LncRNA UCA1 elevates the resistance of human leukemia cells to daunorubicin by the PI3K/AKT pathway via sponging miR-613

Acute leukemia is a hematological malignant tumor. Long non-coding RNA urothelial cancer-associated 1 (UCA1) is involved in the chemo-resistance of diverse cancers, but it is unclear whether UCA1 is associated with the sensitivity of acute leukemia cells to daunorubicin (DNR). DNR (100 nM) was selected for functional analysis. The viability, cell cycle progression, apoptosis, and invasion of treated acute leukemia cells (HL-60 and U-937) were evaluated by cell counting kit-8 (CCK-8) assay, flow cytometry assay, or transwell assay. Protein levels were detected with Western blot analysis. Expression patterns of UCA1 and miR-613 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between UCA1 and microRNA-613 (miR-613) was verified by dual-luciferase reporter assay. We observed that UCA1 expression was elevated in HL-60 and U-937cells. DNR constrained viability, cell cycle progression, invasion, and facilitated apoptosis of HL-60 and U-937 cells in a dose-dependent manner, but these impacts mediated by DNR were reverted after UCA1 overexpression. MiR-613 was down-regulated in HL-60 and U-937 cells, and UCA1 was verified as a miR-613 sponge. MiR-613 inhibitor reversed DNR treatment-mediated effects on viability, cell cycle progression, apoptosis, and invasion of HL-60 and U-937 cells, but these impacts mediated by miR-613 inhibitor were counteracted after UCA1 inhibition. Notably, the inactivation of the PI3K/AKT pathway caused by DNR treatment was reversed after miR-613 inhibitor introduction, but this influence mediated by miR-613 inhibitor was offset after UCA1 knockdown. In conclusion, UCA1 up-regulation facilitated the resistance of acute leukemia cells to DNR via the PI3K/AKT pathway by sponging miR-613.     

MiR-214 Targets β-Catenin Pathway to Suppress Invasion, Stem-Like Traits and Recurrence of Human Hepatocellular Carcinoma

The down-regulation of miR-214 has previously been observed in human hepatocellular carcinoma (HCC). Here, we demonstrated the down-regulation of miR-214 is associated with cell invasion, stem-like traits and early recurrence of HCC. Firstly, we validated the suppression of miR-214 in human HCC by real-time quantitative RT-PCR (qRT-PCR) in 20 paired tumor and non-tumor liver tissues of HCC patients and 10 histologically normal liver tissues from colorectal cancer patients with liver metastases. Further qRT-PCR analysis of 50 HCC tissues from an independent cohort of HCC patients of whom 29 with early recurrent disease (<2 years) and 21 with late recurrent disease demonstrated that the suppression of miR-214 was significantly more suppressed in samples from HCC patients with early recurrent disease compared those from patients with no recurrence. Re-expression of miR-214 significantly suppressed the growth of HCC cells in vitro and reduced their tumorigenicity in vivo. The enhancer of zeste homologue 2 (EZH2) and β-catenin (CTNNB1) was identified as two potential direct downstream targets of miR-214 through bioinformatics analysis and experimentally validated the miRNA-target interactions with a dual-firefly luciferase reporter assay. In corroborate with this, both EZH2 and CTNNB1 are found to be significantly overexpressed in human HCC biopsies. Since EZH2 can regulate CTNNB1, CTNNB1 can also be an indirect target of miR-214 through EZH2. Silencing EZH2 or CTNNB1 expression suppressed the growth and invasion of HCC cells and induced E-cadherin (CDH1), known to inhibit cell invasion and metastasis. Furthermore, the silencing of miR-214 or overexpression of EZH2 increased EpCAM(+) stem-like cells through the activation of CTNNB1. Interestingly, the up-regulation of EZH2, CTNNB1 and the down-regulation of CDH1 in HCC patients correlated with early recurrent disease and can be an independent predictor of poor survival. Therefore, miR-214 can directly or indirectly target CTNNB1 to modulate the β-catenin signaling pathway in HCC.     

LncRNA UCA1 Suppresses the Inflammation Via Modulating miR-203-Mediated Regulation of MEF2C/NF-κB Signaling Pathway in Epilepsy

Although many advances have been made in the pathogenesis of epilepsy recently, the pathological mechanisms of epilepsy are still largely unknown. Exploring the pathological mechanisms and developing novel therapeutic strategies for epilepsy are urgently needed. A SD rat model of epilepsy was established with lithium chloride-pilocarpine. Astrocytes were isolated, cultured from 8 to 12 week rats and identified by flow cytometry and immunofluorescence. Immunohistochemical staining was used for MEF2C and NF-κB in paraffin-embedded sections. RT-qPCR and western blot were used to analyze gene expression. ELISA was used to analyze the concentration of IL-6, TNF-α and Cox-2. Cells were transfected with pcDNA-MEFC2, sh-MEFC2, pcDNA-UCA1, sh-UCA1, miR-203 mimic or miR-203 inhibitor. Cell viability was assessed by MTT assay. Dual luciferase assay was used to determine the direct interaction of lncRNA UCA1/miR-203 and miR-203/MEF2C. MEF2C was down-regulated and inhibited NF-κB expression and the secretion of IL-6 and TNF-α in epilepsy. LncRNA UCA1 was also down-regulated in epilepsy. LncRNA UCA1 over-expression increased the expression of MEF2C and its knock-down decreased MEF2C expression. Luciferase activity showed lncRNA UCA1 directly targeted miR-203 and miR-203 directly targeted MEF2C. MiR-203 suppressed the expression of MEF2C, and promoted NF-κB, phosphorylated IκB/IKK and inflammatory effectors, which was reversed by MEF2C knock-down. Moreover, lncRNA UCA1 could increase the expression of MEF2C to inhibit NF-κB, phosphorylated IκB/IKK and inflammatory effectors, which was also reversed by miR-203 mimic transfection. LncRNA UCA1 inhibited the inflammation via regulating miR-203 mediated regulation of MEF2C/NF-κB signaling in epilepsy. Our investigation elucidated novel pathological mechanisms and provided potential therapeutic targets for epilepsy.

     

Circular RNA circMTO1 suppressed the progression of renal cell carcinoma progression by sponging miR-211 and miR-204

Despite considerable improvements in renal cell carcinoma (RCC) diagnostic and therapeutic strategy, the clinical prognosis of patients is far from satisfactory due to its recurrence and metastasis. Here, we attempted to explore the role of circMTO1 in RCC progression, and the underlying mechanism was further elucidated. We first detected the expression of circMTO1 in 90 pairs of RCC tissues and adjacent normal tissues using qRT-PCR. Besides, circMTO1, miR-211, miR-204 and KLF6 expression levels in RCC cells were also measured using qRT-PCR. MTT assay, cell migration, flow cytometry analysis, qRT-PCR and western blotting analysis were applied to evaluating the effect of circMTO1 in RCC cells. The bioinformatics analysis and the rescue experiment were devoted to the underlying mechanism. The results demonstrated CircMTO1 expression was significantly down-regulated in RCC tissues and cell lines. Besides, CircMTO1 inhibited cell proliferation, migration and invasion, induced apoptosis in RCC cells. In addition, CircMTO1 serves as a sponge for miR-211 and miR-204, KLF6 is a direct target of miR-211 and miR-204. Furthermore, circMTO1 and KLF6 overexpression rescued the suppression of miR-211/204 in RCC cell proliferation. In short, circMTO1 repressed RCC progression by regulating KLF6 via sponging miR-211 and miR-204, which may provide new idea of diagnosis and treatment in renal cell carcinoma.

     

Upregulated lncRNA-UCA1 contributes to metastasis of bile duct carcinoma through regulation of miR-122/CLIC1 and activation of the ERK/MAPK signaling pathway

In the present study, we aimed to identify specific lncRNAs and miRNAs, as well as mRNAs, involved in bile duct carcinoma (BDC) and to further explore the way in which lncRNA UCA1 regulates cell metastasis ability. Differentially expressed RNAs were screened out from the TCGA database. In in vitro experiments, qRT-PCR was used to measure lncRNA UCA1, miR-122 and CLIC1 expression. We performed a dual luciferase assay to validate the target relationships among UCA1, CLIC1 and miR-122. The cell migration ability was measured by a wound healing assay, and Transwell assays were applied to detect cell invasive ability. Western blot analysis was employed to detect the expression of related proteins in the MAPK signaling pathway. According to the bioinformatics analysis, lncRNA UCA1 and CLIC1 were both significantly upregulated in BDC, while the expression of miR-122 declined compared with the normal group. The target relationship among UCA1, CLIC1 and miR-122 was verified. UCA1 promoted BDC cell migration and invasiveness, while miR-122 inhibited their progression. CLIC1 served as the downstream target gene of miR-122 and had opposite effects. The ERK/MAPK signaling pathway was activated after upregulating UCA1. LncRNA-UCA1 promoted the metastasis of BDC cells by regulating the expression of miR-122 and its downstream gene mRNA CLIC1 and promoted the activation of the ERK/MAPK pathway, which expanded the horizons of targeted therapy of cholangiocarcinoma.     

Effect of dietary epigallocatechin-3-gallate on cytochrome P450 2E1-dependent alcoholic liver damage: enhancement of fatty acid oxidation

This study was designed to determine whether dietary epigallocatechin-3-gallate (EGCG), the most abundant catechin polyphenol in green tea, can protect the liver from cytochrome P450 2E1 (CYP2E1)-dependent alcoholic liver damage. Compared with an ethanol group, when EGCG was present in the ethanol diet, the formation of a fatty liver was significantly reduced and the serum aspartate transaminase (AST) and alanine transaminase (ALT) levels were much lower. Ethanol treatment significantly elevated hepatic CYP2E1 expression while simultaneously reducing hepatic phospho-acetyl CoA carboxylase (p-ACC) and carnitine palmitoyl-transferase 1 (CPT-1) levels. While EGCG markedly reversed the effect of ethanol on hepatic p-ACC and CPT-1 levels, it had no effect on the ethanol-induced elevation in CYP2E1 expression. EGCG prevents ethanol-induced hepatotoxicity and inhibits the development of a fatty liver. These effects were associated with improvements in p-ACC and CPT-1 levels. The use of EGCG might be useful in treating patients with an alcoholic fatty liver.     

Knockdown of differentiation antagonizing non-protein coding RNA exerts anti-tumor effect by up-regulating miR-214 in endometrial carcinoma

Differentiation antagonizing non-protein coding RNA (DANCR) is a valuable long noncoding RNA (lncRNA) that involves in the progress of various cancers. However, the functions of DANCR in endometrial carcinoma (EC) have not been validated. In the present study, we aimed to evaluate the roles of DANCR in EC and explore the underlying mechanism. Expression patterns of DANCR in EC specimens and normal control specimens were determined using qRT-PCR. DANCR was knocked down in EC cell lines (AN3CA and HEC-1B) through transfection with small interfering RNA (siRNA) targeting DANCR (si-DANCR). Cell proliferation was examined using the cell counting kit-8 (CCK-8) assay. Cell apoptosis was measured by flow cytometry. Online software starBase was used to predict the target gene of DANCR. Luciferase reporter assay was carried out to confirm the association between DANCR and the predicted target microRNA (miRNA). DANCR expression was up-regulated in EC tissues as compared to the normal control tissues. Knockdown of DANCR in AN3CA and HEC-1B cells markedly suppressed cell proliferation and induced cell apoptosis. miR-214 was found to be a target miRNA of DANCR and its expression was significantly decreased in EC tissues. Suppression of miR-214 abolished the effects of si-DANCR on cell proliferation and apoptosis in AN3CA and HEC-1B cells. DANCR played an important role in promoting tumorigenesis of EC via sponging miR-214. DANCR might serve as a therapeutic target for the treatment of EC.

     

Silencing of lncRNA UCA1 curbs proliferation and accelerates apoptosis by repressing SIRT1 signals by targeting miR‐204 in pediatric AML

The long noncoding RNA urothelial carcinoma‐associated 1 (UCA1) has been reported to sustain the proliferation of acute myeloid leukemia (AML) cells through downregulating cell cycle regulators p27^kip1. Yet, the foundational mechanism of UCA1 in AML pathologies remains unclear. Herein, we found an escalation of UCA1 expression and suppression of miR‐204 expression in pediatric AML patients and cells. UCA1 silencing suppressed cell proliferative abilities, promoted apoptotic rates, decreased Ki67, and increased cleaved caspase‐3 in AML cells. Moreover, UCA1 sponged miR‐204 and suppressed its expression. UCA1 overexpression inversed the miR‐204 suppressed proliferation and promoted apoptosis. UCA1 also boosted the expression of SIRT1, a miR‐204 target, via the sponging interaction. Furthermore, miR‐204 inhibited inducible nitric oxide synthase and cyclooxygenase‐2 expression, while UCA1 overexpression inversed the inhibitory effects in AML cells. Our findings concluded that UCA1 downregulation repressed cell proliferation and promoted apoptosis through inactivating SIRT1 signals by upregulating miR‐204 in pediatric AML.     

Long noncoding RNA UCA1 regulates HCV replication and antiviral response via miR-145-5p/SOCS7/IFN pathway

Hepatitis C virus (HCV) infection involves a variety of viral and host factors, which leads to the dysregulation of number of relevant genes including long noncoding RNAs (LncRNAs). LncRNA urothelial carcinoma-associated 1 (UCA1) has been reported to be upregulated in HCV-infected individuals. In a bid to elucidate on the contribution of UCA1 on HCV replication, we infected Huh7.5 cells with cell culture-derived HCV and found that UCA1 expression was elevated in time- and dose-dependent manners. Functionally, UCA1 knockdown by siRNA upregulated interferon (IFN) responses, thereby increasing the expression of interferon-stimulating genes (ISGs), and subsequently suppressing HCV replication. Bioinformatics analysis and experimental results indicated that, functioning as competitive endogenous RNA, UCA1 could sponge microRNA (miR)-145-5p, which targeted suppressor of cytokine signaling 7 (SOCS7) mRNA and subsequently mediated SOCS7 silencing. Moreover, SOCS7 protein exerted an inhibitory effect on IFN responses, thereby facilitating HCV replication. Taken together, at first, our findings demonstrate that UCA1 can counteract the expression of miR-145-5p, thereby upregulating the level of SOCS7, and in turn leading to the suppression of antiviral response in Huh7.5 cells.     

MicroRNA-449b-5p targets HMGB1 to attenuate hepatocyte injury in liver ischemia and reperfusion

MicroRNAs (miRNAs) participate in the pathological process of liver ischemia/reperfusion (I/R) injury. MiR-449b-5p is the target miRNA of high mobility group box 1 (HMGB1). Its role and molecular mechanism in liver I/R injury remain unidentified. In this study, we found a protective effect of miR-449b-5p against hepatic I/R injury. HMGB1 expression significantly increased, whereas miR-449b-5p dramatically decreased in patients after liver transplant and in L02 cells exposed to hypoxia/reoxygenation (H/R). A dual-luciferase reporter assay confirmed the direct interaction between miR-449b-5p and the 3′ untranslated region of HMGB1 messenger RNA. We also found that overexpression of miR-449b-5p significantly promoted cell viability and inhibited cell apoptosis of L02 cells exposed to H/R. Moreover, miR-449b-5p repressed HMGB1 protein expression and nuclear factor-κB (NF-κB) pathway activation in these L02 cells. In an in vivo rat model of hepatic I/R injury, overexpression of miR-449b-5p significantly decreased alanine aminotransferase and aspartate aminotransferase and inhibited the HMGB1/NF-κB pathway. Our study thus suggests that miR-449b-5p alleviated hepatic I/R injury by targeting HMGB1 and deactivating the NF-κB pathway, which may provide a novel and promising therapeutic target for hepatic I/R injury.     

磷脂酰胆碱对酒精性肝病保护作用的研究进展

酒精性肝病已逐渐成为第二大肝脏疾病,威胁着人类的健康。乙醇引发肝损伤的机制主要包括其代谢产物乙醛对肝细胞造成的毒性作用、自由基损伤及对肝脏线粒体产生的毒性作用等。已证实,磷脂酰胆碱对酒精肝损伤具有明显的保护作用。简要介绍了酒精肝病机制及磷脂酰胆碱对酒精性肝损伤的保护作用研究进展,以期为治疗酒精性肝病提供帮助。     

长非编码RNA SNAI3-AS1调控人软骨细胞C28/I2增殖能力及退变的机制研究

摘要 目的：为了探究长非编码RNA SNAI3-AS1（LncRNA SNAI3-AS1,即SNAI3-AS1）在骨性关节炎（osteoarthritis,OA）进展中的作用与机制。方法：通过全转录组测序筛选出在OA中差异表达的lncRNA SNAI3-AS1,并通过实时荧光定量PCR（qRT-PCR）检测SNAI3-AS1在软骨细胞退变模型中的表达情况。在软骨细胞C28/I2中分别转染SNAI3-AS1特异性siRNA或真核过表达质粒,分别敲低或过表达SNAI3-AS1,通过MTT、平板克隆形成和EdU掺入实验检测细胞增殖活力,Western Blot检测炎症和细胞外基质蛋白的表达情况。通过生物信息学网站预测SNAI3-AS1相互作用的miRNA和下游靶基因,并通过双荧光素酶报告基因和RIP实验进行验证。结果：相较于正常软骨细胞, SNAI3-AS1的表达水平在OA中显著下调。敲低正常软骨细胞中SNAI3-AS1的表达后,软骨细胞的增殖能力减弱并促进了软骨细胞的退变,而在OA模型的软骨细胞中过表达SNAI3-AS1后,软骨细胞的增殖活力加强并抑制了软骨细胞的退变。在机制上,SNAI3-AS1可充当竞争性内源性RNA（ceRNA）,经海绵吸附miR-2278间接上调PRELP,发挥促进软骨细胞增殖和抑制其退变的作用。结论：LncRNA SNAI3-AS1通过LncRNA SNAI3-AS1/ miR-2278/PRELP轴参与骨性关节炎的发生发展过程。     

LncRNA H19-elevated LIN28B promotes lung cancer progression through sequestering miR-196b

LncRNA H19 is involved in the development of multiple cancers. Here, we firstly provide new evidence that H19 can induce LIN28B, a conserved RNA binding protein, to accelerate lung cancer growth through sponging miR-196b. Abundance in LIN28B was observed in clinical lung cancer samples. A positive link was observed between H19 and LIN28B in clinical lung cancer samples. In lung cancer cells, H19 was capable of increasing LIN28B expression. Mechanistically, miR-196b directly targeted LIN28B to inhibit LIN28B expression. H19 was capable of promoting LIN28B expression through sequestering miR-196b. Functionally, H19-increased LIN28B conferred the cell proliferation of lung cancer. Our finding indicates that H19 depresses miR-196b to elevate LIN28B, resulting in accelerating cell proliferation in lung cancer.