Association of low p16INK4a and p15INK4b mRNAs expression with their CpG islands methylation with human hepatocellular carcinogenesis

Abstract

Aim: To study the significance of p16 and p15 transcription suppression with hypermethylation of their genes' 5'CpG islands during human hepatocellular carcinogenesis.

Methods: The mRNA expression levels of p16 and p15 genes were evaluated in cancerous, para-cancerous and non-cancerous tissues of 20 HCC, 3 normal liver tissues from 3 accidentally died healthy adults using semi-quantitatively Northern blot. The methylation status was also assessed with methylation specific PCR.

Results: p16 mRNA expression level was decreased in the cancerous tissues in 60% (12/20) of HCC patients, of which 2 cases had no p16 mRNA detected, 5 cases (25%) displayed variation in the order of cancerous<para-cancerous<non-cancerous liver tissues. p15 mRNA expression level was decreased in the cancerous tissues in 50% (10/20) HCC patients, of which one case had no p15 mRNA detected, 4 cases (20%) displayed variation in the order of cancerous<para-cancerous<non-cancerous liver tissues. In cancerous, para-cancerous and non-cancerous tissues, p16 promoter CpG islands hypermethylation occurred 65%, 60% and 35%, while p15 promoter CpG islands hypermethylation occurred 50%, 40% and 25%. Of 12 HCCs with lower p16 mRNA expression level, 11 cases showed p16 promoter CpG islands methylation (91.6%). Hundred percent (10/10) HCCs with lower p15 mRNA expression level showed p15 promoter CpG islands methylation. Significant correlation between 5'CpG islands methylation and p16/p15 mRNA expression suppression was found. The decreased expression of p16/p15 mRNA or methylation of p16/p15 promoters 5'CpG island was significantly correlate with poor differentiation of HCC (P=0.0083, 0.0102, 0.00271, 0.0218, respectively, P<0.05).

Conclusion: p16 and p15 genes transcriptional inactivation might play an important role in hepatocarcinogenesis. 5'CpG islands methylation might be the major mechanism of p16 and p15 genes inactivation in primary HCC in the studied population. 5'CpG islands methylation of p16 and p15 genes might be an early event in hepatocarcinogenesis.

Figure 1

Northern blot analysis of the p16 and p15 genes in human primary heparocarcinoma. Total RNA from tissues of human primary heparocarcinoma was hybridized with cDNA fragment probes of p16, p15 and γ-actin labeled with non-radiated digoxinin. N: Normal liver tissue control; C, P, N represent RNA from cancerous, para-cancerous, non-cancerous liver tissues respectively; 1, 2 represent the No. of HCC patient.

Figure 2

Methylation analysis of p16 gene in human primary heparocarcinoma. MSP product of p16 gene from HCC tissues was electrophoresed on a 25 g/L agarose gel. M: pBR322/ HeaIII DNA molecular marker; N: Normal liver tissue DNA; HS: HS-Sultan DNA (positive control); C, P, N represent RNA from cancerous, para-cancerous, non-cancerous liver tissue respectively; 1, 2 mark the HCC patient number; m: PCR prod-ucts from methylation specific primers, u: PCR products from unmethylation specific primers.

Figure 3

Methylation analysis of p15 gene in human primary heparocarcinoma. MSP product of p15 gene from HCC tissues was electrophoresed on a 2.5% agarose gel. M: pBR322/HeaIII DNA molecular marker (in graph m) or PBR322/Msp I DNA marker (in graph u); N: Normal liver tissue DNA; HS: HS-Sul-tan DNA (positive control); C, P, N represent RNA from cancerous, paracancerous, non-cancerous liver tissue respectively; 1, 2 mark the HCC patient number; m: PCR products from methylation specific primers, u: PCR products from unmethylation specific primers.