Kui Hong

Ubiquitin-like Protein FAT10 Promotes the Invasion and Metastasis of Hepatocellular Carcinoma by Modifying β-Catenin Degradation

The ubiquitin-like protein FAT10 and the homeobox protein HOXB9 each promote metastatic progression in hepatocellular carcinoma (HCC). In this study, we investigated the clinicopathologic significance of FAT10 and HOXB9 in HCC and investigated a mechanistic role for FAT10 in HOXB9-mediated invasiveness and metastasis. Relative to adjacent normal tissues, FAT10 and HOXB9 were markedly overexpressed in HCC, where a positive correlation in their expression and associated malignant characteristics were found. RNAi-mediated silencing of FAT10 decreased HOXB9 expression and inhibited HCC invasion and metastasis in vitro and in vivo. The effects of FAT10 silencing were reversed by HOXB9 overexpression, whereas RNAi-mediated silencing of HOXB9 decreased HCC invasion and metastasis driven by FAT10 overexpression. Mechanistically, FAT10 regulated HOXB9 expression by modulating the β-catenin/TCF4 pathway, directly binding to β-catenin and preventing its ubiquitination and degradation. Together, our results identified a novel HCC regulatory circuit involving FAT10, β-catenin/TCF4, and HOXB9, the dysfunction of which drives invasive and metastatic character in HCC. Cancer Res; 74(18); 5287-300. ©2014 AACR.

FAT10 protects cardiac myocytes against apoptosis

FAT10 is a new member of the ubiquitin-like protein family with yet-to-be defined biological functions in the heart. Our objective was to determine the role of FAT10 in the heart. FAT10 is expressed in the normal human and murine hearts, as detected by qPCR and Western blotting. Expression of FAT10 is increased in the heart at the border zone of myocardial infarction and in cultured neonatal rat cardiac myocytes (NRCM) subjected to hypoxia/reoxygenation (H/R) stress. Lentiviral-mediated overexpression of FAT10 in NRCM reduced p53 (TP53) and its target miR-34a levels, while BCL2 level, a target of miR-34a, was increased and BAX level, a pro-apoptotic protein, was reduced. These changes were associated with reduced apoptosis, detected by FACS analysis of annexin-V expression and TUNEL assay, in response to H/R injury. Knock down of FAT10 by shRNA targeting had the opposite effects. Likewise, lentiviral mediated expression of miR-34a was associated with reduced BCL2 and increased BAX levels in NRCM and also reversed changes in BCL-2 and BAX levels observed upon over-expression of FAT10. Treatment of NRCM with proteasome inhibitor MG132 increased p53 and miR-34a levels and reduced BLC2/BAX ratio. These changes were not reversed upon over-expression of FAT10. Thus, FAT10 is upregulated in the heart and NRCM in response to H/R stress, which protects cardiac myocytes against apoptosis. The anti-apoptotic effects of FAT10 are associated with suppression of p53, probably through fatylation and proteasomal degradation, reduced miR-34a expression, and a shift in the BCL2/BAX proteins against apoptosis. Thus, FAT10 is a cardioprotective protein.

Arrhythmogenic cardiomyopathy in a patient with a rare loss-of-function KCNQ1 mutation.

Background Ventricular tachycardia (VT) is a common manifestation of advanced cardiomyopathies. In a subset of patients with dilated cardiomyopathy, VT is the initial and the cardinal manifestation of the disease. The molecular genetic basis of this subset of dilated cardiomyopathy is largely unknown. Methods and Results We identified 10 patients with dilated cardiomyopathy who presented with VT and sequenced 14 common causal genes for cardiomyopathies and arrhythmias. Functional studies included cellular patch clamp, confocal microscopy, and immunoblotting. We identified nonsynonymous variants in 4 patients, including a rare missense p.R397Q mutation in the KCNQ1 gene in a 60‐year‐old man who presented with incessant VT and had mild cardiac dysfunction. The p.R397Q mutation was absent in an ethnically matched control group, affected a conserved amino acid, and was predicted by multiple algorithms to be pathogenic. Co‐expression of the mutant KCNQ1 with its partner unit KCNE1 was associated with reduced tail current density of slowly activating delayed rectifier K+ current (IKs). The mutation reduced membrane localization of the protein. Conclusions Dilated cardiomyopathy with an initial presentation of VT may be a forme fruste of arrhythmogenic cardiomyopathy caused by mutations in genes encoding the ion channels. The findings implicate KCNQ1 as a possible causal gene for arrhythmogenic cardiomyopathy.

Identification of a Novel Binding Protein of FAT10: Eukaryotic Translation Elongation Factor 1A1

BackgroundFAT10 is known to execute its functions mainly through conjugation to different substrates, and these known functions include cytokine responses, apoptosis, mitosis, and tumorigenesis. Nonetheless, the known binding proteins of FAT10 cannot explain all its known functions. As such, the aim of this study was to identify unidentified conjugation proteins of FAT10.MethodsThe yeast two-hybrid system was employed in this study. FAT10 was used as the bait protein for screening of a cDNA library from a human hepatocellular carcinoma cell line, Hep3B. Protein interactions were confirmed based on localization studies and co-immunoprecipitation assays. The expression of mRNA and protein was determined using real-time polymerase chain reaction and western blot analyses, respectively.ResultsIn this study, we identified eukaryotic elongation factor 1A1 (eEF1A1) as a FAT10-specific binding protein. The binding between FAT10 and eEF1A1 was confirmed both in vivo and in vitro. We also found that, when the expression of FAT10 was reduced by siRNA knockdown, this resulted in downregulation of eEF1A1 expression at both the mRNA and protein levels in human hepatocellular carcinoma cells.ConclusionsWe propose a model in which eEF1A1 serves as a substrate of FAT10 to accomplish, in part, its functions in regulating the biological behavior of tumor cells. Since both eEF1A1 and FAT10 are important for tumorigenesis and development, comprehending the mechanisms of this interaction can provide clues for identification of novel strategic targets for drug screening and molecular typing, and possibly in the development of new effective therapeutic strategies against hepatocellular carcinoma.

A Rare Loss-of-Function SCN5A Variant is Associated With Lidocaine-induced Ventricular Fibrillation

The human genome contains over 4 million variant sites, as compared with the reference genome, including rare sequence variants, which have the potential to exert large phenotypic effects, such as susceptibility to drug toxicity. We report identification and functional characterization of a rare non-synonymous (p.A1427S) variant in the SCN5A gene that was associated with incessant and lethal ventricular tachycardia and fibrillation after administration of lidocaine to a patient with acute myocardial infarction. The variant, located in a highly conserved domain distinct from the predicted lidocaine-binding site, decreased peak current density of the sodium channel. With the increasing availability of the whole exome and whole genome sequencing data, it would be possible to identify and characterize rare variants in SCN5A that might predispose to lethal ventricular arrhythmias.

De Novo FGF12 (Fibroblast Growth Factor 12) Functional Variation Is Potentially Associated With Idiopathic Ventricular Tachycardia

Background Idiopathic ventricular tachycardia (VT) is a type of cardiac arrhythmia occurring in structurally normal hearts. The heritability of idiopathic VT remains to be clarified, and numerous genetic factors responsible for development of idiopathic VT are as yet unclear. Variations in FGF12 (fibroblast growth factor 12), which is expressed in the human ventricle and modulates the cardiac Na+ channel NaV1.5, may play an important role in the genetic pathogenesis of VT. Methods and Results We tested the hypothesis that genetic variations in FGF12 are associated with VT in 2 independent Chinese cohorts and resequenced all the exons and exon–intron boundaries and the 5′ and 3′ untranslated regions of FGF12 in 320 unrelated participants with idiopathic VT. For population‐based case–control association studies, we chose 3 single‐nucleotide polymorphisms—rs1460922, rs4687326, and rs2686464—which included all the exons of FGF12. The results showed that the single‐nucleotide polymorphism rs1460922 in FGF12 was significantly associated with VT after adjusting for covariates of sex and age in 2 independent Chinese populations: adjusted P=0.015 (odds ratio: 1.54 [95% CI, 1.09–2.19]) in the discovery sample, adjusted P=0.018 (odds ratio: 1.64 [95% CI, 1.09–2.48]) in the replication sample, and adjusted P=2.52E‐04 (odds ratio: 1.59 [95% CI, 1.24–2.03]) in the combined sample. After resequencing all amino acid coding regions and untranslated regions of FGF12, 5 rare variations were identified. The result of western blotting revealed that a de novo functional variation, p.P211Q (1.84% of 163 patients with right ventricular outflow tract VT), could downregulate FGF12 expression significantly. Conclusions In this study, we observed that rs1460922 of FGF12 was significantly associated with VT and identified that a de novo variation of FGF12 may be an important genetic risk factor for the pathogenesis of VT.

Erratum to: Identification of a Novel Binding Protein of FAT10: Eukaryotic Translation Elongation Factor 1A1.

Figure 5 and the text reported erroneous data regarding the influence of FAT10 on mRNA expression of eEF1A1. The corrected Fig. 5 and caption are depicted here. Text corrections: 1. Last sentence of Abstract: Substitute with: ‘‘Knockdown of FAT10 with siRNA downregulated eEF1A1 expression at the protein level but not at the mRNA level in human hepatocellular carcinoma cells.’’ 2. Fourth sentence in the final paragraph of Introduction Substitute with: ‘‘Although siRNA knockdown of FAT10 significantly downregulated eEF1A1 at the protein level, eEF1A1 mRNA expression was not affected in HCC cells.’’ 3. Results, third subheading: Substitute for existing: ‘‘FAT10 siRNA Downregulates eEF1A1 Protein Expression, but Does Not Affect eEF1A1 mRNA Abundance.’’ 4. First sentence of the first paragraph after the third subheading in ‘‘Results’’ Substitute: ‘‘affect’’ for ‘‘effect.’’ 5. Final sentence of the first paragraph after the third subheading in ‘‘Results’’ Substitute with: ‘‘In Fig. 5, 0.2–0.3 of the FAT10 expression level was relative to the expression level of FAT10 (ratio to GAPDH). To better display the mRNA expression relationship between FAT10 and eEF1A1, the expressions of FAT10 and eEF1A1 in the control group were, respectively, set to 1 in the redone experiment. Then, the relative expression differences of FAT10 between the shFAT10 group and the control group were calculated by the way of -DDCt, while the relative expression differences of eEF1A1 between the shFAT10 group and the control group were also calculated. The results show that, compared with the control group, FAT10 expression in the shFAT10 group is decreased about 5 times, while the eEF1A1 expression remains unchanged. Thus, only FAT10 mRNA was significantly downregulated in the FAT10 siRNA-transfected cells (p\ 0.01), indicating that FAT10 siRNA did not downregulate eEF1A1 mRNA expression.’’ 6. First sentence of the second paragraph after the third subheading in ‘‘Results’’ Substitute: ‘‘affect eEF1A1 protein expression’’ for ‘‘effect eEF1A1 expression.’’ 7. First paragraph of the ‘‘Discussion’’ Substitute last sentence with: ‘‘Furthermore, siRNA knockdown of FAT10 significantly reduced eEF1A1 protein expression without affecting the abundance of eEF1A1 mRNA.’’ The online version of the original article can be found under doi:10.1007/s10620-012-2189-1.

Effect of Notch signal pathway on H9c2 cardiomyocytes apoptosis induced by hypoxia/reoxygenation via ROCK2

Background It is very important to explore the new strategies of prevention and treatment of Ischemic heart disease (IHD) in molecular level. Notch signaling pathways are very expedient in terms of the protection and recovery of myocardial when the heart muscle is impaired. It is also known that ROCKs are closely related to the apoptosis of cardiomyocytes. We infer that there are interactions between Notch signaling pathways and ROCKs on cell apoptosis in H9c2 cardiomyocytes model of H/R.

Genetic study of ischemia-induced ventricular arrhythmia associated with potassium channels

Objective Recent reports and our preliminary research showed that sodium channel gene is one of the disease-causing gene in ischemia-induced ventricular arrhythmia. In this study, candidate potassium genes were screened using direct sequencing to reveal the genetics of ischemia-induced ventricular arrhythmia. Methods DNA were extracted from 23 patients with ischemia-induced ventricular tachycardia/ventricular fibrillation. Eleven candidate genes were screened with direct sequencing methods. Gene variation was compared with 100 normal control subjects to ensure gene mutation or SNP. Results Mutation from 11 potassium channels has not been found in 23 patients. 10 single nucleotide polymorphisms in 11 potassium channel genes (KCNJ12-G216G, KCNJ12-Q192H, KCNJ12-P156L, HERG-F513F, HERG-I489I, KV1.5-P513P, Kir2.1-L382L, Kir6.2-V337I, KCNQ1-P448R, KCNQ1-S546S) have been found. The G216G is a novel SNP in KCNJ12 gene. F513F was found in five patients while in 44 normal con individuals. There is a difference in the incidence of F513F between the two groups (p<0.05). Conclusion A novel SNP of G216G was discovered in Chinese people. The SNP of F513F might be a predisposing factor in ischemia-induced ventricular arrhythmia.