| Objective：To investigate the effect of (5-Aza-CdR) on PTEN expression and AKTactivation in hepatocellular carcinoma HepG2cells, and thus explore its potentialmechanism in liver cancer development.Methods：Human hepatocellular carcinoma HepG2cells were treated with5-Aza-CdR, andcytotoxic effects on HepG2cells were determined by Lactate dehydrogenase releaseexperiments. Then0.4,1.6,6.4,25.6,102.4μmol/L of5-Aza-CdR was administratedfor24h,48h and72h, and the cell growth inhibition rate was determined by MTTassay; The mRNA level of phosphatase and tensin homolog (PTEN) were detected byRT-PCR; Protein level of PTEN and Phosphorylation of Akt were determined byWestern blot. Methylation of PTEN was analyzed by methylation-specific PCR(MSP).Results:1. Lactate dehydrogenase release experiment showed that102.4μmol/L5-Aza-CdRhad no obvious influence on HepG2cells (P>0.05). MTT assay showed that0.4μmol/L,1.6μmol/L,6.4μmol/L,25.6μmol/L and102.4μmol/L of5-Aza-CdRcould inhibit HepG2cells growth in a time and concentration-dependent mannerfor48h； The inhibition rates were (18.32±3.87)%，(38.15±1.72)%，(47.67±0.47)%，(63.41±1.34)%and (71.88±2.04)%， respectively, there wasstatistic significant between the groups (P<0.05).2. RT-PCR demonstrated that5-Aza-CdR treatment could up-regulate PTEN mRNAexpression in time-and dose-dependent manner. Gray scale scanning showed therewas statistic significant between the groups (P<0.05)3. Western blot showed that5-Aza-CdR could induce HepG2cells expression of PTEN, as the concentration of5-Aza-CdR increases, the expression level increased(P<0.05).4. MSP revealed that5-Aza-CdR could inhibit PTEN methylation, and could alsoincrease the non-methylated protein level (P<0.05).5. Western blot demonstrated that5-Aza-CdR could inhibit Akt phosphorylation(P<0.05).Conclusion:5-Aza-CdR can increase the expression of PTEN, decrease the methylated PTEN, anddown-regulated p-AKT protein expression in HepG2cells. |
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