MicroRNA-122 (miR-122), which accounts for 70% of the liver's 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.

Our study suggests that miR-122, a tumor suppressor microRNA affecting hepatocellular carcinoma intrahepatic metastasis by angiogenesis suppression, exerts some of its action via regulation of ADAM17. Restoration of miR-122 has a far-reaching effect on the cell. Using the concomitant down-regulation of its targets, including ADAM17, a rational therapeutic strategy based on miR-122 may prove to be beneficial for patients with hepatocellular carcinoma.

G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Emerging evidence suggests that G9a is required to maintain the malignant phenotype, but the role of G9a function in mediating tumor metastasis has not been explored. Here, we show that G9a is expressed in aggressive lung cancer cells, and its elevated expression correlates with poor prognosis. RNAi-mediated knockdown of G9a in highly invasive lung cancer cells inhibited cell migration and invasion in vitro and metastasis in vivo. Conversely, ectopic G9a expression in weakly invasive lung cancer cells increased motility and metastasis. Mechanistic investigations suggested that repression of the cell adhesion molecule Ep-CAM mediated the effects of G9a. First, RNAi-mediated knockdown of Ep-CAM partially relieved metastasis suppression imposed by G9a suppression. Second, an inverse correlation between G9a and Ep-CAM expression existed in primary lung cancer. Third, Ep-CAM repression was associated with promoter methylation and an enrichment for dimethylated histone H3K9. G9a knockdown reduced the levels of H3K9 dimethylation and decreased the recruitment of the transcriptional cofactors HP1, DNMT1, and HDAC1 to the Ep-CAM promoter. Our findings establish a functional contribution of G9a overexpression with concomitant dysregulation of epigenetic pathways in lung cancer progression. Cancer Res; 70(20); 7830-40. ©2010 AACR.

The up-regulation of fucosyltransferase 8 (FUT8), the only enzyme catalyzing α1,6-fucosylation in mammals, has been observed in several malignant cancers including liver, ovarian, thyroid, and colorectal cancers. However, the pathological role and the regulatory mechanism of FUT8 in cancers remain largely unknown. In the current study, we report that the expression of FUT8 is up-regulated in nonsmall cell lung cancer (NSCLC) and correlates with tumor metastasis, disease recurrence, and poor survival in patients with NSCLC. Knocking down FUT8 in aggressive lung cancer cell lines significantly inhibits their malignant behaviors including in vitro invasion and cell proliferation, as well as in vivo metastasis and tumor growth. The results of glycoproteomic and microarray analyses show that FUT8 globally modifies surface antigens, receptors, and adhesion molecules and is involved in the regulation of dozens of genes associated with malignancy, suggesting that FUT8 contributes to tumor progression through multiple mechanisms. Moreover, we show that FUT8 is up-regulated during epithelial-mesenchymal transition (EMT), a critical process for malignant transformation of tumor, via the transactivation of β-catenin/lymphoid enhancer-binding factor-1 (LEF-1). These results provide a model to illustrate the relation between FUT8 expression and lung cancer progression and point to a promising direction for the prognosis and therapy of lung cancer.TGF-beta | E-cadherin | fucose F ucosylation, the transfer of fucose from GDP-fucose to glycoconjugates such as glycoproteins and glycolipids, is catalyzed by a family of enzymes called fucosyltransferases (FUTs). So far, 13 FUTs are known to be encoded by the human genome, including FUT1 to 11, protein O-fucosyltransferase 1 (POFUT1), and POFUT2. Through these FUTs, fucoses could be attached to N-, O-, and lipid-linked glycans through an α1,2-(by FUT1 and 2), α1,3-(by FUT3 to 7 and FUT9 to 11), α1,4-(by FUT3 and 5), or α1,6-(by FUT8) linkage, or directly link to the serine/threonine residues of EGF-like or thrombospondin repeats (by POFUT1 and 2, respectively) (1, 2). In mammals, fucosylated glycans have pivotal roles in many aspects of biological processes such as lymphocyte homing, immune responses, fertilization, and development (3). Moreover, aberrant fucosylation, which results from the deficiency or overexpression of FUTs, is associated with a variety of human diseases, including cystic fibrosis, leukocyte adhesion deficiency type II, and cancers (3, 4).Unlike other FUTs, which are functionally redundant, FUT8 is the only enzyme responsible for the α1,6-linked (core) fucosylation by adding fucose to the innermost GlcNAc residue of an N-linked glycan. A growing body of evidence indicates that core fucosylation is important for regulating protein functions. For example, deletion of the core fucose from the Fc region of IgG1 greatly improves its binding affinity to Fcγ receptor IIIa, which in turn enhances antibody-dependent cell-mediated cytotoxicity for over 50 folds (5, 6). Co...

A comprehensive microarray analysis of hepatocellular carcinoma (HCC) revealed distinct synexpression patterns during intrahepatic metastasis. Recent evidence has demonstrated that synexpression group member genes are likely to be regulated by master control gene(s). Here we investigate the functions and gene regulation of the transcription factor SOX4 in intrahepatic metastatic HCC. SOX4 is important in tumor metastasis as RNAi knockdown reduces tumor cell migration, invasion, in vivo tumorigenesis and metastasis. A multifaceted approach integrating gene profiling, binding site computation and empirical verification by chromatin immunoprecipitation and gene ablation refined the consensus SOX4 binding motif and identified 32 binding loci in 31 genes with high confidence. RNAi knockdown of two SOX4 target genes, neuropilin 1 and semaphorin 3C, drastically reduced cell migration activity in HCC cell lines suggesting that SOX4 exerts some of its action via regulation of these two downstream targets. The discovery of 31 previously unidentified targets expands our knowledge of how SOX4 modulates HCC progression and implies a range of novel SOX4 functions. This integrated approach sets a paradigm whereby a subset of member genes from a synexpression group can be regulated by one master control gene and this is exemplified by SOX4 and advanced HCC.

Trans-splicing is a post-transcriptional event that joins exons from separate pre-mRNAs. Detection of trans-splicing is usually severely hampered by experimental artifacts and genetic rearrangements. Here, we develop a new computational pipeline, TSscan, which integrates different types of high-throughput long-/short-read transcriptome sequencing of different human embryonic stem cell (hESC) lines to effectively minimize false positives while detecting trans-splicing. Combining TSscan screening with multiple experimental validation steps revealed that most chimeric RNA products were platformdependent experimental artifacts of RNA sequencing. We successfully identified and confirmed four trans-spliced RNAs, including the first reported trans-spliced large intergenic noncoding RNA (''tsRMST ''). We showed that these trans-spliced RNAs were all highly expressed in human pluripotent stem cells and differentially expressed during hESC differentiation. Our results further indicated that tsRMST can contribute to pluripotency maintenance of hESCs by suppressing lineagespecific gene expression through the recruitment of NANOG and the PRC2 complex factor, SUZ12. Taken together, our findings provide important insights into the role of trans-splicing in pluripotency maintenance of hESCs and help to facilitate future studies into trans-splicing, opening up this important but understudied class of post-transcriptional events for comprehensive characterization.

No abstract

MicroRNAs (miRNAs) make up a novel class of gene regulators; they function as oncogenes or tumor suppressors by targeting tumor-suppressor genes or oncogenes. A recent study that analysed a large number of human cancer cell lines showed that miR-330 is a potential tumorsuppressor gene. However, the function and molecular mechanism of miR-330 in determining the aggressiveness of human prostate cancer has not been studied. Here, we show that miR-330 is significantly lower expressed in human prostate cancer cell lines than in nontumorigenic prostate epithelial cells. Bioinformatics analyses reveal a conserved target site for miR-330 in the 3 0 -untranslated region (UTR) of E2F1 at nucleotides 1018-1024. MiR-330 significantly suppressed the activity of a luciferase reporter containing the E2F1-3 0 -UTR in the cells. This activity could be abolished with the transfection of anti-miR-330 or mutated E2F1-3 0 -UTR. In addition, the expression level of miR-330 and E2F1 was inversely correlated in cell lines and prostate cancer specimens. After overexpressing of miR-330 in PC-3 cells, cell growth was suppressed by reducing E2F1-mediated Akt phosphorylation and thereby inducing apoptosis. Collectively, this is the first study to show that E2F1 is negatively regulated by miR-330 and also show that miR-330 induces apoptosis in prostate cancer cells through E2F1-mediated suppression of Akt phosphorylation.