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BACKGROUND AIM Anemia is commonly associated with acute and chronic inflammation, but the mechanisms of their interaction are not clear. We investigated whether microRNA 122 (MIR122), which is generated in the liver and is secreted into the blood, is involved in the development of anemia associated with inflammation. METHODS We characterized the primary transcript of the human liverspecific MIR122 using northern blot, quantitative real-time PCR, and 3' and 5' RACE analyses. We studied regulation of MIR122 in human hepatocellular carcinoma (HCC) cell lines (Huh7 and HepG2) as well as in C57BL/6 and mice with disruption of the tumor necrosis factor gene (Tnf). Liver tissues were collected and analyzed by bioluminescence imaging or immunofluorescence. Inflammation in mice was induced by lipopolysaccharide (LPS) or by cerulein injections. Mice were given 4 successive injections of LPS, leading to inflammation-induced anemia. Steatohepatitis was induced with a choline-deficient high-fat diet. Hemolytic anemia was stimulated by phenylhydrazine injection. MIR122 was inhibited in mice by tail-vein injection of antogomiR-122 (an oligonucleotide antagonist of MIR122). MicroRNA and mRNA levels were determined by quantitative real time PCR. RESULTS The primary transcript of MIR122 spanned 5 kb, comprising 3 exons; the third encodes MIR122. Within the MIR122 promoter region we identified a nuclear factor-B (NF-B) binding site and demonstrated that RELA, as well as activators of NF-B (TNF and LPS), increased promoter activity of MIR122. Administration of LPS to mice induced secretion of MIR122 into blood, which required TNF. Secreted MIR122 reached the kidney and reduced expression of erythropoietin (Epo), which we identified as a MIR122 target gene. Injection of mice with antagomiR-122 increased blood levels of EPO, reticulocytes, and hemoglobin. We found an inverse relationship between blood levels of MIR122 and EPO in mice with acute pancreatitis or steatohepatitis, and also in patients with acute inflammation. CONCLUSION In mice, we found that LPS-induced inflammation increases blood levels of MIR122, which reduces expression of Epo in the kidney; this is a mechanism of inflammation-induced anemia. Strategies to block MIR122 in patients with inflammation could reduce the development or progression of anemia. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT AbstractBackground & Aim: Anemia is commonly associated with acute and chronic inflammation, but the mechanisms of their interaction are not clear. We investigated whether microRNA 122 (MIR122),...

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The oncofetal long noncoding RNA (lncRNA) H19 is postnatally repressed in most tissues, and re-expressed in many cancers, including hepatocellular carcinoma (HCC). The role of H19 in carcinogenesis is a subject of controversy. We aimed to examine the role of H19 in chronic inflammation-mediated hepatocarcinogenesis using the Mdr2/Abcb4 knockout (Mdr2-KO) mouse, a well-established HCC model. For this goal, we have generated Mdr2-KO/H19-KO double knockout (dKO) mice and followed spontaneous tumor development in the dKO and control Mdr2-KO mice. Cellular localization of H19 and effects of H19 loss in the liver were determined in young and old Mdr2-KO mice. Tumor incidence and tumor load were both significantly decreased in the liver of dKO versus Mdr2-KO females. The expression levels of H19 and Igf2 were variable in nontumor liver tissues of Mdr2-KO females and were significantly downregulated in most matched tumors. In nontumor liver tissue of aged Mdr2-KO females, H19 was expressed mainly in hepatocytes, and hepatocyte proliferation was increased compared to dKO females. At an early age, dKO females displayed lower levels of liver injury and B-cell infiltration, with higher percentage of binuclear hepatocytes. In human samples, H19 expression was higher in females, positively correlated with cirrhosis (in nontumor liver samples) and negatively correlated with CTNNB1 (beta-catenin) mutations and patients' survival (in tumors). Our data demonstrate that the lncRNA H19 is pro-oncogenic during the development of chronic inflammation-mediated HCC in the Mdr2-KO mouse model, mainly by increasing liver injury and decreasing hepatocyte polyploidy in young mice.

Our findings indicate that genomic instability derived during the IL6-mediated liver regenerative response within a milieu of chronic inflammation links partial hepatectomy to accelerated hepatocarcinogenesis; this suggests a new therapeutic approach through the usage of an anti-IL6 treatment to extend the tumor-free survival of patients undergoing surgical resection. (Hepatology 2017;65:1600-1611).