The role of LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis in Memantine mediated protective effects on blood‐brain barrier in AD microenvironment

The dysfunction of the blood‐brain barrier (BBB) is one of the main pathological features of Alzheimer's disease (AD). Memantine (MEM), an N‐methyl‐d ‐aspartate (NMDA) receptor antagonist, has been reported that been used widely for AD therapy. This study was performed to demonstrate the role of the MEM in regulating BBB permeability in AD microenvironment as well as its possible mechanisms. The present study showed that LINC00094 was dramatically increased in Abeta_1‐42‐incubated microvascular endothelial cells (ECs) of BBB model in vitro. Besides, it was decreased in MEM‐incubated ECs. Silencing LINC00094 significantly decreased BBB permeability, meanwhile up‐regulating the expression of ZO‐1, occludin and claudin‐5. Furthermore, silencing LINC00094 enhance the effect of MEM on decreasing BBB permeability in AD microenvironment. The analysis of the mechanism demonstrated that reduction of LINC00094 inhibited Endophilin‐1 expression by up‐regulating miR‐224‐4p/miR‐497‐5p, promoted the expression of ZO‐1, occludin and claudin‐5, and ultimately alleviated BBB permeability in AD microenvironment. Taken together, the present study suggests that the MEM/LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis plays a crucial role in the regulation of BBB permeability in AD microenvironment. Silencing LINC00094 combined with MEM provides a novel target for the therapy of AD.     

Atractylenolide II reverses the influence of lncRNA XIST/miR‐30a‐5p/ROR1 axis on chemo‐resistance of colorectal cancer cells

This investigation was conducted to elucidate whether atractylenolide II could reverse the role of lncRNA XIST/miR‐30a‐5p/ROR1 axis in modulating chemosensitivity of colorectal cancer cells. We totally collected 294 pairs of colorectal cancer tissues and adjacent normal tissues and also purchased colorectal cancer cell lines and human embryonic kidney cell line. 5‐fluorouracil, cisplatin, mitomycin and adriamycin were designated as the chemotherapies for colorectal cell lines, and atractylenolides were arranged as the Chinese drug. The expressions of XIST, miR‐30a‐5p and ROR1 were quantified with aid of qRT‐PCR or Western blot, and luciferase reporter gene assay was implemented to determine the relationships among XIST, miR‐30a‐5p and ROR1. Our results demonstrated that XIST and ROR1 expressions were dramatically up‐regulated, yet miR‐30a‐5p expression was down‐regulated within colorectal cancer tissues (P < 0.05). The overexpressed XIST and ROR1, as well as under‐expressed miR‐30a‐5p, were inclined to promote viability and proliferation of colorectal cells under the influence of chemo drugs (P < 0.05). In addition, XIST could directly target miR‐30a‐5p, and ROR1 acted as the targeted molecule of miR‐30a‐5p. Interestingly, atractylenolides not only switched the expressions of XIST, miR‐30a‐5p and ROR1 within colorectal cancer cells but also significantly intensified the chemosensitivity of colorectal cancer cells (P < 0.05). Finally, atractylenolide II was discovered to slow down the viability and proliferation of colorectal cancer cells (P < 0.05). In conclusion, the XIST/miR‐30a‐5p/ROR1 axis could be deemed as pivotal markers underlying colorectal cancer, and administration of atractylenolide II might improve the chemotherapeutic efficacy for colorectal cancer.     

LncRNA SNHG1 functions as a ceRNA to antagonize the effect of miR‐145a‐5p on the down‐regulation of NUAK1 in nasopharyngeal carcinoma cell

How lncRNA SNHG1 influences the aggressiveness of nasopharyngeal carcinoma cells as well as the underlying mechanism was studied. The lncRNA differences were analysed by GSE12452 gene microarray. The expression of SNHG1, MiR‐145‐5p and NUAK1 was identified by qRT‐PCR and western blot. Transfection was conducted to construct nasopharyngeal carcinoma cells with different expressions of SNHG1, miR‐145‐5p and NUAK1. Dual‐luciferase reporter assay was performed to explore the relationship between SNHG1, miR‐145‐5p and NUAK1. Wound‐healing assay and transwell invasion experiments were employed to study changes in cell migration capacity and cell invasion, respectively. Tumour xenografts were performed to observe lung metastasis of nude mice inoculated with transfected CNE cells. SNHG1 is highly expressed in nasopharyngeal carcinoma tissues and in cell lines. Down‐regulation of SNHG1 facilitated the expression of miR‐145‐5p and further suppressed the level of NAUK1 in CNE and HNE‐1 cells. Silencing of SNHG1, up‐regulation of miR‐145‐5p and inhibition of NAUK1 by relative transfection all attenuated the aggressiveness of CNE and HNE‐1 cells both in vivo and in vitro. Moreover, the impaired cell migration and invasion by SNHG1 siRNA could be rescued by cotransfection of miR‐145‐5p in CNE and HNE‐1 cells. LncRNA SNHG1 promoted the expression of NUAK1 by down‐regulating miR‐145‐5p and thus promoted the aggressiveness of nasopharyngeal carcinoma cells through AKT signalling pathway and induced epithelial‐mesenchymal transition (EMT).     

MicroRNA‐503 promotes angiotensin II‐induced cardiac fibrosis by targeting Apelin‐13

Cardiac fibrosis is a major cause of heart failure. MicroRNAs (miRs) are important epigenetic regulators of cardiac function and cardiovascular diseases, including cardiac fibrosis. This study aimed to explore the role of miR‐503 and its mechanisms in regulating cardiac fibrosis. miR‐503 was found up‐regulated in the mouse LV tissues subjected to transverse aortic constriction (TAC) and in neonatal cardiac fibroblasts (CFs) cultured with Angiotension II. The role of miR‐503 in regulating CF cell proliferation and/or collagen production in mice neonatal CFs were determined using an MTT assay and RT‐PCR respectively. Forced expression of miR‐503 increased the cellular proliferation and collagen production in mice neonatal CFs. The effects were abrogated by cotransfection with AMO‐503 (a specific inhibitor of miR‐503). Injection of antagomiR‐503 elevated cardiac function and inhibited the expression of connective tissue growth factor (CTGF) and transforming growth factor (TGF)‐β in the TAC mice. Additional analysis revealed that Apelin‐13 is a direct target of miR‐503, as the overexpression of miR‐503 decreased the protein and mRNA expression levels of Apelin‐13. In the CFs with pre‐treatment of AngII, we transfected AMO‐503 into the cells treated with siRNA‐APLN. siRNA‐APLN abolished the effects of AMO‐503 on the production of collagen I and III and the expression of TGF‐β and CTGF. Furthermore, pre‐treatment of CFs with Apelin‐13 (1–100 nmol/l) inhibited angiotensin II‐mediated collagen production and activation of CTGF and TGF‐β. So we conclude that miR‐503 promotes cardiac fibrosis via miR‐503‐Apelin‐13‐TGF‐β‐CTGF‐collagen production pathway. Thus, miR‐503 is a promising therapeutic target for reducing cardiac fibrosis.     

LncRNA HOXA11‐AS regulates calcium oxalate crystal–induced renal inflammation via miR‐124‐3p/MCP‐1

Long noncoding RNA (lncRNA) has been suggested to play an important role in a variety of diseases over the past decade. In a previous study, we identified a novel lncRNA, termed HOXA11‐AS, which was significantly up‐regulated in calcium oxalate (CaOx) nephrolithiasis. However, the biological function of HOXA11‐AS in CaOx nephrolithiasis remains poorly defined. Here, we demonstrated that HOXA11‐AS was significantly up‐regulated in CaOx nephrolithiasis both in vivo and in vitro. Gain‐/loss‐of‐function studies revealed that HOXA11‐AS inhibited proliferation, promoted apoptosis and aggravated cellular damage in HK‐2 cells exposed to calcium oxalate monohydrate (COM). Further investigations showed that HOXA11‐AS regulated monocyte chemotactic protein 1 (MCP‐1) expression in HK‐2 cell model of CaOx nephrolithiasis. In addition, online bioinformatics analysis and dual‐luciferase reporter assay results showed that miR‐124‐3p directly bound to HOXA11‐AS and the 3'UTR of MCP‐1. Furthermore, rescue experiment results revealed that HOXA11‐AS functioned as a competing endogenous RNA to regulate MCP‐1 expression through sponging miR‐124‐3p and that overexpression of miR‐124‐3p restored the inhibitory effect of proliferation, promotion effects of apoptosis and cell damage induced by HOXA11‐AS overexpression. Taken together, HOXA11‐AS mediated CaOx crystal–induced renal inflammation via the miR‐124‐3p/MCP‐1 axis, and this outcome may provide a good potential therapeutic target for nephrolithiasis.     

Down‐regulated lncRNA SBF2‐AS1 in M2 macrophage‐derived exosomes elevates miR‐122‐5p to restrict XIAP,thereby limiting pancreatic cancer development

Evidence has indicated that M2 macrophages promote the progression of cancers, but few focus on the ability of M2 macrophage‐derived exosomes in pancreatic cancer (PC). This study aims to explore how M2 macrophages affect malignant phenotypes of PC through regulating long non‐coding RNA SET‐binding factor 2 antisense RNA 1 (lncRNA SBF2‐AS1)/microRNA‐122‐5p (miR‐122‐5p)/X‐linked inhibitor of apoptosis protein (XIAP) axis. THP‐1 cells were transformed into M1 macrophages by lipopolysaccharide and interferon‐γ treatment, and into M2 macrophages after interleukin‐4 treatment. The PANC‐1 PC cell line with the largest lncRNA SBF2‐AS1 expression was selected, and M2 macrophage‐derived exosomes were isolated and identified. A number of assays were applied for the examination of lncRNA SBF2‐AS1 expression, PC cell biological functions and subcellular localization of lncRNA SBF2‐AS1. XIAP expression was detected, along with the interaction among lncRNA SBF2‐AS1, miR‐122‐5p and XIAP. M2 macrophage exosomal lncRNA SBF2‐AS1 expression's effects on the tumorigenic ability of PANC‐1 cells in nude mice were also investigated. M2 macrophage‐derived exosomes promoted progression of PC cells. Overexpressed lncRNA SBF2‐AS1 promoted progression of PC cells. LncRNA SBF2‐AS1 was found to act as a competing endogenous RNA to repress miR‐122‐5p and up‐regulate XIAP. Constrained lncRNA SBF2‐AS1 in M2 macrophage‐derived exosomes contributed to restraining tumorigenic ability of PC cells. Collectively, our study reveals that constrained lncRNA SBF2‐AS1 in M2 macrophage‐derived exosomes increases miR‐122‐5p expression to restrain XIAP expression, which further inhibits PC progression.     

Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p

Long non‐coding RNAs (lncRNAs) could regulate growth and metastasis of hepatocellular carcinoma (HCC). In this study, we aimed to investigate the mechanism of lncRNA F11‐AS1 in hepatitis B virus (HBV)–related HCC. The relation of lncRNA F11‐AS1 expression in HBV‐related HCC tissues to prognosis was analysed in silico. Stably HBV‐expressing HepG2.2.15 cells were established to explore the regulation of lncRNA F11‐AS1 by HBx protein, as well as to study the effects of overexpressed lncRNA F11‐AS1 on proliferation, migration, invasion and apoptosis in vitro. Subsequently, the underlying interactions and roles of lncRNA F11‐AS1/miR‐211‐5p/NR1I3 axis in HBV‐related HCC were investigated. Additionally, the influence of lncRNA F11‐AS1 and miR‐211‐5p on tumour growth and metastasis capacity of HepG2.2.15 cells were studied on tumour‐bearing nude mice. Poor expression of lncRNA F11‐AS1 was correlated with poor prognosis in patients with HBV‐related HCC, and its down‐regulation was caused by the HBx protein. lncRNA F11‐AS1 was proved to up‐regulate the NR1I3 expression by binding to miR‐211‐5p. Overexpression of lncRNA F11‐AS1 reduced the proliferation, migration and invasion, yet induced apoptosis of HepG2.2.15 cells in vitro, which could be abolished by overexpression of miR‐211‐5p. Additionally, either lncRNA F11‐AS1 overexpression or miR‐211‐5p inhibition attenuated the tumour growth and metastasis capacity of HepG2.2.15 cells in vivo. Collectively, lncRNA F11‐AS1 acted as a modulator of miR‐211‐5p to positively regulate the expression of NR1I3, and the lncRNA F11‐AS1/miR‐211‐5p/NR1I3 axis participated in HBV‐related HCC progression via interference with the cellular physiology of HCC.     

LncRNA‐SULT1C2A regulates Foxo4 in congenital scoliosis by targeting rno‐miR‐466c‐5p through PI3K‐ATK signalling

Congenital scoliosis (CS) is the result of anomalous vertebrae development, but the pathogenesis of CS remains unclear. Long non‐coding RNAs (lncRNAs) have been implicated in embryo development, but their role in CS remains unknown. In this study, we investigated the role and mechanisms of a specific lncRNA, SULT1C2A, in somitogenesis in a rat model of vitamin A deficiency (VAD)‐induced CS. Bioinformatics analysis and quantitative real‐time PCR (qRT‐PCR) indicated that SULT1C2A expression was down‐regulated in VAD group, accompanied by increased expression of rno‐miR‐466c‐5p but decreased expression of Foxo4 and somitogenesis‐related genes such as Pax1, Nkx3‐2 and Sox9 on gestational day (GD) 9. Luciferase reporter and small interfering RNA (siRNA) assays showed that SULT1C2A functioned as a competing endogenous RNA to inhibit rno‐miR‐466c‐5p expression by direct binding, and rno‐miR‐466c‐5p inhibited Foxo4 expression by binding to its 3′ untranslated region (UTR). The spatiotemporal expression of SULT1C2A, rno‐miR‐466c‐5p and Foxo4 axis was dynamically altered on GDs 3, 8, 11, 15 and 21 as detected by qRT‐PCR and northern blot analyses, with parallel changes in Protein kinase B (AKT) phosphorylation and PI3K expression. Taken together, our findings indicate that SULT1C2A enhanced Foxo4 expression by negatively modulating rno‐miR‐466c‐5p expression via the PI3K‐ATK signalling pathway in the rat model of VAD‐CS. Thus, SULT1C2A may be a potential target for treating CS.     

SCFFbx2‐E3‐ligase‐mediated degradation of BACE1 attenuates Alzheimer’s disease amyloidosis and improves synaptic function

BACE1 (β‐secretase) plays a central role in the β‐amyloidogenesis of Alzheimer’s disease (AD). The ubiquitin–proteasome system, a major intracellular protein quality control system, has been implicated recently in BACE1 metabolism. We report that the SCF^Fbx2‐E3 ligase is involved in the binding and ubiquitination of BACE1 via its Trp 280 residue of F‐box‐associated domain. Physiologically, we found that Fbx2 was expressed in various intracellular organelles in brain neurons and that BACE1 is colocalized with Fbx2 and the amyloid precursor protein (APP), mainly at the early endosome and endoplasmic reticulum. The former are believed to be the major intracellular compartments where the APP is cleaved by BACE1 and β‐amyloid is produced. Importantly, we found that overexpression of Fbx2 in the primary cortical and hippocampal neurons derived from Tg2576 transgenic mice significantly promoted BACE1 degradation and reduced β‐amyloid production. In the search for specific endogenous modulators of Fbx2 expression, we found that PPARγ coactivator‐1α (PGC‐1α) was capable of promoting the degradation of BACE1 through a mechanism involving Fbx2 gene expression. Interestingly, we found that the expression of both Fbx2 and PGC‐1α was significantly decreased in the brains of aging Tg2576 mice. Our in vivo studies using a mouse model of AD revealed that exogenous adenoviral Fbx2 expression in the brain significantly decreased BACE1 protein levels and activity, coincidentally reducing β‐amyloid levels and rescuing synaptic deficits. Our study is the first to suggest that promoting Fbx2 in the brain may represent a novel strategy for the treatment of AD.     

LncRNA‐DANCR contributes to lung adenocarcinoma progression by sponging miR‐496 to modulate mTOR expression

Long non‐coding RNAs (lncRNAs) have emerged as new and important regulators of pathological processes including tumour development. In this study, we demonstrated that differentiation antagonizing non‐protein coding RNA (DANCR) was up‐regulated in lung adenocarcinoma (ADC) and that the knockdown of DANCR inhibited tumour cell proliferation, migration and invasion and restored cell apoptosis rescued; cotransfection with a miR‐496 inhibitor reversed these effects. Luciferase reporter assays showed that miR‐496 directly modulated DANCR; additionally, we used RNA‐binding protein immunoprecipitation (RIP) and RNA pull‐down assays to further confirm that the suppression of DANCR by miR‐496 was RISC‐dependent. Our study also indicated that mTOR was a target of miR‐496 and that DANCR could modulate the expression levels of mTOR by working as a competing endogenous RNA (ceRNA). Furthermore, the knockdown of DANCR reduced tumour volumes in vivo compared with those of the control group. In conclusion, this study showed that DANCR might be an oncogenic lncRNA that regulates mTOR expression through directly binding to miR‐496. DANCR may be regarded as a biomarker or therapeutic target for ADC.     

let‐7e replacement yields potent anti‐arrhythmic efficacy via targeting beta 1‐adrenergic receptor in rat heart

Beta‐adrenoceptor (β‐AR) exerts critical regulation of cardiac function. MicroRNAs (miRNAs) are potentially involved in a variety of biological and pathological processes. This study aimed to investigate the role of miRNA let‐7e in the up‐regulation of β₁‐AR and arrhythmogenesis in acute myocardial infarction (AMI) in rats. β₁‐AR expression was significantly up‐regulated and let‐7a, c, d, e and i were markedly down‐regulated in the infarcted heart after 6 and 24 hrs myocardial infarction. Forced expression of let‐7e suppressed β₁‐AR expression at the protein level, without affecting β₁‐AR mRNA level, in neonatal rat ventricular cells (NRVCs). Silencing of let‐7e by let‐7e antisense inhibitor (AMO‐let‐7e) enhanced β₁‐AR expression at the protein level in NRVCs. Administration of the lentivirus vector containing precursor let‐7e (len‐pre‐let‐7e) significantly inhibited β₁‐AR expression in rats, whereas len‐AMO‐let‐7e up‐regulated β₁‐AR relative to the baseline control level, presumably as a result of depression of tonic inhibition of β₁‐AR by endogenous let‐7e. Len‐negative control (len‐NC) did not produce significant influence on β₁‐AR expression. Len‐pre‐let‐7e also profoundly reduced the up‐regulation of β₁‐AR induced by AMI and this effect was abolished by len‐AMO‐let‐7e. Importantly, len‐pre‐let‐7e application significantly reduced arrhythmia incidence after AMI in rats and its anti‐arrhythmic effect was cancelled by len‐AMO‐let‐7e. Notably, anti‐arrhythmic efficacy of len‐pre‐let‐7e was similar to propranolol, a non‐selective β‐AR blocker and metoprolol, a selective β₁‐AR blocker. Down‐regulation of let‐7e contributes to the adverse increase in β₁‐AR expression in AMI and let‐7e supplement may be a new therapeutic approach for preventing adverse β₁‐AR up‐regulation and treating AMI‐induced arrhythmia.     

Sl‐lncRNA15492 interacts with Sl‐miR482a and affects Solanum lycopersicum immunity against Phytophthora infestans

Long non‐coding RNAs (lncRNAs) are involved in the resistance of plants to infection by pathogens via interactions with microRNAs (miRNAs). Long non‐coding RNAs are cleaved by miRNAs to produce phased small interfering RNAs (phasiRNAs), which, as competing endogenous RNAs (ceRNAs), function as decoys for mature miRNAs, thus inhibiting their expression, and contain pre‐miRNA sequences to produce mature miRNAs. However, whether lncRNAs and miRNAs mediate other molecular mechanisms during plant resistance to pathogens is unknown. In this study, as a positive regulator, Sl‐lncRNA15492 from tomato (Solanum lycopersicum Zaofen No. 2) plants affected tomato resistance to Phytophthora infestans. Gain‐ and loss‐of‐function experiments and RNA ligase‐mediated 5′‐amplification of cDNA ends (RLM‐5′ RACE) also revealed that Sl‐miR482a was negatively involved in tomato resistance by targeting Sl‐NBS‐LRR genes and that silencing of Sl‐NBS‐LRR1 decreased tomato resistance. Sl‐lncRNA15492 inhibited the expression of mature Sl‐miR482a, whose precursor was located within the antisense sequence of Sl‐lncRNA15492. Further degradome analysis and additional RLM‐5′ RACE experiments verified that mature Sl‐miR482a could also cleave Sl‐lncRNA15492. These results provide a mechanism by which lncRNAs might inhibit precursor miRNA expression through antisense strands of lncRNAs, and demonstrate that Sl‐lncRNA15492 and Sl‐miR482a mutually inhibit the maintenance of Sl‐NBS‐LRR1 homeostasis during tomato resistance to P. infestans.     

LncRNA MEG3 inhibits the progression of prostate cancer by modulating miR‐9‐5p/QKI‐5 axis

This study was designed to detecting the influences of lncRNA MEG3 in prostate cancer. Aberrant lncRNAs expression profiles of prostate cancer were screened by microarray analysis. The qRT‐PCR and Western blot were employed to investigating the expression levels of lncRNA MEG3, miR‐9‐5p and QKI‐5. The luciferase reporter assay was utilized to testifying the interactions relationship among these molecules. Applying CCK‐8 assay, wound healing assay, transwell assay and flow cytometry in turn, the cell proliferation, migration and invasion abilities as well as apoptosis were measured respectively. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer tissues and cells and could inhibit the expression of miR‐9‐5p, whereas miR‐9‐5p down‐regulated QKI‐5 expression. Overexpressed MEG3 and QKI‐5 could decrease the abilities of proliferation, migration and invasion in prostate cancer cells effectively and increased the apoptosis rate. On the contrary, miR‐9‐5p mimics presented an opposite tendency in prostate cancer cells. Furthermore, MEG3 inhibited tumour growth and up‐regulated expression of QKI‐5 in vivo. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer and impacted the abilities of cell proliferation, migration and invasion, and cell apoptosis rate, this regulation relied on regulating miR‐9‐5p and its targeting gene QKI‐5.     

Schisandrin B down‐regulated lncRNA BCYRN1 expression of airway smooth muscle cells by improving miR‐150 expression to inhibit the proliferation and migration of ASMC in asthmatic rats

Objective

The mechanism of Schisandrin B on the proliferation and migration of airway smooth muscle cells (ASMCs) in asthmatic rats was explored.

Methods

SD rats were divided into three groups: control (group 1), model (group 2) and model + Schisandrin B (group 3). miR‐150 and lncRNA BCYRN1 levels were measured by qRT‐PCR. The combination of BCYRN1 and miR‐150 was detected by RNA pull down. ASMCs’ viability/proliferation/migration were examined by WST‐1 assay and 24‐well Transwell system.

Results

Schisandrin B up‐regulated miR‐150 expression and down‐regulated BCYRN1 expression in sensitized rats. Schisandrin B reversed the expression of miR‐150 and BCYRN1 in MV‐treated ASMCs. In addition, Schisandrin B inhibited the viability, proliferation and migration of MV‐induced ASMCs. We also found miR‐150 inhibited BCYRN1 expression which was proved by experiments using ASMCs transfected with miR‐150 inhibitor.

Conclusion

Schisandrin B increased miR‐150 expression and decreased BCYRN1, and BCYRN1 expression was inhibited by miR‐150, which indicated that Schisandrin B could regulate BCYRN1 through miR‐150.

     

Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E₂ (PGE₂) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE₂ production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE₂ production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE₂ production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE₂ production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.     

Screening the expression characteristics of several miRNAs in G93A‐SOD1 transgenic mouse: altered expression of miRNA‐124 is associated with astrocyte differentiation by targeting Sox2 and Sox9

MicroRNA s (miRNA s) are suspected to be a contributing factor in amyotrophic lateral sclerosis (ALS ). Here, we assess the altered expression of miRNA s and the effects of miR‐124 in astrocytic differentiation in neural stem cells of ALS transgenic mice. Differentially expressed miRNA ‐positive cells (including miR‐124, miR‐181a, miR‐22, miR‐26b, miR‐34a, miR‐146a, miR‐219, miR‐21, miR‐200a, and miR‐320) were detected by in situ hybridization and qRT ‐PCR in the spinal cord and the brainstem. Our results demonstrated that miR‐124 was down‐regulated in the spinal cord and brainstem. In vitro , miR‐124 was down‐regulated in neural stem cells and up‐regulated in differentiated neural stem cells in G93A‐ superoxide dismutase 1 (SOD 1 ) mice compared with WT mice by qRT ‐PCR . Meanwhile, Sox2 and Sox9 protein levels showed converse change with miR‐124 in vivo and vitro . After over‐expression or knockdown of miR‐124 in motor neuron‐like hybrid (NSC 34) cells of mouse, Sox2 and Sox9 proteins were noticeably down‐regulated or up‐regulated, whereas Sox2 and Sox9 mRNA s remained virtually unchanged. Moreover, immunofluorescence results indicated that the number of double‐positive cells of Sox2/glial fibrillary acidic protein (GFAP) and Sox9/glial fibrillary acidic protein (GFAP) was higher in G93A‐SOD 1 mice compared with WT mice. We also found that many Sox2‐ and Sox9‐positive cells were nestin positive in G93A‐SOD 1 mice, but not in WT mice. Furthermore, differentiated neural stem cells from G93A‐SOD 1 mice generated a greater proportion of astrocytes and lower proportion of neurons than those from WT mice. MiR‐124 may play an important role in astrocytic differentiation by targeting Sox2 and Sox9 in ALS transgenic mice.

Cover Image for this issue: doi: 10.1111/jnc.14171 .