Improvement effect and mechanism of total flavonoids from Alpinia zerumbet on gastric mucosa injury induced by absolute ethanol / 中国药房

OBJECTIVE To explore the improvement effect and potential mechanism of total flavonoids from Alpinia zerumbet on gastric mucosa injury induced by absolute ethanol through microRNA-146a-5p （miR-146a-5p）. METHODS Using human gastric mucosa GES-1 cells as objects， the acute gastric ulcer model was established by absolute ethanol； based on the investigation of the effects of different concentrations of total flavonoids from A. zerumbet on cell activity and the selection of action concentration， the relative expression level of miR-146a-5p in GES-1 cells was detected， the protein expressions of tumor necrosis factor （TNF） receptor-associated factor 6（TRAF6）， nuclear factor-κB p65 （NF-κB p65） and TNF-α were detected， and the levels of interleukin- 1β （IL-1β）， IL-6 and prostaglandin E2 （PGE2） in cell supernatant were determined. The targeting relationship between miR-146a- 5p and TRAF6 was verified； the effects of overexpressed miR-146a-5p and TRAF6 knockdown on the levels of IL-1β， IL-6 and PEG2 in supernatant of model cells as well as the effects of miR-146a-5p knockdown on anti-gastric ulcer effect of total flavonoids from A. zerumbet were observed. RESULTS Compared with the blank group， the relative expression of miR-146a-5p in cells and the level of PGE2 in cell supernatant were decreased significantly in the model group （P＜0.01）， while the protein expressions of TRAF6， NF-κB p65 and TNF-α in cells and the levels of IL-1β and IL-6 in cell supernatant were increased significantly （P＜ 0.01）. Compared with the model group， the relative expression of miR-146a-5p in cells and the level of PGE2 in cell supernatant were increased significantly in model+A. zerumbet total flavonoids （60 mg/L） group （P＜0.01）， while the protein expressions of TRAF6， NF-κB p65 and TNF-α in cells and 82260767） the levels of IL-1β and IL-6 in cell supernatant were decreased significantly （P＜0.05 or P＜0.01）. There was a targeted relationship and a negative correlation between miR-146a-5p E-mail：3113836821@qq.com and TRAF6. After overexpression of miR-146a-5p or TRAF6 knockdown， the levels of IL-1β and IL-6 were decreased significantly in cell supernatant， while the level of PGE2 was increased significantly （P＜0.05）. After miR-146a-5p knockdown， the levels of IL-1β and IL-6 in cell supernatant and the protein expression of TRAF6 in cells administered with total flavonoids of A. zerumbet were increased significantly， while the level of PGE2 was decreased significantly （P＜0.05）. CONCLUSIONS Total flavonoids of A. zerumbet can improve the gastric mucosa injury induced by absolute ethanol. The mechanism may be related to up-regulating the expression of miR-146a-5p， inhibiting the expression of TRAF6， and further inhibiting the secretion of related inflammatory factors.

Expressions of miRNA-146a in peripheral mononuclear cells of children with systemic juvenile idiopathic arthritis and its clinical significance / 中华实用儿科临床杂志

Objective:To investigate the role of miR-146a in the pathogenesis of systemic juvenile idiopathic arthritis (sJIA) and its clinical significance.Methods:This article is a prospective clinical cohort study.Twenty-six patients with sJIA (14 cases of initial active group and 12 cases of stable group), 15 patients with multijoint juvenile idiopathic arthritis (JIA) and 15 patients with oligojoint JIA diagnosed in the Department of Rheumatology and Immunology of Anhui Provincial Children′s Hospital from June 2018 to December 2020 were enrolled.Twenty healthy controls from the out-patient clinic were also recruited.The expression level of miR-146a in peripheral blood mononuclear cells (PBMCs) of research objects was detected by real-time fluorescence quantitative polymerase reaction (qPCR), and the serum levels of interleukin (IL) - 6, tumor necrosis factor (TNF) - α and IL-1β in sJIA patients and healthy controls were detected by enzyme-linked immunosorbent assay.The expression levels of miR-146a in PBMCs and cytokines among different groups were compared by analysis of variance. Pearson correlation analysis was used to analyze the correlation of the relative expression level of miR-146a in PBMCs with clinical inflammatory indexes and serum cytokines in sJIA patients. Results:(1) The expression level of miR-146a in PBMCs of early sJIA patients was significantly higher than that in the multijoint JIA group and oligojoint JIA group (8.77±3.15 vs.4.40±1.59, 2.55±1.15, t=6.27, 14.23; all P<0.05). The expression level of miR-146a in PBMCs of sJIA active patients was significantly higher than that in sJIA stable patients (8.77±3.15 vs.3.63±1.37, t=10.27, P<0.05). There was no significant difference in the expression level of miR-146a between the sJIA stable group and healthy control group ( P>0.05). (2) The expression levels of IL-1β, IL-6 and TNF-α were significantly higher in sJIA active patients group than those in sJIA stable group[(58.56±17.47) ng/L vs.(26.32±10.54) ng/L, (73.72±11.16) ng/L vs.(23.20±9.12) ng/L, (70.93±19.97) ng/L vs.(24.25±9.49) ng/L, all P<0.05]. There was no significant difference in the expression levels of IL-1β, IL-6 and TNF-α between the sJIA stable group and healthy control group(all P>0.05). (3)The expression of miR-146a in PBMCs of sJIA patients was positively correlated with serum ferritin levels, platelets, erythrocyte sedimentation rates, C-reactive proteins, IL-1β, IL-6 and TNF-α( r=0.542, 0.433, 0.329, 0.306, 0.333, 0.342, 0.319, all P<0.05). Conclusions:miR-146a may be involved in the inflammatory process of sJIA disease.miR-146a can well distinguish sJIA from multijoint JIA and oligojoint JIA.TNF-α, IL-1β and IL-6 are involved in sJIA inflammatory responses.

MicroRNA-146a regulates the production of cytokines in lymphocytes stimulated by / 华西口腔医学杂志

OBJECTIVES@#This study aimed to investigate the effects of microRNA-146a (miR-146a) on the production of cytokines in lymphocytes stimulated by @*METHODS@#Lymphocytes were harvested from mouse spleen and cultured @*RESULTS@#Compared with non-LPS-stimulated group, @*CONCLUSIONS@#MiR-146a can provide a suitable microenvironment for bone formation by preventing the inflammatory effects of

MicroRNA-146a inhibition promotes total neurite outgrowth and suppresses cell apoptosis, inflammation, and STAT1/MYC pathway in PC12 and cortical neuron cellular Alzheimer's disease models

This study aimed to explore the effect of microRNA (miR)-146a inhibition on regulating cell apoptosis, total neurite outgrowth, inflammation, and STAT1/MYC pathway in Alzheimer's disease (AD). PC12 and cortical neuron cellular AD models were constructed by Aβ1-42 insult. For the former model, nerve growth factor (NGF) stimulation was previously conducted. miR-146a inhibitor and negative-control (NC) inhibitor were transfected into the two cellular AD models, and then cells were named miR-inhibitor group and NC-inhibitor group, respectively. After transfection, cell apoptosis, total neurite outgrowth, supernatant inflammation cytokines, and STAT1/MYC pathway were detected. miR-146a expression was similar between PC12 cellular AD model and control cells (NGF-stimulated PC12 cells), while miR-146a expression was increased in cortical neuron cellular AD model compared with control cells (rat embryo primary cortical neurons). In both PC12 and cortical neuron cellular AD models, miR-146a expression was reduced in miR-inhibitor group compared with NC-inhibitor group after transfection. Furthermore, cell apoptosis was attenuated, while total neurite outgrowth was elevated in miR-inhibitor group compared with NC-inhibitor group. As for supernatant inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-17 levels were lower in miR-inhibitor group than in NC-inhibitor group. Additionally, STAT1 and c-Myc mRNA and protein expressions were attenuated in miR-inhibitor group compared with NC-inhibitor group. In conclusion, miR-146a potentially represented a viable therapeutic target for AD.

Expression and effect of microRNA-146a gene mediated by ultrasound microbubble in rats with ischemia/reperfusion liver injury / 中国中西医结合急救杂志

Objective To evaluate the relationship between the expression of microRNA-146a (miR-146a) in liver tissue and the inflammatory hepatic injury induced by ischemia/reperfusion (I/R) in rats. Methods One hundred and forty-four Sprague-Dawley (SD) rats were randomly divided into three groups: control (group N), sham operation (group S) and group I/R. Each group was subdivided into four subgroups (n = 12), and different substances were respectively injected intravenously to rats in different subgroups at 1 hour before the experiment: 220 μL physiological saline (group A), 20 μL miR-146a mimic + 200 μL physiological saline (group B), 20 μL miR-146a mimic + 200 μL ultrasound microbubble contrast agent (group C) and 20 μL miR-146a inhibitor + 200 μL ultrasound microbubble contrast agent (group D). Before the experiment and after experiment for 24 hours, the plasma concentrations of alanine aminotransferase (ALT), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected, the reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression of miR-146a in liver tissue, and Western Blot was applied to detect protein expressions of Toll-like receptor 4 (TLR4), IL-1 receptor associated kinase 1 (IRAK-1), IL-6 and TNF-α, and the pathological hepatic cell injury was observed. Results Before the experiment and 24 hours after experiment in various subgroups of N and S groups, there were no statistical significant differences in the plasma concentrations of ALT, IL-6 and TNF-α, and the expression of miR-146a level and the protein expressions of TLR4, IRAK-1, IL-6 and TNF-α in liver tissues; the pathological examination also did not show any obvious hepatic cell injury. After the experiment for 24 hours: compared to the group S, the liver tissue miR-146a expression was significantly decreased in the subgroups A and D of group I/R (miR-146a/U6nsRNA: 0.51±0.13, 0.22±0.09 vs. 1.01±0.02, both P < 0.01), and the plasma concentrations of ALT, IL-6 and TNF-α and the protein expressions of TLR4, IRAK-1, IL-6 and TNF-α in liver tissues were significantly increased [ALT (U/L): 103.23±26.64 vs. 44.16±18.55, 176.46±7.26 vs. 49.74±6.83, IL-6 (μg/L): 64.28±16.19 vs. 17.68±7.54, 88.49±3.23 vs. 15.58±2.38; TNF-α (μg/L): 31.28±2.57 vs. 5.58±3.35, 59.12±8.74 vs. 5.27±1.37; TLR4/GAPDH: 2.43±0.36, 3.23±0.71 vs. 0.96±0.24, IRAK-1/GAPDH: 2.34±0.52, 3.14±0.63 vs. 0.76±0.21, IL-6/GAPDH: 1.01±0.22, 1.11±0.16 vs. 0.98±0.37, TNF-α/GAPDH: 2.05±0.48, 2.86±0.27 vs. 0.59±0.16, all P < 0.01], moreover, the hepatic pathological lesions were obvious; the liver tissue expression of miR-146a was significantly increased after being transfected with miR-146a mimic in subgroups B and C of group I/R (miR-146a/U6nsRNA: 1.56±0.31, 2.40±0.53 vs. 1.01±0.02, both P < 0.01), especially in group C combined with ultrasound microbubble (P < 0.01). However, the protein expressions of TLR4, IRAK-1, IL-6 and TNF-α in liver tissues were significantly decreased (TLR4/GAPDH:0.77±0.18, 0.65±0.27 vs. 0.96±0.24, IRAK-1/GAPDH: 0.61±0.14, 0.47±0.20 vs. 0.76±0.21, IL-6/GAPDH:0.80±0.13, 0.54±0.22 vs. 0.98±0.37, TNF-α/GAPDH: 0.41±0.14, 0.16±0.03 vs. 0.59±0.16; all P < 0.01), and the expressions were more significant in the group C combined with ultrasound microbubbles (P < 0.01), and the hepatic pathological damage was mild, however, the plasma concentrations of ALT, IL-6 and TNF-α were of no statistical significant differences. Conclusion Ultrasound microbubble can efficiently transfect miR-146a mimic and inhibitor into the liver tissue, and miR-146a may negatively regulate the I/R inflammatory liver injury mediated by TLR signaling pathway.

Effects of intrauterine exposure to ambient fine particles on immune function-related indicators in neonatal rats / 中华围产医学杂志

Objective@#To investigate the effects of maternal exposure to ambient fine particles (PM2.5) in Fuzhou during pregnancy on immune responses to ovalbumin (OVA) in neonatal rats and the possible mechanisms.@*Methods@#Pregnant Sprague-Dawley rats were randomly assigned into four groups (ten in each): filtered air (FA) plus normal saline (NS), airborne PM2.5 plus NS (PM2.5-NS), FA plus OVA (FA-OVA) and PM2.5 plus OVA (PM2.5-OVA) groups. Pregnant dams in the PM2.5 exposure groups were placed in a PM2.5 exposure chamber in which the PM2.5 concentration was equal to the ambient air from the beginning of gestation till delivery, whereas the other dams inhaling air without particulate matters were put into a clean chamber. OVA sensitization was conducted through intraperitoneal injection of OVA at 50 μg per dam at 4 and 9 days of gestation, followed by inhalation of atomized 1% OVA for 30 min at 18, 19 and 20 days of gestation. Dams without OVA sensitization were given NS in the same way. Levels of interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ) in neonatal rats' plasma were measured by enzyme-linked immunosorbent assay just after birth. Protein levels of transcription factors GATA-3 and T-bet in lung were analyzed by Western-blotting. Changes in microRNA(miR)-146a and miR-146b in spleen were detected by real-time polymerase chain reaction. Histological changes in lung were observed under light microscope. One-way analysis of variance and LSD test were used as statistical methods.@*Results@#(1) IL-4 level in plasma was significantly increased in PM2.5-NS [(18.56±7.04) ng/L], FA-OVA [(34.04±7.06) ng/L] and PM2.5-OVA groups [(45.67±8.18) ng/L] as compared with that in FA-NS group [(10.51±2.88) ng/L], and the level of IL-4 in PM2.5-OVA group was higher than that in PM2.5-NS and FA-OVA groups (F=54.667, P<0.001). Significantly increased IL-5 level in plasma was found in FA-OVA and PM2.5-OVA groups as compared with that in FA-NS group (F=6.253, P=0.023). Among the four groups, FA-OVA group showed significantly increased IFN-γ level in plasma (F=28.604, P<0.001). (2) GATA-3 level in lung tissues was significantly increased in PM2.5-NS (31.09±3.54), FA-OVA (35.24±5.00) and PM2.5-OVA groups (47.81±3.63) as compared with that in FA-NS group (24.19±3.12), and higher in PM2.5-OVA group than in PM2.5-NS and FA-OVA groups (F=96.581, P<0.001). T-bet level was significantly lower in FA-OVA and PM2.5-OVA groups than in FA-NS group. Moreover, PM2.5-OVA group showed decreased T-bet level as compared with that in PM2.5-NS and FA-OVA groups (F=30.852, P<0.001). (3) Expression of miR-146a in spleen was significantly enhanced in PM2.5-NS (1.72±0.27), FA-OVA (1.56±0.37) and PM2.5-OVA groups (3.06±0.52) than in FA-NS group (1.05±0.25). Moreover, PM2.5-OVA group showed enhanced expression of miR-146a as compared with that in PM2.5-NS and FA-OVA groups (F=42.276, P<0.001). Changes in the expressions of miR-146b were similar to those in miR-146a (F=28.776, P<0.001). (4) Stenosis or disappearance of alveolar spaces, accompanied with infiltration of inflammatory cells in interstitial substance and congestion in alveolar septum, was seen in FA-OVA and PM2.5-OVA groups and conditions in the latter group were more severe.@*Conclusions@#Intrauterine exposure to ambient PM2.5 negatively affects fetal lung development and immunological function in rats, especially when the dams are sensitized with OVA during pregnancy.

Effects of intrauterine exposure to ambient fine particles on immune function-related indicators in neonatal rats / 中华围产医学杂志

Objective To investigate the effects of maternal exposure to ambient fine particles (PM2.5) in Fuzhou during pregnancy on immune responses to ovalbumin (OVA) in neonatal rats and the possible mechanisms.Methods Pregnant Sprague-Dawley rats were randomly assigned into four groups (ten in each):filtered air (FA) plus normal saline (NS),airborne PM2.5 plus NS (PM2.5-NS),FA plus OVA (FA-OVA) and PM2.5 plus OVA (PM2.5-OVA) groups.Pregnant dams in the PM2.5 exposure groups were placed in a PM2.5 exposure chamber in which the PM2.5 concentration was equal to the ambient air from the beginning of gestation till delivery,whereas the other dams inhaling air without particulate matters were put into a clean chamber.OVA sensitization was conducted through intraperitoneal injection of OVA at 50 μ g per dam at 4 and 9 days of gestation,followed by inhalation of atomized 1％ OVA for 30 min at 18,19 and 20 days of gestation.Dams without OVA sensitization were given NS in the same way.Levels of interleukin (IL)-4,IL-5 and interferon-γ (IFN-γ) in neonatal rats' plasma were measured by enzyme-linked immunosorbent assay just after birth.Protein levels of transcription factors GATA-3 and T-bet in lung were analyzed by Western-blotting.Changes in microRNA(miR)-146a and miR-146b in spleen were detected by real-time polymerase chain reaction.Histological changes in lung were observed under light microscope.One-way analysis of variance and LSD test were used as statistical methods.Results (1) IL-4 level in plasma was significantly increased in PM2.5-NS [(18.56±7.04) ng/L],FA-OVA [(34.04±7.06) ng/L] and PM2.5-OVA groups [(45.67±8.18) ng/L] as compared with that in FA-NS group [(10.51 ±2.88) ng/L],and the level of IL-4 in PM2.5-OVA group was higher than that in PM2.5-NS and FA-OVA groups (F=54.667,P＜0.001).Significantly increased IL-5 level in plasma was found in FA-OVA and PM2.5-OVA groups as compared with that in FA-NS group (F=6.253,P=0.023).Among the four groups,FA-OVA group showed significantly increased IFN-γ level in plasma (F=28.604,P＜0.001).(2) GATA-3 level in lung tissues was significantly increased in PM2.5-NS (31.09 + 3.54),FA-OVA (35.24± 5.00) and PM2.5-OVA groups (47.81 ±3.63) as compared with that in FA-NS group (24.19±3.12),and higher in PM2.5-OVA group than in PM2.5-NS and FA-OVA groups (F=96.581,P＜0.001).T-bet level was significantly lower in FA-OVA and PM2.5-OVA groups than in FA-NS group.Moreover,PM2.5-OVA group showed decreased T-bet level as compared with that in PM2.5-NS and FA-OVA groups (F=30.852,P＜0.001).(3) Expression of miR-146a in spleen was significantly enhanced in PM2.5-NS (1.72±0.27),FA-OVA (1.56±0.37) and PM2.5-OVA groups (3.06± 0.52) than in FA-NS group (1.05 ±0.25).Moreover,PM2.5-OVA group showed enhanced expression of miR-146a as compared with that in PM2.5-NS and FA-OVA groups (F=42.276,P＜0.001).Changes in the expressions of miR-146b were similar to those in miR-146a (F=28.776,P＜0.001).(4) Stenosis or disappearance of alveolar spaces,accompanied with infiltration of inflammatory cells in interstitial substance and congestion in alveolar septum,was seen in FA-OVA and PM2.5-OVA groups and conditions in the latter group were more severe.Conclusions Intrauterine exposure to ambient PM2.5 negatively affects fetal lung development and immunological function in rats,especially when the dams are sensitized with OVA during pregnancy.

Mechanistic study on the role of microRNA-146a in THP-1 cells-associated inflammatory response induced by Cryptococcus neoformans and Cryptococcus gattii / 中国感染与化疗杂志

Objective To analyze the differential expression of microRNA-146a (miR-146a) in monocyte-macrophage cell line (THP-1 cells) after induction by Cryptococcus neoformans (C.neoformans,reference strain WM148) or Cryptococcus gattii (C.gattii,reference strain R265),and investigate the mechanism of miR-146a in regulating the inflammatory response of cryptococcal meningitis.Methods The cultured THP-1 cells were divided into two groups to be induced by C.neoformans or C.gattii,respectively.THP-1 cells were induced with inactivated WN148 (or R265) strains at multiplicity of infection (MOI) of 5 in all experiments.The supematant and the cell pellet were collected separately after incubation.The expression of miR-146a was measured by real-time quantitative PCR (qRT-PCR) technique.The levels of TNF-u and IL-6 release were assayed by ELISA.Results The expression of miR-146a increased significantly in the C.neoformans induction group compared to 0 h.It reached peak at 3 h (P＜0.01),and then declined gradually.The level of TNF-α increased in supematant and reached peak at 12 h.The expression of IL-6 did not change significantly at each time point.The expression of miR-146a and TNF-α increased gradually and reached peak at 12 h in the C.gattii induction group (P ＜0.01),but the change did not reach statistical significance at 3 h,6 h time points.The expression of IL-6 gradually increased,and reached peak at 12 h time point.Conclusions Following stimulation with C.neoformans or C.gattii,the expression ofmiR-146a in THP-1 cells showed different patterns over time.The expression levels of TNF-α and IL-6 showed different patterns.These findings suggest that there may be different regulatory mechanisms in the THP-1 cells-associated inflammatory response after stimulation by inactivated C.neoformans and C.gattii strains.

Development of a universal probe based microRNA assay and detection of plasma microRNA-146a of human brucellosis / 中国人兽共患病学报

We developed a universal probe based microRNA detection assay and applied it to detect microRNA-146a in human brucellosis,testing the possibility of using it as diagnosis signature.By using orthogonal design,the annealing temperature,probe concentration and commercial kits were optimized and the assay was developed.Total RNAs were isolated from plasma of human brucellosis and healthy control,and microRNA-146a was detected and compared.Results reveal that the optimized universal probe assay was established,which was more specific than the SYBR GreenI assay,and had a wider range of amplification.Compared with healthy control,the application of universal probe assay for the detection of serum microRNA146a in patients with brucellosis was significantly inhibited (P＜0.01).Implying the potential of microRNA-146a as biomarker in diagnosis of brucellosis.It is suggested that universal probe based assay is a universal,specific and sensitive method for microRNA detection.MicroRNA-146a represents a potential biomarker for human brucellosis diagnosis.

MicroRNA-146a promotes apoptosis of gastric cancer SGC-7901 cells by targeting TAK1 / 中国病理生理杂志

AIM: To explore the effect of microRNA-146a (miR-146a) on apoptosis of human gastric cancer SGC-7901 cells and the underluing mechanism.METHODS: miR-146a mimic (up-regulated miR-146a expression) and miR-146a inhibitor (down-regulated miR-146a expression) were transfected into the SGC-7901 cells by liposome method.At the same time, miRNA nonsense sequence transfection group as the negative control group (NC group) was set up.RT-qPCR was used to evaluate the levels of miR-146a in the SGC-7901 cells after transfection.The effects of miR-146a on the cell apoptosis and growth were assessed by flow cytometry analysis and CCK-8 assay, respectively.The effect of over-expression or knockdown of miR-146a on transforming growth factor-β-activated kinase 1 (TAK1)/ nuclear factor-kappa B (NF-κB) signaling was evaluated by RT-qPCR and Western blot.RESULTS: miR-146a modulated apoptosis of SGC-7901 cells.Over-expression of miR-146a significantly increased apoptosis, whereas knockdown of miR-146a inhibited the apoptosis of SGC-7901 cells.The expression of TAK1 at mRNA and protein levels was significantly decreased when miR-146a mimic was transfected into the SGC-7901 cells (P<0.05).On the contrast, the expression of TAK1 at mRNA and protein were significantly higher in miR-146a inhibitor transfection group than that in NC group (P<0.05), suggesting that miR-146a negatively regulated TAK1 expression.Moreover, knockdown of TAK1 enhanced the apoptosis of SGC-7901 cells (P<0.01), while over-expression of TAK1 inhibited the apoptosis of SGC-7901 cells(P<0.01).Additionally, both over-expression of miR-146a and knockdown of TAK1 led to a prominent increase in the expression of NF-κB inhibitor protein alpha (IκBα) and a significat decrease in B cell lymphoma-2 (Bcl-2) level in the SGC-7901 cells.CONCLUSION: miR-146a significantly promotes apoptosis of SGC-7901 cells by inhibition of NF-κB pathway via targeting TAK1.

Effect and molecular mechanism of mir-146a on proliferation of lung cancer cells by targeting and regulating MIF gene

OBJECTIVE@#To discuss the effect and molecular mechanism of miR-146a on the proliferation of lung cancer cells by targeting and regulating the macrophage migration inhibitory factor (MIF) gene.@*METHODS@#RT-PCR was employed to detect expression of miR-146a; immunohistochemistry was used to detect the expression of MIF. The luciferase reporter gene technique was adopted to verify that MIF was the specific reverse target gene of miR-146a and the liposome Lipofectamine™2000 was employed to transfer the modeled miR-146a mimics, and miR-146a negative control (NC) in NSCLC cells to detect the expression of MIF mRNA and protein. MTT assay was used to detect cell viability, cloning technique to detect cell proliferation ability, AnnexinV-PI to detect cell apoptosis, UV spectrophotometry to detect viability of cysteinyl aspartate specific proteinase 3 (Caspase 3), and western blot to detect expression of nuclear factor-κB (NF-κB) in cells.@*RESULTS@#The expression of miR-146a in NSCLC lung tissues was lower than that in the normal lung tissues besides the lung cancer; while the expression of miR-146a in NSCLC cells was lower than that in normal human embryonic lung tissues. It was chosen as the subsequent cell line for its appropriate expression in A549. The expression of MIF protein in NSCLC lung tissues was higher than that in the normal lung tissues besides the lung cancer. The luciferase reporter gene proved that MIF was the reverse target gene of miR-146a. The miR-146a mimics were transfected into A549 cells through the liposome. Compared with NC group, the expression of MIF protein and mRNA was significantly decreased (P < 0.01), with the decrease in the cell viability (P < 0.01), the decrease in the number of clones (P < 0.01), cell apoptosis (P < 0.01), the increase in the activity of Caspase 3 (P < 0.01), and decrease in the phosphorylation of NF-κB p65 (P < 0.01).@*CONCLUSIONS@#miR-146a has low expression in NSCLC tissues and cell lines, while MIF has the over expression in NSCLC tissues. The increased expression of miR-146a can inhibit the expression of MIF via the gene targeting and thus inhibit the proliferation of A549 cells and induce the apoptosis of cancer cells, which may be realized through NF-κB signaling pathway.

Local injection of the microRNA-146a recombinant lentiviral vector into the inner ear for immune-mediated inner ear disease in guinea pigs / 医学研究生学报

Objective Autoimmune inner ear disease is one of the inner ear diseases that can be effectively treated clinically, and researches on it have significant clinical and practical value.This study explored the immunoregulating and therapeutic effects of the mi-croRNA-146a recombinant lentiviral vector (RLV) on immune-medicated inner ear disease (IMIED) in guinea pigs so as to avoid the adverse reactions of the currently used immunodepressants. Methods IMIED models were established in 30 guinea pigs by immuni-zation with keyhole limpet hemocyanin and divided into three groups: microRNA-146a RLV, empty lentiviral vector ( ELV) control, and surgery simulation control, microinjected via the scala tympani with microRNA-146a RLV, ELV, and PBS, respectively.Before and after immunization and at 7 days after microinjection, the auditory function and the level of specific anti-KLH antibodies in the ser-um were measured by auditory brainstem response ( ABR) audiometry and ELISA, respectively.Seven days after microinjection, 3 ani-mals in each group were subjected to HE staining and light microscopy, another 3 to fluoro-autography for realizing the situations of the inner ear transfected by the lentiviral vector, and the other 4 to measurement of the contents of microRNA-146a in the inner ear tissue. Results Compared with the baseline, immunization significantly increased the level of specific anti-KLH antibodies in the serum (0.09 ±0.01 vs 1.90 ±0.74 in the RLV group, 0.11 ±0.02 vs 2.20 ±0.75 in the ELV group, and 0.11 ±0.02 vs 2.10 ±0.64 in the surgery simulation control) as well as the average threshold of the ABRⅢwave (left ear 11.67 ±2.58 vs 61.67 ±5.16 and right ear 12.50 ±2.73 vs 60.00 ±4.47 in the RLV group;left ear 14.16 ±3.76 vs 64.33 ±9.17 and right ear 13.33 ±2.58 vs 60.83 ± 4.92 in the ELV group;left ear 15.83 ±3.76 vs 64.17 ±10.2 and right ear 15.00 ±5.47 vs 62.50 ±9.35 in the surgery simulation control) .The average threshold of the ABRⅢwave was decreased after local injection into the inner ear as compared with that after immunization, with no statistically significant difference between the ELV and surgery simulation control groups.Fluoro-autographic ob-servation showed that the main parts of the inner ear transfected with microRNA-146a recombinant lentiviruses were the spiral limbus, spiral ganglion afferent fibers, basal cells of the spiral ligament, Corti organ, and psalterial cords.The immune inflammatory reaction of the inner ear was significantly reduced in the RLV group as compared with the ELV and surgery simulation control groups, while the content of microRNA-146a in the inner ear tissue was obviously increased in the former group than in the latter two. Conclusion The microRNA-146a recombinant lentiviral vectors are widely distributed and transfected in the inner ear tissue after injected via the scala tympani.Injecting recombinant microRNA-146a lentiviral vectors into the inner ear can significantly reduce pathological damage and auditory dysfunction caused by inner ear immune inflammatory reaction.

Expression pattern of MiR-146a and its correlation with antibody titers in human brucellosis / 中国免疫学杂志

Objective:To investigate the expression pattern of microRNA-146a in Brucella patients and its correlation with antibody titers.Methods: By using real time PCR assay, expression levels of microRNA-146a in sera samples from 20 brucellosis patients and 20 healthy volunteers were analyzed.The correlation between expression level of microRNA-146a and serum antibody titers were analyzed with SPSS17.0.Results: A quantification curve of microRNA-146a was constructed with synthesized standard.Expression levels of microRNA-146a among brucellosis patients were significantly lower than those in 20 healthy volunteers (P<0.001).For brucellosis patients,the expression level of microRNA-146a was negatively related with antibody titers (P<0.05). Conclusion:Expression of miRNA-146a in brucellosis patients was significantly inhibited and negatively related with antibody titer.

Effect and molecular mechanism of mir-146a on proliferation of lung cancer cells by targeting and regulating MIF gene

Objective To discuss the effect and molecular mechanism of miR-146a on the proliferation of lung cancer cells by targeting and regulating the macrophage migration inhibitory factor (MIF) gene. Methods RT-PCR was employed to detect expression of miR-146a; immunohistochemistry was used to detect the expression of MIF. The luciferase reporter gene technique was adopted to verify that MIF was the specific reverse target gene of miR-146a and the liposome Lipofectamine™2000 was employed to transfer the modeled miR-146a mimics, and miR-146a negative control (NC) in NSCLC cells to detect the expression of MIF mRNA and protein. MTT assay was used to detect cell viability, cloning technique to detect cell proliferation ability, AnnexinV-PI to detect cell apoptosis, UV spectrophotometry to detect viability of cysteinyl aspartate specific proteinase 3 (Caspase 3), and western blot to detect expression of nuclear factor-κB (NF-κB) in cells. Results The expression of miR-146a in NSCLC lung tissues was lower than that in the normal lung tissues besides the lung cancer; while the expression of miR-146a in NSCLC cells was lower than that in normal human embryonic lung tissues. It was chosen as the subsequent cell line for its appropriate expression in A549. The expression of MIF protein in NSCLC lung tissues was higher than that in the normal lung tissues besides the lung cancer. The luciferase reporter gene proved that MIF was the reverse target gene of miR-146a. The miR-146a mimics were transfected into A549 cells through the liposome. Compared with NC group, the expression of MIF protein and mRNA was significantly decreased (P < 0.01), with the decrease in the cell viability (P < 0.01), the decrease in the number of clones (P < 0.01), cell apoptosis (P < 0.01), the increase in the activity of Caspase 3 (P < 0.01), and decrease in the phosphorylation of NF-κB p65 (P < 0.01). Conclusions miR-146a has low expression in NSCLC tissues and cell lines, while MIF has the over expression in NSCLC tissues. The increased expression of miR-146a can inhibit the expression of MIF via the gene targeting and thus inhibit the proliferation of A549 cells and induce the apoptosis of cancer cells, which may be realized through NF-κB signaling pathway.

Expressions and its clinical significance of microRNA-21 and microRNA-146a in colorectal neoplasms / 中华消化杂志

Objective To investigate the differences of miRNA-21 and miRNA-146a expression between colorectal neoplasms tissues and serum of the patients with colorectal neoplasm,and the clinical significance was analyzed.Methods The endoscopic biopsy tissues and serum samples of 100 colorectal cancer (CRC),80 colorectal adenoma (CRA)patients and 65 healthy controls were collected.The expressions of miRNA-21 and miRNA-146a were detected by quantitative real time polymerase chain reaction.The relationship between the expression and clinicopathological features were analyzed.And then,the diagnostic value of expression difference of serum miRNA in colorectal neoplasm were evaluated. The Mann-WhitneyU test and Kruskal-Wallis test were performed for comparisons between groups,and the correlation of miRNA expression between tissues and serum was analyzed by Spearman test.Results The expressions of miRNA-21 in the tissues of CRC and CRA were 8.573 ±0.898 and 7.746 ±1 .183, respectively,which were significantly higher than that of healthy controls 6.160 ±0.835 (U =120.129 and 33.230,both P 0.05 ).The expression of miRNA-146a in the tissues and serum of CRC patients were both lower than those of CRA patients (U =73.809 and 21 .123, both P <0.01).The degree of decreased expression of miRNA-146a in CRC patients was correlated with TNM staging and tumor differentiation degree,however there was no correlation between the expression in CRA and clinical features.According to receiver operating characteristic (ROC)curve analysis,the AUC of miRNA-21 ,miRNA-146a and a combination of them in CRC and health individuals was 0.889, 0.791 and 0.863,respectively;in CRA and health individuals was 0.784,0.692 and 0.761 ,respectively;in CRC and CRA was 0.705 ,0.820 and 0.713,respectively.Conclusion The different expressions of miRNA-21 and miRNA-146a had potential values in early detection of colorectal cancer.

The protective effects of transfected microRNA-146a on mice with sepsis-induced acute lung injury in vivo / 中华危重病急救医学

ObjectiveTo investigate the protective effect of transfected microRNA-146a (miR-146a) on mice with sepsis-induced acute lung injury (ALI) in vivo.Methods Twenty-four healthy male BALB/C mice were randomly divided into sham group, sepsis group, transfection group and transfection control group, eachn = 6. Mice in transfection group were given miR-146a agomir loaded by in vivo-jetPEITM via airway before reproduction of model, and mice in transfection control group were given negative control loaded by in vivo-jetPEITM only via airway. The septic model was reproduced by cecal ligation and puncture (CLP) 12 hours after transfection , and the mice in the sham group underwent laparotomy and closure only without ligation and puncture of the cecum. The mice of each group were sacrificed at 24 hours post-operation. The expression of miR-146a in lung tissue was determined by real time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the quantity of tumor necrosis factor-α (TNF-α) in the bronchial alveolar lavage fluid (BALF) was determined with enzyme-linked immunosorbent assay (ELISA). The wet/dry ratio of lung (W/D) was determined. The pathohistological changes in the lung were observed and scored. Results The expression of miR-146a showed a significant increase in sepsis group, transfection group and transfection control group, which were (3.56±0.43), (27.64±3.46) and (3.72±0.54) folds of that in sham group, respectively (P 0.05). Compared with the sham group, higher level of TNF-αin the BALF was found in the sepsis group, transfection group and transfection control group (ng/L: 511.65±43.47, 305.74±34.76, 492.27±42.21 vs. 50.72±7.23, allP< 0.01). The level of TNF-α in transfection group was significantly lower than that in sepsis group and transfection control group (bothP< 0.01). Compared with the sham group, the W/D ratio of lung in sepsis group, transfection group and transfection control group showed a significant increase (6.11±0.32, 5.02±0.29, 6.05±0.43 vs. 4.18±0.10, allP< 0.01). The W/D ratio of lung in transfection group was significantly lower than that of sepsis group and transfection control group (bothP< 0.01). The lung injury score of transfection group was significantly lower than that of sepsis group and transfection control group (6.12±0.75 vs. 10.53±1.52, 9.73±1.08, bothP< 0.01).Conclusions miR-146a agomir loaded by in vivo-jetPEITM instillation into airway was able to increase the expression of miR-146a in the lung tissue of septic mice. Up-regulation of miR-146a inhibit the release of the inflammatory cytokine TNF-α stimulated by sepsis, and alleviate inflammatory reaction and lung tissue injury in mice with sepsis-induced ALI.

Influence of Gleevec on expression of miR-146a,miR-29b and DNMT in K562 cells / 重庆医学

Objective To investigate the changes of microRNA (miR)-146a ,miR-29b expression levels and the 3 kinds of meth-ylase DNMT1 ,DNMT3a and DNMT3b levels in K562 cell lines after BCR/ABL inhibitor Gleevec treatment .Methods The half maximal inhibitory concentration(IC50 ) of Gleevec on K562 cells was detected by the MTT method .The stem loop primers method and the fluorogenic quantitative PCR were adopted to detect miRNAs and the methylase gene level .Results IC50 of Gleevec acting on K562 cells was 40 .85μmol/L .After Gleevec action ,miR-29b showed the increasing trend ,but 3 kinds of methylase expression level were decreased to some extent .Gleevec could significantly increase the miR-146a level in K562 cells(P<0 .05) .Conclusion Gleevec can influence the expression of miR-146a ,miR-29b and DNMTs levels in K562 cells .

Study on the levels of microRNA-146b, microRNA-155 and microRNA-30b in human milk and their correlation with maternal and infant health / 中华实用儿科临床杂志

Objective To explore the expression levels of immune-related microRNA-146b (miR-146b),microRNA-155 (miR-155) and microRNA-30b(miR-30b) in human breast milk and its relationship with maternal and infant's health.Methods One hundred and thirty-four mothers and their infants from obstetrical department were recruited in the study after delivery.The subjects were divided into 2 groups,breast feeding group(n =86) and formula-feeding group(n =48),and were followed up 3 months after delivery.Breast milk samples were collected at 2-5 days after delivery(colostrum) and 3 months after delivery(mature milk).The expression levels of microRNAs in milk samples were detected by real-time PCR.The relationship between levels of microRNAs and maternal and infant-related factors was analyzed.Results 1.MiR-146b,miR-155 and miR-30b expressions were abundant both in human colostrums (5.950 ± 0.823,3.899 ± 0.920,4.057 ± 0.604) and mature milk (4.840 ± 0.805,2.128 ± 0.969,4.929 ± 0.566).The levels of miR-146b and miR-155 were higher in colostrum than that of mature milk (t =7.716,10.215,all P ＜ 0.01),while the level of miR-30b was higher in mature milk than that of colostrums(t =-8.626,P ＜ 0.0l).2.Additionally,the level of miR-30b was negatively correlated with maternal pre-pregnancy body mass index (r =-0.298,P ＜ 0.01).3.The levels of miR-146b and miR-30b were higher in mothers giving birth by vaginal delivery than those who underwent cesarean section(t =2.356,3.108,all P ＜0.05).4.The levels of miR-146b and miR-155 were higher in colostrum-fed girls than boys (t =-2.204,-2.985,all P ＜ 0.05).5.The level of miR-146b in mature milk was negatively correlated with 3-month-old infant' s Z score of body weight (r =-0.425,P ＜ 0.05) and body length (r =-0.569,P ＜ 0.01).6.During follow-up,the incidence of baby eczema in breast feeding group (8.82％,3/34 cases) was lower than that in formula milk feeding group(29.17％,14/48 cases) (x2 =5.012,P =0.025).Conclusions The levels of immunocompetent microRNAs in human milk are influenced by the lactation period,maternal prepregnancy body mass index,mode of delivery and infant sex.The immune-related microRNAs in human milk could be involved in the regulation of infant's immunity and growth.

Expression changes in miR-16 and miR-146a in rat lungs after fast buoyancy ascent escape or diving decompression sickness / 军事医学

Objective To study the expression levels of microRNA (miR)-16 and miR-146a in rat lungs of decompres-sion sickness (DCS) caused by fast buoyancy ascent escape or diving .Methods At 0.5 h after fast buoyancy ascent es-cape or diving, the pathological changes in rat lungs and expression levels of miR-16,and miR-146a were detected by re-verse transcription-quantitive polymerase chain reaction and compared with normal control group .Results The pathological characteristics of lungs in two DCS groups were tissue damage .At 0.5 h after DCS caused by fast buoyancy ascent escape , the lung tissue expression levels of miR-16 and miR-146a did not significantly change compared with normal control and diving DCS groups ,but the rat lung tissue expression level of miR-146 a in diving DCS group was obviously increased , com-pared with normal control group .Conclusion miR-146a may play a role in post-transcriptional regulation in the process of diving DCS .

Effects of KLF2 on ox-LDL induced expression of microRNA-146a and proinflammatory cytokines in human umbilical vein endothelial cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of Kr üppel-like factor 2 ( KLF2 ) on oxidized low density lipoprotein (ox-LDL) induced expression of microRNA-146a (miR-146a) and proinflammatory cyto-kines (MCP-1 and IL-6) in human umbilical vein endothelial cells (HUVECs).Methods Human umbili-cal vein endothelial cells (HUVECs) were cultured and then stimulated with 50μg/ml ox-LDL for 24 hours. HUVECs were infected with adenovirus vectors over-expressing human KLF2 at an appropriate multiplicity of infection.KLF2-siRNA duplexes were transfected into HUVECs to silence the gene expression .HUVECs were collected at time points of 0 h, 3 h, 6 h, 12 h, 24 h and 48 h after infection.Real-time quantitative-PCR was performed to measure the expression of miR-146a at mRNA level.Silenced endogenous miR-146a using LNA-anti-miR-146 a was transfected into HUVECs with lipofectamine 2000 .The levels of MCP-1 and IL-6 in supernatants were detected by ELISA .Results KLF2 remarkably inhibited the expression of miR-146 a in unstimulated and ox-LDL-stimulated HUVECs in a time-dependent manner .The ox-LDL induced ex-pression of miR-146a, MCP-1 and IL-6 in HUVECs were significantly decreased by KLF2.Silenced expres-sion of miR-146a downr-egulated the ox -LDL induced expression of MCP-1 and IL-6 in HUVECs.Moreover, silenced miR-146 a could partly reverse the inhibitory effects of KLF 2 on ox-LDL induced expression of MCP-1 and IL-6 in HUVECs.Conclusion KLF2 inhibited ox-LDL induced expression of MCP-1 and IL-6 in HUVECs partly through down-regulating the expression of miR-146a.