Long noncoding RNA lnc-sox5 modulates CRC tumorigenesis by unbalancing tumor microenvironment

<p>Long non-coding RNAs (LncRNAs) have been recently regarded as systemic regulators in multiple biologic processes including tumorigenesis. In this study, we observed the expression of lncRNA lnc-sox5 was significantly increased in colorectal cancer (CRC). Despite the CRC cell growth, cell cycle and cell apoptosis was not affected by lnc-sox5 knock-down, lnc-sox5 knock-down suppressed CRC cell migration and invasion. In addition, xenograft animal model suggested that lnc-sox5 knock-down significantly suppressed the CRC tumorigenesis. Our results also showed that the expression of indoleamine 2,3-dioxygenase 1 (IDO1) was significantly reduced by lnc-sox5 knock-down and therefore modulated the infiltration and cytotoxicity of CD3⁺CD8⁺T cells. Taken together, these results suggested that lnc-sox5 unbalances tumor microenvironment to regulate colorectal cancer progression.</p

Impacts of Nb on grain refinement in a simulated coarse-grained-heat-affected-zone of ultra-high-strength steels

The impact of Nb contents on grain refinement in a simulated coarse-grained heated-affected zone (CGHAZ) in steels was investigated. The austenite grain grew in multiple directions and then impinged against each other during the α→γ transformation. Subsequently, austenite grains coarsened via the coalescence of small grains and boundary migration. With the increased Nb content, the austenite in CGHAZ became coarser due to the decreased quantity and increased size of Nb precipitates. Furthermore, the austenite decomposed into bainite during the cooling process. Compared with the low-Nb specimen, the crystallographic grain size in CGHAZ of high-Nb specimen is slightly coarser, relating to the coarser prior austenite grain. However, higher Nb content delays the bainite transformation start temperature, leading to multiple variants selection of bainite, increasing the refinement ratio.</p

MiR-21 Simultaneously Regulates ERK1 Signaling in HSC Activation and Hepatocyte EMT in Hepatic Fibrosis

<div><p>Background</p><p>MicroRNA-21 (miR-21) plays an important role in the pathogenesis and progression of liver fibrosis. Here, we determined the serum and hepatic content of miR-21 in patients with liver cirrhosis and rats with dimethylnitrosamine-induced hepatic cirrhosis and examined the effects of miR-21 on <i>SPRY2</i> and <i>HNF4α</i> in modulating ERK1 signaling in hepatic stellate cells (HSCs) and epithelial-mesenchymal transition (EMT) of hepatocytes.</p><p>Methods</p><p>Quantitative RT-PCR was used to determine miR-21 and the expression of <i>SPRY2, HNF4α</i> and other genes. Immunoblotting assay was carried out to examine the expression of relevant proteins. Luciferase reporter assay was performed to assess the effects of miR-21 on its predicted target genes <i>SPRY2</i> and <i>HNF4α</i>. Primary HSCs and hepatocytes were treated with miR-21 mimics/inhibitors or appropriate adenoviral vectors to examine the relation between miR-21 and <i>SPRY2</i> or <i>HNF4α.</i></p><p>Results</p><p>The serum and hepatic content of miR-21 was significantly higher in cirrhotic patients and rats. <i>SPRY2</i> and <i>HNF4α</i> mRNA levels were markedly lower in the cirrhotic liver. MiR-21 overexpression was associated with enhanced ERK1 signaling and EMT in liver fibrosis. Luciferase assay revealed suppressed <i>SPRY2</i> and <i>HNF4α</i> expression by miR-21. Ectopic miR-21 stimulated ERK1 signaling in HSCs and induced hepatocyte EMT by targeting <i>SPRY2</i> or <i>HNF4α</i>. Downregulating miR-21 suppressed ERK1 signaling, inhibited HSC activation, and blocked EMT in TGFβ1-treated hepatocytes.</p><p>Conclusions</p><p>MiR-21 modulates ERK1 signaling and EMT in liver fibrosis by regulating <i>SPRY2</i> and <i>HNF4α</i> expression. MiR-21 may serve as a potentially biomarker as well as intervention target for hepatic cirrhosis.</p></div

Clinical features of the patients providing cirrhotic liver tissues.

<p>Clinical features of the patients providing cirrhotic liver tissues.</p

MiR-21 and <i>HNF4α</i> mutually modulate the expression of each other in regulating EMT of primary hepatocytes.

<p>Primary rat hepatocytes were treated with miR-21 mimics for 48 h. MiR-21 and the mRNA transcript levels of <i>HNF4α</i>, EMT associated genes <i>E-cadherin</i>, <i>vimentin</i> and <i>slug</i> (A), <i>MMP-2</i> and <i>MMP-9</i>, fibrogenesis-associated genes α<i>-SMA</i> and <i>TIMP1</i> and liver specific genes <i>ALB</i>, <i>CYP1a2</i> and <i>AFP</i> (B) were examined by quantitative RT-PCR. <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i>, ALB, <i>MMP-2</i> and <i>TIMP1</i> were examined by Western blotting assays (C). GADPH was used as a loading control. Blots representative of at least three independent experiments are shown. Primary hepatocytes were treated with TGFβ1 for 48 h followed by AdHNF4α infection 48 h later (D). Primary hepatocytes were transfected with miR-21 mimics followed by AdHNF4α (E) or AdshHNF4α (F) infection 48 h later. MiR-21 and the mRNA transcript levels of <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i> were then examined by quantitative RT-PCR. Each value in (A), (B), (D), (E) and (F) represents the mean with the SD (error bars) for triplicate samples of at least three independent experiments and miR-21 expression was normalized against <i>Rnu6-2</i> and mRNA expression was normalized against <i>β-actin</i>. *<i>P</i><0.05 or **<i>P</i><0.01 vs. controls.</p

MiR-21 inhibitors target ERK1 signaling and <i>SPRY2</i> in HSC activation and block EMT of TGFβ1-treated hepatocytes by targeting <i>HNF4α</i>.

<p>Primary HSCs were treated with TGFβ1 for 5 d followed by transfection with miR-21 inhibitors. MiR-21 and the mRNA transcript levels of <i>SRPY2</i>, <i>ERK1</i> and <i>RSK2</i> (A), <i>α-SMA</i>, <i>TIMP1</i>, <i>MMP-2</i> and <i>MMP-13</i> (B) were examined by quantitative RT-PCR. Immunoblotting assays were done to detect the expression of <i>SPRY2</i>, <i>phospho-ERK1</i>, <i>phospho-ERK2</i> and <i>RSK2</i> (C). HSC-T6 cells were transfected with miR-21 inhibitors. MiR-21 and the mRNA transcript levels of <i>SRPY2</i>, <i>ERK1</i> and <i>RSK2</i>, <i>MMP-9</i>, <i>MMP-13</i>, fibrogenesis-associated genes <i>TGFβ1</i>, α<i>-SMA</i>, <i>Col I</i>, <i>Col III</i> and <i>TIMP1</i> were examined by quantitative RT-PCR (D, E). Immunoblotting assays were done to detect the expression of <i>SPRY2</i>, <i>phospho-ERK1</i>, <i>phospho-ERK2</i> and <i>RSK2</i>, <i>Col I</i> and <i>TIMP1</i> (F). HSC-T6 cells were transfected with miR-21 inhibitors and transwell cell migration assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108005#s2" target="_blank">methods</a> (G, H). Hepatocytes were treated with TGFβ1 for 48 h followed by transfection with miR-21 inhibitors. MiR-21 and the mRNA transcript levels of <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i>, <i>MMP-2</i>, <i>MMP-9</i> and α<i>-SMA</i>, liver specific genes <i>CYP1a2</i> and <i>AFP</i> were examined by quantitative RT-PCR (I, J). Immunoblotting assays were done to detect the expression of <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i>, <i>MMP2</i>, <i>α-SMA</i> and <i>TIMP1</i> (K). GADPH was used as a loading control. Blots representative of at least three independent experiments are shown. MiR-21 expression was normalized against <i>Rnu6-2</i> and mRNA expression was normalized against <i>β-actin</i>. Each value in (A), (B), (D), (E), (H), (I) and (J) represents the mean with the SD (error bars) for triplicate samples of at least three independent experiments. *<i>P</i><0.05 or **<i>P</i><0.01 vs. controls.</p

Liver fibrosis is associated with broad changes in the expression of miR-21 and its targeted genes, ERK1 signaling and EMT-associated genes.

<p>Serum miR-21 contents in cirrhotic patients (n = 20) and normal subjects (n = 20) (A) and in rats with dimethylnitrosamine-induced liver cirrhosis (B). MiR-21 expression was normalized against miR-238 in serum. **<i>P</i><0.01 vs. normal controls. Quantitative RT-PCR examination of the expression of miR-21 and its targeted genes <i>SPRY2</i> and <i>HNF4α</i>, <i>ERK1</i> and its downstream target <i>RSK2</i>, and epithelial-mesenchymal transition (EMT)-associated genes <i>E-cadherin</i> and <i>vimentin</i> in the liver tissues of cirrhotic patients (n = 7) (C) and rats (n = 10) (D). *<i>P</i><0.05 and **<i>P</i><0.01 vs. normal subjects or rats. Primary HSCs were treated with TGFβ1 for 7 days (E) and primary hepatocytes for 48 h (F) as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108005#s2" target="_blank">methods</a>. Quantitative RT-PCR examination of the expression of miR-21 and its targeted genes <i>SPRY2</i> and <i>HNF4α, ERK1</i> and <i>RSK2</i>, <i>E-cadherin</i>, <i>vimentin</i> and <i>MMP-9</i>, liver fibrogenesis-associated genes α<i>-SMA</i> and <i>TIMP1</i>, and liver specific genes <i>ALB</i> and <i>CYP1a2</i>. MiR-21 expression was normalized against <i>Rnu6-2</i> and mRNA expression was normalized against <i>β-actin</i>. *<i>P</i><0.05 and **<i>P</i><0.01 vs. non-stimulated HSCs or hepatocytes. Each value represents the mean with the SD (error bars) for triplicate samples.</p