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【Objective】 To investigate the molecular mechanism of microRNA-101-5p (miR-101-5p) affecting the proliferation and invasion of lung squamous cell carcinoma (LUSC) cells. 【Methods】 We downloaded the miRNA mature expression data and total RNA sequencing data of TCGA-LUSC from TCGA database to identify differentially expressed genes. The signal pathway enriched in ATAD2 was analyzed. The mRNA expressions of miR-101-5p and ATAD2 in the LUSC cells were detected by qRT-PCR. The effects of miR-101-5p on the proliferation and invasion of LUSC cells were detected by MTT assay, cloning assay, and invasion assay. The effects of ATAD2 on the cell cycle of LUSC cells were detected by flow cytometry. Western blotting was used to detect the expression of ATAD2 protein. Double luciferase experiment was used to verify whether miR-101-5p could bind to ATAD2 target. Finally, we detected the changes in the proliferation, cloning and invasion ability of LUSC cells by co-transfection with oe-ATAD2 and miR-101-5p mimic, and further explored whether miR-101-5p could regulate the biological function of LUSC cells through ATAD2. 【Results】 The miR-101-5p was significantly downregulated in LUSC tissues and cells. Overexpression of miR-101-5p could significantly inhibit the proliferation and invasion of LUSC cells. ATAD2, its downstream regulatory target gene, was significantly upregulated in LUSC, and miR-101-5p and ATAD2 expressions were inversely correlated. GSEA enrichment results showed that ATAD2 was significantly enriched in the cell cycle signal pathway. The double luciferase experiment proved that miR-101-5p targeted ATAD2, and the recovery experiment showed that miR-101-5p regulated the proliferation and invasion of LUSC cells by targeting ATAD2. 【Conclusion】 In this study, we found that miR-101-5p had low expression in LUSC, and that miR-101-5p decreased the proliferation and invasion of LUSC cells by targeted inhibition of ATAD2.
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Objective:To investigate the effect of microRNA-101 (miR-101) on proliferation, apoptosis and invasion of PANC1 cells of pancreatic cancer and its potential mechanism.Methods:PANC1 cells in logarithmic growth period were divided into 5 groups, including transfected miRNA mimic negative control group (mimic-NC group), transfected miRNA-101 mimic group (miR-101 mimic group), transfected miRNA inhibitor negative control group (inhibitor-NC group), and transfected miR-101 inhibitor group (miR-101 inhibitor group) besides control group. The relative expression level of miR-101 was detected by qRT-PCR, cell proliferation rate was determined by MTT assay, cell apoptosis rate was detected by flow cytometry, and cell invasion ability was detected by Transwell assay. The relative expression levels of IL-6, JAK2, p-JAK2, STAT3 and p-STAT3 mRNA and proteins were detected by qRT-PCR and Western blot. The targeting relationship between miR-101 and IL-6 was detected by dual luciferase reporter gene method.Results:After 48 h of transfection, the expression levels of miR-101 in PANC1 cells in control group, mimic-NC group, miR-101 mimic group, inhibitor-NC group and miR-101 inhibitor group were 1.98±0.12, 2.01±0.18, 6.73±0.23, 2.16±0.22 and 1.34±0.13; the proliferation rates were (32.75±2.43)%, (33.17±2.77)%, (15.68±1.17)%, (31.57±2.65)% and (45.75±3.16)%, respectively; the apoptosis rates were (3.82±0.57) %, (3.54±0.55)%, (28.61±0.78)%, (3.57±0.63)% and (1.03±0.62) %, respectively; the number of penetrating cells was (125.82±3.55), (132.17±4.28), (58.83±3.24), (128.77±5.06), (248.42±5.64)/high power field, respectively. The expression level of miR-101 and apoptosis rate in miR-101 mimic group were significantly higher than those in control group and mimic-NC group, and the proliferation rate and the number of transmembrane cells were significantly lower than those in control group and mimic-NC group. The expression level of miR-101 and apoptosis rate in miR-101 inhibitor group were significantly lower than those in control group, inhibitor-NC group and miR-101 mimic group, and the proliferation rate and the number of transmembrane cells were significantly higher than those in control group, inhibitor-NC group and miR-101 mimic group. The expression levels of IL-6, JAK2, STAT3 mRNA and protein in PANC1 cells of miR-101 mimic group were significantly lower than those in control group and mimic-NC group, while those in miR-101 inhibitor group were significantly higher than those in control group, miR-101 mimic group and inhibitor-NC group. The differences above were statistically significant ( all P value <0.001). Dual luciferase reporter gene assay showed that miR-101 could target IL-6. Conclusions:miR-101 can inhibit the proliferation and invasion of pancreatic cancer cells and promote their apoptosis, possibly through the regulation of IL-6/JAK2/STAT3 signaling pathway.
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Objective:To examine the plasma expression levels and clinical significances of microRNA(miR)-101-3p and miR-141-3p in children with sepsis.Methods:One hundred and fifty-three children with sepsis admitted in Sanya People′s Hospital from January 2016 to October 2019 were divided into sepsis without shock group (94 cases) and septic shock group (59 cases). In addition, they were further divided into survival group (107 cases) and death group (46 cases) according to the 28-day survival.Another 60 healthy children were selected as the healthy control group.Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was performed to detect plasma levels of miR-101-3p and miR-141-3p in all subjects.Receiver operating characteristic curve(ROC) were depicted to identify the diagnostic and prognostic potentials of plasma miR-101-3p, miR-141-3p and procalcitonin(PCT) in sepsis. Pearson′ s correlation analysis was performed to analyze the correlation between the expression levels of miR-101-3p, miR-141-3p and PCT with Acute Physiology and Chronic Health Evaluation Ⅱ(APACHE Ⅱ)score, Sequential Organ Failure Assessment(SOFA)score, leukocyte count and C-reactive protein level in children with sepsis. Results:Plasma levels of miR-101-3p, miR-141-3p and PCT in septic shock group and sepsis without shock group were significantly higher than those in the healthy control group (all P<0.001). Moreover, plasma levels of miR-101-3p (4.25±1.46 vs.1.86±0.75), miR-141-3p (3.17±1.08 vs.1.20±0.52) and PCT [(20.75±9.36) μg/L vs.(5.80±2.40) μg/L] in septic shock group were significantly higher than those in sepsis without shock group (all P<0.001). In addition, plasma levels of miR-101-3p, miR-141-3p and PCT in survival group and death group were significantly higher than those in the healthy control group (all P<0.001). Notably, plasma levels of miR-101-3p (4.83±1.62 vs.1.40±0.58), miR-141-3p (3.50±1.13 vs.0.96±0.47), and PCT [(26.30±11.72) μg/L vs.(3.25±2.16) μg/L] in death group were significantly higher than those in the survival group (all P<0.001). ROC curve analysis showed that the area under the curve (AUC) and 95% confidence interval (95% CI) of the combined diagnosis of sepsis with miR-101-3p, miR-141-3p and PCT were significantly higher than that of miR-101-3p, miR-141-3p or PCT alone [0.908 (0.850-0.970) vs.0.810 (0.748-0.873), 0.784 (0.723-0.844) and 0.825 (0.764-0.883), respectively; Z1=4.682, Z2=5.380 and Z3=4.417, all P<0.05]. The sensitivity and specificity of the combined diagnosis was 92.5% and 84.0%, respectively.The AUC and 95% CI of the combined prediction of miR-101-3p, miR-141-3p and PCT in the mortality of children with sepsis children with were significantly higher than those with miR-101-3p, miR-141-3p or PCT alone [0.930 (0.872-0.986) vs.0.848 (0.786-0.907), 0.792 (0.730-0.853) and 0.820 (0.762-0.878), respectively; Z1=4.537, Z2=5.728 and Z3=5.106, all P<0.05]. The sensitivity and specificity of the combined prediction in the mortality was 94.6%, and 87.0%, respectively.Correlation analysis showed that miR-101-3p and miR-141-3p levels were positively correlated with PCT ( r=0.804, 0.773, all P<0.001), APACHE Ⅱ score ( r=0.738, 0.695, P<0.001) and SOFA score ( r=0.752, 0.764, all P<0.001). Conclusions:Plasma levels of miR-101-3p and miR-141-3p in children with sepsis significantly increased, which are correlated with the severity of sepsis.A combination detection of miR-101-3p, miR-141-3p and PCT has high diagnostic and prognostic potentials in children with sepsis.
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Objective:To investigate the role of microRNA (miRNA)-101a in the liver fibrosis induced by activated hepatic stellate cell (HSC), through upregulating IRE1α signaling pathway.Methods:Carbon tetrachloride (CCl 4) induced liver fibrosis model of mice was established. RT-PCR was used to measure the mRNA level of miRNA-101a in liver tissue of mice. Protein level of α smooth muscle actin(αSMA), collagen I and IRE1α were investigated by Western blot. It was divide into Vehicle, TGFβ1, TGFβ1+ miRNA-NC and TGFβ1+ miRNA-M groups. TGFβ1+ miRNA-NC and TGFβ1+ miRNA-M group were transfected with miRNA-101a mimic negative control and miRNA-101a mimic, respectively. After the corresponding treatments, mRNA level of miRNA-101a was detected by RT-PCR, the protein level of αSMA、collagen I and IRE1α were measured by Western blot. Results:Compared to normal mice, the fibrotic deposition in liver tissue of CCl 4 group was increased significantly [(0.17±0.06) vs. (2.09±0.39), P<0.001)]. Protein level of αSMA, collagen I and IRE1α was increased significantly in the model group [(1.00±0.23) vs. (4.09±0.80), (1.00±0.21) vs. (4.98±1.19), (1.00±0.24) vs. (3.27±0.65), all P<0.001)]. While the mRNA level of miRNA-101a was decreased (1.00±0.05) vs. (0.43±0.05), P<0.001). In vitro study, we found that TGFβ1 could inhibit the mRNA expression of miRNA-101a, induced HSC-T6 activation and then up-regulated protein expression of αSMA, collagen I and IRE1α. Compared to TGFβ1+ miRNA-NC group, the expression of miRNA-101a in TGFβ1+ miRNA-M group increased significantly [(0.59±0.19) vs. (1.89±0.20), P<0.001)]. The protein levels of αSMA, collagen I were reduced by over expression of miRNA-101a [(2.65±0.69) vs. (0.84±0.13), (3.15±0.59) vs. (1.31±0.25), all P<0.05)], and the protein content of IRE1α was down-regulated [ (2.63±0.47) vs. (1.03±0.15), P<0.001)]. Conclusion:miRNA-101a may play a critical role in the inhibition of HSC activation and liver fibrogenesis by blocking IRE1α signaling pathway.
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Objective To investigate the effect ofmicroRNA-101(miR-101) overexpression on proliferation and metastasis of human hepatoma HepG2 cells and the molecular mechanism.Methods HepG2 cells were divided into blank group,negative control group and miR-101 transfection group.HepG2 cell line stably overexpressing miR-101 was established by lentiviral vector.The overexpression of miR-101 was detected by chemiluminescence method.The expression of vascular endothelial growth factor (VEGF) protein was detected by Western Blot.Scratch experiments was used to analyze the cell migration and the Transwell assay was used to detect cell proliferation.Results The expression of miR-101 in HepG2 cells was significantly increased after transfection with miR-101,and there was a direct targeting relationship between miR-101 and VEGF.Compared with the negative control group,the VEGF protein level in the miR-101 transfected group was significantly down-regulated,and the difference was statistically significant (P＜0.01).Moreover,the cell scratch healing ability and invasion ability were decreased in the miR-101 transfected group.Conclusions Overexpression of miR-101 can inhibit invasion and migration of human hepatoma HepG2 cells by targeting VEGF.
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Objective To observe the effect ofmicroRNA-101 (miR-101) over-expression on proliferation,apoptosis and cycle of human glioma cell line U251.Methods The human glioma cell line U251 was routinely cultured in vitro,and randomly divided into miR-101 group,negative control group and blank control group.Negative controls or miR-101 mimics were transfected into U251 cells by lipofectamine RNAiMAX in the cells from negative control group and miR-101 group.MiR-101 expression levels were detected by real-time fluorescence quantificative PCR 48 h after transfection.Cell viability was determined by cell counting kit-8 (CCK-8) 12,24,36,48,60 and 72 h after transfection.Flow cytometry was used to monitor the changes in cell apoptosis and cycle,and the protein expression levels of BCL-2 and P21 were detected by Western blotting 48 h after transfection.Results As compared with that in the negative control group and blank control group,miR-101 expression level in the miR-101 group was significantly up-regulated (P＜0.05).The survival rate of cells from miR-101 group began to decrease 12 h after transfection,and was significantly lower than that of blank control group and negative control group 48 h after transfection (P＜0.05).As compared with blank control and negative control groups,miR-101 group had significantly increased cell apoptosis rate ([13.73±2.60]％ vs.[4.40±0.57]％ and [4.23±0.44]％),significantly higher percentage of G0/G1 stage cells,statistically decreased stage S cells percentage,significantly decreased inhibitor of apoptosis protein BCL-2 expression and increased expression of key protein regulating cell cycle P21 (P＜005).Conclusion Over-expression of miR-101 in U251 glioma cells can suppress proliferation,block cell cycle and induce apoptosis,and miR-101 may serve as a new potential target of treatment for glioma.
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Objective: To investigate the effects of epithelial cell transformingsequence 2 oncogene (ECT 2) gene-silencing on the proliferation and apoptosis of human breast cancer cells, as well as its potential molecular mechanism. Methods: Firstly, the expressions of ECT2 mRNA and protein in normal mammary epithelial cells (MCF-10A) and breast cancer cells (MDA-MB-231, SK-BR-3, MCF-7, and BT474) were detected by real-Time fluorescent quantitative PCR and Western blotting, respectively. After transfection with the specific siRNA targeting ECT 2 gene (ECT2 siRNA) and treatment with extracellular regulated protein kinase (ERK) pathway activator or transfection with microRNA-101 (miR-101) inhibitor, the proliferation and apoptosis of MDA-MB-231 and MCF-7 cells were detected by CCK-8 and FCM assay, respectively. Then the levels of phospho-ERK (p-ERK), Ras-related C3 botulinum toxin substrate 1 (Rac1) and miR-101 in MDA-MB-231 and MCF-7 cells were detected by Western blotting and real-Time fluorescent quantitative PCR. Results: The expression levels of ECT2 mRNA and protein in breast cancer cells were significantly increased as compared with those in normal mammary epithelial cells (both P 0.05). Conclusion: ECT 2 gene-silencing may affect the proliferation and apoptosis of breast cancer cells by ERK-miR-101-Rac1 signaling pathway.
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Objective To investigate the effect of microRNA-101 (miRNA-101) on atrial fibrosis in human chronic atrial fibrillation (AF). Methods Right atrial appendages were obtained from 59 patients (30 with AF) undergoing cardiac surgery, including 47 patients with valve heart disease and 12 patients with congenital heart disease. The expression of miRNA-101 was determined by quantitative real-time PCR in the right atrial appendages of patients with and without AF. The cell-specific localization of miRNA-101 was detected by in situ hybridization assay. The mRNA and protein expression levels of transforming growth factor β typeⅠreceptor (TGFβRⅠ) and collagen type I (COL1) were determined by quantitative real-time PCR and Western-blot assay, respectively. Collagen in the right atrial appendages was observed by Masson staining assay. Results The expression of miRNA-101 was found to be significantly down-regulated in AF patients compared with patients with sinus rhythm (SR) (P 0.05). But the protein expression of TGFβRI in patients with AF was significantly higher than that of patients with SR (P < 0.05). The mRNA and protein expressionsl of COL1 were significantly higher in patients with AF than thoset of patients with SR (P < 0.05). The collagen was significantly increased in patients with AF than that of patients with SR (P < 0.05). Conclusions Downregulation of miRNA-101 may contribute to atrial fibrosis in human atrial fibrillation by targeting TGFβRⅠ.