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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.
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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.
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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.
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Humans , MAP Kinase Signaling System , Apoptosis , Sirtuins/genetics , MicroRNAs/genetics , Human Umbilical Vein Endothelial Cells , Lipoproteins, LDL/pharmacologyABSTRACT
Purpose To investigate the effect of down-regulation of miR-92 a on the proliferation and angiogenesis of nonsmall cell lung cancer (NSCLC). Methods Human NSCLC cell A549 was divided into three groups: A549 group (non-transfected A549 cells), sc-siRNA group (A549 cells transfected with sc-siRNA) and miR-92a-siRNA group (A 5 4 9 cells trans-fected with miR-92a-siRNA). The relative expression level of miR-92 a, PTEN and vascular endothelial growth factor (VEGF) in A549 cells and human bronchial epithelial (HBE) cells were detected by RT-PCR and Western blot respectively. The proliferation ability of A549 cells in each group was detected by living cell count and crystal violet staining experiment. Results The relative expression of miR-92 a in A549 cells was significantly higher than that in HBE cells (P < 0.05), the expression level of PTEN protein in A549 cells was significantly lower than that in HBE cells (P < 0.05), and the expression level of VEGF protein was significantly higher than that in HBE cells (P < 0.05).In the miR-92a-siRNA group, the relative expression of miR-92 a decreased (P < 0.05), the expression level of PTEN protein in-creased (P < 0.05), and the expression level of VEGF protein decreased (P < 0.05). The expression levels of PI3 K and Akt in miR-92a-siRNA group decreased (P < 0.05). the number of cells and cell proliferation ability in miR-92a-siRNA group reduced. Conclusion The expression of miR-92 a in NSCLC A549 cells is up-regulated, miR-92 a gene silencing can significantly inhibit cell proliferation and inhibit cell angiogenesis, PTEN and VEGF related PI3K/Akt signaling pathways may play an important role in this process.
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Objective To analyze miR-92a expression and its clinical significance in the plasma of systemic lupus erythematosus (SLE) patients.Methods Plasma samples from 44 SLE patients,16 rheumatoid arthritis (RA) patients and 20 healthy controls were collected.The small RNAs in these plasma samples were isolated and reversely transcribed.Using cel-miR-39 as the external reference,the levels of miR-92a expression were detected by real-time polymerase chain reaction (PCR) method.MiR-92a and cel-miR-39 were analyzed by real-time fluorescence quantitative PCR and agarose gel electrophoresis.The sensitivity and specificity of miR-92a as SLE were analyzed by receiver-operating characteristic (ROC) curve.The correlation between the levels of miR-92a expression and the clinic pathological features of SLE and biological significance of miR-92a expression in SLE were further analyzed by Pearson or Chi-square test.Results Our data indicated that the plasma levels of miR-92a expression was 49.20 (5.33,95.17) in SLE patients,411.30 (320.84,504.69) in healthy controls,and 25.59(11.20,30.54) in RA patients.The difference was significant (x2=40.77,P＜0.01).The area under the ROC curve (AUC) was 0.958 for discriminating between SLE patients and normal subjects and 1.00 for discriminating between RA patients and healthy controls.The levels of miR92a expression cutoff values were set the as 198.59 for healthy control and 85.35 for RA patients,the diagnostic sensitivity and specificity were 93.2％,90％,and 100％,100％,res-pectively.The analysis of the correlation between miR-92a expression and the clinic pathological features of SLE had shown that the levels of plasma miR-92a expressions were much lower in SLE patients with down-regulated complement C3,and up-regulated urea nitrogen,creatinine,LDH,ATH (all P＜0 05).Conclusion Down-regulated miR-92a expression in plasma of SLE may be involved in the SLE disease occurrence or development and could be used as a novel potential diagnostic biomarker for SLE.
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Background and purpose:Osteosarcoma is the most common primary malignant bone tumor in the children and young adults. Until now, there is no prognostic indicator with high speciifcity and high sensitivity. This study investigated the relationship between miR-92a level in palsma and clinical pathological characteristics, as well as the prognosis in osteosarcoma.Methods:The plasma from 30 healthy volunteers who underwent physical examination and from 45 cases of osteosarcoma before and after operation were collected. The miR-92a level was detected by real-time lfuorescent quantitative polymerase chain reaction (RTFQ-PCR) method. According to the relative expression≥5 of miR-92a, the osteosarcoma patients were divided into 2 groups: miR-92a high expression group and low expression group.Results:The miR-92a level in palsma from osteosarcoma is signiifcantly higer than that from healthy volunteers. Before surgery, the miR-92a level was markedly higher than after surgery. There was no correlation between the miR-92a level and the gender, age, tumor location, tumor size and histological type. However, signiifcant correlation was found with Enneking grade, tumor cell necrosis rate and lung metastasis. Compared with the low-expression group, high miR-92a expression was associated with a signiifcantly shorter overall survival using Kaplan-Meier analysis (P=0.035). Conclusion:The results suggest a signiifcant relationship between miR-92a and survival ratio. miR-92a expression level may be a useful prognostic indicator in osteosarcoma.
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Objective:To investigate the expression of miR-92a in colorectal cancer (CRC) and its effect on the regulation mechanisms of tumor angiogenesis. Methods:The miRNA-92a expression in 25 CRC tissues and HT29, SW620, SW480, and HCT116 CRC cells was detected using quantitative real-time polymerase chain reaction. The CD31 positive expression of blood microvessels in CRC tissues was measured by immunohistochemistry. Pearson correlation analysis was used to analyze the relationship between miR-92a and tu-mor angiogenesis. The miR-92a mimic or inhibitor was transfected into HCT116 and SW620 cells in vitro to upregulate or downregu-late the miR-92a expression. The effect of miR-92a overexpression or suppression on the formation of human umbilical vein endotheli-al cell (HUVEC) tubules was detected by tube formation assay. Changes in phosphatase and tensin homolog (PTEN) protein expression were measured by Western blot. Results:The expression level of miR-92a in CRC tissues was significantly higher than that in matched adjacent tissues (P<0.01). The expression levels of miR-92a in HT29, SW480, SW620, and HCT116 colon cancer cell lines were signifi-cantly higher than that of the normal colorectal epithelium control (P<0.05). The number of CD31 positive expression of microvessel density (MVD) in CRC tissues was significantly higher than that in adjacent tissues (P<0.01), and the miR-92a expression level was posi-tively correlated with the MVD in CRC tissues (r=0.580, P=0.01). The cell culture supernatant of HCT116 with miR-92a overexpression could significantly promote the formation of HUVEC tubules (P<0.05). The upregulation of miR-92a expression could significantly inhib-it the expression of PTEN protein in CRC cells (P<0.01). Conclusion:The miR-92a was highly expressed in CRC cells and tissues, which was closely related to the formation of tumor angiogenesis in CRC. The miR-92a could promote tumor angiogenesis in CRC by inhibit-ing PTEN expression.