Circulating miR‐145 is associated with plasma high‐sensitivity C‐reactive protein in acute ischemic stroke patients

Stroke is a major cerebrovascular disease threatening human health and life with high morbidity, disability and mortality. We aimed to find effective biomarkers for the early diagnosis on stroke. Nine previously reported stroke‐associated miRNAs (miR‐21, miR‐23a, miR‐29b, miR‐124, miR‐145, miR‐210, miR‐221, miR‐223 and miR‐483‐5p) were measured by quantitative real time‐PCR, and plasma high‐sensitivity C‐reactive protein (hs‐CRP) and serum interleukin 6 (IL‐6), the pro‐inflammation markers in brain injury, were examined by enzyme‐linked immunosorbent assay in 146 acute ischemic stroke patients and 96 healthy blood donors. We found that serum miR‐145 was significantly increased within 24 h after stroke onset and serum miR‐23a and miR‐221 were decreased in patients. Moreover, serum miR‐145 was strong positively correlated with plasma hs‐CRP and moderate positively correlated with serum IL‐6. Meanwhile, serum miR‐23a and miR‐221 were moderate negatively correlated with plasma hs‐CRP but not serum IL‐6. Importantly, the combination of hs‐CRP and serum miR‐145 gained a better sensitivity/spectivity for prediction of acute ischemia stroke (area under receiver operating characteristic curve from 0.794 to 0.896). Conclusively, our preliminary findings indicate that serum miR‐145 upregulated in acute ischemic stroke might be a new biomarker for acute ischemia stroke evaluation. Copyright © 2015 John Wiley & Sons, Ltd.     

INPP1 up‐regulation by miR‐27a contributes to the growth,migration and invasion of human cervical cancer

Inositol polyphosphate‐1‐phosphatase (INPP1) is an enzyme that is responsible for glycolysis and lipid metabolism. Here, we discovered that INPP1 expression was up‐regulated in CC tissues compared to that in adjacent normal tissues by RT‐qPCR. Inositol polyphosphate‐1‐phosphatase overexpression promoted and INPP1 knockdown suppressed cell viability, cellular migration/invasion and EMT in CC cells. To explore the mechanism of dysregulation, INPP1 was predicted to be a target of miR‐27a, and a pmiRGLO dual‐luciferase reporter assay showed that miR‐27a bound to the 3′ UTR of INPP1. RT‐qPCR revealed that miR‐27a was also up‐regulated and had a positive correlation with INPP1 expression in CC tissues. Furthermore, shR‐INPP1 could favour the malignant phenotype reversion induced by miR‐27a, suggesting that miR‐27a up‐regulates INPP1 to promote tumorigenic activities. Altogether, our findings show that the up‐regulation of INPP1 by miR‐27a contributes to tumorigenic activities and may provide a potential biomarker for CC.     

LUCAT1 contributes to MYRF‐dependent smooth muscle cell apoptosis and may facilitate aneurysm formation via the sequestration of miR‐199a‐5p

The overwhelming number of interrogations reveals the implication of long noncoding RNAs (lncRNAs) in diverse malignancies, little is unveiled about lncRNAs participation in the abdominal aortic aneurysm (AAA). The study aimed to monitor the role and responsible mechanism of LUCAT1 in AAA. The cellular function of LUCAT1 on smooth muscle cells (SMCs) proliferation and apoptosis were examined through the conduction of CCK‐8, EdU, TUNEL, and caspase‐3 activity assays. LUCAT1 depletion was observed to boost SMCs proliferation or suppress SMCs apoptosis. The opposite results on SMCs proliferation and apoptosis were achieved in response to LUCAT1 promotion. The abundance of LUCAT1 in the cytoplasm was ascertained by subcellular fractionation and FISH analyses on the basis of LncLocator prediction. The binding of LUCAT1 to miR‐199a‐5p predicted by DIANA and starbase was certified by luciferase reporter assay and RIP analysis. Besides, multiple prediction tools unveiled the interaction between miR‐199a‐5p and myelin regulatory factor (MYRF). Quantitative real‐time polymerase chain reaction uncovered the suppressive effect of miR‐199a‐5p and the positive regulation of LUCAT1 on MYRF expression. Rescue experiments revealed that LUCAT1 depletion pose suppression on SMCs apoptosis and MYRF elevation abrogated this suppression induced by LUCAT1 inhibition. These findings unmasked that the pro‐apoptosis impact of LUCAT1 in SMCs via directly targeting miR‐199a‐5p to elevate MYRF expression, which may provide valuable information on AAA prevention.     

Down‐regulation of miR‐10a‐5p in synoviocytes contributes to TBX5‐controlled joint inflammation

MicroRNAs are considered to play critical roles in the pathogenesis of human inflammatory arthritis, including rheumatoid arthritis (RA). The purpose of this study was to determine the relationship between miR‐10a‐5p and TBX5 in synoviocytes and evaluate their contribution to joint inflammation. The expression of miR‐10a‐5p and TBX5 in the synovium of RA and human synovial sarcoma cell line SW982 stimulated by IL‐1β was determined by RT‐qPCR and Western blotting. The direct interaction between miR‐10a‐5p and TBX5 3′UTR was determined by dual‐luciferase reporter assay in HeLa cells. Mimics and inhibitors of miR‐10a‐5p were transfected into SW982 cells. TBX5 was overexpressed by plasmid transfection or knocked down by RNAi. Proinflammatory cytokines and TLR3 and MMP13 expressions were determined by RT‐qPCR and Western blotting. Down‐regulated expression of miR‐10a‐5p and up‐regulation of TBX5 in human patients with RA were found compared to patients with OA. IL‐1β could reduce miR‐10a‐5p and increase TBX5 expression in SW982 cells in vitro. The direct target relationship between miR‐10a‐5p and 3′UTR of TBX5 was confirmed by luciferase reporter assay. Alterations of miR‐10‐5p after transfection with its mimic and inhibitor caused the related depression and re‐expression of TBX5 and inflammatory factors in SW982 cells. Overexpression of TBX5 after pCMV3‐TBX5 plasmid transfection significantly promoted the production of TLR3, MMP13 and various inflammatory cytokines, while this effect was rescued after knocking down of TBX5 with its specific siRNA. We conclude that miR‐10a‐5p in a relation with TBX5 regulates joint inflammation in arthritis, which would serve as a diagnostic and therapeutic target for RA treatment.     

UVA influenced the SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in human skin primary fibroblasts

Both SIRT1 and UVA radiation are involved in cellular damage processes such as apoptosis, senescence and ageing. MicroRNAs (miRNAs) have been reported to be closely related to UV radiation, as well as to SIRT1. In this study, we investigated the connections among SIRT1, UVA and miRNA in human skin primary fibroblasts. Our results showed that UVA altered the protein level of SIRT1 in a time point–dependent manner. Using miRNA microarray, bioinformatics analysis, we found that knocking down SIRT1 could cause up‐regulation of miR‐27a‐5p and the latter could down‐regulate SMAD2, and these results were verified by qRT‐PCR or Western blot. Furthermore, UVA radiation (5 J/cm²), knocking down SIRT1 or overexpression of miR‐27a‐5p led to increased expression of MMP1, and decreased expressions of COL1 and BCL2. We also found additive impacts on MMP1, COL1 and BCL2 under the combination of UVA radiation + Sirtinol (SIRT1 inhibitor), or UVA radiation + miR‐27a‐5p mimic. SIRT1 activator resveratrol could reverse damage changes caused by UVA radiation. Besides, absent of SIRT1 or overexpression of miR‐27a‐5p increased cell apoptosis and induced cell arrest in G2/M phase. Taken together, these results demonstrated that UVA could influence a novel SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in skin primary fibroblasts, and may provide potential therapeutic targets for UVA‐induced skin damage.     

MiR‐128‐1‐5p regulates tight junction induced by selenium deficiency via targeting cell adhesion molecule 1 in broilers vein endothelial cells

Vein endothelial cells (VECs) constitute an important barrier for macromolecules and circulating cells from the blood to the tissues, stabilizing the colloid osmotic pressure of the blood, regulating the vascular tone, and rapidly changing the intercellular connection, and maintaining normal physiological function. Tight junction has been discovered as an important structural basis of intercellular connection and may play a key role in intercellular connection injuries or vascular diseases and selenium (Se) deficiency symptoms. Hence, we replicated the Se‐deficient broilers model and detected the specific microRNA in response to Se‐deficient vein by using quantitative real time‐PCR (qRT‐PCR) analysis. Also, we selected miR‐128‐1‐5p based on differential expression in vein tissue and confirmed its target gene cell adhesion molecule 1 (CADM1) by the dual luciferase reporter assay and qRT‐PCR in VECs. We made the ectopic miR‐128‐1‐5p expression for the purpose of validating its function on tight junction. The result showed that miR‐128‐1‐5p and CADM1 were involved in the ZO‐1‐mediated tight junction, increased paracellular permeability, and arrested cell cycle. We presumed that miR‐128‐1‐5p and Se deficiency might trigger tight junction. Interestingly, miR‐128‐1‐5p inhibitor and fasudil in part hinder the destruction of the intercellular structure caused by Se deficiency. The miR‐128‐1‐5p/CADM1/tight junction axis provides a new avenue toward understanding the mechanism of Se deficiency, revealing a novel regulation model of tight junction injury in vascular diseases.     

Synthesis of some novel quinone diimine derivatives of benzo‐15‐crown‐5 for application in Hg2+ recognition

A series of novel fluoroionophore bearing derivatives of benzo‐15‐crown‐5 were synthesized by the amination of benzo‐15‐crown‐5 followed by condensation with different quinones in the presence of titanium tetrachloride (TiCl₄) and 1,4‐diazabicyclo‐[2.2.2]octane. The compounds were characterized by infrared, ¹H and ¹³C nuclear magnetic resonance, mass spectroscopy and elemental analysis. Absorption and fluorescence spectral characteristics of these compounds were studied. It was observed that the anthraquinone derivative was acting as an Hg²⁺ ion sensor. Copyright © 2013 John Wiley & Sons, Ltd.     

Selective recognition of thymidine homopolymer (poly T) oligonucleotide with cobalt(II)–4‐[(5‐chloro‐2‐pyridyl)azo]‐1,3‐diaminobenzene complex

The interactions of cobalt(II)–4‐[(5‐chloro‐2‐pyridyl)azo]‐1,3‐diaminobenzene (5‐Cl‐PADAB) complex with different kinds of homopolymer oligonucleotides in basic medium were investigated based on the measurements of resonance light scattering, UV–vis, circular dichroism spectra and dark field light‐scattering imaging. Experiments showed that only thymidine homopolymer (poly T) oligonucleotides with the length in the range of poly T₆ to poly T₁₈ could interact with the Co(II)–5‐Cl‐PADAB complex in alkaline conditions and cause evident color and spectral change. Thus, the binary complex of Co(II)–5‐Cl‐PADAB could be employed as a visual probe for selectively recognizing the poly T oligonucleotides. Copyright © 2009 John Wiley & Sons, Ltd.     

Caveolin‐1 down‐regulation is required for Wnt5a‐Frizzled 2 signalling in Ha‐RasV12‐induced cell transformation

Caveolin‐1 (Cav1) is down‐regulated during MK4 (MDCK cells harbouring inducible Ha‐Ras^V12 gene) transformation by Ha‐Ras^V12. Cav1 overexpression abrogates the Ha‐Ras^V12‐driven transformation of MK4 cells; however, the targeted down‐regulation of Cav1 is not sufficient to mimic this transformation. Cav1‐silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction‐related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I‐CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha‐Ras^V12‐inducing MK4 cells increased exosome‐like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I‐CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I‐CM (MK4+I‐EXs). Wnt5a, a downstream product of Ha‐Ras^V12, was markedly secreted by MK4+I‐CM and MK4+I‐EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha‐Ras^V12‐ and MK4+I‐CM‐induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down‐regulation, either by Ha‐Ras^V12 or targeted shRNA, increased frizzled‐2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I‐EXs in MDCK cells. These data suggest that Cav1‐dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha‐Ras^V12‐Wnt5a‐Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha‐Ras^V12‐driven cell transformation.     

Nitric oxide‐mediated regulation of ‐amyloid clearance via alterations of MMP‐9/TIMP‐1

Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2^?/? mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.     

MiR‐133a‐3p Inhibits Oral Squamous Cell Carcinoma (OSCC) Proliferation and Invasion by Suppressing COL1A1

The aim of our study was to investigate the effects of miR‐133a‐3p on human oral squamous cell carcinoma (OSCC) cells by regulating gene COL1A1. OSCC tissues, adjacent tongue epithelial tissues, the immortalized oral epithelial cell line HIOEC, and OSCC cell lines (CAL‐27, TCA‐8113, SCC‐4, SCC‐9, and SCC‐15) were used in this research. Quantitative real‐time PCR (RT‐qPCR) was employed to determine the expression of miR‐133a‐3p and COL1A1. Dual luciferase reporter gene assay and Western blot were applied to verify the binding relationship between miR‐133a‐3p and COL1A1. Functional assays were also conducted in this study, including CCK‐8 assay, colony formation assay, flow cytometry analysis as well as Transwell assay. MiR‐133a‐3p was found low‐expressed both in OSCC tissues and cells lines compared with normal tissues and cell line, respectively, whereas COL1A1 was just the opposite. The over‐expression of miR‐133a‐3p or the down‐regulation of COL1A1 suppressed the proliferation, invasion, and mitosis of OSCC cells, whereas simultaneous down‐regulation of miR‐133a‐3p and up‐regulation of COL1A1 led to no significant alteration of cell activities. MiR‐133a‐3p could inhibit the proliferation and migration of OSCC cells through directly targeting COL1A1 and reducing its expression. J. Cell. Biochem. 119: 338–346, 2018. © 2017 Wiley Periodicals, Inc.     

MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.