Figure 2 Conserved increase in expression of the miR-17�C92 polycistron and miR-21 in both human and woodchuck HCCs. Northern blot analysis of the expression of miRNAs in human and woodchuck peri-tumor liver and primary HCCs. A: Human samples. B: Woodchuck selleck … Interestingly, expression of the major liver miRNA, miR-122, which is down-regulated in a rat HCC model,28 was maintained in all of the human HCCs samples. Although miR-122 expression was substantially reduced in some of the HBV-positive HCCs (Figure 2A), there was no statistically significant difference in matched tumor and peri-tumor samples (P = 0.86) (supplemental Figure 1, see http://ajp.amjpathol.org).

HBV-Associated Cirrhotic Livers Overexpresses Members of the miR-17�C92 Polycistron and miR-21 Compared to Normal Liver Cirrhosis is considered to be a significant aetological factor that precedes HCC in humans, whether it is associated with chronic HBV infection or not. qRT-PCR analysis of human liver and human HCC (Ambion) demonstrates that miR-17�C92 polycistron and miR-21 were overexpressed in HCC, a result that is consistent with our Northern blot and cloning data (Figure 3). To determine whether these miRNAs are overexpressed in precancerous liver, we analyzed their levels in three cirrhotic human livers from individuals that were undergoing human liver transplant surgery, and in which primary HCC had not been identified. The severe nodular cirrhosis of these patients was reflected in their liver function tests (supplemental Table 2, see http://ajp.amjpathol.org).

Each of the three cirrhotic livers showed variable levels of elevation of the miRNAs as compared with normal liver (Figure 3). Out of the four members of the miR-17�C92 polycistron (miR-17, miR-19a, miR-20, and miR-92), each cirrhotic liver has statistically significant over-expression of at least two of the miRNAs; however, although miR-19a showed a trend of increased expression in cirrhotic liver these changes were not statistically significant (Figure 3). As expected, the expression of these oncogenic miRNAs was variable and not generally as high as that determined for HCC. We attribute this miRNA expression pattern to the multinodular precancerous nature of the liver cirrhosis samples that were used to make the RNAs. For example, in liver cirrhosis 1, miR-17 and miR-20 exhibited respective 3.

5- and 12-fold increases over liver (Figure 3). Furthermore, miR-21 expression was significantly enhanced in all of the cirrhotic livers (~ 15-fold Dacomitinib increase) (Figure 3). However, the fold increase (relative to normal liver) of these miRNAs in cirrhotic livers was less than that observed for the same miRNAs in HCC. These data support the hypothesis that activation of the miR-17�C92 miRNAs and miR-21 precedes HCC, and suggests that these genetic elements may represent important etiological agents in tumor initiation or progression and not just products of the transformed state.