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Abstract Objective This study aimed to investigate the clinical significance of serum microRNA-146a and pro-inflammatory factors in children with Mycoplasma pneumoniae pneumonia after azithromycin treatment. microRNA-146a is known to regulate inflammatory responses, and excessive inflammation is a primary characteristic of MPP. Methods Children with MPP received conventional symptomatic therapy along with intravenous administration of azithromycin for one week. Serum levels of microRNA-146a and pro-inflammatory factors were measured using RT-qPCR and ELISA kits, respectively. The correlation between microRNA-146a and pro-inflammatory factors was analyzed by the Pearson method. Pulmonary function indexes were assessed using a pulmonary function analyzer, and their correlation with microRNA-146a and pro-inflammatory factors after treatment was evaluated. Children with MPP were divided into effective and ineffective treatment groups, and the clinical significance of microRNA-146a and pro-inflammatory factors was evaluated using receiver operating characteristic curves and logistic multivariate regression analysis. Results Serum microRNA-146a was downregulated in children with MPP but upregulated after azithromycin treatment, contrasting with the trend observed for pro-inflammatory factors. MicroRNA-146a showed a negative correlation with pro-inflammatory cytokines. Pulmonary function parameters were initially reduced in children with MPP, but increased after treatment, showing positive/inverse associations with microRNA-146a and pro-inflammatory factors. Higher microRNA-146a and lower pro-inflammatory factors predicted better efficacy of azithromycin treatment. MicroRNA-146a, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and forced expiratory volume in the first second/forced vital capacity (FEV1/FVC) were identified as independent factors influencing treatment efficacy. Conclusion Azithromycin treatment in children with MPP upregulates microRNA-146a, downregulates pro-inflammatory factors, and effectively improves pulmonary function.
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Objective:To investigate the role of miR-146a in regulating the homeostasis and function of epidermal Langerhans cells (LCs).Methods:Fresh and in vitro cultured epidermal LCs were isolated and purified by flow cytometry (FCM). The expression of miR-146a in LCs was detected by quantitative PCR (qPCR). The percentages of epidermal LCs in wild-type (WT) and miR-146a conventional knockout (miR-146a cKO) mice were analyzed by FCM. The expression of major histocompatibility complex Ⅱ (MHCⅡ) and co-stimulatory molecules (CD86 and CD80) was analyzed by FCM to evaluate the effect of miR-146a on the maturation of LCs. The percentage of Dextran-FITC + LCs was detected by FCM to evaluate the effect of miR-146a on the phagocytic function of LCs. In vitro and in vivo experiments were used to analyze the ability of miR-146a-deficient and -sufficient LCs to stimulate the proliferation of CD8 + OT-ⅠT cells and CD4 + OT-Ⅱ T cells. Results:The expression of miR-146a was significantly increased in mature LCs than in the freshly isolated LCs. There was no significant difference in the number of epidermal LCs between wild-type (WT) and miR-146a cKO mice. After a 48 h culture in vitro, the expression of MHCⅡ, CD86 and CD80 in the epidermal LCs of miR-146a cKO mice was similar to that of WT mice. Moreover, miR-146a deletion had no significant influence on antigen uptake by LCs. However, miR-146a deficiency enhanced the antigen-presenting ability of LCs that could stimulate the proliferation of OVA-specific CD8 + OT-Ⅰ T cells and CD4 + OT-Ⅱ T cells. Conclusions:miR-146a had no influence on the homeostasis, maturation and phagocytosis of LCs, but enhanced the antigen-presenting function.
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OBJECTIVE@#To explore the effect of atorvastatin (AVT) on biological behaviors and the miR-146a/PI3K/Akt signaling pathway in human glioma cells.@*METHODS@#Human glioma U251 cells were treated with 8.0 μmol/L AVT or transfected with a miR-146a inhibitor or a negative control fragment (miR-146a NC) prior to AVT treatment. RT-PCR was used to detect miR-146a expression in the cells, and the changes in cell proliferation rate, apoptosis, cell invasion and migration were detected using MTT assay, flow cytometry, and Transwell assay. Western blotting was performed to detect the changes in cellular expressions of proteins in the PI3K/Akt signaling pathway.@*RESULTS@#AVT treatment for 48 h resulted in significantly increased miR-146a expression and cell apoptosis (P < 0.01) and obviously lowered the cell proliferation rate, invasion index, migration index, and expressions of p-PI3K and p-Akt protein in U251 cells (P < 0.01). Compared with AVT treatment alone, transfection with miR-146a inhibitor prior to AVT treatment significantly reduced miR-146a expression and cell apoptosis (P < 0.01), increased the cell proliferation rate, promoted cell invasion and migration, and enhanced the expressions of p-PI3K and p-Akt proteins in the cells (P < 0.01); these effects were not observed following transfection with miR-146a NC group (P>0.05).@*CONCLUSION@#AVT can inhibit the proliferation, invasion and migration and promote apoptosis of human glioma cells possibly by up-regulating miR-146a expression and inhibiting the PI3K/Akt signaling pathway.
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Humans , Apoptosis , Atorvastatin/pharmacology , Cell Line, Tumor , Cell Proliferation , Glioma/pathology , MicroRNAs/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal TransductionABSTRACT
OBJECTIVE: This study aims to identify the effect of miR-146a-5p on trophoblast cell invasion as well as the mechanism in preeclampsia (PE). METHODS: Expression levels of miR-146a-5p and Wnt2 in preeclamptic and normal placentae were quantified. Trophoblast cells (HTR-8) were separately transfected with miR-146a-5p mimic, miR-146a-5p inhibitor, pcDNA3.1-Wnt2 or sh-Wnt2, and then the expression levels of miR-146a-5p, Wnt2, and epithelial-mesenchymal transition (EMT)-related proteins (Vimentin, N-cadherin and E-cadherin) were measured. Moreover, the proliferative, migratory and invasive capacities of trophoblast cells were detected, respectively. Dual luciferase reporter assay determined the binding of miR-146a-5p and Wnt2. RESULTS: Compared with normal placental tissues, the placentae from PE patients showed higher miR-146a-5p expression and lower Wnt2 expression. Transfection of miR-146a-5p inhibitor or pcDNA3.1-Wnt2 exerted pro-migratory and pro-invasive effects on HTR-8 cells and encouraged EMT in HTR-8 cells; transfection with miR-146a-5p mimic or sh-Wnt2 weakened the proliferative, migratory and invasive capacities as well as reduced EMT process of HTR-8 cells. Moreover, Wnt2 overexpression could partially counteract the suppressive effects of miR-146a-5p overexpression on the progression and EMT of HTR-8 cells. CONCLUSION: miR-146a-5p mediates trophoblast cell proliferation and invasion through regulating Wnt2 expression.
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Humans , Female , Pregnancy , Pre-Eclampsia , Trophoblasts/cytology , MicroRNAs/genetics , Epithelial-Mesenchymal Transition , Placenta , Cell Movement , Cell ProliferationABSTRACT
BACKGROUND: Porous hydroxyapatite scaffolds have good osteogenesis in vivo and in vitro. However, little research has been done on the complex regulation mechanisms of miRNAs involved. OBJECTIVE: To investigate the changes of related miRNA expression in rat bone marrow mesenchymal stem cells during osteogenic mineralization by porous hydroxyapatite scaffolds. METHODS: Rat bone marrow mesenchymal stem cells were isolated, cultured and identified in vitro. Bone marrow mesenchymal stem cells co-cultured with porous hydroxyapatite scaffold were as experimental group, and bone marrow mesenchymal stem cells cultured alone served as blank control group, both of which underwent osteogenic induction for 7 days. During the osteogenic mineralization, miRNA high-throughput sequencing technology was used to analyze the changes of miRNA expression profiles followed by GO analysis. The miRNA molecules with obvious expression differences were screened and verified by qRT-PCR. RESULTS AND CONCLUSION: (1) Compared with the blank control group, in the experimental group, the expression levels of BMP2, ALP and Runx2 mRNA were up-regulated, and the expression level of BMP2 was up-regulated significantly (P < 0.05). (2) Results of miRNA high-throughput sequencing showed that 13 miRNAs such as miR-210-3p and miR-146a-5p were up-regulated, and 17 miRNAs such as let-7c-3p and let-3615 were down-regulated significantly. (3) GO analysis revealed that up-regulated miRNA target genes were mainly involved in biological regulation, cellular gene expression, and gene expression regulation, mainly including nuclear factor-κB, Toll-like receptor 9, intercellular adhesion, interleukin-1 regulation, and signaling pathways such as angiogenesis and Hippo. (4) Real-time fluorescence quantitative qPCR results showed that miRNA-210 was up-regulated 15 times and miR-146a-5p was up-regulated 10 times in the experimental group (P < 0.05). These results indicate that the new microchannel porous hydroxyapatite scaffold can promote the differentiation of bone marrow mesenchymal stem cells by up-regulating miRNA-210-3p and miR-146a.
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Objective@#To investigate the effect of lncRNA GIHCG on the radiosensitivity of glioma cells and its mechanism.@*Methods@#The expression levels of GIHCG and miR-146a-3p in human brain normal glial cells HEB and glioma cell lines U251, A172, SHG139 and U87 were quantitatively measured by qRT-PCR assay. U251 and SHG139 cells were used for subsequent experiment. After silencing the expression of GIHCG or overexpressing miR-146a-3p in U251 and SHG139 cells, cell proliferation was detected by MTT assay, cell apoptosis was detected by flow cytometry, cell radiosensitivity was detected by colony formation assay and the expression levels of CDK1, CyclinD1, Bcl-2 and Bax proteins were measured by Western blot. The bioinformatics software predicted the presence of a binding site for GIHCG and miR-146a-3p. Dual luciferase reporter gene assay and qRT-PCR assay were adopted to verify the targeting relationship between GIHCG and miR-146a-3p.@*Results@#Compared with HEB cells, the expression of GIHCG was significantly up-regulated in glioma U87, U251, A172 and SHG139 cells (all P<0.05), whereas that of miR-146a-3p was remarkably down-regulated (P<0.05). Silencing GIHCG expression or overexpression of miR-146a-3p significantly decreased the U251 and SHG139 cell survival rate, survival fraction and the expression of CDK1, CyclinD1 and Bcl-2 proteins (all P<0.05), whereas considerably increased the apoptotic rate and expression of Bax protein (both P<0.05). GIHCG performed targeted negative regulation of miR-146a-3p expression in U251 and SHG139 cells and inhibition of miR-146a-3p expression reversed the effect of silencing GIHCG on proliferation, apoptosis and radiosensitivity of glioma cells.@*Conclusion@#Silencing GIHCG expression up-regulates the expression of miR-146a-3p, thereby enhancing the radiosensitivity of glioma cells.
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Objective To investigate the effect of lncRNA GIHCG on the radiosensitivity of glioma cells and its mechanism.Methods The expression levels of GIHCG and miR-146a-3p in human brain normal glial cells HEB and glioma cell lines U251,A172,SHG139 and U87 were quantitatively measured by qRT-PCR assay.U251 and SHG139 cells were used for subsequent experiment.After silencing the expression of GIHCG or overexpressing miR-146a-3p in U251 and SHG139 cells,cell proliferation was detected by MTI assay,cell apoptosis was detected by flow cytometry,cell radiosensitivity was detected by colony formation assay and the expression levels of CDK1,CyclinD1,Bcl-2 and Bax proteins were measured by Western blot.The bioinformatics software predicted the presence of a binding site for GIHCG and miR-146a-3p.Dual luciferase reporter gene assay and qRT-PCR assay were adopted to verify the targeting relationship between GIHCG and miR-146a-3p.Results Compared with HEB cells,the expression of GIHCG was significantly up-regulated in glioma U87,U251,A172 and SHG139 cells (all P＜0.05),whereas that of miR-146a-3p was remarkably down-regulated (P＜0.05).Silencing GIHCG expression or overexpression of miR-146a-3p significantly decreased the U251 and SHG139 cell survival rate,survival fraction and the expression of CDK1,CyclinDl and Bcl-2 proteins (all P＜0.05),whereas considerably increased the apoptotic rate and expression of Bax protein (both P＜0.05).GIHCG performed targeted negative regulation of miR-146a-3p expression in U251 and SHG139 cells and inhibition of miR-146a-3p expression reversed the effect of silencing GIHCG on proliferation,apoptosis and radiosensitivity of glioma cells.Conclusion Silencing GIHCG expression up-regulates the expression of miR-146a-3p,thereby enhancing the radiosensitivity of glioma cells.
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Background: Gastric cancer is one of the most significant reasons for cancer-related death. miR-146a is one of the dysregulated factors associated with gastric tumorigenesis. However, deregulation of this microRNA (miRNA) has become controversial. Moreover, the inflammation-mediating role of this miRNA implies that miR-146a might be dysregulated by gastric cancer-related pathogens, such as Helicobacter pylori. However, the dysregulation of miR-146a in H. pylori-infected gastric tumors has not been widely studied. Objectives: We aimed to analyze the expression level of miR-146a in gastric cancer tissues and then to assess any potential association between miR-146a and H. pylori infection and other clinical characteristics. Materials and Methods: miR-146a expression level was quantitatively studied by reverse transcription quantitative polymerase chain reaction, in 144 fresh tissues including 44 normal and 100 gastric cancer samples. Results: A dramatic overexpression of miR-146a was observed in primary gastric tumors. miR-146a showed lower expression in progressed tumors with greater stages and lymph node metastasis. Conclusion: miR-146a is highly expressed in primary gastric tumor independent of H. pylori infection. It is highly expressed in the lower stages and lymph node-negative tumors. It might suggest the importance of upregulation and downregulation of this miRNA in the initiating/promoting and progressive steps of gastric tumorigenesis, respectively
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PURPOSE: Acute leukemia (AL) is classified as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). This study aimed to investigate the effect of miR-146a on childhood AL and its underlying molecular mechanisms. MATERIALS AND METHODS: Bone marrow samples were obtained from 39 AL children and 10 non-cancer controls. The expressions of miR-146a and ciliary neurotrophic factor receptor (CNTFR) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in ALL and AML pediatric patients, as well as ALL (Jurkat) and AML (HL-60) cells. Correlations between miR-146a and clinical indicators were explored. A targeting relationship between miR-146a and CNTFR was detected by dual luciferase reporter gene assay. Cell proliferation, apoptosis, migration, and invasion of Jurkat and HL-60 cells were measured by MTT assay, flow cytometry, and transwell assay, respectively. LIF expression was detected by qRT-PCR in Jurkat and HL-60 cells. The expression of p-JAK2, JAK2, p-STAT3, and STAT3 in HL-60 cells was measured by Western blot. RESULTS: miR-146a was increased in ALL and AML pediatric patients, while CNTFR was decreased. miR-146a expression was associated with immunophenotype, karyotype, fusion gene, and SIL-TAL1. CNTFR was a target gene of miR-146a. miR-146a could promote cell proliferation, migration, and invasion, as well as inhibit cell apoptosis in Jurkat and HL-60 cells by downregulating CNTFR. Meanwhile, miR-146a inhibited the expression of LIF and activated JAK2/STAT3 pathway by downregulating CNTFR. CONCLUSION: miR-146a could promote the proliferation, migration, and invasion and inhibit the apoptosis of AL Jurkat and HL-60 cells by downregulating CNTFR and activating the JAK2/STAT3 pathway.
Subject(s)
Child , Humans , Apoptosis , Blotting, Western , Bone Marrow , Cell Proliferation , Ciliary Neurotrophic Factor , Flow Cytometry , Genes, Reporter , HL-60 Cells , Karyotype , Leukemia , Leukemia, Myeloid, Acute , Luciferases , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Real-Time Polymerase Chain Reaction , Receptor, Ciliary Neurotrophic FactorABSTRACT
Objective To study the effect of miR-146a on hepatic INSR gene expression in F1 offspring of paternal rats with high-glucose-high-fat diet ( HGFD) . Methods 5-week-old male SD rats were randomly divided into normal diet ( ND) and HGFD groups. Male rats with ND or HGFD feeding for three months mated with normal female ones. Blood glucose concentration, glucose tolerance, and insulin tolerance in newborn male offspring rats were detected respectively. Differential miRNAs between ND and HGFD groups were compared using next generation sequencing and were then confirmed by real-time quantitative PCR ( qPCR) . The relative expression of INSR mRNA and the methylation level of INSR promoter in liver tissues of the offspring newborn rat were detected and the correlations between them and the relative expression of differential microRNA were analyzed respectively. In vitro, effects of miR-146a on expression and methylation of INSR gene in BRL-3A cells were detected using Western-blot assay and qPCR respectively. Results The fasting glucose concentration of different groups were without significant difference, but glucose tolerance and insulin tolerance of neonatal male rats in HGFD group were decreased significantly . Next generation sequencing has revealed 45 up-regulated miRNAs and 15 down-regulated miRNAs in HGFD group. Among them, differences of 8 miRNAs expression in the enlarged samples were confirmed by qPCR. miR-146a was up-regulated for more than 10 times in the liver of the offspring of HGFD group. Expression level of miRNA-146a was negatively correlated with the relative expression of INSR and positively correlated with the methylation level of INSR in livers of neonatal rats in HGFD group. In vitro, miR-146a ( mimics ) promoted the methylation of INSR gene and inhibited the expression of INSR in BRL-3A cells. Conclusion HGFD feeding to male SD rats leads to the inhibition of hepatic INSR gene expression in neonatal offspring via upregulating miR-146a.
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Objective To investigate the association between miR146a(rs2910164)G>C polymorphism and susceptibility to acute lymphoblastic leukemia(ALL)in children.Methods Two hundred blood specimens were ob-tained from children with ALL as patient group and 100 blood specimens were obtained from healthy children as healthy control group,who were all from Baoding First Central Hospital between March 2010 and October 2016.There were no significant differences in sex and age between patient group and healthy control group(χ2=0.430,P=0.512;χ2=2.839,P=0.092).The distribution of gene frequency of patient group and healthy control group conformed to Hardy-Weinberg equilibrium.miR146a(rs2910164)G>C polymorphism was identified by adopting restriction fragment length polymorphism(RFLP).The relation of genotype and ALL was demonstrated by odds ratio(OR)and 95% credibility interval(CI). Results Gene frequency of miR146a(rs2910164)GG,GC and CC genotypes in patient group and healthy control group was 16.0%,44.5%,39.5% and 29.0%,41.0%,30.0%,respectively.The GC/CC genotypes were significantly higher in patient group than those in healthy control group(GG genotype as reference,GC genotype:OR=1.967,95%CI:1.054-3.672,P=0.037;CC genotype:OR=2.386,95%CI:1.239-4.595,P =0.012). Conclusion miR146a(rs2910164)G>C polymorphism is significantly associated with susceptibility to ALL in chil-dren.
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@#AIM:To investigate the correlation of serum miR-146a with nuclear factor-κB(NF-κB)and vascular endothelial growth factor(VEGF)in diabetic retinopathy patients. <p>METHODS: A total of 100 patients with T2DM treated in our hospital from July 2016 to December 2017 were assigned into T2DM patients with DR(DR group, <i>n</i>=32)and T2DM patients without DR(T2DM group, <i>n</i>=68). Thirty healthy volunteers were selected as control group. Real-time PCR was used to examine the expression of miR-146a. Enzyme linked immunosorbent assay was used to detect the levels of NF-κB and VEGF. The correlation between miR-146a and NF-κB and VEGF was analyzed. <p>RESULTS: Compared with the control group, HbA1c in T2DM group and DR group increased(<i>t</i>=6.822, 5.709; <i>P</i><0.001), FBG increased(<i>t</i>=8.889, 7.923; <i>P</i><0.001), 2hPBG increased(<i>t</i>=6.646, 5.514; <i>P</i><0.001). Compared with T2DM group, the duration of diabetes in DR group was longer(<i>t</i>=2.431, <i>P</i>=0.017). Compared with the control group, serum miR-146a in T2DM and group DR significantly decreased(<i>t</i>=3.967, 7.169; <i>P</i><0.001), and the DR group was lower than that in the T2DM group(<i>t</i>=4.444, <i>P</i><0.001). Compared with the control group, the serum NF-κB in the T2DM and DR group increased significantly(<i>t</i>=6.063, 14.851; <i>P</i><0.001), VEGF increased significantly(<i>t</i>=7.613, 12.943; <i>P</i><0.001), NF-κB and VEGF in DR group were larger than those in T2DM group(<i>t</i>=11.406, 7.560; <i>P</i><0.001). Pearson analysis showed that miR-146a was negatively correlated with NF-κB and VEGF(<i>r</i>=-0.503, -0.574; <i>P</i><0.05). <p>CONCLUSION: The serum miR-146a in DR patients significantly decreased, the NF-κB and VEGF significantly increased. MiR-146a may be involved in the pathogenesis of DR by mediating inflammatory reaction and vascular proliferation.
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Objective To investigate the changes of miR-146a expression in serum,hippocam-pus and prefrontal cortex in postoperative cognitive dysfunction (POCD) mice.Methods Fifty healthy male C57BL/6 mice,aged 12-14 weeks,weighing 25-30 g,were randomly divided into two groups (n =25 each)using a random number table:control group (group C)and anesthesia plus sur-gery group (group AS).Mice in group AS underwent open tibial fracture of the left hind paw with in-tramedullary fixation in aseptic conditions under general anesthesia with 2.1% isoflurne.Ten mice in each group received the fear conditioning test (FCT)on the 1,3 and 7 days after anesthesia/surgery. The rest of mice were sacrificed 24 h before (baseline),and 6,12,24,48 h after anesthesia/surgery, and then the serum,prefrontal cortex and hippocampus were collected or removed for detection of the expression of miR-146a using quantitative reverse transcription polymerase chain reaction (qRT-PCR).Results Compared with group C,the percentage of freezing time in contextual FCT was sig-nificantly decreased (P <0.05)in group AS,while no significant change in freezing time percentage was found in tone-cued FCT.In serum,compared with group C,miR-146a expression at 6,12,24, 48 h after anesthesia/surgery was significantly up-regulated in group AS (P < 0.05 );and in group AS,the expression of miR-146a was significantly decreased 6,24,48 h as compared to that at 12 h after anesthesia/surgery (P <0.05).In hippocampus,compared with group C,miR-146a expression at 6,12,24,48 h after surgery was significantly up-regulated in group AS (P <0.05);and in group AS,the expression of miR-146a at 6,48 h after surgery was significantly decreased as compared to that at 12 h after anesthesia/surgery (P <0.05).In prefrontal cortex,compared with group C,miR-146a expression at 24,48 h after surgery was significantly up-regulated in group AS (P <0.05);and in group AS,the expression of miR-146a was significantly increased at 48 h as compared to that at 24 h after anesthesia/surgery (P <0.05).Conclusion The expression of miR-146a in serum,hippocam-pus and prefrontal cortex in POCD mice was up-regulated,and changes of miR-146a expression may be related to the development of POCD.
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AIM To explore the effects of Shexiang Wulong Pills (Moschus Artifactus,Aconiti Radix Cocta,Pheretima,etc.) drug serum on the expression levels of miR-146a,miR-130 and miR-223 in peripheral blood mononuclear cells (PBMCs) of patients with rheumatoid arthritis (RA).METHODS PBMCs were extracted from blood of 30 cases of RA patients,cells were divided into three groups,blank control group,serum-free groupand Shexiang Wulong Pills drug serum group.After 48 hours,total RNA was extracted,then the expression levels of three miRs were detected by RT-PCR.RESULTS Compared with the serum-free group,the expression levels of miR-146a,miR-130 and miR-223 in the Shexiang Wulong Pills drug serum group were significantly decreased (P ＜ 0.01).CONCLUSION Shexiang Wulong Pills can inhibit inflammation in RA patients by down-regulating the expression levels of miR-223,miR-130 and miR-146a in PBMCs.
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OBJECTIVE:To study the effect and its mechanism of dexmedetomidine(Dex)on inflammatory response in septic mice. METHODS:Mice were randomly divided into normal control group,model group,miR-146a inhibitor (50 mg/kg)+Dex (50 μg/kg)group,Dex low-dose,medium-dose,high-dose groups(10,30,50 μg/kg),10 in each group. Except for normal con-trol group,other groups were intraperitoneally injected lipopolysaccharide to induce septic models,intraperitoneally injected rele-vant medicines after 0.5 h. After drug intervention for 6 h,miR-146a expression,IRAK1 and TRAF6 mRNA expressions in periph-eral blood mononuclear cells in each group were detected by real-time fluorescence quantitative polymerase chain reaction method. IRAK1,TRAF6 protein expressions in peripheral blood mononuclear cells in each group were detected by Western blot method. TNF-α,IL-6 levels in serum were detected by enzyme-linked immunosorbent method. RESULTS:Compared with normal control group,miR-146a expression,TNF-α and IL-6 levels,IRAK1,TRAF6 mRNA and protein expressions in peripheral blood mononu-clear cells in model group were increased(P0.05). Compared with Dex high-dose group,miR-146a expression,in peripheral blood mononuclear cells in miR-146a inhibitor+Dex group was de-creased;TNF-α and IL-6 levels,IRAK1,TRAF6 protein expressions were increased (P0.05). CONCLUSIONS:Dex can inhibit the inflammatory response in sep-tic mice. The mechanism may associate with inducing miR-146a expression and inhibiting the 2 important adaptor proteins IRAK1, TRAF6 expressions in Toll-like receptor 4/nuclear factor-κB pathway.
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Objective To construct the has-miR 146a eukaryotic overexpression vector pmR 146a and to explore its effect on the expression of c-Myc gene in HepG2.2.15 cells.Methods The has-miR-146a precursor gene fragment pre-has-miR-146a was amplified by PCR,then connected to the pmR-mCherry plasmid vector after double enzyme digestion,the accuracy of recombinant vector was verified by colony PCR,double enzyme digestion and sequencing;then the recombinant vector was transfected into HepG2.2.15 cells as the experimental group,meanwhile the empty vector group (transfecting pmR-mCherry empty plasmid group) and blank group(transfecting reagent lip2000+PBS),then the fluorescent protein expression amount was observed under the fluorescence microscopy at 24,48 h;the expression of has miR-146a was evaluated by qPCR;at 24,48 h after transfection,the expression levels of c-Myc gene mRNA were detected by qPCR,and the c-Myc protein expression level after 48 h was detected by Western blot.Results The colony PCR,double enzyme digestion and sequencing verified that the pre-has-miR-146a gene fragment was inserted into the pmR-mCherry vector;at 24,48 h after transfection in the experimental group and empty vector group,intracellular strong fluorescence was seen by fluorescent microscope,the transfection efficiency was at 50％-60％ contrasting without fluorescence;the has-miR-146a expression level in the experimental group was significantly higher than that in the empty vector group and blank group (P＜0.01);the c-Myc mRNA expression at 24,48 h after tranfection was significantly lower than that in the empty vector group and blank group (P＜0.05);the protein expression amount at 48 h after transfection was lower than that in the empty vector group and blank group (P＜0.01).Conclusion The pmR-146a eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miR-146a stably;miR-146a can down-regulate c-Myc cancer gene expression,which can serve as one of potential targets for treating hepatocellular carcinoma.
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Objective To study the expression of miR-146a in the serum of sepsis and the diagnostic value.Methods Serum miR-146a expression was detected in 98 patients diagnosed with sepsis,69 patients with severe sepsis and 30 healthy controls.ROC plots were used to evaluate the diagnostic value.Results The expression of miR-146a was decreased successively in healthy group,general sepsis group and severe sepsis group.In the diagnosis of sepsis,at the optimal expression cutoff miR-146a value of 0.805,the sensitivity was 0.967 and specificity 0.940,with an AUC of 0.983.In the diagnosis of sepsis severity degree,at the optimal expression cutoff miR-146a value of 0.530,the sensitivity was 0.796 and specificity 0.986,with an AUC of 0.943.Conclusion miR-146a can be a potential marker in diagnosis of sepsis and severity degree .
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Objective To explore the expression of miR-146a and its effect on the biological characteristics in gastric carcinoma. Methods The expressions of miR-146a in gastric cancer tissue and carcinoma adjacent tissue were detected by RT-qPCR , and human gastric cancer cell line MKN-28, SGC-7901, MKN-45 and immortalized gastric epithelial cell GES-1 were cultured,GES-1 as a reference, the expressions of miR-146a in each cell line was detected by RT-qPCR; miR-146a mimics and miR-146a control were transfected into gastric cancer cell line MKN-45, and the expression of miR-146a was detected by RT-qPCR;CCK8 was used to detect the effect of miR-146a on the proliferation of cells;flow cytometry was used to detect the effect of miR-146a on cell apoptosis. Western blot was used to detect the expressions of the apoptosis-related proteins. Results The expression of miR-146a in gastric cancer tissue was significantly lower than carcinoma adjacent tissue(P<0.01); expression of miR-146a in gastric cancer cell MKN-45 was the lowest campared to GES-1, so the gastric cancer cell MKN-45 was selected as a follow-up study. The expression of miR-146a in miR-146a mimics group was significantly higher than control group(P<0.01); CCK8results showed that the proliferation rateof miR-146a mimics group was significantly lower thanmiR-146a control on 24 hour (P<0.05),and was significantly lower than miR-146a control on 48 hour and 72 hour (P<0.01).The results of flow cytometry showed that the apoptosis rate in miR-146a mimics group was significantly higher than miR-146a control;Western blot showed that the expressions of Cleaved-caspase-3 and Bax protein in miR-146a mimics group was significantly lower than those in miR-146a control, and Bcl-2 protein expression was significantly higher than miR-146a control (P< 0.05). Conclusion The expression of miR-146a in gastric carcinoma was lower than carcinoma adjacent tissue, and the miR-146a could inhibit cell proliferation and promote cell apoptosis after transfected.
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Objective@#To investigate the association between miR-146a single nucleotide polymorphism and genetic susceptibility to hepatocellular carcinoma (HCC).@*Methods@#PubMed, Web of Science, Cochrane Library, Wanfang Data, and Google Scholar were searched for case-control studies on the association between miR-146a single nucleotide polymorphism and genetic susceptibility to HCC published up to October, 2016 in Chinese or English. The Q-statistics test was used to evaluate the heterogeneity of these articles.@*Results@#A total of 18 articles with 5 610 cases and 7 531 controls were included for the meta-analysis. There was no significant association between miR-146a single nucleotide polymorphism and genetic susceptibility to HCC. The odds ratio (OR), 95% confidence interval (95% CI), and P values for the five genetic models were as follows: the allele model C/G (OR = 0.99, 95% CI 0.88-1.06, P = 0.440); the heterozygous model CG/GG (OR = 0.99, 95% CI 0.90-1.10, P = 0.898); the homozygous model CC/GG (OR = 0.91, 95% CI 0.75-1.10, P = 0.314); the dominant model CC+CG/GG (OR = 0.97, 95% CI 0.79-1.19, P = 0.759); the recessive model CG+GG/CC (OR = 1.05, 95% CI 0.94-1.18, P = 0.405). A subgroup analysis of race, source of control population, and Hardy-Weinberg equilibrium were performed in these five genetic models, and miR-146a single nucleotide polymorphism increased the susceptibility to HCC only in the control population-based subgroups of the recessive model CG+GG/CC (OR = 1.20, 95% CI 1.02-1.40, P = 0.024). There was no association between miR-146a rs2910164 polymorphism and susceptibility to HCC in all the other subgroups. A stratified analysis of HBV infection revealed that miR-146a rs2910164 polymorphism increased the risk of HBV-positive HCC (OR = 1.26, 95% CI 1.10-1.49, P = 0.001).@*Conclusion@#There is no significant association between miR-146a rs2910164 polymorphism and the risk of HCC, but miR-146a rs2910164 polymorphism may increase the risk of HBV-positive HCC.
ABSTRACT
Background:1 α,25-dihydroxyvitamin D3 [1,25 (OH) 2 D3],the active form of vitamin D,is reported in some studies having antifibrotic potential in liver fibrosis,however,its mechanism is not fully clarified.MicroRNAs (miRNAs) have recently been shown could regulate the proliferation and activation of hepatic stellate cells (HSCs),and are involved in the promotion or inhibition of liver fibrosis.Aims:To explore whether the inhibiting effect of 1,25 (OH) 2 D3 on activation of HSCs is by regulating miRNAs expression.Methods:Literature review and qPCR method were used to screen out the differentially expressed miRNAs between transforming growth factor-β1 (TGF-β1)-stimulated (activated) HSCs and the inactivated HSCs.Then the HSCs were co-cultured with TGF-β1 and the mimic of differentially expressed miRNA,the negative control mimic,1,25(OH)2D3 and DMSO,respectively,and the cell viability and apoptosis were determined by CCK-8 assay and flow cytometry.Results:Expression of miR-146a was down-regulated in activated HSCs (P ＜ 0.05).Compared with HSCs in DMSO group,the expression of miR-146a was significantly up-regulated in HSCs treated with 1,25 (OH) 2 D3;meanwhile,the cell viability was decreased and the apoptosis was increased (P all ＜ 0.05).In HSCs transfected with miR-146a mimic,the expression of miR-146a was up-regulated,the cell viability was decreased,and the apoptosis was increased similarly with HSCs in 1,25 (OH)2D3 group (P all ＜ 0.05).Conclusions:Regulation of miR-146a expression might be one of the important mechanisms of 1,25 (OH) 2 D3 in inhibiting TGF-β1-stimulated HSC activation and inducing apoptosis in HSCs.