Inhibition of renal clear cell carcinoma proliferation by avasimibe through SOAT1-mediated cholesterol metabolism remodeling
Objective To identify small molecule inhibitors which inhibit the cell proliferation of clear cell renal cell carcino-ma(ccRCC),and to clarify the role of avasimibe in inhibiting cell proliferation by targeting SOAT1 in ccRCC.Methods Cell counting kit-8(CCK8)method was used to screen small molecular inhibitors which could significantly inhibit the cell viability of ccRCC in the Lipid Metabolism Compound Library,and the changes of the cell viability of ccRCC in the control group and the SOAT1 knock-down/over-expression group were determined.The total cholesterol and cholesterol ester flu-orescence determination kit was used to detect the content of cholesterol ester in ccRCC cells of control group,avasimibe group and SOAT1 knock-down group;quantitative real-time polymerase chain reaction(RT-qPCR)was used to detect the changes of SOAT1 mRNA expression;western blot was used to detect the expression of SOAT1;plate clone formation ex-periment was used to detect the changes of cell clone formation.The independent sample t test was used for comparison between the two groups.Results In the two rounds of drug screening,compared with the control group(1.00),the viabil-ity of 769-P(round 1:0.01±0.02,t=75.50,P＜0.001;round 2:0.02±0.00,t=348.67,P＜0.001)and Caki-1(round 1:0.53±0.01,t=62.28,P＜0.001;round 2:0.02±0.00,t=440.25,P＜0.001)cells in avasimibe group decreased.The results of 50％inhibition concentration determination of avasimibe in ccRCC showed that 769-P(IC50:43.94 μmol/L)and Caki-1(IC50:48.01 μmol/L)were more sensitive to avasimibe than HK-2(IC50:49.65 μmol/L),ACHN(IC50:55.36μmol/L),786-O(IC50:56.29 μmol/L)and A498(IC50:61.75 μmol/L),P＜0.001.Compared with the control group,the content of cholesterol ester in 769-P[(855.27±17.49)μmol/L vs(798.97±15.49)μmol/L,t=4.17,P=0.014]and Caki-1[(825.77±10.10)μmol/L vs(665.88±32.40)μmol/L,t=8.16,P=0.001]cells in avasimibe group decreased.Compared with the control group(1.00±0.05),knocking down SOAT1 significantly inhibited the expression of SO AT1 mRNA in 769-P(0.46±0.02)and Caki-1(0.33±0.02)cells,with statistically significant differences,with t values of 17.15 and 20.39,respectively,both P＜0.001;compared with the control group(1.00±0.00),the expression of SOAT1 protein decreased in both 769-P(0.38±0.14,P=0.001)and Caki-1(0.41±0.07,P＜0.001),with t values of 7.95 and 15.26 respectively;after knocking down SOAT1,compared with the control group(1.00),the cell proliferation ability of 769-P(0.63±0.06)and Caki-1(0.58±0.07)decreased with statistical significance,with t values of 10.54 and 10.95,respectively,both P＜0.001;cloning ability(769-P:178.00±4.00 vs 85.33±4.16;Caki-1:235.67±6.81 vs 140.67±6.43)decreased with statistical significance,with t values of 27.80 and 17.54 respectively,both P＜0.001;and the intra-cellular cholesterol ester content[769-P:(1 047.45±20.16)μmol/L vs(885.88±14.07)μmol/L;Caki-1:(1 273.24±29.51)μmol/L vs(1 102.49±36.17)μmol/L]decreased with statistical significance,with t values of 11.38 and 6.34,and P values of＜0.001 and 0.003,respectively.The cell viability of 769-P(0.69±0.03 vs 0.44±0.03)was increased in the group of overexpression of SOAT1,t=9.89,P＜0.001.Conclusion Avasimibe inhibits the proliferation of ccRCC by remodeling cholesterol metabolism through SO AT1,which may be a new therapeutic target of ccRCC.
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