Expression and Correlation Analysis of Serum miR-497-5p and FGF-2 in Patients with Alzheimer's Disease / 医学研究杂志

Objective To investigate the expression level,diagnostic value and correlation of miR-497-5p and human fibroblast growth factor-2(FGF-2)in patients with Alzheimer's disease(AD).Methods The clinical data of 50 patients with first diagnosed AD and 37 normal subjects(control group)were collected,among which AD patients were divided into mild AD group(n=18),moder-ate AD group(n=18)and severe AD group(n=14).The expression level of miR-497-5p was detected by real-time quantitative polymerase chain reaction(RT-qPCR)and FGF-2 was detected by enzyme-linked immunosorbent assay(ELISA).Mini-mental state examination(MMSE)was used to evaluate the cognitive function of AD patients,and the correlation between miR-497-5p and MMSE and FGF-2 levels was analyzed.The diagnostic efficacy of miR-497-5p and FGF-2 levels for AD was evaluated using receiv-er operator characteristic(ROC)curve.Results Compared with the control group and mild AD group,the expression levels of miR-497-5p in moderate and severe AD groups were significantly increased(P＜0.01),and the level of FGF-2 was significantly decreased(P＜0.01).MiR-497-5p in AD group was negatively correlated with MMSE score and FGF-2 level(r were-0.724 and-0.748,P＜0.01).ROC curve analysis results showed that miR-497-5p,FGF-2 and their combined indexes had higher area under the curve,sensitivity and specificity in the diagnosis of moderate and severe AD and in the differentiation of mild and moderate AD,as well as mild and severe AD,and the combined indexes of miR-497-5p and FGF-2had the best diagnostic and differential efficacy.Conclusion Serum miR-497-5p is up-regulated and FGF-2 level is down-regulated in patients with moderate and severe AD.The combined detection of miR-497-5P and FGF-2has certain diagnostic value for moderate and severe AD and provides certain reference.

Effect and mechanism of lncRNA GACAT3 on the radiation resistance of gliomas by regulating the miR-497/YAP1 axis / 中华放射肿瘤学杂志

Objective:To investigate the role and mechanism of long non-coding RNA (lncRNA) gastric cancer associated transcript 3 (GACAT3) in glioma radioresistance.Methods:Real-time reverse transcription PCR (RT-qPCR) was used to detect the expression of lncRNA GACAT3 and miR-497 in human astrocyte NHA cells and glioma cells U251. NC-siRNA and GACAT3-siRNA were transfected into U251 cells, and the cells were treated with X-ray irradiation. Colony formation assay was used to detect the survival fraction of U251 cells. The apoptosis of U251 cells was detected by flow cytometry. Western blot was used to detect the expression of cysteine containing aspartate specific protease 3 (Caspase-3) in U251 cells. Bioinformatics software and dual luciferase reporter gene assay were used to predict and verify the targeting relationship between lncRNA GACAT3 and miR-497, and between miR-497 and Yes-associated protein 1 (YAP1), respectively. NC mimic, miR-497 mimic, GACAT3-siRNA and NC inhibitor, GACAT3-siRNA and miR-497 inhibitor were co-transfected into U251 cells. Colony formation assay, flow cytometry and Western blot were adopted to evaluate the effect of miR-497 overexpression and lncRNA GACAT3 on the radiosensitivity of U251 cells by regulating miR-497.Results:Compared with NHA cells, the expression of lncRNA GACAT3 in U251 cells was significantly up-regulated, and the expression of miR-497 in U251 cells was significantly down-regulated (both P<0.05). After knockdown of GACAT3, the survival fraction of irradiated U251 cells was significantly decreased, while the apoptosis rate and Caspase-3 protein expression were significantly increased (all P<0.05). lncRNA GACAT3 targeted and negatively regulated the expression of miR-497. Overexpression of miR-497 significantly reduced the survival fraction of U251 cells after irradiation, and increased the apoptosis rate and Caspase-3 protein expression. Inhibition of miR-497 significantly reversed the promoting effect of lncRNA GACAT3 knockdown on the radiosensitivity of U251 cells. miR-497 targeted and negatively regulated the expression of YAP1. Conclusion:Knockdown of lncRNA GACAT3 can enhance the radiosensitivity of glioma cells by regulating the miR-497/YAP1 axis.

Identification of MicroRNAs Binding Site in the 3’Untranslated Region of Long Non-Coding RNA, MIR497HG: A Bioinformatic Prediction

@#Introduction: Prediction and identification of miRNAs target genes are crucial for understanding the biology of miRNAs. Amidst reported long-coding RNA (lncRNA), the microRNA 195-497 cluster host gene (MIR497HG) regulation is mediated by multiple non-coding RNAs (ncRNAs) such as microRNAs (miRNAs). MIR497HG has been implicated as a tumour suppressor in various cancers. However, the impact of MIR497HG and its derived miRNAs is largely unknown and still needs to be further explored. Employing an experimental approach is often challenging since some lncRNAs are difficult to identify and isolate by the current isolation technique. Thus, bioinformatic tools are introduced to aid these problems. This study sought to search and identify the miRNAs targeting the 3’untranslated region (3’UTR) of MIR497HG. Methods: Here, bioinformatic tools were adopted to identify a unique list of miRNAs that potentially target the 3’UTR of MIR497HG. Results: A total of 57 candidate miRNAs that target the 3’UTR of MIR497HG were extracted using the miRDB. Meanwhile, STarMir predicted 291 miRNAs that potentially target the 3’UTR of MIR497HG. A common list of 36 miRNAs was obtained using the Venny 2.1.0 and further narrowed down using the LogitProb score of StarMir. Finally, a total 4 miRNAs (hsa-miR-3182, hsa-miR-7156-5p, hsa-miR-452-3p and hsa-miR-2117) were identified. The mRNA target of identified miRNAs was identified by TargetScan. Finally, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of mRNA target was done using Enrichr. Conclusion: This finding could be useful in understanding the complex interaction between MIR497HG and its regulatory miRNA. In addition, a comparative analysis of computational miRNA-target predictions is provided in this study would potentially lay the foundations for miRNAs to be used for biomarkers in cancer research.

Effect of aluminum exposure on abnormal phosphorylationof tau protein in PC12 cells of rats / 预防医学

Objective @#To investigate the effect of aluminum exposure on expression of miR-497-5p, wingless murine breast cancer virus integration site family member 3a (Wnt3a), β-catenin protein, glycogen synthase kinase-3β (GSK-3β) protein and tau protein in rat adrenal pheochromocytoma PC12 cells, so as to provide insight into unraveling the mechanisms underlying aluminum exposure-induced abnormal phosphorylation of tau protein.@* Methods@# PC12 cells were exposed to Al(mal)3 at concentrations of 0, 100, 200, 400 μmol/L for 24 h. The viability of PC12 cells was measured using cell counting kit-8 (CCK-8) assay. The relative expression of miR-497-5p and Wnt3a was detected using a real-time fluorescent quantitative PCR (RT-qPCR) assay, and the expression of Wnt3a, β-catenin, GSK-3β, P-GSK-3β (Ser9), tau and p-tau (Ser396) proteins were determined using Western blotting. @*Results @#The viability of PC12 cells appeared a tendency towards a decline with the increase of aluminum dose (Ftrend=323.473, P=0.001). RT-qPCR assay detected that the relative miR-497-5p expression appeared a tendency towards a rise with the increase of aluminum dose (Ftrend=14.888, P=0.031), and the relative Wnt3a expression appeared a tendency towards a decline with the increase of aluminum dose (Ftrend=165.934, P<0.001). The miR-497-5p expression negatively correlated with the relative Wnt3a expression (r=-0.693, P=0.012). The expression of Wnt3a (Ftrend=357.656, P=0.001), β-catenin (Ftrend=208.750, P=0.001) and p-GSK-3β (Ser9) proteins (Ftrend=512.583, P<0.001) appeared a tendency towards a decline with the increase of aluminum dose, and the expression of GSK-3β (Ftrend=39.965, P<0.001), tau (Ftrend=277.929, P=0.006) and p-tau (Ser396) proteins (Ftrend=96.247, P=0.002) appeared a tendency towards a rise with the increase of aluminum dose. @*Conclusion@# Up-regulation of miR-497-5p and GSK-3β expression and down-regulation of Wnt3a and β-catenin expression may be a mechanism underlying aluminum exposure-induced abnormal phosphorylation of tau protein.

Mechanism of LncRNA DDX11-AS1 in Regulating Proliferation, Migration and Apoptosis of Gastric Cancer Cells by Targeting MiR-497-5p / 胃肠病学

Background: LncRNA expression was up-regulated or down-regulated in gastric cancer, but the mechanism of role of LncRNA in the development and progression of gastric cancer was not been fully clarified. Aims: To investigate the mechanism of LncRNA DDX11-AS1 in regulating the proliferation, migration and apoptosis of gastric cancer cells by targeting the expression of miR-497-5p. Methods: qRT-PCR was used to detect the expressions of DDX11-AS1 and miR-497-5p in gastric cancer tissue and cell lines. Pearson method was used to analyze the correlation between DDX11-AS1 and miR-497-5p in gastric cancer tissue. Gastric cancer HGC-27 cells were randomly divided into NC group, si-con group, si-DDX11-AS1 group, miR-con group, miR-497-5p group, si-DDX11-AS1+anti-miR-con group, si-DDX11-AS1+anti-miR-497-5p group. The cell proliferation was detected by MTT. The cell apoptosis was detected by flow cytometry. Transwell assay was used to detect the migration and invasion ability. The dual luciferase reporter system assay was used to verify the targeted regulatory relationship between DDX11-AS1 and miR-497-5p. The protein expressions of cyclin D1, cleaved caspase-3, MMP-2 and MMP-9 were detected by Western blotting. Results: The expression of DDX11-AS1 in gastric cancer tissue and cell lines was significantly increased (P<0.05), while the expression of miR-497-5p was significantly decreased (P<0.05), DDX11-AS1 was negatively correlated with miR-497-5p (r=-0.754, P<0.05). Interfering the expression of DDX11-AS1 or up-regulating the expression of miR-497-5p significantly inhibited cell proliferation, migration and invasion (P<0.05), apoptosis rate was significantly increased (P<0.05), and the expressions of cyclin D1, MMP-2 and MMP-9 were significantly decreased (P<0.05), and the expression of cleaved caspase-3 was significantly increased (P<0.05). The dual luciferase reporter assay demonstrated that DDX11-AS1 negatively regulated the expression and activity of miR-497-5p. Inhibition of miR-497-5p expression reversed the effect of interference with DDX11-AS1 expression on the biological behavior of gastric cancer cells. Conclusions: Interference with LncRNA DDX11-AS1 expression can inhibit the proliferation, migration, invasion and induce apoptosis of gastric cancer cells by targeting and up-regulating the expression of miR-497-5p.

miR-497 over-expression inhibits epithelial-mesenchymal transition in lung cancer A549 cells by targeting cyclin E1 / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨过表达miR-497靶向细胞周期蛋白E1（cyclin E1，CCNE1）对肺癌A549细胞上皮间质转化（epithelial-mesenchymal transition，EMT）的影响。方法：常规培养人肺癌A549细胞，细胞实验分为正常组（不加干预）、对照组（转染miR-497 mimics-NC）、实验组（转染miR-497 mimics）。采用Transwell小室实验、免疫荧光染色、qPCR、Western blotting法分别检测各组细胞迁移和侵袭能力、蛋白间质标志物α-SMA和上皮标志物E-cadherin的表达、miR-497和CCNE1的表达水平，荧光素酶基因基因报告实验验证miR-497和CCNE1的靶向关系。结果：与对照组和正常组相比，实验组A549细胞迁移和侵袭的数量明显减少（均P<0.05），细胞的间质标志物α-SMA的绿色荧光强度明显减弱[（36.95±5.81） vs （98.69±2.36）、（97.94±2.63），均P<0.05]，上皮标志物E-cadherin的绿色荧光强度明显增强[（388.41±10.93） vs （100.95±6.37）、（102.55±3.18），均P<0.05]，miR-497的表达明显升高（均P<0.05），CCNE1的表达均明显下降（均P<0.05）。miR-497能够靶向调控CCNE1的表达。结论：在肺癌A549细胞中miR-497能够靶向调控CCNE1的表达，上调miR-497的表达后能明显抑制A549细胞迁移和侵袭能力，影响EMT相关蛋白的表达。

FGFR1 is an independent prognostic factor and can be regulated by miR-497 in gastric cancer progression

Fibroblast growth factor receptor 1 (FGFR1) has been reported in gastric cancer to be a prognostic factor. However, miR-497-targeted FGFR1 has not been explored in the carcinogenesis of gastric cancer. The present study intended to revalidate the prognostic significance of FGFR1 in patients with gastric cancer, and the mechanism of miR-497-regulated FGFR1 was investigated in gastric cancer cell proliferation and apoptosis. The messenger RNA (mRNA) and protein levels were assayed by RT-qPCR and western blotting, respectively. The targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. Cell proliferation was analyzed by CCK-8 assay. Annexin V-FITC/PI staining was used to evaluate the apoptosis in AGS and SGC-7901 cells. FGFR1 was frequently up-regulated in gastric cancer tissues and associated with poor overall survival in patients with gastric cancer. Interestingly, FGFR1 loss-of-function resulted in a significant growth inhibition and apoptosis in AGS and SGC-7901 cells. In addition, we found that miR-497 was inhibited in gastric cancer tissues and cell lines, while overexpression of miR-497 could suppress proliferation and induce apoptosis in AGS and SGC-7901 cells. Importantly, bioinformatics analysis and experimental data suggested that FGFR1 was a direct target of miR-497, which could inhibit FGFR1 expression when transfected with miR-497 mimics. Furthermore, we found that overexpression of FGFR1 reversed the growth inhibition and apoptosis of miR-497 mimics in AGS and SGC-7901 cells. These findings suggested that overexpression of miR-497 inhibited proliferation and induced apoptosis in gastric cancer through the suppression of FGFR1.

Research on miR-497 inhibiting the expression of inflammation-related genes in colon cancer cell line HCT116 using gene expression microarray / 肿瘤研究与临床

Objective To analyze the effect of miR-497 high expression on the gene expression profile of colon cancer cell line HCT116. Methods MiR-497 high expressing colon cancer cell model HCT116-497 and negative control HCT116-CON were established by lentiviral transduction. The human (V2) gene expression microarray was used to identify genes that were differentially expressed between colon cancer cells overexpressing miR-497 and the controls. The candidates were subjected to the gene ontology (GO) and KEGG pathway enrichment analysis by Molecule Annotation System 3.0 (MAS3.0). The differential expression of representative genes relative to inflammation were confirmed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results Of all the differently expressed genes, 582 genes were down-regulated by at least 3-folds, which were enriched in inflammation-related signaling pathways in colon cancer cells overexpressing miR-497. The decrease in 15 representative genes was validated by qPCR. Compared with those in HCT116-CON cells, expressions of 10 genes in HCT116-497 cells, including CACNB1, FOS, IL-29, RPS6KA2, TNFSF15, IL-11, INHBC, CSF1R, JAK3 and IL-2Rβ, were decreased significantly, and there were statistical differences (all P< 0.05) Conclusion MiR-497 inhibits the mRNA expression of inflammation-related genes in colon cancer cell line HCT116.

Expression Difference and Its Mechanisms of miR-497 and miR-34a in Platinum-sensitive and Platinum-re-sistant Epithelial Ovarian Carcinoma Patients / 中国药房

OBJECTIVE:To investigate the expression difference and its mechanism of miR-497 and miR-34a in platinum-sen-sitive and platinum-resistant epithelial ovarian carcinoma(EOC)patients. METHODS:A total of 72 EOC patients underwent ovari-an cancer staging surgery or cytoreductive surgery were selected from department of gynaecology and obstetriscs of our hospital dur-ing Jan. 2008-Jan. 2012. They received standardized platinum chemotherapy after surgery and were followed up (during Jul.2008-Jul.2016). According to the sensitivity to platinum,those patients were divided into platinum-sensitive group (42 cases) and platinum-resistant group (30 cases) . Real-time fluorescent quantitative PCR was adopted to detect the expression of miR-497 and miR-34a in tumor tissue,and the relationship of it with total survival period was investigated. The levels of DNA methylation of miR-497 and miR-34a promoter region were determined by nest type land type methylation specific PCR. Western blot assay was used to detect the H3K9 dimethylation(H3K9me2)levels. The H3K9me2 levels of miR-497 and miR-34a promoter region were de-termined by chromatin immunoprecipitation method. RESULTS:The expression levels of miR-497 and miR-34a in platinum-sensi-tive group were significantly higher than platinum-resistant group,with statistical significance (P0.05). CONCLUSIONS:The expression of miR-497 and miR-34a in tumor tissue of EOC patients are related to the sensitivity of platinum chemotherapy and the survival time of patients. DNA methylation and histone methylation of promoter region may be one of the mechanisms of their expression changes.

miR-497 regulates cell proliferation and apoptosis by targeting Bcl-2 in liver cancer cells / 中国肿瘤临床

Objective:To evaluate the effect of miR-497 on regulating cell proliferation and apoptosis by targeting Bcl-2 in liver cancer cells. Methods:We tested liver cancer tissue and para-carcinoma tissue and used RT-PCR or Western blot to detect the expression of miR-497and Bcl-2 protein. We also tested the liver cancer cel HepG2 transfected with miR-497 mimics and mimic control. The expressions of miR-497and Bcl-2 protein were detected by RT-PCR or Western blot. Cell proliferation activity was detected by the MTT method, cell apoptosis was detected by flow cytometry, and cel luciferase activity was detected by the dual-luciferase reporter gene experiment. Results:1) Compared with para-carcinoma tissue, the miR-497 expression of liver cancer tissue significantly decreased (P<0.05), whereas the Bcl-2 protein expression of liver cancer tissue significantly increased (P<0.05). 2) Compared with transfection mimic control, transfection miR-497 mimics could increase the miR-497 expression of liver cancer cel HepG2 (P<0.05) and decrease the Bcl-2 protein expression of liver cancer cel HepG2 (P<0.05). 3) Compared with transfection mimic control, co-transfection with miR-497 mimics and Bcl-2-WT could significantly decrease the luciferase activity of liver cancer cell HepG2 (P<0.05). 4) Compared with transfection mimic control, the proliferative activity of liver cancer cell HepG2 significantly decreased after transfection with miR-497 mimics (P<0.05). Compared with transfection miR-497 mimics, the proliferative activity of liver cancer cell HepG2 significantly increased after transfection with miR-497 mimics+Bcl-2 (P<0.05). 5) Compared with transfection mimic control, the total apoptosis rate of liver cancer cell HepG2 significantly increased after transfection with miR-497 mimics (P<0.05). Compared with transfection miR-497 mimics, the total apoptosis rate of liver cancer cell HepG2 significantly decreased after transfection with miR-497 mimics+Bcl-2 (P<0.05). Conclusion:In liver cancer cel s, miR-497 could target Bcl-2 to inhibit cel proliferation and enhance cell apoptosis.

Changes of serum miR-145 and miR-497 levels and their correlations with high sensitive C-reactive protein and matrix metalloproteinase-9 in patients with ischemic stroke / 中华神经医学杂志

Objective To explore the changes of serum miR-145 and miR-497 levels and their correlations with high sensitive C-reactive protein (Hs-CRP) and matrix metalloproteinase-9 (MMP-9) in patients with ischemic stroke.Methods One hundred and twenty-six patients with ischemic stroke,including 28 mild neurologic impairment (NI) patients,61 moderate NI patients and 37 severe NI patients,and 107 normal controls,were collected in our hospital from January 2013 to October 2013.Expressions of serum miR-145 and miR-497 were measured by real-time fluorescent quantitative PCR,protein markers Hs-CRP and MMP-9 were determined by immune turbidimetry and ELISA assay,respectively,correlations between microRNAs (miRNAs) and protein markers were analyzied by partial correlation analysis.Results Both expressions of serum miR-145 and miR-497,and Hs-CRP and MMP-9 levels in ischemic stroke patients were significantly elevated as compared with those in the normal controls (P＜0.05).Positive correlations were noted between of serum miR-145 expression and both serum miR-497 expression and expressions of serum Hs-CRP and MMP-9 (r=0.718,P=0.000;r=0.658,P=0.000;r=0.455,P=0.000);meanwhile,serum miR-497 expression and expressions of serum Hs-CRP and MMP-9 were also positively correlated (r=0.845,P=0.000;r=0.787,P=0.000).Severe NI patients had significantly higher expressions of serum miR-145 and miR-497,and Hs-CRP and MMP-9 levels as compared with mild NI patients and moderate NI patients (P＜0.05),and these levels in the moderate NI patients were significantly higher than those in the mild NI patients (P＜ 0.05);these miRNAs and protein markers showed a different correlation strength depended on the different severities of neurologic impairment.Conclusion Both of serum miR-145 and miR-497 levels may be useful biomarkers for screening ofischemic stroke;and miR-145 and miR-497 may have synergistic effect on regulating the pathogenesis of ischemic stroke.