Effect Of Demethylating Agent On RUNX3 Expression And The Growth And Drug Sensitivity Of Human Hepatocarcinoma Cell Line HepG2

Objective:RUNX3 was previously identified as a candidate tumor suppressor gene whose loss of erpression by DNA hypermethylation had been demonstrated to frequently associate with human tumorignesis. This study was to investingate the effects of demethylating agent, 5-Aza-2'-deoxycytidine (5-Aza-CdR), on the proliferation and the sensitivity to chemotherapeutic drugs of hepatocarcinoma cell line HepG2, and to explore the probability of re-expression of the hypermethylated and silenced RUNX3 gene by 5-Aza-CdR in HepG2 cells.Methods :The expression change of RUNX3 mRNA was semi-quantifed by RT-PCR before and after 5-Aza-CdR treatment. Cell proliferation of HepG2 cells and IC50 values of chemotherapeutic drugs on them were detected by MTT assay. The sensitivity of HepG2 cells to 5-fluorouracil (5-FU) was analyzed by clonogenic assays. Cell cycle of HepG2 cells and fluorescence intensity of intracellular adiamycin (ADM) in them was analyzed by flow cytometry. Apoptosis morphology was observed by transmitsing electron microscopy.Results :RUNX3 gene was reactivated in RUNX3 non-expression HepG2 cells treated with two different doses of 5-Aza-CdR, which could also inhibit the proliferation of HepG2 cells in a dose-dependent manner (P<0.05) .γ24h=0.894,γ48h=0.973,γ 72h=0.967, 24h,48h and 72h IC₅₀ values of 5-Aza-CdR on HepG2 cells at 24h, 48 and 72h were 87.5, 84.7 and 59.0μmol/L, respectively. 5-Aza-CdR could induce typical apoptotic morphology change of the cells. Flow cytometry assay showed that the cells were arrested in S phase by 5.0 or 10.0μmol/L 5-Aza-CdR. MTT assay indicated the IC₅₀ of 5-FU and ADM on HepG2 cells was decreased when co-treated with 5-Aza-CdR (P<0.05) . MFI and accumulation of ADM in HepG2 cells was increased by 5-Aza-CdR (P<0.05) . Drug sensitivity assay showed that HepG2 cells became more sensitive to 5-FU and ADM after 5-Aza-CdR treatment.Conclusion :5-Aza-CdR could exert its inhibition of proliferation, induction of apopotosis and increasing the sensitivity to chemotherapeutic drugs of hepatocarcinoma cell line HepG2 via reactivating the RUNX3 gene transcription silenced by aberrant hypermethylation.