The Role And Mechanism Of Cholesterol And 27-hydroxycholesterol In The Proliferation And Metastasis Of Esophageal Squamous Cell Carcinoma

ObjectiveThe purpose of this research was to investigate effects of cholesterol and 27-hydroxycholesterol on the changes of the biological behaviors of esophageal squamous cell carcinoma in its proliferation,invasion and migration capacity,as well as the effects on the secretion of inflammatory cytokines mcp-1.MethodsIn vivo experiments of animals:Through the establishment of an animal model of esophageal squamous cell carcinoma in nude mice,the effects of cholesterol on the growth of esophageal squamous cell carcinoma in vivo were observed by giving them different diets(n=4).At the end of the fifth week,the tumor volume of the two groups of animals was measured and the tumor inhibition rate was calculated Cell experiment:Cholesterol(0,0.123,0.148,0.185,0.269,0.370mg/ml)and 27-hydroxycholesterol(0,1,5,10,20?m/ml)were observed in normal esophageal squamous cell carcinoma cells(ECA109)and the gene of CYP27A1 and CYP7B1 knockout cells.However,the methods did not affect the proliferation ability of ECA109 cells after the gene of CYP27A1 and CYP7B1 knockout,and the survival rate of ECA109 cells after the intervention of different concentrations of cholesterol and 27-hydroxycholesterol was detected by CCK-8 method.The invasion effect of tumor cells was determined by scratch test and invasion test(cholesterol 0.185mg/ml,27-hydroxy cholesterol ?m/ml).Lentivirus was used to transfer to knockout the upstream synthetic gene CYP27A1 and the downstream metabolic.gene CYP7B1 of 27-hydroxy cholesterol.The above experiments were repeated respectively,and the effect of gene knockout on the secretion of cytokine mcp-1 was detected by ELISA.The tumor volume of the two groups of animals was analyzed by repeated measurement ANOVA,the absorbance value measured by CCK-8 experiment was analyzed by two-factor ANOVA,and the simple effect analysis was performed when the interaction was statistically significant.One-way ANOVA was used for the comparison of mean differences between groups,and LSD method was used for pairwise multiple comparison.Results1.Animal experiments showed that in the fifth week,the tumor volume of xenotransplantation in nude mice of high-cholesterol diet group is(5.055±0.774)cm3 and the control group is(5.055±0.774)cm3,respectively,and the tumor inhibition rate was-170.79%,with statistically significant difference(P<0.05).2.Cell experiments showed that:(1)Cholesterol can promote the proliferation of ECA109 cells and ECA109 cells after the knockout of upstream gene CYP27A1,and the effect of promoting the proliferation of ECA109 cells at low cholesterol concentration is significant(P<0.05).27-hydroxy cholesterol can inhibit the proliferation of ECA109 cells,but promote the proliferation of ECA109 cells after the knockdown of downstream gene CYP7B1(P<0.05).(2)When the upstream gene CYP27A1 and the downstream gene CYP7B1 are knocked out,the proliferation of the upstream gene CYP27A1 is more sensitive to cholesterol concentration than that of normal ECA109 cells,and cell proliferation can be promoted when the cholesterol concentration is 0.123mg/ml,0.148mg/ml(P<0.05).The proliferation activity of CYP7B1 knockout cells was more sensitive to 27-hydroxycholesterol concentration,and cell proliferation was promoted when the concentration of 27-hydroxycholesterol was 5.m/ml and 10?m/ml.But inhibit cell proliferation when the concentration was 20?m/ml(P<0.05).(3)Cholesterol and 27-hydroxycholesterol had no effects on the migration ability of ECA109 cells(Fdrug type=2.418,Pdrug type=0.170),the deletion of upstream gene CYP27A1 and downstream gene CYP7B1 of 27-hydroxycholesterol had no effects on the migration ability of ECA109 cells(Fcell type=0.602,Pcell type=0.578).Compared with the control group after gene knockout,the invasion ability of cells was changed(F=3.992,P=0.047).Synthesis gene CYP27A1 knockout had no effect on the invasion ability of cells(P>0.05),but the invasion ability of the knockout group of downstream metabolic gene CYP7B1 was inhibited compared with the normal ECA109 cell group(P<0.05).(4)Gene knockout can also change the secretion of mcp-1 factor(F=553.538,P<0.001).When the upstream gene CYP27A1 was knocked out,the secretion of mcp-1 increased,while the downstream gene CYP7B1 was knocked out,the secretion of mcp-1 decreased(P<0.001).Conclusion(1)Cholesterol can stimulate the proliferation of ECA109 cells both in vivo and in vitro.(2)27-hydroxycholesterol inhibited the proliferation of ECA109 cells,and promoted the proliferation of ECA109 cells,both of them in a concentration-dependent manner.(3)The knockdown of the upstream gene CYP27A1 can enhance the effect of 27-hydroxycholesterol on cell proliferation activity but did not affect the cell invasion ability.While the knockdown of the downstream metabolic gene CYP7B1 can enhance the effect of 27-hydroxycholesterol on cell proliferation activity and inhibited the cell invasion ability.(4)The secretion of mcp-1 factor in cells may be increased by gene cyp27al knockout and inhibited by gene cyp7bl knockout.