Differential Expression Of Aldo-Keto Reductase AKR1C Isoforms In Hepatic Carcinoma Cells And Enzymatic Analysis

There are four subtypes of human Aldo-Keto Reductases AKR1 C,which have been shown to be related to the occurrence and development of cancer.In recent years,whether AKR1 C is a tumor marker and the role of inhibitors in assisting anti-cancer and reversing tumor tolerance have become the focus of research by experts and scholars in the fields of biology,clinical medicine and pharmacy.Four types of AKR1C enzymes were only expressed simultaneously in liver tissue,so HepG2 and HL-7702 corresponding to normal liver cells were selected as the research object in this paper.Real-time PCR and western-blot were used to study the expression differences of these four enzymes in different cells at the level of mRNA and protein expression;The four enzymes were expressed in vitro and analyzed by enzymology;AutoDockTools molecular docking software was used to screen small-molecule inhibitors that extensively inhibited 4 enzymes.The research results include the following aspects:1)The transcription level of mRNA showed that the expression of AKR1C1 in HepG2 was 3.01 times higher than that in HL-7702(*P<0.05),and the expression of AKR1C2 in HepG2 was 2.83 times higher than that in HL-7702(*P<0.05).The expression of AKR1C3 in HepG2 was 3.27 times higher than that in HL-7702(*P<0.05),and the expression of AKR1C4 in HepG2 was 1.04 times higher than that in HL-7702(*P<0.05).2)The expression level of protein showed that the expression of AKR1C1 in HepG2 was 1.51 times higher than that of HL-7702(*P<0.05),the expression of AKR1C2 in HepG2 was 1.54 times higher than that of HL-7702(*P<0.05),and the expression of AKR1C3 in HepG2 was 1.56 times higher than that of HL-7702(*P<0.05).The expression of AKR1C4 in HepG2 was 0.49 times higher than that in HL-7702(*P<0.05).3)Four prokaryotic expression vectors were successfully constructed,and the specific activities of the four enzymes were AKR1C1:95659U/mg,AKR1C1:61495U/mg,AKR1C3:66720U/mg,AKR1C4:51045U/mg.4)The optimum conditions for the determination of four enzymes with DLglyceraldehyde as substrate were as follows: pH 7.0,37 ?,substrate concentration 5 mM,NAPDH concentration 0.35 mM.5)Computational biology AutoDockTools software analysis for 10 inhibitors:Medroxyprogesterone acetate?Ursdeoxycholate?2'-hydroxyflavanone?Methyliasmonate?Indomethacin?Flufenaminic acid?Mefenamic acid?Sulindac?Meclofenamic acid?Naproxen,It was concluded that medroxyprogesterone acetate was an effective inhibitor of the four enzymes of AKR1 C,and then the IC50 values of the inhibitors of the four enzymes of AKR1 C were determined,which was in accordance with the above prediction.The results of this study provide an experimental basis for the further study of the mechanism of the occurrence and development of AKR1 C four subtypes of enzymes and hepatoma cells,as well as the theoretical basis and possible mechanism for screening the small molecular inhibitors of the four subtypes of AKR1 C as therapeutic drugs.