Ting-Fen Tsai

MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis

MicroRNA-122 (miR-122), which accounts for 70% of the livers total miRNAs, plays a pivotal role in the liver. However, its intrinsic physiological roles remain largely undetermined. We demonstrated that mice lacking the gene encoding miR-122a (Mir122a) are viable but develop temporally controlled steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). These mice exhibited a striking disparity in HCC incidence based on sex, with a male-to-female ratio of 3.9:1, which recapitulates the disease incidence in humans. Impaired expression of microsomal triglyceride transfer protein (MTTP) contributed to steatosis, which was reversed by in vivo restoration of Mttp expression. We found that hepatic fibrosis onset can be partially attributed to the action of a miR-122a target, the Klf6 transcript. In addition, Mir122a(-/-) livers exhibited disruptions in a range of pathways, many of which closely resemble the disruptions found in human HCC. Importantly, the reexpression of miR-122a reduced disease manifestation and tumor incidence in Mir122a(-/-) mice. This study demonstrates that mice with a targeted deletion of the Mir122a gene possess several key phenotypes of human liver diseases, which provides a rationale for the development of a unique therapy for the treatment of chronic liver disease and HCC.

Genetics of Angelman syndrome.

We apologize to the many authors whose primary data could not be cited because of limitations of space. We thank Grace Watson for great assistance in preparation of the manuscript. This work is supported by NIH grant HD 37283.

Cisd2 deficiency drives premature aging and causes mitochondria-mediated defects in mice

CISD2, the causative gene for Wolfram syndrome 2 (WFS2), is a previously uncharacterized novel gene. Significantly, the CISD2 gene is located on human chromosome 4q, where a genetic component for longevity maps. Here we show for the first time that CISD2 is involved in mammalian life-span control. Cisd2 deficiency in mice causes mitochondrial breakdown and dysfunction accompanied by autophagic cell death, and these events precede the two earliest manifestations of nerve and muscle degeneration; together, they lead to a panel of phenotypic features suggestive of premature aging. Our study also reveals that Cisd2 is primarily localized in the mitochondria and that mitochondrial degeneration appears to have a direct phenotypic consequence that triggers the accelerated aging process in Cisd2 knockout mice; furthermore, mitochondrial degeneration exacerbates with age, and the autophagy increases in parallel to the development of the premature aging phenotype. Additionally, our Cisd2 knockout mouse work provides strong evidence supporting an earlier clinical hypothesis that WFS is in part a mitochondria-mediated disorder; specifically, we propose that mutation of CISD2 causes the mitochondria-mediated disorder WFS2 in humans. Thus, this mutant mouse provides an animal model for mechanistic investigation of Cisd2 protein function and help with a pathophysiological understanding of WFS2.

IMPRINTING IN ANGELMAN AND PRADER-WILLI SYNDROMES

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by deficiencies of gene expression from paternal or maternal chromosome 15q11-q13, respectively. Many advances have occurred during the past year. The gene for necdin was mapped in the PWS candidate region and found to be paternally expressed in mouse and human. The bisulfite method for analysis of methylation was established for genomic sequencing and diagnostics, and the methylation of Snrpn was studied in detail in the mouse. A region near the Snrpn promoter was shown to function as a silencer in Drosophila. Point mutations were found in the gene for E6-AP ubiquitin-protein ligase (UBE3A) identifying it as the AS gene, and tissue-specific imprinting (maternal expression) was shown in the human brain and in hippocampal neurons and Purkinje cells in the mouse.

The SNRPN promoter is not required for genomic imprinting of the Prader-Willi/Angelman domain in mice

In mice and humans, the locus encoding the gene for small nuclear ribonucleoprotein N (SNRPN/Snrpn), as well as other loci in the region are subject to genomic imprinting. The SNRPN promoter is embedded in a maternally methylated CpG island, is expressed only from the paternal chromosome and lies within an imprinting center that is required for switching to and/or maintenance of the paternal epigenotype. We show here that a 0.9-kb deletion of exon 1 of mouse Snrpn did not disrupt imprinting or elicit any obvious phenotype, although it did allow the detection of previously unknown upstream exons. In contrast, a larger, overlapping 4.8-kb deletion caused a partial or mosaic imprinting defect and perinatal lethality when paternally inherited.

Necdin-deficient mice do not show lethality or the obesity and infertility of Prader-Willi syndrome.

Necdin-deficient mice do not show lethality or the obesity and infertility of Prader-Willi syndrome

Autophagy suppresses tumorigenesis of hepatitis B virus‐associated hepatocellular carcinoma through degradation of microRNA‐224

In hepatocellular carcinoma (HCC), dysregulated expression of microRNA‐224 (miR‐224) and impaired autophagy have been reported separately. However, the relationship between them has not been explored. In this study we determined that autophagy is down‐regulated and inversely correlated with miR‐224 expression in hepatitis B virus (HBV)‐associated HCC patient specimens. These results were confirmed in liver tumors of HBV X gene transgenic mice. Furthermore, miR‐224 was preferentially recruited and degraded during autophagic progression demonstrated by real‐time polymerase chain reaction and miRNA in situ hybridization electron microscopy after extraction of autophagosomes. Our in vitro study demonstrated that miR‐224 played an oncogenic role in hepatoma cell migration and tumor formation through silencing its target gene Smad4. In HCC patients, the expression of low‐Atg5, high‐miR‐224, and low‐Smad4 showed significant correlation with HBV infection and a poor overall survival rate. Autophagy‐mediated miR‐224 degradation and liver tumor suppression were further confirmed by the autophagy inducer amiodarone and miR‐224 antagonist using an orthotopic SD rat model. Conclusion: A noncanonical pathway links autophagy, miR‐224, Smad4, and HBV‐associated HCC. These findings open a new avenue for the treatment of HCC. (Hepatology 2014;59:505–517)

Enhanced expression of vascular endothelial growth factor‐A in ground glass hepatocytes and its implication in hepatitis B virus hepatocarcinogenesis

Ground glass hepatocytes (GGH) in chronic hepatitis B virus (HBV) infection harbor HBV pre‐S deletion mutants in endoplasmic reticulum (ER) and exhibit complex biologic features such as ER stress, DNA damage, and growth advantage. The presence of pre‐S mutants in serum has been shown to predict the development of hepatocellular carcinoma (HCC) in HBV carriers. GGHs hence represent a potentially preneoplastic lesion. Whether a specific growth factor is overexpressed and activated in GGHs remains to be clarified. In this study, growth factor(s) up‐regulated by pre‐S mutants was identified using a growth factor array in HuH‐7 cells. Immunohistochemistry, reverse‐transcriptase polymerase chain reaction, and Western blot analysis were performed to study the participation of these genes and their signal pathways in HuH‐7 cells and liver tissues. We demonstrate that vascular endothelial growth factor‐A (VEGF‐A) was up‐regulated by pre‐S mutants in HuH‐7 cells and further confirmed in GGHs by immunostaining. The VEGF‐A up‐regulation by pre‐S mutants could be suppressed by vomitoxin, an ER stress inhibitor. Furthermore, pre‐S mutants‐expressed HuH‐7 cells exhibited activation of Akt/mTOR (mammalian target of rapamycin) signaling and increased growth advantage, which could be inhibited by VEGF‐A neutralization. Consistent with this notion, enhanced expression of VEGF‐A and activation of Akt/mTOR signaling, comparable to the levels of paired HCC tissues, were also detected in HBV‐related nontumorous livers. Conclusion: The enhanced expression of VEGF‐A in GGHs provides potential mechanism to explain the progression from preneoplastic GGHs to HCC in chronic HBV infection. (HEPATOLOGY 2009;49:1962–1971.)

Glycine N-methyltransferase-/- mice develop chronic hepatitis and glycogen storage disease in the liver.

Glycine N‐methyltransferase (GNMT) affects genetic stability by regulating DNA methylation and interacting with environmental carcinogens. To establish a Gnmt knockout mouse model, 2 lambda phage clones containing a mouse Gnmt genome were isolated. At 11 weeks of age, the Gnmt−/− mice had hepatomegaly, hypermethioninemia, and significantly higher levels of both serum alanine aminotransferase and hepatic S‐adenosylmethionine. Such phenotypes mimic patients with congenital GNMT deficiencies. A real‐time polymerase chain reaction analysis of 10 genes in the one‐carbon metabolism pathway revealed that 5,10‐methylenetetrahydrofolate reductase, S‐adenosylhomocysteine hydrolase (Ahcy), and formiminotransferase cyclodeaminase (Ftcd) were significantly down‐regulated in Gnmt−/− mice. This report demonstrates that GNMT regulates the expression of both Ftcd and Ahcy genes. Results from pathological examinations indicated that 57.1% (8 of 14) of the Gnmt−/− mice had glycogen storage disease (GSD) in their livers. Focal necrosis was observed in male Gnmt−/− livers, whereas degenerative changes were found in the intermediate zones of female Gnmt−/− livers. In addition, hypoglycemia, increased serum cholesterol, and significantly lower numbers of white blood cells, neutrophils, and monocytes were observed in the Gnmt−/− mice. A real‐time polymerase chain reaction analysis of genes involved in the gluconeogenesis pathways revealed that the following genes were significantly down‐regulated in Gnmt−/− mice: fructose 1,6‐bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose‐6‐phosphate transporter. Conclusion: Because Gnmt−/− mice phenotypes mimic those of patients with GNMT deficiencies and share several characteristics with GSD Ib patients, we suggest that they are useful for studies of the pathogenesis of congenital GNMT deficiencies and the role of GNMT in GSD and liver tumorigenesis. (HEPATOLOGY 2007.)

HURP Permits MTOC Sorting for Robust Meiotic Spindle Bipolarity, Similar to Extra Centrosome Clustering in Cancer Cells

Similar to clustering of extra centrosomes in cancer cells, HURP promotes microtubule stability and sorts MTOCs into distinct poles during meiosis.