Alcoholic hepatitis and HCV interactions in the modulation of liver disease

Most HCV‐infected patients regularly consume alcohol. Alcoholic liver disease (ALD) and chronic hepatitis C virus (HCV) infection together are the most common causes of liver disease worldwide. Although both factors independently cause liver disease, they synergistically promote rapid liver disease progression with devastating outcomes for patients. This review focuses on the prevalence, clinical characteristics and molecular pathophysiologic mechanisms of HCV infection associated with alcohol abuse. Recent findings have centred on the synergistic effect of alcohol and HCV on viral replication, hepatocyte apoptosis, oxidative stress, alcohol‐induced ‘leaky gut’, miR‐122 and immune dysregulation. Clinical and basic research findings presented here summarize key scientific findings with the aim of highlighting potential areas for new therapies and identifying ways of optimizing current treatments for alcoholics with HCV infection.     

Exosomal levels of miRNA-21 from cerebrospinal fluids associated with poor prognosis and tumor recurrence of glioma patients

Glioma is a most common type of primary brain tumors. Extracellular vesicles, in the form of exosomes, are known to mediate cell–cell communication by transporting cell-derived proteins and nucleic acids, including various microRNAs (miRNAs). Here we examined the cerebrospinal fluid (CSF) from patients with recurrent glioma for the levels of cancer-related miRNAs, and evaluated the values for prognosis by comparing the measures of CSF-, serum-, and exosome-contained miR-21 levels. Samples from seventy glioma patients following surgery were compared with those from brain trauma patients as a non-tumor control group. Exosomal miR-21 levels in the CSF of glioma patients were found significantly higher than in the controls; whereas no difference was detected in serum-derived exosomal miR-21 expression. The CSF-derived exosomal miR-21 levels correlated with tumor spinal/ventricle metastasis and the recurrence with anatomical site preference. From additional 198 glioma tissue samples, we verified that miR-21 levels associated with tumor grade of diagnosis and negatively correlated with the median values of patient overall survival time. We further used a lentiviral inhibitor to suppress miR-21 expression in U251 cells. The results showed that the levels of miR-21 target genes of PTEN, RECK and PDCD4 were up-regulated at protein levels. Therefore, we concluded that the exosomal miR-21 levels could be demonstrated as a promising indicator for glioma diagnosis and prognosis, particularly with values to predict tumor recurrence or metastasis.     

miR-122-5p调节创伤性脑外伤后小胶质细胞极化减弱炎症反应北大核心CSCD

目的探讨miR-122-5p对创伤性脑外伤后小胶质细胞凋亡、极化和炎症反应的影响。方法建立创伤性脑外伤(traumatic brain injury,TBI)小鼠模型和细胞模型。使用TBI小鼠脑匀浆刺激星型胶质细胞产生外泌体,microRNA微阵列分析外泌体中显著改变的microRNA。实时荧光定量PCR检测TBI小鼠模型和TBI细胞模型中miR-122-5p表达。使用TUNEL凋亡染色、免疫荧光共聚焦、蛋白质印迹研究miR-122-5p抑制剂在TBI神经炎症中对小胶质细胞凋亡、小胶质细胞M1/M2表型转化、NLRP3通路及NFκB磷酸化的作用。结果通过microRNA微阵列分析发现有83个下调miRNA(改变2倍以上,P<0.05),其中miR-122-5p显著下调(P<0.01),miR-122-5p在TBI小鼠及细胞模型中表达显著下降[(1.0±0.00)vs.(0.41±0.15),P<0.001];[(1.0±0.00)vs.(0.34±0.07),P<0.001]。TUNEL凋亡检测、免疫荧光染色结果表明抑制miR-122-5p表达,可以显著减轻LPS诱导的小胶质细胞凋亡[(8.03±1.30)vs.(3.17±0.34),P<0.001],促进小胶质细胞M1向M2表型转化,即M1表型极化减少[(56.96±13.70)vs.(34.70±3.47),P=0.002],M2表型极化增加[(30.46±3.67)vs.(40.74±2.49),P=0.005]。蛋白质印迹结果表明miR-122-5p抑制剂降低NLRP3炎症小体活化[(0.77±0.10)vs.(0.51±0.11),P=0.02],降低NFκB的磷酸化[(0.73±0.08)vs.(0.50±0.07),P=0.003]。结论miR-122-5p在TBI星形胶质细胞分泌的外泌体及小胶质细胞中表达下调,miR-122-5p抑制剂可以通过抑制TBI后NLRP3炎症小体通路的活化及NFκB的磷酸化,促进小胶质细胞M1向M2表型转化,减少小胶质细胞凋亡,从而减轻TBI后小胶质细胞炎症损伤。     

Induction of delayed hypersensitivity to protein antigens in mice without Freund adjuvants

Three ways for inducing tuberculin-type delayed hypersensitivity (DH) in mice to purified protein antigens without Freund adjuvant are described. Four strains of mice compared for susceptibility to sensitization with several antigens ranked SJL > CF-1 > CAF₁ = C57B1. Females were more easily sensitized than males. The first way of sensitizing without Freund adjuvant was simply to inject antigen intradermally in saline, but it could be used only with the potent antigen methylated human serum albumin (HSA). Six-mercaptopurine injections during the first 5 days of sensitization enhanced such sensitization in CF-1 but inhibited it in CAF₁ mice. The second way sensitized with a weaker antigen, chicken conalbumin (CA), and thus is more versatile. It consisted of injecting a saline solution of antigen into a 24-hr strong DH dermal reaction to an unrelated antigen (dextran). The third way was simplest and best: Freund adjuvant was replaced with aqueous Evans blue dye. Thus, distilled water solutions of antigen (CA or chicken ovalbumin) and dye injected subcutaneously induced DH indistinguishable from that induced with the same antigens in Freund adjuvant. Neither antigen in distilled water alone sensitized. The dye also intensified Arthus sensitization. This diazo dye therefore may be a practical, harmless water-soluble substitute for Freund adjuvant for inducing DH or cell-mediated immunity and for intensifying Arthus sensitization.     

MiR-122在肝细胞癌中的表达及其与肿瘤表型标志物的相关性研究

目的：检测肝细胞癌（HCC）组织中 miR-122和上皮性钙粘蛋白（E-cadherin）、转化生长因子-β1（TGF-β1）的表达,并分析miR-122与E-cadherin、TGF-β1之间的相关性。方法应用实时荧光定量PCR技术检测45例HCC及其配对癌旁组织中miR-122的表达情况;免疫组织化学法检测上述HCC及其配对癌旁组织中E-cadherin、TGF-β1的表达,分析miR-122的表达量与E-cadherin、TGF-β1之间的相关性。结果 miR-122在肝癌组织低表达,肝癌组织中miR-122表达量低于其癌旁组织者有41例,表达量相仿或高于其癌旁组织者4例,差异具有统计学意义（P＜0.05）。E-cadherin在HCC组织中的表达显著低于癌旁组织（P＜0.05）,HCC组织中TGF-β1表达显著高于癌旁组织（P＜0.05）。HCC组织中miR-122表达与E-cadherin呈正相关（r＝0.827）,与TGF-β1表达呈负相关（r＝-0.356）。结论 miR-122在HCC组织中低表达,其表达量与肝癌分化程度正相关。     

miR-122对乙型肝炎病毒抗原表达的影响

目的:探讨miR-122对乙型肝炎病毒HBsAg、HBeAg表达的影响.方法:设计合成2务miR-122的反义寡核苷酸(anti-miR-122,LNA-antimiR-122)、hsa-miR-122以及阴性对照anti-GFP,脂质体介导转染HepG2.2.15细胞.取细胞转染24h、48 h后的培养液,时间分辨荧光免疫法检测anti-miR-122组、LNA-antimiR-122组、hsa-miR-122组以及anti-GFP组HBsAg和HBeAg的表达,定量PCR检测各组HBV DNA的表达.转染48 h后,提取细胞内总RNA,Taqman技术实时荧光定量PCR检测各组miR-122的表达.结果:①转染48 h后anti-miR-122组、LNA-antimiR-122组miR-122表达明显受抑制,低于阴性对照组(P ＜0.001).hsa-miR-122组miR-122水平较阴性对照组升高(P＜0.001).②hsa-miR-122组HBsAg、HBeAg表达均低于阴性对照组(P ＜0.001);anti-miR-122组、LNA-antimiR-122组HBsAg、HBeAg表达均高于阴性对照组(P ＜0.001).③转染24 h与48 h后各组HBV DNA表达与阴性对照组比较无统计学意义(P＞0.05).结论:miR-122可调节乙型肝炎病毒抗原表达.     

LINC00205 promotes proliferation,migration and invasion of HCC cells by targeting miR-122-5p

Long non-coding RNAs (lncRNAs) have been identified as crucial regulators in the tumorigenesis and progression of hepatocellular carcinoma (HCC). Recently, long intergenic non-protein coding RNA 205 (LINC00205) has been identified as a prognostic biomarker in HCC. However, the biological role of LINC0205 and its potential molecular mechanism are poorly investigated. Here, we found that the expression of LINC00205 was dramatically up-regulated in HCC tissues compared to adjacent nontumor tissues. Furthermore, the level of LINC00205 in both Hep3B and Huh7 cells was prominently higher than that in normal hepatic cell line LO2. Notably, the high expression of LINC00205 was strongly correlated with tumor size ≥5 cm, venous infiltration and advanced tumor stages. Functionally, LINC00205 knockdown obviously repressed the proliferation, migration and invasion of Hep3B and Huh7 cells in vitro. An inverse correlation between LINC00205 and miR-122-5p was detected in HCC tissues. Interestingly, LINC00205 knockdown increased the level of miR-122-5p in both Hep3B and Huh7 cells. Mechanistically, luciferase reporter assay demonstrated LINC00205 acted as a competing endogenous RNA (ceRNA) by directly interacting with miR-122-5p. More importantly, miR-122-5p overexpression significantly restrained the proliferation, migration and invasion of HCC cells. Collectively, our study provides solid evidence to support the oncogenic role of LINC00205 in HCC, which may be benefit for the improvement of HCC therapy.