| Objectives:The expression of AKR1B10 in hepatocellular carcinoma(HCC)is increased,which is closely related to the early diagnosis and prognosis of HCC.This paper intends to explore the effect of AKR1B10 on cell cycle and its corresponding mechanism.Methods:1)L02,HepG2 and Huh-7 cells were cultured;2)Hep G2 cells were infected with lentivirus LV-AKR1B10-shRNA,and the transfection efficiency of AKR1B10 was detected by fluorescence microscopy,Western blot and real-time quantitative PCR.The control group was named HepG2/NC,and the target group was named HepG2/AKR1B10;3)The enzyme activity of AKR1B10 was detected by reaction of the decrease of optical density of NADPH at 340 nm.4)Cell Counting Kit-8 assay was used to assess the proliferation of HepG2 cells treated with different concentrations of AKR1B10 inhibitor epalrestat(0,50,75,100,125 and 150 ?M)and down expressed for 24?48 and 72 hours.5)Flow cytometry was used to assess the cell cycle of HepG2 cells treated with different concentrations of AKR1B10 inhibitor epalrestat(0,50,100 ?M)and down expressed for 24 hours.6)Western blot was used to measure the protein expression of p-Rb,cyclinD1,cylinE1,p27 to investigate the molecular mechanism underlying the effect on cell cycle.7)Data were shown as the mean ± standard deviation.Student's t-test was used to compare two samples.All the experiments were repeated three times.SPSS 24.0 software was used for statistical analysis,P < 0.05 was statistically significant.Results: 1)The expression of AKR1B10 was increased in Hepatocellular carcinoma cells.The relative expression level of AKR1B10 protein in HepG2 was 6.71±1.11(P=0.012).The relative expression level of AKR1B10 protein in Huh-7 cells was 3.32±0.39(P=0.009).2)The AKR1B10 cell line with low expression was established.Compared with the control group,the expression level of AKR1B10 RNA was 0.19 ± 0.03(P=0.001).AKR1B10 protein level was 0.24±0.07(P=0.002),and the transfection efficiency was about 70% ? 80%.3)Inhibition of the expression of AKR1B10 and treated with different concentrations of AKR1B10 inhibitor epalrestat(0,50,75,100,125 and 150 ?M)for 24,48,72 h inhibited the cell proliferation.The optical density of HepG2/NC cells cultured for 24,48 and 72 hours were 0.67±0.06,1.34±0.05,1.82±1.11,respectively;The optical density of HepG2 / AKR1B10 cells cultured for 24,48 and 72 hours were 0.54 ± 0.06,1.02 ± 0.09,1.36 ± 0.08,significantly lower than the control group(P < 0.05).4)Epalrestat inhibited AKR1B10 activity in a dose-dependent manner.However,compared with the control group,AKR1B10 expression of HepG2 cells treated with different concentrations of epalrestat did not significant change.5)Inhibition of the expression of AKR1B10 and treated with different concentrations of AKR1B10 inhibitor epalrestat(0,50,100 ?M)for 24,48,72 h promoted G0/G1 cell cycle arrest of HepG2 cells.Compared with the control group,100 ?M epalrestat significantly increased the G1 phase of HepG2 cells(65.44±1.78 vs 61.29±1.81,P=0.047).Compared with HepG2/NC cells,the proportion of G1 phase in HepG2 / AKR1B10 cells increased significantly(58.24±1.14 vs 49.33±2.94,P=0.008).6)Western blot showed that 50 and 100 ?M epalrestat could significantly inhibit the expression of cyclin D1,cyclin E1 and p-Rb,and promote the expression of p27 in a concentration dependent manner.Compared with HepG2/NC cells,the expression of cyclin D1,cyclin E1 and p-Rb in HepG2/AKR1B10 cells decreased,while the expression of p27 increased.Conclusions:Inhibiting the expression and activity of AKR1B10 can promote the G0/G1 cell cycle arrest of HepG2 cells,which is related to the regulation of p27/p-Rb pathway. |
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