Non-coding RNAs in hepatitis C-induced hepatocellular carcinoma:Dysregulation and implications for early detection,diagnosis and therapy

Hepatitis C virus(HCV)infection is one of main causes of hepatocellular carcinoma(HCC)and the prevalence of HCV-associated HCC is on the rise worldwide.It is particularly important and helpful to identify potential markers for screening and early diagnosis of HCC among high-risk individuals with chronic hepatitis C,and to identify target molecules for the prevention and treatment of HCV-associated-HCC.Small noncoding RNAs,mainly microRNAs(miRNAs),and long non-coding RNAs(lncRNAs)with size greater than 200nucleotides,are likely to play important roles in a variety of biological processes,including development and progression of HCC.For the most part their underlying mechanisms of action remain largely unknown.In recent years,with the advance of high-resolution of microarray and application of next generation sequencing techniques,a significant number of non-coding RNAs(ncRNAs)associated with HCC,particularly caused by HCV infection,have been found to be differentially expressed and to be involved in pathogenesis of HCVassociated HCC.In this review,we focus on recent studies of ncRNAs,especially miRNAs and lncRNAs related to HCV-induced HCC.We summarize those ncRNAs aberrantly expressed in HCV-associated HCC and highlight the potential uses of ncRNAs in early detection,diagnosis and therapy of HCV-associated HCC.We also discuss the limitations of recent studies,and suggest future directions for research in the field.miRNAs,lncRNAs and their target genes may represent new candidate molecules for the prevention,diagnosis and treatment of HCC in patients with HCV infection.Studies of the potential uses of miRNAs and lncRNAs as diagnostic tools or therapies are still in their infancy.

Legalon-SIL downregulates HCV core and NS5A in human hepatocytes expressing full-length HCV

AIM: To determine the effect of Legalon-SIL (LS) on hepatitis C virus (HCV) core and NS5A expression and on heme oxygenase-1 (HMOX-1) and its transcriptional regulators in human hepatoma cells expressing full length HCV genotype 1b. METHODS: CON1 cells were treated with 50 μmol/L or 200 μmol/L LS. Cells were harvested after 2, 6 and 24 h. HCV RNA and protein levels were determined by quantitative real-time polymerase chain reaction and Western blotting, respectively. RESULTS: HCV RNA (core and NS5A regions) wasdecreased after 6 h with LS 200 μmol/L (P < 0.05). Both 50 and 200 μmol/L LS decreased HCV RNA levels [core region (by 55% and 88%, respectively) and NS5A region (by 62% and 87%, respectively) after 24 h compared with vehicle (dimethyl sulphoxide) control (P < 0.01). Similarly HCV core and NS5A protein were decreased (by 85%, P < 0.01 and by 65%, P < 0.05, respectively) by LS 200 μmol/L. Bach1 and HMOX-1 RNA were also downregulated by LS treatment (P < 0.01), while Nrf2 protein was increased (P < 0.05).CONCLUSION: Our results demonstrate that treatment with LS downregulates HCV core and NS5A expression in CON1 cells which express full length HCV genotype 1b, and suggests that LS may prove to be a valuable alternative or adjunctive therapy for the treatment of HCV infection.

Iron increases HMOX1 and decreases hepatitis C viral expression in HCV-expressing cells

AIM： To investigate effects of iron on oxidative stress, heme oxygenase-1 （HMOX1） and hepatitis C viral （HCV） expression in human hepatoma ceils stably expressing HCV proteins. METHODS： Effects of iron on oxidative stress, HMOX1, and HCV expression were assessed in CON1 cells. Measurements included mRNA by quantitative reverse transcription-polymerase chain reaction, and protein levels by Western blots. RESULTS： Iron, in the form of ferric nitrilotriacetate,increased oxidative stress and upegulated HMOX1 gene expression. Iron did not affect mRNA or protein levels of Bach1, a repressor of HMOXl. Silencing the up-regulation of HMOXl nuclear factor-erythroid 2-related factor 2 （Nrf2） by Nrf2-siRNA decreased FeNTA-mediated up-regulation of HMOXl mRNA levels. These iron effects were completely blocked by deferoxamine （DFO）. Iron also significantly decreased levels of HCV core mRNA and protein by 80%-90%, nonstructural 5A mRNA by 90% and protein by about 50% in the Con1 full length HCV replicon cells, whereas DFO increased them. CONCLUSION： Excess iron up-regulates HMOXl and down-regulates HCV gene expression in hepatoma cells. This probably mitigates liver injury caused by combined iron overload and HCV infection.

DNA methylation patterns in alcoholics and family controls

AIM: To assess whether DNA methylation patterns in chronic alcoholics are different from non-alcoholic sibling controls. METHODS: We examined the methylation patterns in DNA samples from 25 chronic alcoholics and 22 matched siblings as controls (one per family). DNA was extracted from peripheral blood and analyzed for differences in the methylation patterns after bisulfite-conversion. We used the Illumina GoldenGate Methylation Cancer Panel I (Illumina, San Diego, CA), which probes the methylation profile at 1505 CpG sites from 807 cancer related genes. We excluded the 84 X-chromosome CpG sites and 134 autosomal CpG sites that failed to show a within sample reliability score of at least 95% for all samples, leaving 1287 autosomal CpG sites (associated with 743 autosomal genes) with reliable signals for all samples. A methylation score was calculated as the average methylation for the 1287 CpG sites examined. Differences were assessed by a two-sample t-test. We also examined the average sib pair differences in methylation scores at each of the 1287 sites. All analyses were performed using SPSS, version 9.0, P < 0.05 was considered significant. RESULTS: Methylation levels at the 1287 CpG sites averaged 28.2% for both alcoholics and controls. The mean difference in methylation scores between alcoholic and non-alcoholic sibs by CpG site was < 1% with small inter-individual variances; and only 5 CpG sites had an average sib difference > 5%. Subgroup analysis showed that methylation scores were significantly lower for the alcoholic-dependent subjects who smoked compared to their non-smoking unaffected siblings. Specifically, among smokers who are alcoholic, global methylation indices were significantly lower than in non-alcoholic sib controls, whereas among non-smoking alcoholics, the global indices were significantly higher (P = 0.008). CONCLUSION: Although we observed no effect of alcoholism alone on DNA methylation, there is a decrease in alcoholics who smoke, suggesting a mechanism for alcohol-tobacco synergy for carcinogenesis.

Heme status affects human hepatic messenger RNA and microRNA expression

AIM:To assess effects of heme on messenger RNA(mRNA) and microRNA(miRNA) profiles of liver cells derived from humans.METHODS:We exposed human hepatoma cell line Huh-7 cells to excess iron protoporphyrin(heme)(10 μmol/L) or induced heme deficiency by addition of 4,6-dioxoheptanoic acid(500 μmol/L),a potent inhibitor of aminolevulinic acid dehydratase,for 6 h or 24 h.We harvested total RNA from the cells and performed both mRNA and miRNA array analyses,with use of Affymetrix chips,reagents,and instruments(human genome U133 plus 2.0 and miRNA 2.0 arrays).We assessed changes and their significance and interrelationships with Target Scan,Pathway Studios,and Ingenuity software.RESULTS:Changes in mRNA levels were most numerous and striking at 6 h after heme treatment but were similar and still numerous at 24 h.After 6 h of heme exposure,the increase in heme oxygenase 1 gene expression was 60-fold by mRNA and 88-fold by quantitative reverse transcription-polymerase chain reaction.We found striking changes,especially up-regulation by heme of nuclear erythroid-2 related factor-mediated oxidative stress responses,protein ubiquitination,glucocorticoid signaling,P53 signaling,and changes in RNAs that regulate intermediary metabolism.Fewer mRNAs were down-regulated by heme,and the fold decreases were less exuberant than were the increases.Notable decreases after 24 h of heme exposure were patatin-like phospholipase domain-containing protein 3(-6.5-fold),neuronal PAS domain protein 2(-1.93-fold),and protoporphyrinogen oxidase(-1.7-fold).CONCLUSION:Heme excess exhibits several toxic effects on liver and kidney,which deserve study in humans and in animal models of the human porphyrias or other disorders.

用密度梯度离心法富集源于小鼠胚胎干细胞的运动神经元样细胞前体(英文)

目的：我们的目标是从源于胚胎体（EBs）的小鼠胚胎干细胞（mESCs）中分离运动神经元样细胞前体（MNLCPs）,以用于发展针对运动神经元疾病的药物筛选试验和移植疗法。天然Shh蛋白（或Shh通路激动剂）和维甲酸诱导的胚胎体中,MNLCPs和未分化细胞的含量是不确定的。如果不把未分化的细胞从细胞培养中充分去除,其可能会干涉药物筛选试验或在移植后增生。我们开发了一种以密度梯度离心法为基础的富集MNLCPs的方法。方法：我们用Wichterle等人2008年改进的方法,将mESCs（HBG3：eGFP：HB9）扩大和分化。通过化学和酶学的无研磨处理,含有绿色荧光蛋白的MNLCPs和未分化细胞的胚胎体被小心轻轻地离解成单细胞。利用OptiprepTM8%~20%逐步梯度离心技术将MNLCPs回收。拥有绿色荧光蛋白的MNLCPs的含量由流式细胞仪检测。结果：我们的结果表明,在胚胎体形成前,mESCs在明胶包被的培养板上生长,其分化为MNLCPs的能力减少。比较mESCs在明胶,明胶与PMEFs,及PMEFs包被的培养板上的生长发现,mESCs在PMEFs包被的培养板上产生含绿色荧光蛋白的MNLCPs的得率为（54.1±11.0）%（x±s;n=12）,在明胶包被的培养板上的得率为（2.8±1.1）%（x±s;n=9）。用密度梯度离心法获得的含绿色荧光蛋白的MNLCPs的平均含量为（87.7±5.5）%（x±s;n=3）。结论：我们的数据表明,不使用细胞分选器,无研磨解离和密度梯度离心法也能用于富集具有高存活率的MNLCPs。有必要对MNLCPs在体外、体内和表型上进行进一步的生理学意义（如神经轴突的生长及形成神经肌肉接头的能力）上的鉴定。