Matrine inhibits inflammatory response induced by TNF-α in human umbilical vein endothelial cells through miR-25-3p-mediated Klf4 pathway / 中国中药杂志

This study aimed to analyze the effect of matrine on tumor necrosis factor-α(TNF-α)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs) and explore whether the underlying mechanism was related to the miR-25-3p-mediated Krüppel-like factor 4(Klf4) pathway. The HUVEC cell inflammation model was induced by TNF-α stimulation. After 24 or 48 hours of incubation with different concentrations of matrine(0.625, 1.25, and 2.5 mmol·L~(-1)), CCK-8 assay was used to detect cell proliferation. After treatment with 2.5 mmol·L~(-1) matrine for 48 h, the expression of TNF-α, interleukin-6(IL-6), interleukin-1β(IL-1β), and Klf4 mRNA and miR-25-3p was detected by real-time fluorescence-based quantitative PCR, and the protein expression of TNF-α, IL-6, IL-1β, and Klf4 was detected by Western blot. The anti-miR-25-3p was transfected into HUVECs, and the effect of anti-miR-25-3p on TNF-α-induced cell proliferation and inflammatory factors was detected by the above method. The cells were further transfected with miR-25-3p and incubated with matrine to detect the changes in proliferation and expression of related inflammatory factors, miR-25-3p, and Klf4. The targeting relationship between miR-25-3p and Klf4 was verified by bioinformatics analysis and dual luciferase reporter gene assay. The results displayed that matrine could inhibit TNF-α-induced HUVEC proliferation, decrease the mRNA and protein expression of TNF-α, IL-6, and IL-1β, increase the mRNA and protein expression of Klf4, and reduce the expression of miR-25-3p. Bioinformatics analysis showed that there were specific complementary binding sites between miR-25-3p and Klf4 sequences. Dual luciferase reporter gene assay confirmed that miR-25-3p negatively regulated Klf4 expression in HUVECs by targeting. The inhibition of miR-25-3p expression can reduce TNF-α-induced cell proliferation and mRNA and protein expression of TNF-α, IL-6, and IL-1β. MiR-25-3p overexpression could reverse the effect of matrine on TNF-α-induced cell proliferation and the mRNA and protein expression of TNF-α, IL-6, IL-1β, and Klf4. This study shows that matrine inhibits the inflammatory response induced by TNF-α in HUVECs through miR-25-3p-mediated Klf4 pathway.

miR-92a-3p and miR-25-3p Sponges Up-regulate Kiss1 and Affect the Onset of Puberty and Estrous Cycle of Female Mice / 中国生物化学与分子生物学报

Kisspeptin, the neuropeptide produced by Kiss1 neurons in the hypothalamus, is involved in the neuroendocrine regulation of puberty initiation, reproductive system maturation, ovulation and other processes by influencing the secretion of gonadotropin-releasing hormone. Kiss1 gene expression is regulated by multiple trans-regulatory factors and epigenetics. Prediction and preliminary experiments have shown that the seed sequences of miR-92a-3p and miR-25-3p can directly bind to the 3′-UTR of Kiss1 and inhibit the expression of Kiss1. In order to further study the role of miR-92a-3p and miR-25-3p in the regulation of Kiss1, specific absorptive sponge vectors (sponge-miR-92a and sponge-miR-25) with inhibitory effects on miR-92a-3p and miR-25-3p were constructed to realize the functional loss of miRNA. Flow cytometry and dual luciferase reporter assays both confirmed that both sponge vectors could adsorb exogenous or endogenous target miRNAs very effectively. The sponge-miR-92a and sponge-miR-25 vectors are further packaged into the lentivirus LV-sponge-miR-92a and LV-sponge-miR-25. The results of real-time fluorescence quantitative PCR showed that the expression level of Kiss1 in the hypothalamic primary neurons infected by LV-sponge-miR-92a and LV-sponge-miR-25 was significantly up-regulated (P < 0. 05). After injecting LV-sponge-miR-92a into the hypothalamus, the time of female mouse vulva opening was significantly earlier (P<0. 05). The normal oestrus cycle of female mice with was disrupted by injections of LV-sponge-miR-92a and LV-sponge-miR-25 in the hypothalamus. In conclusion, we successfully constructed sponge vectors capable of effectively adsorbing miR-92a-3p and miR-25-3p, and demonstrated their role in removing the inhibition of miR-92a-3p and miR-25-3p on Kiss1. Hypothalamic sponge injection had a certain effect on both the time of vulva opening and the estrus cycle of female mice, suggesting that miR-92a-3p and miR-25-3p may play an important role in the initiation of puberty and reproductive maturity.

The relationship of peripheral blood single uncoding RNA-25-3p expression level and the sertraline efficacy in patients with panic disorder / 中华行为医学与脑科学杂志

Objective@#To identify potential relationship between single uncoding RNA-25-3p (miR-25-3p) expression level and the sertraline efficacy in patients with panic disorder.@*Methods@#Sixty cases of patients with panic disorder(case group) and sixty healthy-controls(control group) were collected with demographic data and peripheral venous blood before and after treatment.All the patients were evaluated using the 14-item Hamilton Anxiety Rating Scale (HAMA) and Panic Disorder Severity Scale (PDSS) at baseline, and then received sertraline treatment for 6 weeks.After six-week treatment, each patient was evaluated again with HAMA and PDSS.RT-PCR was used to detect the level of miR-25-3p expression.@*Results@#There was no significant difference in the miR-25-3p levels between control group (1.27±0.32) and case group (1.73±1.09) before treatment(t=1.53, P=0.14), but the levels in case group were much higher than that in control group after the treatment (5.72±4.13 vs 1.73±1.09, t=-2.15, P=0.04). Besides, the changes of the miR-25-3p levels were positively related with both the changes of PDSS3 and PDSS7 items before and after the treatment (r=0.60, P=0.02 for PDSS3 and r=0.61, P=0.02 for PDSS7).@*Conclusions@#miR-25-3p is associated with the drug efficacy and the outcome of some clinical symptoms of panic disorder.These findings might provide some evidence for the individualized treatment of patients with panic disorder according to regulation of gene expression in the future.

The relationship of peripheral blood single uncoding RNA-25-3p expression level and the sertraline efficacy in patients with panic disorder / 中华行为医学与脑科学杂志

Objective To identify potential relationship between single uncoding RNA-25-3p (miR-25-3p) expression level and the sertraline efficacy in patients with panic disorder.Methods Sixty cases of patients with panic disorder(case group) and sixty healthy-controls(control group) were collected with demographic data and peripheral venous blood before and after treatment.All the patients were evaluated using the 14-item Hamilton Anxiety Rating Scale (HAMA) and Panic Disorder Severity Scale (PDSS) at baseline,and then received sertraline treatment for 6 weeks.After six-week treatment,each patient was evaluated again with HAMA and PDSS.RT-PCR was used to detect the level of miR-25-3p expression.Results There was no significant difference in the miR-25-3p levels between control group (1.27±0.32) and case group (1.73±1.09) before treatment(t=1.53,P=0.14),but the levels in case group were much higher than that in control group after the treatment (5.72±4.13 vs 1.73±1.09,t=-2.15,P=0.04).Besides,the changes of the miR-25-3p levels were positively related with both the changes of PDSS3 and PDSS7 items before and after the treatment (r=0.60,P=0.02 for PDSS3 and r=0.61,P=0.02 for PDSS7).Conclusions miR-25-3p is associated with the drug efficacy and the outcome of some clinical symptoms of panic disorder.These findings might provide some evidence for the individualized treatment of patients with panic disorder according to regulation of gene expression in the future.

Expression levels of serum miR-25 in the patients with non-small cell lung cancer and its clinical significance / 临床检验杂志

Objective To determine the expression levels of serum miR-25 in the patients with non-small cell lung cancer (NSCLC),and evaluate its clinical significance in the screening of NSCLC.Methods Serum samples from 82 untreated NSCLC patients,including 4 with TNM satge Ⅰ,10 with TNM stage Ⅱ,11 with TNM stage Ⅲ,53 with TNM stage Ⅳ and 4 with unknown stage,and 82 healthy controls with matched age and gender were collected,and the levels of miR-25 in these samples were determined by quantitative reverse transcription PCR (qRT-PCR).The diagnostic value of miR-25 in NSCLC was evaluated by the receiver operating characteristic (ROC) curve.Results Serum miR-25 levels in NSCLC patients were significantly higher than that in healthy controls (0.017 ± 0.028 vs 0.004 ±0.004,t =4.098,P ＜0.01).The area under the ROC curve (AUGROC),sensitivity and specificity of miR-25 for the diagnosis of NSCLC were 0.818 (95％ CI:0.753-0.882),70.7％ and 80.7％,respectively.In addition,the AUCROC,sensitivity and specificity of serum miR-25 for the screening of stage Ⅰ/Ⅱ NSCLC were 0.852 (95％ CI:0.728-0.976),78.6％ and 87.8％,respectively.Logistic regression analysis showed that miR-25 was an independent risk factor of NSCLC (OR =10.84,95％ CI:5.07-23.19,P ＜ 0.01).Conclusion Serum miR-25 levels in NSCLC patients increase significantly,indicating that it may be a novel molecular biomarker for the screening of NSCLC.

Study on the Relationship of MiR-2 5 Targeting FBXO3 3 with Cell Apoptosis and Prognosis in Renal Cell Carcinoma / 现代检验医学杂志

Objective To explore the correlation between miR-25 and FBXO33 in renal cell carcinoma (RCC),and to analyze the relationship with apoptosis and prognosis of renal cell carcinoma.Methods The 511 RCC chip results,from 1998 to 2013,were downloaded from the Cancer Genome Atlas (TCGA)database,and were analyzed for the correlation between miR-25 and FBXO33 by Pearson test.The expression of fluorescein were detected with the FBXO33 3’UTR wild-type,mu-tant and blank control luciferase reporter gene treated by miR-25.The viability of cells transient translated by the miR-25 mimic,siRNA and the controls were detected by CCK8 method.The apoptosis of cells transient translated by the miR-25 mimic,siRNA and the controls were detected by flow cytometry.58 cases with follow-up data were screened from TCGA by expression of FBXO33 negative correlation miR-25.The survival was analyzed between low expression of miR-25 combined with FBXO33 high expression group (n=34)with high expression of miR-25 combined with FBXO33 low expression group (n=24),using Log-rank test and Gehan-Breslow-Wilcoxon test.Results FBXO33 was negatively correlated with miR-25 in RCC tissue (r=-0.161 1,Pearson test).Compared with the control group,miR-25 could reduce the RLU of wild type group to 80.2%±2.6%,the difference was statistically significant (t=6.539,P=0.006).The RLU of mutation group was 103.5%±8.4% compared with that of blank control group,the difference was not statistically significant (t=0.041 3,P=0.968 4),compared with the blank group in 72h for the cell varibility,miR-25 siRNA group were elevated by 32.7%± 3.5%,the difference was statistically significant (P<0.05).The miR-25 mimic group were reduced by 23.3%±1.7%,the difference was statistically significant (P<0.05),and compared with the control group,the early apoptosis rate was de-creased in mimic-miR-25-3p group (8.83 ± 0.09 vs 12.83 ± 0.14),while the difference was statistically significant (t=42.17,P=0.005).The late apoptosis rate was slightly escalated (0.41±0.10 vs 0.33±0.15),while the difference was not statistically significant (t=0.75,P=0.639).Compared with the control group,the early apoptosis rate was increased in siR-NA-miR-25-3p group (19.05 ± 1.64 vs 13.68 ± 0.78),while the difference was statistically significant (t=5.12,P=0.006).But the late apoptosis rate was reduced (0.56±0.10 vs 0.62±0.08),while the difference was not statistically sig-nificant (t=0.83,P=0.376).The survival rate was higher in patients with low expression of miR-25 combined with high expression of FBXO33 (n=34)than that of miR-25 high expression combined with low expression of FBXO33 (n=24),the difference was statistically significant (Log-rank test P=0.025 2,Gehan-Breslow-Wilcoxon test P=0.004 9).Conclusion MiR-25 can inhibite FBXO33 in renal cell carcinoma,improve the cell activity,inhibit apoptosis and reduce the prognosis.

miR-25 promotes gastric cancer cells proliferation by directly targeting KLF4 / 实用医学杂志

ObjectiveTo investigate whether miR-25 targets KLF4 to regulate the growth of gastric cancer cells and lucidate the mechanism of miR-25 producing functional effects in gastric cancer cells. Methods The expression levels of miR-25 and KLF4 were detected by qRT-PCR in 35 pairs of gastric cancer tissues of patients. Target genes of miR-25 were predicted using Targetscan and verified though dual luciferase activity assay and west-ern blot. MTT assay and clone formation assay were detected after downregulated miR-25 and overexpressed KLF4 respectively. Results miR-25 was highly expressed in gastric cancer tissues with KLF4 downexpresstion. Downreg-ulated miR-25 and overexpressed KLF4 respectively can significantly reduce the proliferation of gastric cancer cells,the results were opposite when overexpression of KLF4. Conclusion miR-25 promotes gastric cancer cells proliferation by targeting KLF4.

Target genes of the hsa-miR-106b-25 cluster:Prediction and bioinformatic analysis / 医学研究生学报

Objective The hsa-miR-106b-25 gene cluster is involved in various biological processes of carcinoma .This study aims at a prediction and function analysis of the hsa-miR-106b-25 gene cluster, miR-106b, miR-93, and miR-25, so as to provide some evidence for further studies on the functions of the three miRNAs and the mechanisms of their interaction . Methods We obtained the sequences of miR-106b, miR-93, and miR-25 from the miRBase, predicted their target genes with TargetScan , PicTar, and miRanda, and used 3 or more experimentally verified target genes from the miRTarbase as the gene set for further bioinformatic analysis .We predic-ted the biological processes of the target genes by GeneOntology analysis and enriched KEGG ( Kyoto Encyclopedia of Genes and Genomes) by pathway analysis, produced protein-protein interaction ( PPI) networks with STRING . Results The target genes of the miR-106b-25 gene cluster were significantly enriched in such biological processes as the regulation of macromolecule metabolism , regulation of metabolic process , and cell cycle process , while the KEGG pathway mainly in glioma, melanoma, prostate cancer , and gallbladder carcino-ma.The proteins encoded by the targeted genes of showed complicated interactions , and those encoded by the KAT2B, PTEN, TP53, CDH1, MDM2, E2F1, RB1, and SMAD7 plaid a core role in the interac-tion network. Conc lusoi n MiRNAs of the miR-106b-25 gene cluster regulate the downstream target proteins involved in tumorigenesis by participating in the cell cycle and cancer signaling pathway .

MiR-25-3p attenuates the proliferation of tongue squamous cell carcinoma cell line Tca8113

OBJECTIVE@#To investigate the effects of miR-25-3p on the occurrence, development and proliferation of tongue squamous cell carcinoma cells.@*METHODS@#To establish tongue squamous cell carcinoma cell line Tca8113 that stably and highly express miR-25-3p using recombinant retroviral vector-mediated gene transfer method. The proliferation of transfected Tca8113 was detected by thiazolyl blue tetrazolium bromide (MTT) and cell colony formation assays. cyclinD1, p21(cip1) and p27(kip1) mRNA expressions in the transfected Tca-8113 were detected by quantitative PCR. cyclinD1, p21, p27(kip1), AKT, p-AKT, FOXO1 and p-FOXO1 expressions in the transfected Tca8113 were detected by western blot analysis. In addition, miR-25-3p expression in the tongue squamous cell carcinoma cell line and tissue specimen was also detected by quantitative PCR.@*RESULTS@#Quantitative PCR showed that miR-25-3p expression in the tongue squamous cell carcinoma cell lines and tissue specimen was significantly lower than that in the adjacent tissue. MTT and cell colony formation assays showed that after miR-25-3p overexpression, the proliferation of transfected Tca8113 was obviously attenuated. Western blot analysis and quantitative PCR showed that after miR-25-3p overexpression, p21(cip1) and p27(kip1) expressions were upregulated, while cyclinD1, AKT, FOXO1 expressions were downregulated, and AKT and FOXO1 phosphorylation was inactivated in the transfected Tca8113 cells.@*CONCLUSIONS@#MiR-25-3p inhibited the proliferation of tongue squamous cell carcinoma cells and regulated cell cycle-related protein expression, playing an important role in the occurrence and development of squamous cell carcinoma of the tongue.