MiR-122, an abundant liver-specific microRNA (miRNA), regulates cholesterol metabolism and promotes hepatitis C virus (HCV) replication. Reduced miR-122 expression in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Nevertheless, the consequences of sustained loss of function of miR-122 in vivo have not been determined. Here, we demonstrate that deletion of mouse Mir122 resulted in hepatosteatosis, hepatitis, and the development of tumors resembling HCC. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF. Moreover, delivery of miR-122 to a MYC-driven mouse model of HCC strongly inhibited tumorigenesis, further supporting the tumor suppressor activity of this miRNA. These findings reveal critical functions for miR-122 in the maintenance of liver homeostasis and have important therapeutic implications, including the potential utility of miR-122 delivery for selected patients with HCC and the need for careful monitoring of patients receiving miR-122 inhibition therapy for HCV.

Phospholipids are major constituents of biological membranes. The fatty acyl chain composition of phospholipids determines the biophysical properties of membranes and thereby affects their impact on biological processes. The composition of fatty acyl chains is also actively regulated through a deacylation and reacylation pathway called Lands’ cycle. Recent studies of mouse genetic models have demonstrated that lysophosphatidylcholine acyltransferases (LPCATs), which catalyze the incorporation of fatty acyl chains into the sn-2 site of phosphatidylcholine, play important roles in pathophysiology. Two LPCAT family members, LPCAT1 and LPCAT3, have been particularly well studied. LPCAT1 is crucial for proper lung function due to its role in pulmonary surfactant biosynthesis. LPCAT3 maintains systemic lipid homeostasis by regulating lipid absorption in intestine, lipoprotein secretion, and de novo lipogenesis in liver. Mounting evidence also suggests that changes in LPCAT activity may be potentially involved in pathological conditions, including nonalcoholic fatty liver disease, atherosclerosis, viral infections, and cancer. Pharmacological manipulation of LPCAT activity and membrane phospholipid composition may provide new therapeutic options for these conditions.

MicroRNAs are negative regulators of protein coding genes. The liver-specific microRNA-122 (miR-122) is frequently suppressed in primary hepatocellular carcinomas (HCCs). In situ hybridization demonstrated that miR-122 is abundantly expressed in hepatocytes but barely detectable in primary human HCCs. Ectopic expression of miR-122 in nonexpressing HepG2, Hep3B, and SK-Hep-1 cells reversed their tumorigenic properties such as growth, replication potential, clonogenic survival, anchorage-independent growth, migration, invasion, and tumor formation in nude mice. Further, miR-122-expressing HCC cells retained an epithelial phenotype that correlated with reduced Vimentin expression. ADAM10 (a distintegrin and metalloprotease family 10), serum response factor (SRF), and insulin-like growth factor 1 receptor (Igf1R) that promote tumorigenesis were validated as targets of miR-122 and were repressed by the microRNA. Conversely, depletion of the endogenous miR-122 in Huh-7 cells facilitated their tumorigenic properties with concomitant up-regulation of these targets. Expression of SRF or Igf1R partially reversed tumor suppressor function of miR-122. Further, miR-122 impeded angiogenic properties of endothelial cells in vitro. Notably, ADAM10, SRF, and Igf1R were up-regulated in primary human HCCs compared with the matching liver tissue. Co-labeling studies demonstrated exclusive localization of miR-122 in the benign livers, whereas SRF predominantly expressed in HCC. More importantly, growth and clonogenic survival of miR-122-expressing HCC cells were significantly reduced upon treatment with sorafenib, a multi-kinase inhibitor clinically effective against HCC. Collectively, these results suggest that the loss of multifunctional miR-122 contributes to the malignant phenotype of HCC cells, and miR-122 mimetic alone or in combination with anticancer drugs can be a promising therapeutic regimen against liver cancer.

MicroRNAs (miR) are a class of small (∼21 nucleotide) noncoding RNAs that, in general, negatively regulate gene expression. Some miRs harboring CGIs undergo methylation-mediated silencing, a characteristic of many tumor suppressor genes. To identify such miRs in liver cancer, the miRNA expression profile was analyzed in hepatocellular carcinoma (HCC) cell lines treated with 5-azacytidine (DNA hypomethylating agent) and/or trichostatin A (histone deacetylase inhibitor). The results showed that these epigenetic drugs differentially regulate expression of a few miRs, particularly miR-1-1, in HCC cells. The CGI spanning exon 1 and intron 1 of miR-1-1 was methylated in HCC cell lines and in primary human HCCs but not in matching liver tissues. The miR-1-1 gene was hypomethylated and activated in DNMT1−/− HCT 116 cells but not in DNMT3B null cells, indicating a key role for DNMT1 in its methylation. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas compared with matching normal liver tissues. Ectopic expression of miR-1 in HCC cells inhibited cell growth and reduced replication potential and clonogenic survival. The expression of FoxP1 and MET harboring three and two miR-1 cognate sites, respectively, in their respective 3′-untranslated regions, was markedly reduced by ectopic miR-1. Up-regulation of several miR-1 targets including FoxP1, MET, and HDAC4 in primary human HCCs and down-regulation of their expression in 5-AzaC–treated HCC cells suggest their role in hepatocarcinogenesis. The inhibition of cell cycle progression and induction of apoptosis after re-expression of miR-1 are some of the mechanisms by which DNA hypomethylating agents suppress hepatocarcinoma cell growth. [Cancer Res 2008;68(13):5049–58]

To identify microRNAs (miRNAs) that may have a causal role in hepatocarcinogenesis, we used an animal model in which C57BL/6 mice fed choline-deficient and amino acid defined (CDAA) diet develop preneoplastic lesions at 65 weeks and hepatocellular carcinomas after 84 weeks. miRNA expression profiling showed significant upregulation of miR-181b and miR-181d in the livers of mice as early as 32 weeks that persisted at preneoplastic stage. The expression of tissue inhibitor of metalloprotease 3 (TIMP3), a tumor suppressor and a validated miR-181 target, was markedly suppressed in the livers of mice fed CDAA diet. Upregulation of hepatic transforming growth factor (TGF)β and its downstream mediators Smad 2, 3 and 4 and increase in phospho-Smad2 in the liver nuclear extract correlated with elevated miR-181b/d in mice fed CDAA diet. The levels of the precursor and mature miR-181b were augmented on exposure of hepatic cells to TGFβ and were significantly reduced by small interference RNA-mediated depletion of Smad4, showing the involvement of TGFβ signaling pathway in miR-181b expression. Ectopic expression and depletion of miR-181b showed that miR-181b enhanced matrix metallopeptidases (MMP)2 and MMP9 activity and promoted growth, clonogenic survival, migration and invasion of hepatocellular carcinoma (HCC) cells that could be reversed by modulating TIMP3 level. Further, depletion of miR-181b inhibited tumor growth of HCC cells in nude mice. miR-181b also enhanced resistance of HCC cells to the anticancer drug doxorubicin. On the basis of these results, we conclude that upregulation of miR-181b at early stages of feeding CDAA diet promotes hepatocarcinogenesis.