Plasma microRNAs are sensitive indicators of inter-strain differences in the severity of liver injury induced in mice by a choline- and folate-deficient diet

MicroRNAs (miRNAs) are a class of small, conserved, tissue-specific regulatory non-coding RNAs that modulate a variety of biological processes and play a fundamental role in the pathogenesis of major human diseases, including nonalcoholic fatty liver disease (NAFLD). However, the association between inter-individual differences in susceptibility to NAFLD and altered miRNA expression is largely unknown. In view of this, the goals of the present study were (i) to determine whether or not individual differences in the extent of NAFLD-induced liver injury are associated with altered miRNA expression, and (ii) assess if circulating blood miRNAs may be used as potential biomarkers for the noninvasive evaluation of the severity of NAFLD. A panel of seven genetically diverse strains of inbred male mice (A/J, C57BL/6J, C3H/HeJ, 129S/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were fed a choline- and folate-deficient (CFD) diet for 12 weeks. This diet induced liver injury in all mouse strains; however, the extent of NAFLD-associated pathomorphological changes in the livers was strain-specific, with A/J, C57BL/6J, and C3H/HeJ mice being the least sensitive and WSB/EiJ mice being the most sensitive. The morphological changes in the livers were accompanied by differences in the levels of hepatic and plasma miRNAs. The levels of circulating miR-34a, miR-122, miR-181a, miR-192, and miR-200b miRNAs were significantly correlated with a severity of NAFLD-specific liver pathomorphological features, with the strongest correlation occurring with miR-34a. These observations suggest that the plasma levels of miRNAs may be used as biomarkers for noninvasive monitoring the extent of NAFLD-associated liver injury and susceptibility to NAFLD.     

microRNA-125b在脓毒症急性肺损伤中的表达及其与炎症因子水平相关性

摘要：目的探讨microRNA-125b（miR-125b）在脓毒症急性肺损伤中的表达及其与炎症因子的相关性。方法 腹腔注射脂多糖（LPS）诱导急性肺损伤大鼠模型,对照组注射等量的生理盐水（n=20）;各模型组（n=20）大鼠注射LPS 4、8、12及24 h后取其肺组织,HE染色观察病理学变化;PCR检测各组各时点肺组织中miR-125b、肿瘤坏死因子-α（TNF-α）与白细胞介素-6（IL-6）变化。Pearson相关性分析实验组肺组织中miR-125b的表达与TNF-α、IL-6水平的相关性。NR8383细胞培养后分组,对照组不进行LPS刺激,实验组加入LPS刺激4 h、8 h、12 h及24 h时收集细胞,检测各组miR-125b、TNF-α与IL-6的含量变化。另NR8383细胞设3组,阴性对照组只转染空表达质粒,miR-125b mimic组转染miR-125b mimic表达质粒,miR-125b inhibitor组转染miR-125b inhibitor质粒。PCR测各组细胞中miR-125b 表达情况,Western blot测Notch1的表达情况。结果LPS各处理组大鼠模型肺组织严重出血、水肿及大量炎症细胞浸润。与对照组比较,在注射LPS 4、8、12以24 h后,实验组大鼠肺组织中miR-125b显著降低;而肺组织中TNF-α与IL-6水平则升高,其中在LPS注射4 h后,TNF-α与IL-6的表达水平达到峰值（P＜0.05）。实验组肺组织中miR-125b的表达与TNF-α及IL-6呈负相关（r 分别为-0.599、-0.580;P＜0.05）。在LPS诱导的第4、8、12、24 h,实验组NR8383细胞系miR-125b表达水平均低于对照组,而TNF-α、IL-6的表达水平均高于对照组（P＜0.05）。与阴性对照组比较,miR-125b mimic组miR-125b 相对表达量升高、Notch1蛋白相对表达量则降低（P＜0.05）,miR-125binhibitor组miR-125b 相对表达量降低、Notch1蛋白相对表达量升高（P＜0.05）;与miR-125b mimic组比较,miR-125binhibitor组miR-125b 相对表达量降低、Notch1蛋白相对表达量升高（P＜0.05）。结论在脓毒症急性肺损伤中,microRNA-125b可能通过调控Notch1蛋白的表达,影响炎症因子TNF-α与IL-6的表达,参与其免疫炎症调控过程。     

Aberrant expression of miRNA-192-5p contributes to N,N-dimethylformamide-induced hepatic apoptosis

Excessive exposure to N,N-dimethylformamide (DMF) can lead to occupational liver poisoning in workers; however, the underlying mechanism is not fully clarified. The importance of microRNAs (miRNAs) in chemical-induced hepatotoxicity has been demonstrated. To determine whether miRNAs are also involved in DMF-induced hepatotoxicity, we systematically analyzed the miRNA expression profiles in DMF-treated (75 and 150 mm ) HL-7702 liver cells and controls by high-throughput sequencing. Among the altered miRNAs, miR-192-5p was the most significantly upregulated in HL-7702 cells after DMF exposure and was involved in DMF-mediated cell apoptosis. By contrast, suppression of miR-192-5p in HL-7702 cells attenuated the apoptosis induced by DMF. Furthermore, the anti-apoptotic gene (NIN1/RPN12 binding protein 1 homolog [NOB1]) was predicted to be a potential miR-192-5p target according to bioinformatics analysis. The direct interaction between miR-192-5p and NOB1 was confirmed by the dual-luciferase activity assay in HEK293FT cells. Overexpression of miR-192-5p efficiently reduced NOB1 mRNA and protein expression in HL-7702 cells. Alteration in NOB1 expression influenced DMF-induced hepatotoxicity by affecting hepatic apoptosis. In addition, the inverse correlation between miR-192-5p expression levels and NOB1 expression was further confirmed in DMF-exposed mouse liver tissue samples. These observations demonstrated that promotion of apoptosis from the suppression of NOB1 by miR-192-5p overexpression was responsible for the DMF-induced hepatotoxicity. This work provides the molecular mechanism at the miRNA level for hepatic apoptosis induced by DMF.     

Altered expression of microRNAs may predict therapeutic response in rheumatoid arthritis patients

BackgroundEpigenetic alternations of microRNAs (miRNAs) can contribute to the pathogenesis and progression of rheumatoid arthritis (RA). This study aimed to measure the expression level of peripheral blood miRNAs, as well as their target mRNAs, in RA patients and healthy controls (HCs), and to evaluate the potential of miRNAs as promising non-invasive biomarkers of treatment response.MethodsThe peripheral expression of miRNAs, including miR-146a, miR-146b, miR-150, miR-155, miR-125a-5p, miR-223, miR-26a, and miR-21, as well as their target mRNAs, was analyzed in 90 RA patients and 30 HCs via quantitative real-time polymerase chain reaction (RT-PCR) assay. We compared differences between the patients in terms of good response (GR; n = 55) and poor response (PR; n = 35) to the conventional therapeutic approach.ResultsAll miRNAs were significantly overexpressed in RA patients. The expression of miR-155, miR-150, miR-146a, miR-146b, miR-125a-5p, and miR-223 increased in both groups of RA patients, compared to HCs, and miR-26a and miR-21 were the only upregulated miRNAs in the GR group versus HCs. Among the upregulated miRNAs, miR-125a-5p expression significantly changed in GR and PR patients (P = 0.047). The ROC curve analysis indicated the potential involvement of miR-125a-5p in the pathogenesis of RA. We also observed the downregulated expression of GATA3, RORC, FOXP3, TBX21, STAT1, and TRAF6 in RA patients versus HCs.ConclusionOur findings indicated that different expression levels of miR-125a-5p in the GR and PR groups of patients may serve as a therapeutic response biomarker, which can be also used as a target for therapeutic interventions.     

Exposure to biomass smoke is associated with an increased expression of circulating miRNA-126 and miRNA-155 in Mexican women: a pilot study

Household air pollution has been associated as a risk factor for cardiovascular diseases (CVD). Therefore, the aim of this study was to assess the expression of vascular inflammation regulators miR-126 and miR-155 in plasma from women that cook with wood and women that cook with liquid petroleum gas (LPG). A cumulative index of exposure to smoke (CIES) was estimated, urinary 1-hydroxypyrene (1-OHP) levels were quantified and miRNAs expression levels were determined by quantitative real-time PCR (qRT-PCR). Biochemical clinical parameters were also evaluated. The average values for CIES and 1-OHP were 140?±?86.8?hours-years (12.0–270?hours-years) and 0.52?±?0.45?µmol/mol creatinine, respectively. miR-126 and miR-155 expression levels were significantly higher (p?<?0.01) in the wood users compared to LPG users. Besides, we found a significant association (p?<?0.01) between miR-126 and miR-155 expression levels and CIES and urinary 1-OHP concentrations. These results contribute to the current evidence about the cardiovascular risk related to biomass smoke exposure, from an epigenetic level.     

FC-99 reduces macrophage tenascin-C expression by upregulating miRNA-494 in arthritis

The excessive production of inflammatory mediators by inflammatory cells contributes to the pathogenesis of rheumatoid arthritis. Tenascin-C (TN-C) is expressed in rheumatoid joint, and is associated with levels of inflammatory mediators. FC-99 (N1-[(4-methoxy)methyl]-4-methyl-1,2-Benzenediamine), a novel 1,2-benzenediamine derivative, was previously reported to block the prolonged expression of key rheumatoid arthritis inflammatory cytokines and relieve zymosan-induced joint inflammation. However, the specific mechanism is unknown. This study aimed to examine the effects of FC-99 on TN-C expression and inflammation and investigate its possible molecular mechanism. The results showed that FC-99 treatment reduced the high expression of TN-C in ankle joints of arthritis mice. Besides, FC-99 reduced the increased number of macrophages in arthritis mice, while did not change the number of synovioblasts. Concomitantly, expression of TN-C in synovial fibroblasts exhibited no difference between control and ZIA groups, and was not apparently altered following FC-99 treatment, while FC-99 decreased TN-C expression in macrophages both in vivo and in vitro. Meanwhile, TargetScan and luciferase assays indicated that TN-C was negatively regulated by miR-494. Transfection assay further demonstrated that FC-99 inhibited TN-C by targeting miR-494. Furthermore, the reduction of miR-494 mimic on expression of TN-C was associated with NF-κB pathway. Similarly, the down-regulation of FC-99 on TN-C was considerably decreased when NF-κB pathway was inhibited. These results indicated that FC-99 relieved macrophages inflammation via the miR-494/TN-C/NF-κB pathway, finally leading to the relief of inflammation in arthritis. The findings suggested that FC-99 might be a potential therapeutic candidate for the treatment of rheumatoid arthritis.     

Myeloid-specific SIRT1 Deletion Aggravates Hepatic Inflammation and Steatosis in High-fat Diet-fed Mice

Sirtuin 1 (SIRT1) is a mammalian NAD⁺-dependent protein deacetylase that regulates cellular metabolism and inflammatory response. The organ-specific deletion of SIRT1 induces local inflammation and insulin resistance in dietary and genetic obesity. Macrophage-mediated inflammation contributes to insulin resistance and metabolic syndrome, however, the macrophage-specific SIRT1 function in the context of obesity is largely unknown. C57/BL6 wild type (WT) or myeloid-specific SIRT1 knockout (KO) mice were fed a high-fat diet (HFD) or normal diet (ND) for 12 weeks. Metabolic parameters and markers of hepatic steatosis and inflammation in liver were compared in WT and KO mice. SIRT1 deletion enhanced HFD-induced changes on body and liver weight gain, and increased glucose and insulin resistance. In liver, SIRT1 deletion increased the acetylation, and enhanced HFD-induced nuclear translocation of nuclear factor kappa B (NF-κB), hepatic inflammation and macrophage infiltration. HFD-fed KO mice showed severe hepatic steatosis by activating lipogenic pathway through sterol regulatory element-binding protein 1 (SREBP-1), and hepatic fibrogenesis, as indicated by induction of connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA), and collagen secretion. Myeloid-specific deletion of SIRT1 stimulates obesity-induced inflammation and increases the risk of hepatic fibrosis. Targeted induction of macrophage SIRT1 may be a good therapy for alleviating inflammation-associated metabolic syndrome.     

Hepatic effects of a methionine-choline-deficient diet in hepatocyte RXRalpha-null mice

Retinoid X receptor-alpha (RXRalpha) is an obligate partner for several nuclear hormone receptors that regulate important physiological processes in the liver. In this study the impact of hepatocyte RXRalpha deficiency on methionine and choline deficient (MCD) diet-induced steatosis, oxidative stress, inflammation, and hepatic transporters gene expression were examined. The mRNA of sterol regulatory element-binding protein (SREBP)-regulated genes, important for lipid synthesis, were not altered in wild type (WT) mice, but were increased 2.0- to 5.4-fold in hepatocyte RXRalpha-null (H-RXRalpha-null) mice fed a MCD diet for 14 days. Furthermore, hepatic mRNAs and proteins essential for fatty acid beta-oxidation were not altered in WT mice, but were decreased in the MCD diet-fed H-RXRalpha-null mice, resulting in increased hepatic free fatty acid levels. Cyp2e1 enzyme activity and lipid peroxide levels were induced only in MCD-fed WT mice. In contrast, hepatic mRNA levels of pro-inflammatory factors were increased only in H-RXRalpha-null mice fed the MCD diet. Hepatic uptake transporters Oatp1a1 and Oatp1b2 mRNA levels were decreased in WT mice fed the MCD diet, whereas the efflux transporter Mrp4 was increased. However, in the H-RXRalpha-null mice, the MCD diet only moderately decreased Oatp1a1 and induced both Oatp1a4 and Mrp4 gene expression. Whereas the MCD diet increased serum bile acid levels and alkaline phosphatase activity in both WT and H-RXRalpha-null mice, serum ALT levels were induced (2.9-fold) only in the H-RXRalpha-null mice. In conclusion, these data suggest a critical role for RXRalpha in hepatic fatty acid homeostasis and protection against MCD-induced hepatocyte injury.     

Regulation of the hepatic multidrug resistance gene expression by endotoxin and inflammatory cytokines in mice

P-glycoprotein (PGP), an ATP-dependent membrane transporter is found in epithelial tissues of the liver, kidneys, intestine and blood-brain barrier. In tumor cells, PGP is often overexpressed and confers multidrug resistance toward cancer chemotherapeutics. It has been previously shown in rats that induction of an inflammatory response evokes a decrease in hepatic expression of PGP. In order to identify the inflammatory mediators involved in this phenomenon, we examined the influence of experimentally induced inflammation and pro-inflammatory cytokines (interleukin (IL)-6, IL-1beta and tumor necrosis factor (TNF)-alpha) on the hepatic expression of PGP in mice. A significant reduction in the hepatic expression of mdr1a, mdr1b, mdr2 and spgp genes were seen in endotoxin (lipopolysaccharide (LPS)) and turpentine-treated mice. Similarly, IL-6-treated mice displayed a 70% reduction in protein expression and a 40-70% reduction in the mRNA levels of all PGP mdr isoforms. Administration IL-1beta caused an increase in both mdr1b mRNA and protein expression, however, mRNA levels of mdr1a, mdr2 and spgp were significantly reduced. Administration of TNF-alpha also caused increases in mdr1b mRNA. These findings indicate that IL-6 plays a principal role in the downregulation of PGP that is observed in the livers of mice during an inflammatory response.     

MiR-130b attenuates vascular inflammation via negatively regulating tumor progression locus 2 (Tpl2) expression

Endothelial cell (EC) activation and dysfunction have been linked to a wide variety of vascular inflammatory diseases. However, the role of microRNAs in EC activation and inflammation remains largely unknown. In this study, we found that miR-130b was significantly decreased in human umbilical vein endothelial cells (HUVECs) after lipopolysaccharides (LPS) treatment. Forced expression of miR-130b inhibited the LPS-induced activation of extracellular signal-regulated kinase (ERK) and the inflammatory genes expression, such as interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α). Furthermore, we identified that tumor progression locus 2 (Tpl2) is a direct target of miR-130b. Finally, in vivo overexpression of miR-130b via miR-130b agomir attenuates acute lung vascular inflammation in the LPS-induced sepsis mouse model. Taken together, our data demonstrated that miR-130b represses vascular inflammation via targeting Tpl2, suggesting that miR-130b mimics might be a promising therapeutic strategy for treatment of vascular inflammatory diseases.     

miR-301a对小鼠坏死性小肠结肠炎的影响及作用机制

目的 观察miR-301a对小鼠坏死性小肠结肠炎（NEC）的影响及作用机制。方法 将60只新生BALB/c小鼠采用随机数字表法分为对照组、NEC组、miR-301a拮抗剂组,每组20只。NEC组、miR-301a拮抗剂组通过人工喂养、缺氧和冷刺激5 d建立NEC模型,miR-301a拮抗剂组给予miR-301a拮抗剂。实验期间记录小鼠体质量,HE染色观察各组肠道组织病理改变,并对肠组织进行损伤评分;采用酶联免疫吸附试验（ELISA）检测各组小鼠血清肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6和IL-8的含量。采用TUNEL法测定不同组小鼠肠组织的细胞凋亡情况,qPCR检测miR-301a和胱天蛋白酶（Caspase）-1基因表达水平,Western blot检测Caspase-1蛋白表达水平。结果 与对照组相比,NEC组小鼠体质量减轻,肠组织出现明显的炎症损伤,损伤评分升高;血清中TNF-α、IL-6和IL-8的含量升高,肠组织细胞凋亡指数增加,miR-301a和Caspase-1的表达水平升高（均P<0.05）。与NEC组相比,miR-301a拮抗剂组小鼠NEC症状明显减...     

Paeonol inhibits NLRP3 mediated inflammatory reaction in rat endothelial cells by elevating hyperlipidemic rats plasma exosomal miRNA-223

OBJECTIVE Atherosclerosis(AS) is featured as a chronic inflammatory disease of vascular stenosis.Paeonol(Pae) is a natural phenolic compounds isolated from a traditional Chinese medicine, Cortex Moutan, which exhibits anti-AS effects in vitro and in vivo. In this study, we aimed to investigate whether the anti-AS efficacy of Pae was regulated through inhibiting NLRP3 inflammasome activityvia elevating hyperlipidemic rats plasma exosomalmicro RNA-223(miR-223). METHODS The Sprague-Dawley rats was induced by a high-fat diet, which was used as AS models.AS aortic pathological morphological in AS mice was examined by HE staining, and serum TC and TG levels were determined by automatic chemistry analyzer. Rat aortic endothelial cells(RAECs) were used during the whole study. After oral administration of Pae, we isolated exosomes from hyperlipidemic rats plasma(Pae-Exos) by ultracentrifugation and characterized by transmission electron, nanoparticle tracking analysis, dynamic light scattering and Western blotting.The activity of RAECs was detected by CCK-8 and trypan blue staining method. IL~(-1)β and IL-6 levels were detected by ELISA method. The expression of miR-223 was detected by q PCR, and the expression of NLRP3, ASC, caspase-1, and ICAM-1 was detected by Western blotting. RESULTS In vivo experiments confirmed that Pae could effectively reduce serum TC and TG levels and decrease serum IL~(-1)β and IL-6 levels, which demonstrated that Pae restricted AS development in hyperlipidemia rats. Both CCK-8 and trypan blue staining showed that the survival rate of RAECs in the PaeExos co-incubation group was higher than that in the model group. We also confirmed via real-time q PCR that Pae-Exos suppressed the expression of the inflammatory cytokines IL~(-1)β and IL-6. Accordingly, Pae-Exos dose-dependently increased the survival rate of RAECs and reduced inflammatory response. Furthermore, compared with the model group, Pae-Exos more successfully increased the expression of miR-223 and inhibited IL~(-1)β and IL-6 expression, which implied that Pae-exo may inhibited the inflammatory response of RAECs by increasing the content of miR-223. Subsequently, we found that Pae-Exos reduced the expressions of NLRP3, ASC, caspase-1 and ICAM-1, which indicated that Pae-Exos may reduced RAECs inflammation by suppressing NLRP3 signaling pathway via promoting miR-223 expression.CONCLUSION Pae can inhibit the downstream NLRP3 inflammatory corpuscle signaling pathway by increasing the level of miR-223 in plasma Exos of hyperlipidemic rats, providing new insights into the anti-atherosclerosis activity of Pae.     

Hepatic effects of a methionine-choline-deficient diet in hepatocyte RXRα-null mice

Retinoid X receptor-α (RXRα) is an obligate partner for several nuclear hormone receptors that regulate important physiological processes in the liver. In this study the impact of hepatocyte RXRα deficiency on methionine and choline deficient (MCD) diet-induced steatosis, oxidative stress, inflammation, and hepatic transporters gene expression were examined. The mRNA of sterol regulatory element-binding protein (SREBP)-regulated genes, important for lipid synthesis, were not altered in wild type (WT) mice, but were increased 2.0- to 5.4-fold in hepatocyte RXRα-null (H-RXRα-null) mice fed a MCD diet for 14 days. Furthermore, hepatic mRNAs and proteins essential for fatty acid β-oxidation were not altered in WT mice, but were decreased in the MCD diet-fed H-RXRα-null mice, resulting in increased hepatic free fatty acid levels. Cyp2e1 enzyme activity and lipid peroxide levels were induced only in MCD-fed WT mice. In contrast, hepatic mRNA levels of pro-inflammatory factors were increased only in H-RXRα-null mice fed the MCD diet. Hepatic uptake transporters Oatp1a1 and Oatp1b2 mRNA levels were decreased in WT mice fed the MCD diet, whereas the efflux transporter Mrp4 was increased. However, in the H-RXRα-null mice, the MCD diet only moderately decreased Oatp1a1 and induced both Oatp1a4 and Mrp4 gene expression. Whereas the MCD diet increased serum bile acid levels and alkaline phosphatase activity in both WT and H-RXRα-null mice, serum ALT levels were induced (2.9-fold) only in the H-RXRα-null mice. In conclusion, these data suggest a critical role for RXRα in hepatic fatty acid homeostasis and protection against MCD-induced hepatocyte injury.     

微RNA-125b靶向调控M2型丙酮酸激酶影响宫颈癌SiHa细胞机制验证

目的探讨宫颈癌细胞中微RNA(miR)-125b和M2型丙酮酸激酶(PKM2)的表达以及miR-125b对PKM2基因的调控作用,从而确定miR-125b下游调控的靶基因,阐明其在宫颈癌发生发展中可能的分子机制。方法培养宫颈癌SiHa细胞及正常上皮细胞(293T细胞),利用细胞转染技术过表达或沉默miR-125b。实验分为实验组:miR-125b mimics(M组)、miR-125b inhibitor(I组);阴性对照组:miR-125b mimics negative con-trol(MNC组)、miR-125b inhibitor negative control(INC组);空白对照组(B组):未进行转染。采用实时荧光定量-聚合酶链反应(qRT-PCR)检测各组细胞中miR-125b的含量。采用免疫荧光及蛋白质印迹法检测各组细胞中PKM2蛋白的表达。结果与293T细胞相比,宫颈癌SiHa细胞中miR-125b的表达下调(0.82±0.05)(P<0.05),PKM2表达上调(3.4±0.5)倍(P<0.05)。SiHa细胞转染成功后,免疫荧光检测miR-125b模拟物组表达PKM2蛋白的细胞数目(1.0±0.7)低于模拟物阴性对照组表达PKM2蛋白的细胞数目(3.6±0.5)(P<0.05);miR-125b抑制物组中表达PKM2蛋白的细胞数目(6.0±0.7)高于抑制物阴性对照组表达PKM2蛋白的细胞数目(3.6±1.3)(P<0.05)。蛋白质印迹法检测miR-125b模拟物组的PKM2蛋白相对表达量(0.239±0.042)低于模拟物阴性对照组的PKM2蛋白相对表达量(0.314±0.018)(P<0.05);miR-125b抑制物组的PKM2蛋白表达水平(0.40±0.03)高于抑制物阴性对照组的PKM2蛋白表达水平(0.34±0.04)(P<0.05)。结论与正常上皮细胞相比,宫颈癌SiHa细胞中miR-125b表达下调,而PKM2蛋白表达上调;宫颈癌细胞中miR-125b表达下调可能会通过增加PKM2的表达,从而增强其糖酵解作用,促进肿瘤的生长。     

Long non-coding RNA THRIL promotes LPS-induced inflammatory injury by down-regulating microRNA-125b in ATDC5 cells

BackgroundOsteoarthritis is an age-related disorder of bone-joint that causes pain and disability in middle and older people. This study aimed to investigate the potential effects of long non-coding RNA (lncRNA) THRIL on lipopolysaccharide (LPS)-induced osteoarthritis cell injury model (ATDC5 cell inflammatory injury), as well as the possible internal molecular mechanisms.MethodsCell viability and apoptosis were assessed using CCK-8 assay and Guava Nexin assay, respectively. Cell transfection was conducted to change the expression of THRIL and microRNA-125b (miR-125b) in ATDC5 cells. qRT-PCR was performed to detect the expression of THRIL, miR-125b and pro-inflammatory cytokines IL-6, TNF-α and monocyte chemotactic protein 1 (MCP-1) in ATDC5 cells. ELISA was used to measure the concentrations of IL-6, TNF-α and MCP-1 in culture supernatant of ATDC5 cells. Finally, the protein expression of key factors involved in cell apoptosis, inflammatory response, JAK1/STAT3 and NF-κB pathways were evaluated using western blotting.ResultsLPS significantly induced ATDC5 cell inflammatory injury and up-regulated the expression of THRIL. Overexpression of THRIL aggravated the LPS-induced ATDC5 cell inflammatory injury. Suppression of THRIL had opposite effects. Moreover, THRIL negatively regulated the expression of miR-125b in ATDC5 cells. miR-125b participated in the effects of THRIL overexpression on LPS-induced ATDC5 cell inflammatory injury. Furthermore, overexpression of THRIL enhanced the LPS-induced JAK1/STAT3 and NF-κB pathways activation by down-regulating miR-125b.ConclusionTHRIL exerted pro-inflammatory roles in LPS-induced osteoarthritis cell injury model. Overexpression of THRIL promoted LPS-induced ATDC5 cell inflammatory injury by down-regulating miR-125b and then activating JAK1/STAT3 and NF-κB pathways.     

MicroRNA delivery by cationic lipoplexes for lung cancer therapy

Lung cancer is the leading cause of cancer deaths in western countries and carries a poor overall five year survival rate. Several studies demonstrate that microRNAs (miRNAs or miRs) are actively involved in tumor development by serving as tumor suppressors, oncogenes or both. In lung cancer, miRNAs may serve as both diagnostic and prognostic biomarkers as well as regulate in vitro and in vivo tumor progression. However, miRNA-based therapy is faced with several challenges including lack of tissue specificity, lack of optimal delivery systems, poor cellular uptake and risk of systemic toxicity. Here, we report a cationic lipid based miRNA delivery system to address some of these challenges. Among many lung cancer related miRNAs, miR-133b, a tumor suppressor, was selected as a therapeutic target because it directly targets the prosurvival gene MCL-1 thus regulating cell survival and sensitivity of lung cancer cells to chemotherapeutic agents. The efficacy of pre-miR-133b containing lipoplexes was evaluated in A549 non-small cell lung cancer (NSCLC) cells. Compared with siPORT NeoFX transfection agent, lipoplexes delivered pre-miR-133b in a more efficient manner with ~2.3-fold increase in mature miR-133b expression and ~1.8-fold difference in MCL-1 protein downregulation in vitro. In the in vivo biodistribution study, lipoplexes achieved ~30% accumulation in lung tissue, which was ~50-fold higher than siPORT NeoFX transfection agent. Mice treated with pre-miR-133b containing lipoplexes had mature miR-133b expression in lung ~52-fold higher than untreated mice. Our results demonstrated that cationic lipoplexes are a promising carrier system for the development of miRNA-based therapeutics in lung cancer treatment.