Charles E. Rogler

A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

Elevated Expression of the miR-17–92 Polycistron and miR-21 in Hepadnavirus-Associated Hepatocellular Carcinoma Contributes to the Malignant Phenotype

Alterations in microRNA (miRNA) expression in both human and animal models have been linked to many forms of cancer. Such miRNAs, which act directly as repressors of gene expression, have been found to frequently reside in fragile sites and genomic regions associated with cancer. This study describes a miRNA signature for human primary hepatitis B virus-positive human hepatocellular carcinoma. Moreover, two known oncomiRs--miRNAs with known roles in cancer--the miR-17-92 polycistron and miR-21, exhibited increased expression in 100% of primary human and woodchuck hepatocellular carcinomas surveyed. To determine the importance of these miRNAs in tumorigenesis, an in vitro antisense oligonucleotide knockdown model was evaluated for its ability to reverse the malignant phenotype. Both in human and woodchuck HCC cell lines, separate treatments with antisense oligonucleotides specific for either the miR-17-92 polycistron (all six members) or miR-21 caused a 50% reduction in both hepatocyte proliferation and anchorage-independent growth. The combination of assays presented here supports a role for these miRNAs in the maintenance of the malignant transformation of hepatocytes.

MicroRNA‐23b cluster microRNAs regulate transforming growth factor‐beta/bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads

Transforming growth factor‐beta / bone morphogenetic protein (TGFβ/BMP) signaling has a gradient of effects on cell fate choice in the fetal mouse liver. The molecular mechanism to understand why adjacent cells develop into bile ducts or grow actively as hepatocytes in the ubiquitous presence of both TGFβ ligands and receptors has been unknown. We hypothesized that microRNAs (miRNAs) might play a role in cell fate decisions in the liver. miRNA profiling during late fetal development in the mouse identified miR‐23b cluster miRNAs comprising miR‐23b, miR‐27b, and miR‐24‐1 and miR‐10a, miR‐26a, and miR‐30a as up‐regulated. In situ hybridization of fetal liver at embryonic day 17.5 of gestation revealed miR‐23b cluster expression only in fetal hepatocytes. A complementary (c)DNA microarray approach was used to identify genes with a reciprocal expression pattern to that of miR‐23b cluster miRNAs. This approach identified Smads (mothers against decapentaplegic homolog), the key TGFβ signaling molecules, as putative miR‐23b cluster targets. Bioinformatic analysis identified multiple candidate target sites in the 3′ UTRs (untranslated regions) of Smads 3, 4, and 5. Dual luciferase reporter assays confirmed down‐regulation of constructs containing Smad 3, 4, or 5, 3′ UTRs by a mixture of miR‐23b cluster mimics. Knockdown of miR‐23b miRNAs during hepatocytic differentiation of a fetal liver stem cell line, HBC‐3, promoted expression of bile duct genes, in addition to Smads, in these cells. In contrast, ectopic expression of miR‐23b mimics during bile duct differentiation of HBC‐3 cells blocked the process. Conclusion: Our data provide a model in which miR‐23b miRNAs repress bile duct gene expression in fetal hepatocytes while promoting their growth by down‐regulating Smads and consequently TGFβ signaling. Concomitantly, low levels of the miR‐23b miRNAs are needed in cholangiocytes to allow TGFβ signaling and bile duct formation. (HEPATOLOGY 2009.)

Multi-miRNA hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown.

A number of natural microRNA (miRNA) hairpins have been found in clusters of multiple identical or different copies, suggesting that effects of miRNAs can be enhanced and multiple genes can be regulated together by encoding multiple miRNA hairpins in a single transcript. Here, we report a simple and effective artificial multi-hairpin method that stimulates production of mature 22-nucleotide small RNAs from modified miRNA hairpins, improves gene knockdown over single-hairpin constructs, and provides linked multi-gene knockdowns.

Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver

Hepatocyte function and regeneration are severely compromised in severe liver disease, and a common sequela is cirrhosis. Structural changes caused by cirrhosis create a cellular environment conducive to the formation of ductular reactions (DRs). Ductular reactions are primarily composed of oval cells also known as “intermediate hepatobiliary cells”. We have conducted single, double, and triple staining to study lineages of oval cells present in DRs. Staining with NCAM, CK19, and HepPar1 has revealed a distinctly bipolar structure to DRs that are embedded in cirrhotic tissue. Spatial analysis of cells that are singly HepPar1‐positive, or CK19‐positive, has revealed hepatocytic and biliary poles, respectively, in the DRs. Also, the location of singly NCAM‐positive cells in DRs suggests that they may be bipotent liver stem/progenitor cells. The locations of other intermediate hepatobiliary cells, which have combinations of markers, suggest that CK19+/NCAM+ cells are transitional cells in the biliary lineage and that rare cells that are negative for all three markers are transitional cells in the hepatocytic lineage. A working cell lineage model for DRs is presented. (HEPATOLOGY 2007;45:716–724.)

Overexpression of miR-21 Promotes an In vitro Metastatic Phenotype by Targeting the Tumor Suppressor RHOB

Metastasis is a multistep process that involves the deregulation of oncogenes and tumor suppressors beyond changes required for primary tumor formation. RHOB is known to have tumor suppressor activity, and its knockdown is associated with more aggressive tumors as well as changes in cell shape, migration, and adhesion. This study shows that oncogenic microRNA, miR-21, represses RHOB expression by directly targeting the 3′ untranslated region. Loss of miR-21 is associated with an elevation of RHOB in hepatocellular carcinoma cell lines Huh-7 and HepG2 and in the metastatic breast cancer cell line MDA-MB-231. Using in vitro models of distinct stages of metastasis, we showed that loss of miR-21 also causes a reduction in migration, invasion, and cell elongation. The reduction in migration and cell elongation can be mimicked by overexpression of RHOB. Furthermore, changes in miR-21 expression lead to alterations in matrix metalloproteinase-9 activity. Therefore, we conclude that miR-21 promotes multiple components of the metastatic phenotype in vitro by regulating several important tumor suppressors, including RHOB. Mol Cancer Res; 8(5); 691–700. ©2010 AACR.

Gene expression pattern in hepatic stem/progenitor cells during rat fetal development using complementary DNA microarrays

To identify new and differentially expressed genes in rat fetal liver epithelial stem/progenitor cells during their proliferation, lineage commitment, and differentiation, we used a high throughput method—mouse complementary DNA (cDNA) microarrays—for analysis of gene expression. The gene expression pattern of rat hepatic cells was studied during their differentiation in vivo: from embryonic day (ED) 13 until adulthood. The differentially regulated genes were grouped into two clusters: a cluster of up‐regulated genes comprised of 281 clones and a cluster of down‐regulated genes comprised of 230 members. The expression of the latter increased abruptly between ED 16 and ED 17. Many of the overexpressed genes from the first cluster fall into distinct, differentially expressed functional groups: genes related to development, morphogenesis, and differentiation; calcium‐ and phospholipid‐binding proteins and signal transducers; and cell adhesion, migration, and matrix proteins. Several other functional groups of genes that are initially down‐regulated, then increase during development, also emerged: genes related to inflammation, blood coagulation, detoxification, serum proteins, amino acids, lipids, and carbohydrate metabolism. Twenty‐eight genes overexpressed in fetal liver that were not detected in adult liver are suggested as potential markers for identification of liver progenitor cells. In conclusion, our data show that the gene expression program of fetal hepatoblasts differs profoundly from that of adult hepatocytes and that it is regulated in a specific manner with a major switch at ED 16 to 17, marking a dramatic change in the gene expression program during the transition of fetal liver progenitor cells from an undifferentiated to a differentiated state. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;39:617–627.)

Current pathogenetic and molecular concepts in viral liver carcinogenesis

SummaryHepatocellular carcinoma (HCC) is one of the most frequent malignancies in humans and in most cases a consequence of chronic infection of the liver by hepatotropic viruses (Hepatitis B Virus (HBV) and possibly Hepatitis C Virus (HCV)). Formation of HCC results from a stepwise process involving different preneoplastic lesions that reflect multiple genetic events, like protooncogene activation, tumor suppressor gene inactivation, and growth factor overor reexpression. Recent investigations have gained new insights into how these factors are activated and may interact. In addition, improved knowledge of the molecular biology of HBV has led to better understanding of its pleiotropic effects on induction and progression in hepatocarcinogenesis

Reactivation of the maternally imprinted IGF2 allele in TGFα induced hepatocellular carcinomas in mice

The Insulin like growth factor 2 (IGF2) gene is expressed in several types of tumors in humans and mice and has been implicated as an important growth factor in tumor progression. IGF2 expression in the TGFα transgenic mice was analysed in liver and tumors from animals which also contained one or two functional IGF2 alleles. In a two by two mating experiment using transgenic mice containing either a TGFα transgene or a IGF2 gene knockout, we have investigated whether IGF2 imprinting is reversed during hepatocarcinogenesis and the consequences of IGF2 expression for tumor growth. We observed that: (1) 100% of the hepatocellular carcinomas expressed IGF2 (2) the normally imprinted maternal allele is active in the tumors in which the paternal allele is knocked out and (3) all three of the murine IGF2 promoters upstream of the reactivated maternal alleles are transcriptionally active in tumors. We also observed that the total tumor burden of animals with two wild type IGF-2 alleles (paternal and maternal) was the same as the tumor burden in animals which contained only a single reactivated maternal allele. The 100% incidence of reactivation of the imprinted maternal allele suggests that IGF2 expression is selected during murine hepatocarcinogenesis and can substitute for the paternal allele when it is inactivated.

Identification of a chromosomal aberration associated with a hepatitis B DNA integration site in human cells

Hepatitis B virus (HBV) DNA integrates into human cellular DNA during long-term persistent infections, implicating integration as one of the steps leading to hepatocarcinogenesis. The present study demonstrates that the integration of HBV DNA can result in or be accompanied by interchromosomal exchange of genomic material containing the integrated DNA. Unique cellular DNA to the left of an HBV DNA integration site cloned from a primary tumor mapped to chromosome 18q (18q11.1-q11.2); right hand flanking DNA mapped to chromosome #17 at a near-terminal region. The presence of chromosomal rearrangements in association with HBV integration may play a role in multistage hepatocarcinogenesis.