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Objective To determine the methylation level of P15INK4B gene promoter in different types of myelodysplastic syndromes (MDS) and its correlation with its prognosis.Methods Methylation frequency of the P15INK4B gene promoter in 44 cases of MDS were determined by methylation-specific polymerase chain reaction (PCR) and pyrosequencing,and its correlation with clinical classification and characteristics of MDS were statistically analyzed.Results Frequency of P15INK4B gene promoter methylation in myelodysplastic syndromes-refractory anemia with excess blasts Ⅱ (MDS-RAEB Ⅱ) patients was (46.89 ± 15.41) ％,significandy higher than that in other types of MDS (P ＜ 0.05),but no difference in promoter methylation frequency was detected among the other types of MDS (P ＞ 0.05) ; frequency of P15INK4B gene promoter methylation was found to be correlated with decline in platelet upon diagnosis (t =9.02,P ＜ 0.01),but showed no significant correlation with drop of hemoglobin or leukopenia (P ＞0.05).As for the correlation between P15INK4B gene promoter methylation and MDS risk stratification,no significant difference was detected between the low-risk and very low-risk groups (P ＞ 0.05),but significant differences were detected among the medium-risk,high-risk,and very high-risk groups (P ＜ 0.05).In addition,frequency of P15INK4B gene promoter methylation was (49.21 ± 8.78)％ in MDS patients that developed leukemia in the following two year,significantly higher than that in MDS patients who didn't (19.64 ± 6.24) ％ (P ＜ 0.05).Conclusions P15INK4B gene promoter methylation frequency is a valuable indicator of prognosis of MDS patients.
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Objective To construct pcDNA3.1 RASSF1 eukaryotic vector and observe the influence of RASSF1 on the apoptosis of hepatocarcinoma cell line HepG2. Methods RASSF1 gene was amplifled from human RASSF1 cDNA by polymerase chain reaction (PCR) and cloned into pcDNA3.1. The recombinant plasmid pcDNA3. 1 RASSF1 was transfected into hepatocarcinoma HepG2 cell line. The expression of RASSF1 was examined by Western blot. The influence of RASSF1 on the cell apoptosis was measured by Annexin V/PI assay. Results DNA enzyme digestion and sequencing results showed that recombinant plasmid pcDNA3. 1-RASSF1 was successfully constructed. RASSF1 protein was overexpressed in HepG2 cell line transfected with pcDNA3. 1-RASSF1 plasmid. The apoptosis rate of blank, pcDNA3. 1 and pcDNA3. 1-RASSF1 group was (5.8 ±0.42)%, (7.48 ±0.68)% and (35. 1 ±3. 15)%, respectively.Conclusion The pcDNA3. 1- RASSF1 eukaryotic vector was successfully constructed, RASSF1 protein overexpression could induce apoptosis in HepG2 cell line.