Dysregulated expression of miR-92a-3p in senile osteoporosis and its diagnostic value in hip fragility fractures / 中华内分泌外科杂志

Objective:To investigate the expression of miR-92a-3p in senile osteoporosis (OP) and its diagnostic value in hip fragility fractures.Methods:With the help of the National Center for Biotechnology Information (NCBI) website, the data sets related to OP and miRNA were retrieved and analyzed and screened to obtain the target miRNA. Serum samples were collectedfrom 53 OP patients and 24 healthy people, and the OP patients were divided into hip fragility fracture group (OP_F; n=30) and no hip fragility fracture group (OP_WF; n=23). The subjects' bone mineral density and serum bone metabolism markers were measured: calcium, phosphorus, parathyroid hormone, 25-hydroxyvitamin D, bone alkaline phosphatase, serum C-terminal peptide. The expression levels of target miRNAs in serum samples were detected by qRT-PCR. Chi-square test, t test, Spearman rank correlation and receiver operating characteristic curve (ROC curve) were used to analyze the potential relationship between the expression level of miR-92a-3p and the clinical characteristics of patients.Results:According to the analysis of NCBI website, the expression of miR-92a-3p in OP patients was higher ( t=3.41, P=0.007) than that in healthy people, and the expression level of miR-1304-5p was lower ( t=5.13, P<0.001). qRT-PCR experiments confirmed the above results, and further found that the expression of miR-92a-3p in the OP_F group was significantly higher than that in the OP_WF group (t=3.01, P=0.004), but there was no significant difference in miR-1304-5p between the two groups (t=0.71, P=0.480). Compared with the healthy control group, the BMI ( t=2.71, P=0.008), bone mineral density score ( t=29.02, P<0.001), calcium ( t=61.20, P<0.001), phosphorus ( t=2.54, P=0.013), 25-hydroxyvitamin D (t=3.01, P=0.004) ,bone alkaline phosphatase ( t=12.56, P<0.001), and serum C-terminal peptide ( t=7.52, P<0.001) levels were statistically different between the OP patient group and the healthy control group. Compared with the OP_WF group, the bone mineral density score ( t=2.08, P=0.042), calcium ( t=15.75, P<0.001), bone alkaline phosphatase ( t=2.02, P=0.049) and serum C-terminal peptide ( t=3.39, P=0.001) levels were statistically different between the OP_F group and the OP_WF group. Bone mineral density score and bone alkaline phosphatase concentration were related to the expression of miR-92a-3p and were negatively and positively correlated ( r=0.416, P=0.022), respectively ( r=-0.403, P=0.027). The area under the ROC curve was 0.723, and the level of miR-92a-3p had potential significance in the diagnosis of hip fragility fractures ( P=0.006) . Conclusion:miR-92a-3p is highly expressed in OP and has biological significance for the diagnosis of hip fragility fractures.

miR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway

Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling 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.