Abstract: Objective To investigate the effects of cholesterol and 27-hydroxycholesterol on the proliferation, invasion and migration of esophageal squamous cell carcinoma(ESCC)and on the secretion of cytokine MCP-1. Methods In vivo animal experiments: After animal models of ESCC were established, they were given high cholesterol diet(high cholesterol diet group, n=4)or normal diet(control group, n=4), to observe the effects of cholesterol on the growth of ESCC in vivo. At the end of the 5th week, the tumor volume of the two groups was measured and tumor inhibition rate was calculated. Cell experiments: Effects of cholesterol(0, 0.123, 0.148, 0.185, 0.269, 0.370 mg/mL)and 27-hydroxycholesterol(0, 1, 5, 10, 20 μmol/mL)on the proliferation of normal ECA109 cells, and ECA109 cells with cyp27a1 and cyp7b1 knockout were observed. The cell proliferation at different concentrations was detected with CCK-8. Cell invasion was determined with scratch test and invasion test(cholesterol 0.185 mg/mL, 27-hydroxycholesterol 1 μmol/mL). The upstream gene cyp27a1 and downstream gene CYP7B1 of 27-hydroxycholesterol were transfected with lentivirus. The above experiments were repeated to observe the effects of gene knockout on proliferation, invasion and migration of cells, and the effect on the secretion of MCP-1 was detected with ELISA. The tumor volume and absorbance measured by CCK-8 in the two groups at different times were analyzed with two-factor ANOVA. When the interaction was statistically significant, simple effect analysis was performed. One-way ANOVA was used for the comparison of mean differences between groups, and LSD method was used for pairwise multiple comparison. Results Animal experiments showed the tumor volume was(5.055±0.774)cm³ in the high cholesterol diet group and(5.055±0.774)cm³ in the control group, and the tumor inhibition rate was-170.79%, with statistically significant difference(P<0.05). Cell experiments showed: (1) Cholesterol promoted the proliferation of ECA109 cells and ECA109 cells with cyp27a1 knockout at low concentration(P<0.05). 27-hydroxycholesterol inhibited the proliferation of ECA109 cells, but promoted the proliferation of ECA109 cells with CYP7b1 knockout(P<0.05). (2) Cholesterol and 27-hydroxycholesterol had no effects on the migration of ECA109 cells(F=2.418, P=0.170), and no effects on the migration of ECA109 cells with cyp27a1 and CYP7b1 knockout(F=0.602, P=0.578). (3) After gene knockout, the cell invasion changed(F=3.992, P=0.047). Cells with CYP27b1 knockout had inhibited invasion(P<0.05), while cells with CYP7a1 knockout had unchanged invasion(P>0.05). (4) Gene knockout affected the secretion of MCP-1(F=553.538, P<0.001). CYP27A1 knockout increased the secretion of MCP-1(213.823±4.572), while CYP7B1 knockout decreased the secretion of MCP-1(107.240±4.121)(P<0.001). Conclusion Cholesterol can stimulate the proliferation of ECA109 cells both in vivo and in vitro. 27-hydroxycholesterol can inhibit the proliferation of ECA109 cells while cholesterol can promote the proliferation of ECA109 cells, both in a concentration-dependent manner. The knockdown of upstream gene cyp27a1 can enhance the effect of cholesterol on the proliferation but does not affect the cell invasion. The knockdown of downstream gene cyp7b1 can enhance the effect of 27-hydroxycholesterol on cell proliferation and inhibit cell invasion. The secretion of MCP-1 is increased by cyp27a1 knockout and inhibited by cyp7b1 knockout.

Key words: 27-Hydroxylcholesterol, Cholesterol, Human esophageal cancer cell, Proliferation, Invasion