon cancer HCT-116 cell line was obtained by Puromycin drug screening.(2)The results of transmission electron microscope showed that there was a significant increase of autophagosomes in the cytoplasm of the cells in the Jianpi Qingre Huoxue recipe group,which could enhance autophagy.The results of MTS showed that on the first day of administration,the treatment group of Jianpi Qingre Huoxue recipe could inhibit the proliferation and growth vitality of HCT-116 cells,which was significantly different from that of the blank control group(HCT-116 group)(P<0.05).On the 2nd and 3rd day,mi R-222-3p I group and Jianpi Qingre Huoxue recipe group significantly inhibited the proliferation of HCT-116 cells and decreased the growth vitality of HCT-116 cells,which were significantly different from those in HCT-116 group(P<0.01).The results of flow cytometry showed that the proportion of S phase cells in mi R-222-3p I group and Jianpi Qingre Huoxue recipe treatment group was lower than that in HCT-116 group and NC group,and the total apoptosis rate was significantly increased.The results of Transwell showed that the number of cell migration in Jianpi Qingre Huoxue recipe treatment group and mi R-222-3p I group was significantly lower than that in HCT-116 group(P<0.05).Conclusion:Jianpi Qingre Huoxue Decoction combined with stable silencing of mi R-222-3p gene can synergistically inhibit the proliferation of coloncancer HCT-116 cells,reduce cell growth activity,cycle progression and migration,enhance cell autophagy,and induce cancer cell apoptosis.