The Study Of Gene RUNX3 Expression And Promoter Aberrant Methylation In Hepatocellular Carcinoma

Objective (1)To investigate the expression level of RUNX3 in human primary hepatocellular carcinoma (HCC) and its relationship with the clinic pathological features of the disease. (2)To determine the relationship between promoter methylation and mRNA expression of RUNX3 in HCC and its relationship with the clinic pathological features of the disease.Methods (1)Total RNA isolated from samples (including sixty-five HCC , adjacent normal tissues, six human liver cancer cell lines and a human normal liver cell line ) were reversely into cDNA. Real-time fluorescent quantitative RT-PCR method was used to analyze the expression level of RUNX3 gene. Some of the quantitative results were confirmed by Western blotting. (2) The methylation status of 10 samples from human normal liver tissues, 75 samples from HCC and adjacent normal tissues was performed by methylation-specific polymerase chain reaction (MSP). Then, we evaluated the expression of mRNA of RUNX3 in all samples to work out the relationship between the methylation status of RUNX3 and its expression in transcriptional and translational levels.Results (1)The expression of RUNX3 mRNA was 4.2-fold lower in HCC tissues than in adjacent normal tissues: 8-fold lower was found in 17 HCC tissues, 4-fold lower was found in 21 HCC tissues and 2-fold lower was found in 3 HCC tissues. The down-regulation of RUNX3 mRNA was found in 66.67 %( 4/6) liver tumor cell lines. Furthermore, Western blot analysis showed that there was also significant difference in RUNX3 protein expression between the HCC and adjacent non-tumor tissues, which was accordant with the results of quantitative RT-PCR analysis of RUNX3 mRNA levels. Lower expression showed significant correlation to cirrhosis (P=0.028) and histologic type (P=0.000). (2) MSP results revealed that abnormal CpG island methylation of RUNX3 gene was found in 34 cases of HCC(45.3%), severn cases (9.3%) of adjacent normal tissues, and no abnormal CpG island methylation of RUNX3 in normal liver tissues, and the result has signifficanct difference (x2=29.18, p<0.001). Compared with the non-methylated group, the relative expression of RUNX3 gene in methylated group significantly reduced. Our research showed that 27 out of 45 (60%) HCC of lower expression, RUNX3 gene was hypermethylated. The level of RUNX3 mRNA in HCC without methylation was over 4–fold higher than that in HCC with methylation. RUNX3 gene CpG island methylation was significant correlation to cirrhosis(x2=5.07, p<0.024).Conclusion (1) The expression of RUNX3 is decreased in HCC. The RUNX3 gene may play an important role in the progression of HCC. (2) Our results indicate that promoter hypermethylation is an important mechanism for low expression of RUNX3 in HCC and was significant correlation to cirrhosis.