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.

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.