Sinomenine reduces MPP+-induced damage in SK-N-SH cells via ANRIL/miR-626 signaling pathway

AIM To investigate the effect of sinomenine (SN) on the damage of human neuroblastoma SK-N-SH cells induced by 1-methyl-4-4 phenylpyridine (MPP⁺) and its mechanism for exploring the pathogenesis of Parkinson disease. METHODS SN was used to treat MPP⁺-induced SK-N-SH cells. The levels of malondialdehyde (MDA) and glutathione (GSH) in cell culture supernatants were measured by ELISA. The apoptosis was analyzed by flow cytometry. The protein expression levels of Bcl-2 and Bax were determined by Western blot. The expression levels of long noncoding RNA ANRIL and microRNA-626 (miR-626) were detected by RT-qPCR. Dual-luciferase reporter assay was used to evaluate the relationship between ANRIL and miR-626. After ANRIL small interfering RNA was transfected into SK-N-SH cells, the effects of ANRIL expression knock-down on MPP⁺-induced SK-N-SH cell apoptosis, the protein expression levels of Bcl-2 and Bax, and the levels of MDA and GSH in cell culture supernatants were examined. RESULTS After treatment with MPP⁺, the apoptotic rate, Bax protein level and ANRIL expression in SK-N-SH cells were increased (P<0.05), and the Bcl-2 protein level and miR-626 expression were decreased (P<0.05). The level of MDA in cell culture supernatants was increased (P<0.05), and the level of GSH was decreased (P<0.05). After SN treatment or ANRIL expression knock-down, decreased apoptotic rate, Bax protein level and ANRIL expression (P<0.05), and increased Bcl-2 protein level and miR-626 expression in MPP⁺-induced SK-N-SH cells were observed (P<0.05). The level of MDA in the cell culture supernatants was decreased (P<0.05), and the level of GSH was increased (P<0.05). CONCLUSION SN attenuates MPP⁺-induced damage in SK-N-SH cells by regulating ANRIL/miR-626 signaling pathway.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

MicroRNA-142-3p modulates atherosclerosis-associated endothelial cell apoptosis via targeting Rictor

AIM To investigate the role of microRNA-142-3p （miR-142-3p） in endothelial cell apoptosis during atherosclerosis （AS） and the underlying mechanism. METHODS Human aortic endothelial cells （HAECs） were treated with oxidized low-density lipoprotein （ox-LDL）. The expression level of miR-142-3p was detected by RT-qPCR. Apoptosis was determined via flow cytometry （FCM） and caspase-3 activity assay. Prediction of the binding site between miR-142-3p and 3’-UTR of Rictor mRNA was performed by bioinformatics analysis and confirmed by dual-luciferase reporter assay. RESULTS The expression of miR-142-3p was substantially up-regulated during the ox-LDL-elicited apoptosis in HAECs （P<0.05,P<0.01）. Forced expression of miR-142-3p exacerbated apoptosis in HAECs whereas inhibition of miR-142-3p partly alleviated apoptotic cell death mediated by ox-LDL. Further analysis identified Rictor as a direct target gene of miR-142-3p, and Rictor knock-down abolished the anti-apoptotic effect of miR-142-3p inhibitor. Moreover, the Akt/endothelial nitric oxide synthase （eNOS） signaling pathway was found to mediate the beneficial effect of miR-142-3p inhibitor on endothelial cells apoptosis. CONCLUSION Down-regulation of miR-142-3p inhibits endothelial cell apoptosis and atherosclerotic development by up-regulating the expression of Rictor and activating the Akt/eNOS signaling pathway.     

miR-485-5p inhibits viability and migration and invasion abilities of hepatocellular carcinoma Hep3B cells by targeting SOX5

AIM: To investigate the effects of microRNA-485-5p (miR-485-5p) on the viability, migration and invasion abilities of hepatocellular carcinoma cells and the underlying mechanism. METHODS: The expression levels of sex determining region Y-box 5 (SOX5) mRNA and miR-485-5p in the hepatocellular carcinoma Hep3B cells were detected by RT-qPCR with normal hepatocyte THLE-3 as control. Western blot was used to measure the expression levels of SOX5, proliferating cell nuclear antigen (PCNA), Ki67, cyclin D1 and matrix metalloproteinase-2 (MMP-2). The viability of Hep3B cells was measured by MTT assay. The migration and invasion abilities of the Hep3B cells were detected by Transwell assay. Dual-luciferase reporter assay system was applied to verify the relationship between miR-485-5p and SOX5. RESULTS: Compared with the control cells, the expression level of miR-485-5p was decreased in hepatocellular carcinoma cells Hep3B, Huh7 and HCCLM3 (P<0.05), while the expression of SOX5 at mRNA and protein levels were significantly increased (P<0.05). Over-expression of miR-485-5p inhibited the viability, migration and invasion of Hep3B cells. miR-485-5p targeted the 3′-UTR of SOX5 and negatively regulated the expression of SOX5. Knocking-down of SOX5 expression inhibited the viability, migration and invasion of Hep3B cells. Over-expression of SOX5 partially reversed the inhibitory effect of miR-485-5p over-expression on the viability, migration and invasion of Hep3B cells. CONCLUSION: miR-485-5p inhibits the viability, migration and invasion of Hep3B cells by targeting SOX5 gene. miR-485-5p is a potential molecular target for hepatocellular carcinoma.     

Scutellarin affects LPS-induced oxidative stress and apoptosis of glomerular epithelial cells by up-regulating miR-7-5p

AIM To investigate the effect of scutellarin （SCU） on oxidative stress and apoptosis induced by lipopolysaccharide （LPS） in human glomerular epithelial cells and its mechanism. METHODS Human glomerular epithelial cells were cultured in vitro, and were treated with LPS （1.0 mg/L） to establish a cell injury model. The cells were divided into normal control （NC） group, LPS group, NC+SCU group, LPS+SCU group, LPS+miR-NC group, LPS+microRNA-7-5p （miR-7-5p） group, LPS+SCU+anti-miR-NC group and LPS+SCU+anti-miR-7-5p group. Cell viability was detected by CCK-8 assay. Apoptosis was detected by flow cytometry. The intracellular malondialdehyde （MDA） content and superoxide dismutase （SOD） activity, and lactate dehydrogenase （LDH） activity in the cell culture supernatant were determined by kit. RT-qPCR was used to detect the expression level of miR-7-5p. RESULTS Compared with NC group, the cell viability, miR-7-5p expression and SOD activity in LPS group were significantly reduced, and the apoptotic rate, MDA content and LDH activity were significantly increased （P<0.05）. Compared with LPS group, the cell viability, miR-7-5p expression and SOD activity in LPS+SCU group were significantly increased, and the apoptotic rate, MDA content and LDH activity were significantly reduced （P<0.05）. Compared with LPS+miR-NC group, the cell viability and SOD activity in LPS+miR-7-5p group were significantly increased, and the apoptotic rate, MDA content and LDH activity were significantly reduced （P<0.05）. Compared with LPS+SCU+anti-miR-NC group, the cell viability and SOD activity in LPS+SCU+anti-miR-7-5p group were significantly reduced, and the apoptotic rate, MDA content and LDH activity were significantly increased （P<0.05）. CONCLUSION Scutellarin inhibits LPS-induced oxidative stress damage and apoptosis in glomerular epithelial cells via up-regulating miR-7-5p expression.     

Paeonol affects viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway

AIM To investigate the effect of paeonol on the viability and migration ability of hepatocellular carcinoma cells and its molecular mechanism. METHODS Human hepatocellular carcinoma Hep3B cells was treated with paeonol at different concentrations (50, 100, 200 and 400 mg/L). The cell viability was measured by CCK-8 assay to determine the optimal drug concentration. The Hep3B cells were divided into normal control (NC) group, paeonol group, miR-NC group, miR-424-3p group, paeonol+anti-miR-NC and paeonol+anti-miR-424-3p group. The expression level of miR-424-3p was detected by RT-qPCR. The migration ability was detected by Transwell assay. The protein levels of cyclin D1, matrix metalloproteinase 2 (MMP2), MMP9 and PI3K/AKT signaling pathway-related molecules were determined by Western blot. RESULTS Paeonol intervention inhibited the viability of Hep3B cells in a concentration-dependent manner (P<0.05). The concentration of paeonol at 200 mg/L was selected for the following study. Paeonol intervention inhibited the protein expression of MMP2 and MMP9 in the Hep3B cells, and inhibited the migration ability of the Hep3B cells. Paeonol intervention promoted the expression of miR-424-3p in the Hep3B cells (P<0.05). Over-expression of miR-424-3p inhibited the expression of cyclin D1, MMP2 and MMP9 in the Hep3B cells and inhibited cell viability and migration ability (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on the viability and migration ability of the Hep3B cells (P<0.05). Paeonol inhibited phosphorylation levels of PI3K and AKT in the Hep3B cells and inhibited the activation of PI3K/AKT signaling pathway (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on PI3K/AKT signaling pathway in the Hep3B cells (P<0.05). CONCLUSION Paeonol inhibits the viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway.     

miR-556-3p regulates viability,migration and invasion of endometrial cancer cells by targeting SASH1

AIM To investigate whether microRNA-556-3p （miR-556-3p） regulates the viability, migration and invasion of endometrial cancer cells by targeting SASH1 gene. METHODS The expression of miR-556-3p, and the mRNA and protein levels of SASH1 in endometrial cancer tissues were detected by RT-qPCR and Western blot. Anti-miR-556-3p or pcDNA-SASH1 was transfected into endometrial cancer Ishikawa cells. The cell viability was detected by MTT assay, the migration and invasion abilities of the cells were detected by Transwell chamber method, and the protein expression levels of cyclin D1, p21, matrix metalloproteinase-2 （MMP-2） and MMP-9 were detected by Western blot. StarBase prediction and dual-luciferase reporter experiments were used to analyze the targeting relationship between miR-556-3p and SASH1. Anti-miR-556-3p and si-SASH1 were co-transfected into the Ishikawa cells, and their effects on cell viability, migration and invasion were examined by the methods described above. RESULTS Compared with adjacent tissues, the expression of miR-556-3p in endometrial cancer tissues was increased significantly, and the expression of SASH1 at mRNA and protein levels was decreased significantly （P<0.05）. Inhibition of miR-556-3p expression or induction of SASH1 over-expression obviously reduced the viability of Ishikawa cells, the number of migratory cells, the number of invasive cells and the protein levels of cyclin D1, MMP-2 and MMP-9, and dramatically increased the protein level of p21 （P<0.05）. miR-556-3p targeted SASH1 and negatively regulated its expression. Knock-down of SASH1 expression reversed the inhibitory effect of miR-556-3p expression inhibition on the viability, migration and invasion of Ishikawa cells. CONCLUSION Inhibition of miR-556-3p expression suppresses the viability, migration and invasion of endometrial cancer cells. The mechanism is related to the regulation of its target gene SASH1.     

Effect of NLRC3 expression knock-down on growth of normal human bronchial epithelial BEAS-2B cells

AIM To investigate the effect of NOD-like receptor family caspase recruitment domain containing 3 (NLRC3) expression knock-down on the viability and apoptosis of normal human bronchial epithelial BEAS-2B cells and its mechanism. METHODS The small interfering RNA (siRNA) fragments of NLRC3 gene were transfected into BEAS-2B cells using Lipofectamine 2000 transfection reagent to knock down the NLRC3 expression. The interference fragment was screened by RT-qPCR. The cell viability was measured by MTT assay. The mitochondrial membrane potential was detected by JC-1 staining. The apoptotic rate was analyzed by flow cytometry with annexin V-FITC/PI staining. The protein expression levels of Bcl-2 and Bax were determined by Western blot. RESULTS The interference segment 3 of NLRC3 gene (siNLRC3-3) displayed the best interference effect on NLRC3 expression in BEAS-2B cells (P<0.01). Knock-down of NLRC3 expression in BEAS-2B cells enhanced the cell viability (P<0.01). Knock-down of NLRC3 increased the mitochondrial membrane potential, and decreased the apoptotic rate (P<0.05). Moreover, knock-down of NLRC3 significantly up-regulated Bcl-2 protein expression and significantly down-regulated Bax protein expression (P<0.01). CONCLUSION Knock-down of NLRC3 expression enhances the viability and inhibits the apoptosis of BEAS-2B cells, which may be related to increase in the expression of Bcl-2 protein and decrease in the expression of Bax protein.     

mTOR kinase inhibitor CC-223 suppresses human breast cancer cell viability

AIM: To investigate the inhibitory effect of CC-223, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on the viability of human breast cancer cells and its mechanism. METHODS: The inhibitory effect of CC-223 on the viability of MCF-7 cells and MDA-MB-231 cells was measured by CCK-8 assay. The cell cycle distribution of breast cancer cells was examined by flow cytometry. The expression of cell cycle-related proteins and oncoproteins c-Myc and survivin was analyzed by Western blot. RESULTS: CC-223 significantly inhibited the viability of both MCF-7 and MDA-MB-231 cells (P<0.05). CC-223 induced cell cycle arrest in both G₁ phase and G₂/M phase in the MCF-7 cells (P<0.05). However, low concentration of CC-223 treatment resulted in the accumulation of MDA-MB-231 cell cycle in the G₂/M phase, and the cell number in G₁ phase was unaffected. Treatment with CC-223 for 24 h clearly inhibited the protein levels of cyclin B1, cyclin D1 and phosphorylated cell division cycle protein 2 in the breast cancer cells (P<0.05). CC-223 suppressed the expression of c-Myc and survivin in both MCF-7 cells and MDA-MB-231 cells (P<0.05). CONCLUSION: CC-223 inhibits cell viability by blocking cell cycle progression and down-regulating expression of c-Myc and survivin in both MCF-7 and MDA-MB-231 cells.     

Effect of Herba Taxilli extracts combined with miR-375 on viability and apoptosis of osteoarthritic chondrocytes

AIM To study the effects of extracts of Herba Taxilli (Sangjisheng, SJS) on the viability and apoptosis of osteoarthritic chondrocytes and the underlying mechanism. METHODS Human primary osteoarticular chondrocytes (RPOC) were divided into control group, interleukin-1β (IL-1β) group, IL-1β+low-dose extracts of SJS (SJS-L) group, IL-1β+medium-dose extracts of SJS (SJS-M) group, IL-1β+high-dose extracts of SJS (SJS-H) group, IL-1β+anti-miR-NC group, IL-1β+anti-miR-375 group, IL-1β+SJS-H+miR-NC group, IL-1β+SJS-H+miR-375 group. The cell viability was measured by MTT assay, apoptosis was analyzed by flow cytometry, miR-375 expression was detected by qPCR, and the protein levels of cyclin D1, P21, Bcl-2, Bax and caspase-3 were determined by Western blot. RESULTS Compared with control group, the viability of RPOC at 24 h, 48 h and 72 h and the protein expression levels of cyclin D1 and Bcl-2 were significantly decreased (P<0.05), the protein levels of P21, Bax and caspase-3, the apoptotic rate and the expression level of miR-375 were remarkably increased in IL-1β group(P<0.05). Compared with IL-1β group, the cell viability at 24 h, 48 h and 72 h and the protein expression of cyclin D1 in the RPOC were greatly increased (P<0.05), while the expression of P21 was significantly decreased in IL-1β+SJS-M group and IL-1β+SJS-H group(P<0.05).The apoptotic rate, Bax, caspase-3 protein and miR-375 expression were obviously decreased (P<0.05), and Bcl-2 protein level was significantly increased in IL-1β+SJS-H group compared with IL-1β group(P<0.05). Compared with IL-1β+anti-miR-NC group, the expression of miR-375, the protein levels of P21, Bax, caspase-3 and the apoptotic rate in the RPOC of IL-1β+anti-miR-375 group were markedly decreased (P<0.05), while the cell viability at 24 h, 48 h and 72 h and the protein levels of cyclin D1 and Bcl-2 were significantly increased (P<0.05). Over-expression of miR-375 reversed the effects of extracts of SJS on the viability and apoptosis of RPOC with IL-1β stimulation. CONCLUSION The extracts of Herba Taxilli promotes the viability and inhibits apoptosis of RPOC treated with IL-1β, which is related to the regulation of miR-375 expression.     

miR-323 regulates hypoxia-induced apoptosis of H9C2 cardiomyocytes through FGF9

AIM: To investigate the effect of microRNA-323 (miR-323) on the apoptosis of hypoxia-induced rat H9C2 cardiomyocytes and its mechanism. METHODS: The hypoxic injury model was established in the H9C2 cells. Anti-miR-323, pcDNA-FGF9 and si-FGF9 were transfected into the H9C2 cells and cultured under hypoxic condition for 48 h. The expression of miR-323 was detected by qPCR. The protein levels of fibroblast growth factor 9 (FGF9), cleaved caspase-3, c-Jun N-terminal kinase (JNK) and p-JNK were determined by Western blot. The cell viability was measured by MTT assay, and the apoptosis was analyzed by flow cytometry. The method of bioinformatics was applied to predict the target gene of miR-323, and dual-luciferase reporter assay was used for further validation. RESULTS: Hypoxia greatly reduced the viability of H9C2 cells at 12 h, 24 h and 48 h (P<0.05), and remarkably increased apoptotic rate and the protein level of cleaved caspase-3 in a time-dependent manner (P<0.05). The expression of miR-323 and the protein level of p-JNK were up-regulated and the expression of FGF9 was down-regulated in the H9C2 cells exposed to hypoxia (P<0.05). Down-regulation of miR-323 and over-expression of FGF9 obviously increased the viability of the H9C2 cells exposed to hypoxia, and decreased the apoptotic rate and the protein level of cleaved caspase-3 (P<0.05). FGF9 was the target gene of miR-323. Down-regulation of FGF9 reversed the attenuating effect of down-regulation of miR-323 on hypoxia-induced H9C2 cell injury. miR-323 regulated FGF9 and affected p-JNK level. CONCLUSION: miR-323 affects the viability and apoptosis of H9C2 cardiomyocytes under hypoxia by targeting FGF9 and regulating JNK signaling pathway.     

Effect of compound of Epimedium, Astragalus and Radix Puerariae on expression of ADAM10 in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin expression knock-down

AIM To explore the effect of compound of Epimedium, Astragalus and Radix Puerariae on the expression of a disintegrin and metalloproteinase 10 （ADAM10） in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin （HAMP） expression knock-down for analyzing the pathogenesis of Alzheimer disease （AD） at cell level. METHODS Hippocampal neuron HT22 cells were cultured in vitro and randomly divided into 7 groups： control group, Aβ group （Aβ_25-35-induced HT22 cells）, RNAi group （HAMP gene was silenced in HT22 cells）, Aβ+RNAi group （HAMP gene expression in Aβ_25-35-induced HT22 cells was silenced）, Aβ+TCM group （Aβ_25-35-induced HT22 cells were treated with Epimedium, Astragalus root and Radix Puerariae effective components）, RNAi+TCM group （HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components） and Aβ+RNAi+TCM group （Aβ_25-35-induced HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components）. The silence efficiency of HAMP siRNA was detected by qPCR and Western blot. The ADAM10 expression in each group was determined by immunofluorescence, qPCR and Western blot. RESULTS The HAMP siRNA-3 sequence had the highest interference efficiency. Compared with control group, the expression levels of ADAM10 in Aβ group, RNAi group and Aβ+RNAi group were decreased （P<0.05）. Compared with Aβ group,the expression levels of ADAM10 in Aβ+RNAi group was also decreased （P<0.05）, and the expression levels of ADAM10 in Aβ+TCM group was increased （P<0.05）. Compared with RNAi group, the expression levels of ADAM10 in Aβ+RNAi group was decreased （P<0.05）, while the expression levels of ADAM10 in RNAi+TCM group was increased （P<0.05）. Compared with Aβ+RNAi group, the expression levels of ADAM10 in Aβ+RNAi+TCM group was increased （P<0.05）. CONCLUSION The effective components of Epimedium, Astragalus and Radix Puerariae compound promotes the expression of ADAM10 in Aβ_25-35-induced HT22 cells, which mechanism may be related to the expression of HAMP.     

Alleviating effect of exenatide on ectopic lipid accumulation in skeletal muscle of ob/ob mice is independent on reducing body weight

AIM To investigate the alleviating effect of exenatide （Exe）, a glucagon-like peptide-1 （GLP-1） receptor agonist, on the ectopic lipid accumulation in skeletal muscle of ob/ob mice and its mechanism. METHODS Eight-week-old male ob/ob mice and their wild-type （WT） littermates were randomly divided into 3 groups, ob/ob group, ob/ob+Exe group and WT group, and treated with Exe at 24 nmol/kg or the same volume of saline intraperitoneally once daily for 4 weeks. The body weight, fasting blood glucose （FBG） and fat content were measured after the 4-week treatment. The oil red O staining and the quantification of triglyceride （TG） were performed on the skeletal muscle. The serum levels of TG, total cholesterol and free fatty acid （FFA） were also measured by ELISA. The expression levels of AMP-activated protein kinase （AMPK） and lipid metabolism-related proteins were determined by Western blot. Mouse myoblast C2C12 cells were used as an in vitro model to further investigate the effects of Exe. RESULTS As compared with the ob/ob mice treated with saline, 4-week Exe treatment did not reduce body weight, FBG, food intake and fat content in ob/ob mice （P>0.05）. However, serum FFA was decreased （P<0.05）. Oil red O staining and the quantification of TG showed that 4-week Exe treatment significantly attenuated the ectopic lipid accumulation in the skeletal muscle of ob/ob mice （P<0.05）. The results of Western blot showed that the levels of phosphorylated AMPK （p-AMPK） and lipolysis-related proteins were up-regulated, while the lipid synthesis-related proteins were down-regulated by Exe （P<0.05）. Treatment with Exe alleviated the lipid accumulation in the C2C12 cells induced by sodium palmate （P<0.05）, and the effects of Exe on the levels of p-AMPK and lipid metabolism-related proteins in the C2C12 cells were consistent with those in the ob/ob mice （P<0.05）. Treatment with Exe also up-regulated the protein expression of glucose transporter 4 and improved the ability of glucose uptake in the C2C12 cells （P<0.05）. CONCLUSION Short-term Exe treatment attenuates the ectopic lipid accumulation in skeletal muscle of ob/ob mice by up-regulating lipolysis-related proteins and down-regulating lipid synthesis-related proteins, which is independent on body weight loss.     

Effect of inhibiting lncRNA LINC01503 on viability,migration and invasion of lung cancer cells by targeted regulation of miR-335-5p

AIM To investigate the effects of long non-coding RNA (lncRNA) LINC01503 on the viability, migration and invasion of lung cancer cells and its mechanism. METHODS Human lung carcinoma H1299 cells were divided into si-NC group (transfected with si-NC), si-LINC01503 group (transfected with si-LINC01503), pcDNA group (transfected with pcDNA), pcDNA-LINC01503 group (transfected with pcDNA-LINC01503), miR-NC group (transfected with miR-NC), miR-335-5p group (transfected with miR-335-5p mimics), si-LINC01503+anti-miR-NC group (co-transfected with si-LINC01503 and anti-miR-NC), si-LINC01503+anti-miR-335-5p group (co-transfected with si-LINC01503 and anti-miR-335-5p), miR-NC+WT-LINC01503 group (co-transfected with miR-NC and WT-LINC01503), miR-NC+MUT-LINC01503 group (co-transfected with miR-NC and MUT-LINC01503), miR-335-5p+WT-LINC01503 group (co-transfected with miR-335-5p and WT-LINC01503) and miR-335-5p+MUT-LINC01503 group (co-transfected with miR-335-5p and MUT-LINC01503). The expression of miR-335-5p and LINC01503 was detected by RT-qPCR. Western blot was used to detect protein expression. MTT assay was used to detect cell viability. Transwell assay was used to detect the migration and invasion abilities. Dual-luciferase reporter assay was used to confirm the targeted relationship between LINC01503 and miR-335-5p. RESULTS Compared with normal tissues, the expression of LINC01503 was significantly increased in the lung cancer tissues, and the expression of miR-335-5p was significantly decreased (P<0.05). Compared with stage I/II , the expression level of LINC01503 in the lung cancer tissues of stage III/IV was significantly increased, and the expression of miR-335-5p was significantly decreased (P<0.05). The patients with high expression of LINC01503 had lower short-term survival rates than those with low expression of LINC01503 (P<0.05). Compared with normal human bronchial epithelial cell line BEAS-2B, the expression of miR-335-5p in lung cancer cell lines H1299, A549 and SPC-A-1 were significantly decreased, and the expression of LINC01503 was significantly increased (P<0.05). Over-expression of miR-335-5p and inhibition of LINC01503 expression inhibited the viability, migration and invasion of H1299 cells, and inhibited the protein expression of cyclin D1, matrix metalloproteinase-2 （MMP-2） and MMP-9 (P<0.05). LINC01503 targeted and regulated miR-335-5p expression, and interfering with miR-335-5p expression reversed the inhibitory effect of inhibiting LINC01503 expression on the viability, migration and invasion of H1299 cells. CONCLUSION Inhibition of lncRNA LINC01503 inhibits the viability, migration and invasion of lung cancer cells. The mechanism may be related to the targeted regulation of miR-335-5p.     

Resveratrol protects cortical neurons in infant rats with bacterial meningitis through miR-223-3p/NLRP3 pathway

AIM To investigate the protective effect of resveratrol (Res) on cortical neurons in rat bacterial meningitis (BM) model. METHODS Group B hemolytic Streptococcus was injected via the posterior cistern to establish a BM model. Resveratrol was administered intranasally and microRNA-223-3p (miR-223-3p) antagomir was administered by intracerebroventricular injection. HE staining was used to observe the pathological changes of the brain tissue. Loeffler scoring method was used to evaluate the neurobehavioral functions. TUNEL staining was used to detect neuronal apoptosis. The expression of interleukin-1β (IL-1β), IL-18, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba1) was detected by immunofluorescence staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3）, cleaved caspase-1, IL-1β and IL-18 were determined by Western blot. The expression level of miR-223-3p was detected by RT-qPCR. Online software TargetScan was used to search for the complementary nucleotide sequences between miR-223-3p and NLRP3 mRNA. RESULTS Compared with sham group, the thickness of meninges in BM model was increased, the neurological score was decreased (P<0.05), and the number of TUNEL positive neurons was increased significantly (P<0.05). Astrocytes and microglia were activated, the fluorescence intensity of IL-1β and IL-18 was increased (P<0.05), and the expression levels of NLRP3, cleaved caspase-1, IL-1β, IL-18 and miR-223-3p were increased (P<0.05). Compared with BM group, after treatment with resveratrol, the neurological score was increased (P<0.05), the number of TUNEL positive neurons was decreased significantly (P<0.05), and the inflammatory response of astrocytes and microglia was suppressed. The fluorescence intensity of IL-1β and IL-18 was decreased (P<0.05), the protein levels of NLRP3, cleaved caspase-1, IL-1β and IL-18 were decreased (P<0.05), and the expression level of miR-223-3p was increased (P<0.05). A nucleotide sequence in the 3'-UTR of NLRP3 mRNA might be targeted by miR-223-3p. In the brain of rat BM model, compared with antagomir control group, the expression of NLRP3 was increased in miR-223-3p antagomir group with resveratrol treatment (P<0.05). CONCLUSION Resveratrol may reduce the inflammatory death of cortical neurons in BM model of infant rats through miR-223-3p/NLRP3 pathway, thus playing a protective role for the neurons.     

IL-33-modified BMSCs attenuate acute kidney injury induced by sepsis in rats through MyD88

AIM To investigate the effect of interleukin-33 (IL-33)-modified bone marrow mesenchymal stem cells (BMSCs) on sepsis-induced acute kidney injury (AKI) in rats and the expression of myeloid differentiation factor 88(MyD88). METHODS A septic rat model was established by cecal ligation and puncture. The SD rats (n=80) were randomly divided into control group, model group, negative transfection group (transplanting untransfected BMSCs) and IL-33 transfection group (transplanting BMSCs transfected with IL-33), with 20 in each group. Survival rates of the rats within 72 h in the 4 groups were compared. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured before, and 24, 48 and 72 h after transplantation. The kidney pathological damage was observed by HE staining, and the apoptosis of renal cells was detected by TUNEL method 72 h after transplantation. Western blot was used to detect the protein expression levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), Toll-like receptor 4 (TLR4) and MyD88. RESULTS The survival rate of the rats in model group was significantly lower than that in control group (P<0.05). The survival rate of the rats in IL-33 transfection group was higher than that in model group and negative transfection group (P<0.05). The levels of SCr and BUN in model group were higher than those in control group (P<0.05). The levels of SCr and BUN in IL-33 transfection group were significantly reduced after transplantation, and were lower than those in model group and negative transfection group (P<0.05). The renal tissue pathological injury score in model group was significantly higher than that in control group (P<0.05). Compared with model group and negative transfection group, the renal tissue pathological injury score in IL-33 transfection group was significantly reduced (P<0.05). The proportion of apoptotic cells in the kidney tissues in model group were higher than that in control group (P<0.05). Compared with model group and negative transfection group, the proportion of apoptotic cells in the kidney tissues in IL-33 transfection group was significantly reduced (P<0.05). The protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in model group were significantly higher than those in control group (P<0.05). Compared with model group and negative transfection group, the protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in IL-33 transfection group were significantly decreased (P<0.05). CONCLUSION IL-33 gene-modified BMSCs significantly improve the renal function of AKI rats with sepsis. The mechanism may be related to IL-33 regulating TLR4/MyD88 signaling pathway and inhibiting renal inflammatory response.     

Mangiferin attenuates hypoxia/reoxygenation-induced injury of human myocardial cells

AIM To investigate the effect of mangiferin on hypoxia/reoxygenation (H/R)-induced injury of human myocardial cells and its mechanism. METHODS Human myocardial AC16 cells were divided into normal group, H/R group and H/R + mangiferin (50, 100 and 200 μmol/L) treatment groups. The mRNA and protein expression levels of Kelch-like epichlorohydrin-associated protein-1 (Keap-1), Bax, Bcl-2, caspase-3, caspase-9 and superoxide dismutase 2 (SOD2) were detected by RT-qPCR and Western blot, respectively. The protein expression of nuclear factor E2-related factor 2 (Nrf-2) in nucleus was determined by Western blot. The expression of microRNA-432-3p (miR-432-3p) was detected by RT-qPCR. The generation of reactive oxygen speciess (ROS) in the cells was measured by DCFH-DA probing. The cell viability was measured by CCK-8 assay. Apoptosis was analyzed by flow cytometry. RESULTS No significant difference in the expression of miR-432-3p and Keap-1 between normal group and H/R group was observed. Compared with normal group, the nuclear translocation of Nrf-2, the ROS level, and the mRNA and protein expression of Bax, caspase-3 and caspase-9 were significantly increased in H/R group （P<0.05）. The mRNA and protein expression of SOD2 and Bcl-2, and the cell viability significantly decreased in H/R group compared with normal group, while the apoptosis was increased significantly （P<0.05）. Treatment with mangiferin resulted in an increase in the miR-432-3p expression and a decrease in the ROS level, and the expression of Keap-1, Bax, caspase-3 and caspase-9 was also inhibited as compared with H/R group （P<0.05）. The Nrf-2 nuclear translocation, and the protein levels of SOD2 and Bcl-2 in mangiferin treatment groups were significantly increased as compared with H/R group （P<0.05）. The cell viability was increased significantly, and the apoptosis was decreased significantly in mangiferin treatment groups as compared with H/R group （P<0.05）. The effects of mangiferin in middle- and high-dose groups were better than those in low-dose group, and no significant difference between middle- and high-dose groups was found. CONCLUSION Mangiferin inhibits the decrease in myocardial cell viability and the apoptosis induced by H/R injury. The mechanism may be related to the up-regulation of Nrf-2 antioxidant stress effect via enhancing the expression of miR-432-3p.