miR-92a inhibits vascular smooth muscle cell apoptosis: role of the MKK4–JNK pathway

Vascular smooth muscle cell (VSMC) apoptosis plays an important role in vascular remodeling and atherosclerotic plaque instability. Oxidative stress in diseased vessels promotes VSMC apoptosis in part by activating the c-Jun N-terminal kinase (JNK) pathway, which has been identified as a molecular target of miR-92a in macrophages. Here, we examined the expression and biological activity of miR-92a in VSMC. Quiescent VSMC exhibited a low basal expression of miR-92a, which was positively regulated by serum stimulation and negatively regulated by H₂O₂. Overexpression of miR-92a decreased H₂O₂-induced VSMC apoptosis as indicated by TUNEL assay and cleaved caspase-3 protein levels. Using 3′UTR-reporter assay, we found that miR-92a overexpression led to suppression of both mitogen-activated protein kinase kinase 4 (MKK4)- and JNK1-dependent luciferase activity. We also found that 10 mer seed match between miRNA:mRNA pair is more efficient than 8 mer seed match for us to identify authentic miRNA target. Protein levels of active phospho-JNK and phospho-c-Jun, downstream targets of the MKK4–JNK1 pathway, were also decreased by overexpressing miR-92a in VSMC under oxidative stress. Consistent with these findings, overexpression of MKK4 reversed the anti-apoptotic effects of miR-92a in oxidatively stressed VSMC. In conclusion, miR-92a overexpression inhibits H₂O₂-induced VSMC apoptosis by directly targeting the MKK4–JNK1 pathway.     

MicroRNA-25 aggravates Aβ1-42-induced hippocampal neuron injury in Alzheimer's disease by downregulating KLF2 via the Nrf2 signaling pathway in a mouse model

Recently, numerous microRNAs (miRNAs) have been considered as key players in the regulation of neuronal processes. The purpose of the present study is to explore the effect of miR-25 on hippocampal neuron injury in Alzheimer's disease (AD) induced by amyloid β (Aβ) peptide fragment 1 to 42 (Aβ1-42) via Kruppel-like factor 2 (KLF2) through the nuclear factor-E2-related factor 2 (Nrf2) signaling pathway. A mouse model of AD was established through Aβ1-42 induction. The underlying regulatory mechanisms of miR-25 were analyzed through treatment of miR-25 mimics, miR-25 inhibitors, or small interfering RNA (siRNA) against KLF2 in hippocampal tissues and cells isolated from AD mice. The targeting relationship between miR-25 and KLF2 was predicted using a target prediction program and verified by luciferase activity determination. MTT assay was used to evaluate the proliferative ability and flow cytometry to detect cell cycle distribution and apoptosis. KLF2 was confirmed as a target gene of miR-25. When the mice were induced by Aβ1-42, proliferation was suppressed while apoptosis was promoted in hippocampal neurons as evidenced by lower levels of KLF2, Nrf2, haem oxygenase, glutathione S transferase α1, glutathione, thioredoxin, and B-cell lymphoma-2 along with higher bax level. However, such alternations could be reversed by treatment of miR-25 inhibitors. These findings indicate that miR-25 may inhibit hippocampal neuron proliferation while promoting apoptosis, thereby aggravating hippocampal neuron injury through downregulation of KLF2 via the Nrf2 signaling pathway.     

MicroRNA-326 attenuates hepatic stellate cell activation and liver fibrosis by inhibiting TLR4 signaling

Hepatic fibrosis is a chronic inflammatory and reversible repair reaction of the liver under the continuous action of virus or various injuries. In this study, we aimed at identifying the role of miR-326 in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. In this study, the liver fibrosis mouse model was developed by injecting CCl₄. Liver tissue morphology was observed and the expression level of α-smooth muscle actin, collagen1α1 and miR-326 was measured. Target gene identification was performed by loss-of-function and gain-of-function. The effect of miR-326 on the expression level of the cytokines associated with the TLR4/MyD88/nuclear factor-κB (NF-κB) pathway was assessed in vitro and in vivo. We show that miR-326 was downregulated in CCl₄-induced fibrotic mice and activated HSCs. The target gene of miR-326 is TLR4. Moreover, miR-326 inhibited the activation of HSCs in vitro through TLR4/MyD88/NF-κB signaling. miR-326 attenuated hepatic fibrosis and inflammation of CCl₄-induced mice in vivo. Our results demonstrate for the first time that miR-326 inhibits HSC activation through TLR4/MyD88/NF-κB signaling. Furthermore, miR-326 plays critical roles in attenuating liver fibrosis and inflammation, suggesting the therapeutic potential of miRNAs.     

MicroRNA-326 suppresses iNOS expression and promotes autophagy of dopaminergic neurons through the JNK signaling by targeting XBP1 in a mouse model of Parkinson's disease

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons, and substantia nigra is primarily one of the damaged brain regions. Evidence indicates that microRNAs (miRNAs) is involved in the pathophysiology of this disease. The present study aimed to investigate the biological function of miR-326 in PD through the JNK signaling pathway by targeting X-box binding protein 1 (XBP1). After liposome complexes were prepared, healthy male C57BL/6 mice were selected to construct a mouse model of PD. The targeting relationship between miR-326 and XBP1 was confirmed. The expression of miR-326 and XBP1 was measured in PD mice, and gain- and loss-function assay was conducted to examine the regulatory effect of miR-326 and XBP1 on inducible nitric oxide synthase (iNOS) expression and autophagy of dopaminergic neurons of PD mice. Mice treated with miR-326 mimic and siRNA-XBP1 showed increased traction test scores, activation of autophagy, expression of LC3-II, c-Jun, and p-α-Syn, but diminished climbing time and expressions of iNOS, α-Syn, and p-c-Jun. The siRNA-XBP1 treatment could reverse the effect of miR-326 inhibitor on PD mice. Overexpression of miR-326 inhibits iNOS expression and promotes autophagy of dopaminergic neurons through JNK signaling by targeting XBP1.     

Retracted: In vivo and in vitro effects of microRNA-221 on hepatocellular carcinoma development and progression through the JAK–STAT3 signaling pathway by targeting SOCS3

Hepatocellular carcinoma (HCC), as the third leading cancer-caused deaths, prevails with high mortality, and affects more than half a million individuals per year worldwide. A former study revealed that microRNA-221 (miR-221) was involved in cell proliferation of liver cancer and HCC development. The current study aims to evaluate whether miR-221 targeting SOCS3 affects HCC through JAK–STAT3 signaling pathway. A series of miR-221 mimic, miR-221 inhibitor, siRNA against SOCS3, and SOCS3 plasmids were introduced to SMMC7721 cells with the highest miR-221 expression assessed. The expression of JAK–STAT3 signaling pathway–related genes and proteins was determined by Western blot analysis. Cell apoptosis, viability, migration, and invasion were evaluated by means of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, and transwell assays, respectively. HCC xenograft in nude mice was performed to measure HCC tumor growth. miR-221 was found to be highly expressed but SOCS3 was poorly expressed in HCC tissues. miR-221 expression was correlated with lymph node metastasis (LNM) and tumor node metastasis (TNM) of HCC, and SOCS3 expression was correlated with LNM, differentiation and TNM of HCC. SOCS3 is a target gene of miR-221. MiR-221 mimic or si-SOCS3 exposure was found to induce cell viability, migration, and invasion, and reduce apoptosis. MiR-221 inhibitor was observed to have inhibitory effects on HCC cell proliferation, migration, and invasion. Moreover, the expression of JAK–STAT3 signaling pathway was suppressed by miR-221 inhibitor. Downregulated miR-221 expression could promote its target gene SOCS3 to inhibit the proliferation, invasion and migration of HCC cells by repressing JAK–STAT3 signaling pathway.     

miR-145-5p targets paxillin to attenuate angiotensin II-induced pathological cardiac hypertrophy via downregulation of Rac 1, pJNK,p-c-Jun,NFATc3, ANP and by Sirt-1 upregulation

Pathological cardiac hypertrophy is associated with many diseases including hypertension. Recent studies have identified important roles for microRNAs (miRNAs) in many cardiac pathophysiological processes, including the regulation of cardiomyocyte hypertrophy. However, the role of miR-145-5p in the cardiac setting is still unclear. In this study, H9C2 cells were overexpressed with microRNA-145-5p, and then treated with Ang-II for 24 h, to study the effect of miR-145-5p on Ang-II-induced myocardial hypertrophy in vitro. Results showed that Ang-II treatment down-regulated miR-145-5p expression were revered after miR-145-5p overexpression. Based on results of bioinformatics algorithms, paxillin was predicted as a candidate target gene of miR-145-5p, luciferase activity assay revealed that the luciferase activity of cells was substantial downregulated the following co-transfection with wild paxillin 3′UTR and miR-145-5p compared to that in scramble control, while the inhibitory effect of miR-145-5p was abolished after transfection of mutant paxillin 3′UTR. Additionally, overexpression of miR-145-5p markedly inhibited activation of Rac-1/ JNK /c-jun/ NFATc3 and ANP expression and induced SIRT1 expression in Ang-II treated H9c2 cells. Jointly, our study suggested that miR-145-5p inhibited cardiac hypertrophy by targeting paxillin and through modulating Rac-1/ JNK /c-jun/ NFATc3/ ANP / Sirt1 signaling, therefore proving novel downstream molecular pathway of miR-145-5p in cardiac hypertrophy

     

mTOR pathway mediates endoplasmic reticulum stress-induced CD4+ T cell apoptosis in septic mice

Endoplasmic reticulum stress (ERS) has been well documented to participate in the pathophysiological processes of apoptosis in many diseases. Inhibition of ERS ameliorates pathological organ injury. However, the upstream signaling pathways and molecular regulatory mechanisms of which are still unknown. mTOR, an evolutionarily conserved protein kinase, is a key regulator of apoptosis. Hence, in this study, a classical cecal ligation and puncture (CLP) sepsis model was constructed by using the T cell-specific knockout mTOR and TSC1 (Tuberous Sclerosis Complex, the inhibitor of mTOR signaling pathway) mice to explore the underlying signaling pathway and molecular mechanism of host immune imbalance caused by apoptosis in sepsis. We found that mTOR may modulate septic T cell apoptosis by regulating Akt–IRE1–JNK pathway. To further clarify the possible mechanism, the specific inhibitors of PI3K-Akt and IRE1–JNK were used to intervene in mice before/after CLP, respectively. By analyzing the proteins of mTOR-ERS signaling pathway and the expression of apoptosis-related proteins and genes, we found that mTOR mediated the ER stress induced CD4⁺ T cell apoptosis in Septic mice by negatively regulating the Akt–IRE1–JNK-Caspase 3 signaling cascades. These results indicate that mTOR–Akt–IRE1α–JNK signaling pathway mediated the Endoplasmic reticulum stress induced CD4⁺ T cell apoptosis in Septic mice.

     

miR-146a down-regulation alleviates H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cytotoxicity of PC12 cells by regulating MCL1/JAK/STAT pathway

Oxidative stress and miRNAs have been confirmed to play an important role in neurological diseases. The study aimed to explore the underlying effect and mechanisms of miR-146a in H₂O₂-induced injury of PC12 cells. Here, PC12 cells were stimulated with 200 μM of H₂O₂ to construct oxidative injury model. Cell injury was evaluated on the basis of the changes in cell viability, migration, invasion, apoptosis, and DNA damage. Results revealed that miR-146a expression was up-regulated in H₂O₂-induced PC12 cells. Functional analysis showed that down-regulation of miR-146a alleviated H₂O₂-induced cytotoxicity in PC12 cells. Dual-luciferase reporter and western blot assay verified that MCL1 was a direct target gene of miR-146a. Moreover, anti-miR-146a-mediated suppression on cell cytotoxicity was abated following MCL1 knockdown in H₂O₂-induced PC12 cells. Furthermore, MCL1 activated JAK/STAT signaling pathway and MCL1 overexpression attenuated H₂O₂-induced cytotoxicity in PC12 cells by JAK/STAT signaling pathway. In conclusion, this study suggested that suppression of miR-146a abated H₂O₂-induced cytotoxicity in PC12 cells via regulating MCL1/JAK/STAT pathway.     

LncRNA CASC11 promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway

In this study, we aimed to investigate the effects of lncRNA CASC11 on gastric cancer (GC) cell progression through regulating miR-340-5p and cell cycle pathway. Expressions of lncRNA CASC11 in gastric cancer tissues and cell lines were determined by qRT-PCR. Differentially expressed lncRNAs, mRNAs and miRNAs were screened through microarray analysis. The relationship among CASC11, CDK1 and miR-340-5p was predicted by TargetScan and validated through dual luciferase reporter assay. Western blot assay examined the protein level of CDK1 and several cell cycle regulatory proteins. GO functional analysis and KEGG pathway analysis were used to predict the association between functions and related pathways. Cell proliferation was determined by CCK-8 assays. Cell apoptosis and cell cycle were detected by flow cytometry assay. CASC11 was highly expressed in GC tissues and cell lines. Knockdown of CASC11 inhibited GC cell proliferation, promoted cell apoptosis and blocked cell cycle. KEGG further indicated an enriched cell cycle pathway involving CDK1. QRT-PCR showed that miR-340-5p was down-regulated in GC cells tissues, while CDK1 was up-regulated. Furthermore, CASC11 acted as a sponge of miR-340-5p which directly targeted CDK1. Meanwhile, miR-340-5p overexpression promoted GC cell apoptosis and induced cell cycle arrest, while CDK1 overexpression inhibited cell apoptosis and accelerated cell cycle. Our study revealed the mechanism of CASC11/miR-340-5p/CDK1 network in GC cell line, and suggested that CASC11 was a novel facilitator that exerted a biological effect by activating the cell cycle signaling pathway. This finding provides a potential therapeutic target for GC.     

microRNA-383 regulates cell viability and apoptosis by mediating Wnt/β-catenin signaling pathway in non–small cell lung cancer

The aim of this study was to investigate the roles of microRNA-383 (miRNA-383) in progression of non–small cell lung cancer (NSCLC) and the potential mechanism. The expressions of miR-383 and Wnt1 protein were detected in lung cancer tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. After the transfection of miR-383 mimics, si-Wnt1 or miR-383+Wnt1, the viability and apoptosis of NSCLC cells were detected by cell counting kit-8 and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling, respectively. The interaction between miR-383 and Wnt1 was investigated by luciferase activity and Western blot analysis. Cells stably transfected with miR-383 mimics were inoculated into the right axillary of nude mice by subcutaneous injection. The tumor volume and weight were measured, and the expressions of miR-383, Wnt1, β-catenin, and cyclin D1 were detected by qRT-PCR and Western blot analysis. The expression of miR-383 was significantly decreased, and the level of Wnt1 was significantly increased (P < 0.05) in lung cancer tissues and cells. Upregulation of miR-383 or inhibition of Wnt1 expression inhibited the cell viability and induce apoptosis in NSCLC cells. Moreover, Wnt1 was the target gene of miR-383, and its overexpression weakened the regulatory effect of miR-383 on cell viability and apoptosis in NSCLC cells. Besides, the addition of miR-383 decreased the tumor volume and size and inhibited the expressions of Wnt1, β-catenin, and cyclin D1 at the protein level in nude mice. Collectively, miR-383 induced apoptosis and inhibited cell viability as well as tumorigenic capacity in nude mice via regulating the Wnt/β-catenin signaling pathway.     

The effect of miR-579 on the PI3K/AKT pathway in human glioblastoma PTEN mutant cell lines

Glioblastoma multiform (GBM) is a type of aggressive brain cancer with limited success in standard treatment. MicroRNAs are one of the most beneficial tools for diagnosis, prognosis, and treatment of cancer. This study aimed to investigate the effect of miR-579 on cellular behaviors and expression of PI3K/AKT signaling pathway in GBM cell lines. In the present study, miR-579 was overexpressed in U251 and A-172 cell lines by using lentil vector, and its effect on cellular behavior such as proliferation and migration was investigated by the cell cycle, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Annexin V, colony formation, Transwell and wound healing assays. MiR-579 predicted target genes (AKT1, Rheb, PDK1, and a few others) were also evaluated by real-time polymerase chain reaction or luciferase assay and Western blot analysis. Our results represented that overexpression of miR-579 could inhibit proliferation, migration, cell cycle and also promoted the apoptosis of GBM cell lines. The luciferase reporter assay showed miR-579 directly targets the 3 UTR of mTOR, Rheb, and PDK1 and repressed their expressions. Furthermore, the Western blot analysis showed that miR-579 could downregulate the AKT1 and Rheb protein expression. Overall, our findings propose that miR-579 functions as a novel tumor suppressor gene in GBM by regulating the PI3K/AKT signaling pathway and may serve as a therapeutic target for clinical therapy of glioblastoma multiform.     

MicroRNA-326 Functions as a Tumor Suppressor in Glioma by Targeting the Nin One Binding Protein (NOB1)

Malignant glioma is the most common type of primary brain tumor in adults, characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. Alterations in the activity of the 26S proteasome have been associated with malignant glioma cells, although the specific defects have not been identified. Recently, microRNA-326 (miR-326) was shown to play an important role in glioblastoma and breast cancer, but the underlying molecular mechanisms remain unclear. In the present study, the human Nin one binding protein (NOB1) was identified as a direct target of miR-326 and a potential oncogene in human glioma. Similar to NOB1 silencing by shRNA, overexpression of miR-326 in human glioma cell lines (A172 and U373) caused cell cycle arrest at the G1 phase, delayed cell proliferation and enhanced apoptosis. MiR-326 inhibited colony formation in soft agar and decreased growth of a xenograft tumor model, suggesting that miR-326 and NOB1 are required for tumorigenesis in vitro and in vivo. Furthermore, these processes were shown to involve the MAPK pathway. NOB1 overexpression in human glioma samples was detected by Affymetrix array analysis, and NOB1 mRNA and protein levels were shown to be increased in high-grade glioma compared to low-grade glioma and normal brain tissue. Furthermore, high levels of NOB1 were associated with unfavorable prognosis of glioma patients. Taken together, these results indicate that miR-326 and NOB1 may play an important role in the development of glioma.     

MiR-137 affects melanin synthesis in mouse melanocyte by repressing the expression of c-Kit and Tyrp2 in SCF/c-Kit signaling pathway

Previously, we created miR-137 overexpressing transgenic mice that produced lighten color phenotypes including gray mice phenotype. However, the miR-137 functional role in coat color regulation is still not well understood. In this study, the quantity of melanin granule and the relative expression of TYRP2 in gray miR-137 overexpression transgenic mouse skin were significantly lower than that in C57BL/6J black mouse skin. The mRNA and protein expression level of c-Kit and c-Kit downstream gene Tyrp2 in miR-137 expression plasmid-transfected melanocytes were significantly down-regulated comparing with that of the control melanocytes. In melanocytes, miR-137 overexpression could decrease the enhanced expression of c-Kit and Tyrp2 and the increased melanin production caused by UV treatment. The target relationship of miR-137 and c-Kit was identified by luciferase assay. The results suggest that miR-137 could inhibit melanogenesis in mouse skin melanocytes by repressing the expression of c-Kit and Tyrp2 in SCF/c-Kit signaling pathway.     

Differential expression of miR-142-3p protects cardiomyocytes from myocardial ischemia-reperfusion via TLR4/NFkB axis

Our research aims to explore the impact of miR-142 on myocardial apoptosis in the mouse ischemia and reperfusion (IR) model and investigate the underlying mechanisms at the molecular level. A considerable downregulation of miR-142 was observed in the cardiac area of mice post IR modeling. To understand the regulatory function of IR-induced miR-142 downregulation, the animals were categorized into four groups: IR model group; IR + agomir-142 group (IR mice treated with agomir-142); IR + antagomir-142 group (IR mice treated with antagomir-142); IR + agomir-142 + negative control (NC) group (IR mice processed with agomir-NC). The results indicated that agomir-142 upregulation was capable of shrinking IR damage-triggered infarction of the ventriculus sinister, strengthening myocardial function, and guarding against cardiomyocyte apoptosis, whereas further decreased miR-142 with antagomir-142 infection displayed negative influence of miR-142 against mice IR damage. In the cellular assay, miR-142 overexpression significantly improved proliferation and inhibited the apoptosis of neonatal rat cardiomyocytes (NRCs). Moreover, we found that miR-142 reduced the Bcl-2/Bax ratio and upregulated hydrogen peroxide (H₂O₂)-induced caspase-3 expression. Furthermore, transfection with an miR-142 mimic prevented the upregulation of TLR4/NFkB expression and activation in H₂O₂-treated NRCs. Our findings also revealed that miR-142 is linked to the 3'-untranslated area of the TLR4 gene. In addition, TLR4 overexpression considerably ablated the protective effects of miR-142 in terms of the cell viability of H₂O₂-treated NRCs. Taken together, miR-142 agomir injection in mice and miR-142 mimic transfection in NRCs plays a role in protecting the heart from IR damage and malfunction via the TLR4/NFkB axis both in vivo and in vitro.     

MiR-150-5p protects against septic acute kidney injury via repressing the MEKK3/JNK pathway

BackgroundSeptic acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients. MicroRNA is reportedly involved in sepsis-induced organ dysfunction, while the role of miR-150 in septic AKI remains ambiguous.MethodsQuantitative real-time PCR (qRT-PCR) was carried out to examine miR-150-5p expression in both septic AKI patients and volunteers without septic AKI. Lipopolysaccharide (LPS) was used to treat renal tubular epithelial cell line HK-2 and C57/BL6 mice to establish in vitro and in vivo sepsis-induced AKI models. Cell apoptosis was determined using TdT-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry. Cell viability was tested using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Renal pathological changes were examined via Hematoxylin-Eosin (H&E) staining, and renal function was measured via blood urea nitrogen (BUN) and creatinine (Cre) measurements. The MEKK3/JNK profile and oxidative stress markers (including COX2 and iNOS) were examined by immunoblot analysis, and the expression levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and oxidative stress markers (MDA, SOD, and CAT) were evaluated by ELISA.ResultsMiR-150-5p was down-regulated in the serum of patients with septic AKI (compared to healthy volunteers). Moreover, miR-150-5p levels were lower in LPS-treated HK-2 cell lines and in the septic AKI mouse model. Additionally, Stat-3 activation mediated the decrease of miR-150-5p. Functionally, miR-150-5p agomir attenuated LPS-induced apoptosis in HK-2 cells, in addition to renal inflammatory responses and oxidative stress. In contrast, inhibition of miR-150-5p aggravated LPS-induced apoptosis, inflammatory reactions and oxidative stress. Furthermore, miR-150-5p agomir decreased BUN and Scr levels in the septic AKI mice model repressed TNF-α, IL-6 and IL-1β, and up-regulated SOD and CAT down-regulated MDA in the kidney tissues. Moreover, miR-150-5p was identified as a target gene for Stat3, and the overexpression of Stat3 partially promoted the effect of down-regulating miR-150-5p on LPS-induced HK2 cell injury. Mechanistically, the MEKK3/JNK pathway was identified as a functional target of miR-150-5p, and the knockdown of MEKK3 showed protective effects against LPS mediated HK-2 cell apoptosis.ConclusionStat3-mediated miR-150-5p exerted protective effects in sepsis-induced acute kidney injury by regulating the MEKK3/JNK pathway.