MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1-extracellular signal-regulated kinase-1/2 axis

BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.

MicroRNA-520a用于异位妊娠血清学诊断的研究

目的评估microRNA-520a作为异位妊娠(ectopic pregnancy,EP)患者非侵入性血清学诊断标志物的临床应用价值。方法收集2017年1月至2018年12月首都医科大学附属北京妇产医院185例孕妇的病例资料及其首次就诊送检的血液标本,包括64例宫腔妊娠(viable intrauterine pregnancy,VIP)、48例自然流产(spontaneous abortion,SA)以及73例EP,采用定量反转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)检测患者血浆中microRNA-520a的表达水平。同时检测血清绒毛膜促性腺激素(human chorionic gonadotrophin,HCG)和孕酮水平,并比较各组间差异。采用ROC曲线分析microRNA-520a浓度作为EP血清学诊断标志物的有效性。结果microRNA-520a在EP组血浆中的表达水平(1.88±0.50)与VIP组(2.67±0.61,P=0.000)及SA组(2.77±0.50,P<0.001)比较,均显著下降。用于VIP与EP的鉴别诊断,ROC曲线的AUC为0.82(95%CI:0.68~0.97,P=0.001)。结论异位妊娠者血浆标本中microRNA-520a表达水平显著下降,microRNA-520a可作为血清学标志物应用于早期EP诊断。

母血microRNA-520a水平与胎儿心脏缺陷关系的研究

目的本研究旨在评估母体血循环microRNA-520a作为先天性心脏缺陷胎儿非侵入性诊断标志物的临床应用价值。方法 44例先天性心脏缺陷胎儿孕妇为研究组,34例正常胎儿孕妇做为对照组。定量RT-PCR检测两组孕妇血浆中的microRNA-520a表达水平。结果 microRNA-520a在先天性心脏缺陷胎儿孕妇血浆中的表达水平与正常对照组相比显著上升(P<0.001),ROC特征曲线下面积(AUC)为0.92(95%可信区间为0.86-0.97;P<0.001)。研究显示,microRNA-520a的血浆表达水平可以相当准确地诊断妊娠期胎儿的先天性心脏缺陷。结论本研究的结果表明,microRNA-520a在先天性心脏缺陷胎儿孕妇的血浆中表达水平显著上升。母体血循环microRNA-520a表达水平可能作为先天性心脏缺陷胎儿的非侵入性诊断标志物。

MicroRNA-520家族与血瘀证相关性探讨

从分子生物学的角度探讨microRNA-520家族与血瘀证的相关性,认为microRNA-520家族是通过介导炎症反应、免疫紊乱、NF-κB信号通路调节血瘀证的形成过程,可以作为血瘀证的生物标记物,为中医药防治血瘀证提供新的治疗依据,,具有重要的临床应用价值。

MicroRNA-520b靶向白介素-2调节肝癌细胞生长、转移和药物敏感性研究

目的:研究microRNA-520靶向调节IL-2对肝癌细胞生长、转移和药物敏感性的影响。方法:选择并收集原发性肝细胞癌患者术后肝癌及其癌旁组织,采用RT-PCR检测miR-520b表达,采用免疫组化的方法检测IL-2的表达。培养肝癌细胞系HepG2,将细胞分为对照组、空白对照组和miR-520b过表达组(简称过表达组),空白对照组和过表达组细胞分别转染miR-NC及miR-520b mimics,3组细胞均加入重组IL-2培养12 h。检测miR-520b、IL-2的表达,双荧光素酶报告基因实验验证miR-520b对IL-2的靶向调控作用。采用MTT法检测细胞增殖率,流式细胞术检测细胞凋亡率,Transwell实验检测细胞转移和侵袭能力。检测细胞药物敏感性。结果:肝癌组织中miR-520b和IL-2表达均显著低于癌旁组织,差异有统计学意义(P<0.05);体外模型中,过表达组细胞野生型IL-2的3′UTR的荧光素酶报告基因的表达较空白对照组降低(P<0.05)。转染miR-520b后,过表达组细胞生长率较对照组和空白对照组显著降低,凋亡率则显著增加,细胞转移和侵袭能力降低(P<0.05);miR-520b联合IL-2培养肝癌细胞的细胞增殖率较单独使用IL-2或miR-520b低(P<0.05)。结论:micro-520b可以靶向调节肝癌细胞中IL-2的表达,抑制癌细胞的生长、转移,增强药物敏感性,micro-520b对肝癌的诊断和治疗具有潜在的价值。

Nix和miR-520h对神经胶质瘤发生的影响及相互作用

目的通过综合分析肿瘤标本中基因和蛋白表达,及体外生物学作用的检测,来评价胶质瘤中Nix蛋白的作用以及Nix蛋白与miR-520h的关系。方法选取46例胶质瘤标本,RT-PCR检测miR-520h基因的表达,Western blotting检测Nix蛋白的表达;培养U251胶质瘤细胞,Western blotting检测Nix蛋白的表达;构建靶向Nix基因的shRNA敲除U251细胞中Nix基因(Nix-kn),Western blotting检测Nix,IKKα,i-κBα和p-NF-κB/p65等蛋白的表达水平;has-miR-520h抑制剂转染U251细胞,Western blotting检测Nix,IKKα,i-κBα和p-NF-κB/p65等蛋白的表达水平。结果胶质瘤标本的基因和蛋白检测表明,miR-520h的高表达伴随着Nix蛋白的高表达;体外实验结果表明,与正常培养的U251相比,缺氧培养的U251细胞Nix表达较高;与Nix-kn组相比,Nix-wt组胶质瘤细胞Nix和p-NF-κB/p65表达均明显增高,而NF-κB活性抑制剂蛋白i-κBα表达降低;200 nmol/L的has-miR-520h抑制剂处理细胞后,与正常对照组比较,Nix蛋白表达显著减少,而p-NF-kappaB/p65蛋白表达显著增加。结论在神经胶质瘤的发生发展中miR-520h与Nix蛋白的表达相关;miR-520h可能通过促进Nix合成,从而成为一个强大的肿瘤激活剂。

Long noncoding RNA X-inactive specific transcript regulates NLR family pyrin domain containing 3/caspase-1-mediated pyroptosis in diabetic nephropathy

BACKGROUND NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology.Long noncoding RNAs(lncRNAs)are active participators of diabetic nephropathy(DN).X inactive specific transcript(XIST)expression has been reported to be elevated in the serum of DN patients.AIM To evaluate the mechanism of lncRNA XIST in renal tubular epithelial cell(RTEC)pyroptosis in DN.METHODS A DN rat model was established through streptozotocin injection,and XIST was knocked down by tail vein injection of the lentivirus LV sh-XIST.Renal metabolic and biochemical indices were detected,and pathological changes in the renal tissue were assessed.The expression of indicators related to inflammation and pyroptosis was also detected.High glucose(HG)was used to treat HK2 cells,and cell viability and lactate dehydrogenase(LDH)activity were detected after silencing XIST.The subcellular localization and downstream mechanism of XIST were investigated.Finally,a rescue experiment was carried out to verify that XIST regulates NLR family pyrin domain containing 3(NLRP3)/caspase-1-mediated RTEC pyroptosis through the microRNA-15-5p(miR-15b-5p)/Toll-like receptor 4(TLR4)axis.RESULTS XIST was highly expressed in the DN models.XIST silencing improved renal metabolism and biochemical indices and mitigated renal injury.The expression of inflammation and pyroptosis indicators was significantly increased in DN rats and HG-treated HK2 cells;cell viability was decreased and LDH activity was increased after HGtreatment. Silencing XIST inhibited RTEC pyroptosis by inhibiting NLRP3/caspase-1. Mechanistically,XIST sponged miR-15b-5p to regulate TLR4. Silencing XIST inhibited TLR4 by promotingmiR-15b-5p. miR-15b-5p inhibition or TLR4 overexpression averted the inhibitory effect ofsilencing XIST on HG-induced RTEC pyroptosis.CONCLUSIONSilencing XIST inhibits TLR4 by upregulating miR-15b-5p and ultimately inhibits renal injury inDN by inhibiting NLRP3/caspase-1-mediated RTEC pyroptosis.

The extracellular secretion of miR-1825 wrapped by exosomes increases CLEC5A expression:A potential oncogenic mechanism in ovarian cancer

Background:Ovarian cancer(OC)is a leading cause of gynecological cancer-linked deaths worldwide.Exosomal miR-1825 and its target gene C-type lectin domain family 5 member A(CLEC5A)are associated with tumorigenesis in cancers that was further probed.Methods:Exosomal miR-1825 expression in exosomes and its impact on overall survival(OS)prediction were determined using Gene Expression Omnibus(GEO)and The Cancer Genome Atlas(TCGA)data.Target genes of miR-1825 were searched in five prediction databases and prognostically significant differentially expressed genes were identified.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were carried out.The ability of CLEC5A to predict OS was evaluated using univariate and multivariate Cox regression analyses and Kaplan-Meier curves.The CLEC5A expression pattern in OC was validated using immunohistochemistry.The CIBERSORT algorithm was used to compare the immune cell landscape,and the results were validated in a GEO cohort.Finally,the predicted half maximal inhibitory concentration(IC50)values for five commonly used chemotherapy agents were also compared.Results:MiR-1825 level was higher in exosomes derived from OC cells and served as a tumor suppressor.The CLEC5A gene was found to be a target of miR-1825,the upregulation of which was correlated with a poor prognosis.M2 macrophage infiltration was significantly enhanced in the CLEC5A high expression group,while T follicular helper cell infiltration was reduced in it.While the predicted IC50 for cisplatin and doxorubicin was higher in the CLEC5A high expression group,that of docetaxel,gemcitabine,and paclitaxel was lower.Conclusion:MiR-1825,a promising OC biomarker,may promote OC progression by increasing CLEC5A expression via exosome-mediated efflux from tumor cells.

MicroRNA-520e通过靶向抑制AEG-1发挥抗结直肠癌特性

目的:探究MicroRNA-520e(miR-520e)在结直肠癌中的表达模式及其对细胞功能的影响。方法:采用qRT-PCR方法检测47例结直肠癌患者的癌组织和癌旁组织中miR-520e和星形胶质细胞上调基因-1(AEG-1)的mRNA表达水平。将SW480细胞分为对照组、miR-520e-mimic组、NC-mimic组、miR-520e-inhibitor组、NC-inhibitor组、miR-520e-mimic+AEG-1-pcDNA3.1组和miR-520e-mimic+NC-pcDNA3.1组。通过MTT法检测SW480细胞的增殖,通过Annexin V-FITC/PI双染色试剂盒检测细胞凋亡,通过Transwell检测细胞迁移和侵袭,通过双荧光素酶报告基因实验验证miR-520e和AEG-1的靶向关系,通过qRT-PCR或Western blotting检测AEG-1、基质金属蛋白酶2(MMP2)、MMP9、NF-κB p65(p65)和磷酸化的NF-κB p65(p-p65)的表达。结果:与癌旁组织相比,结直肠癌组织中miR-520e的表达水平降低(t=9.353,P<0.001)。与对照组相比,miR-520e-mimic组的OD值降低,细胞凋亡率升高,细胞迁移和侵袭数量降低,MMP2、MMP9和p-p65蛋白表达水平降低(P<0.001)。与对照组相比,miR-520e-inhibitor组的OD值升高,细胞凋亡率降低,细胞迁移和侵袭数量升高,MMP2、MMP9和p-p65蛋白表达水平升高。与NC-mimic组相比,miR-520e-inhibitor组的相对荧光素酶活性降低(P<0.001)。与对照组相比,miR-520e-mimic组的AEG-1的mRNA和蛋白表达水平均降低,而miR-520e-inhibitor组均升高(P<0.001)。与miR-520e-mimic+NC-pcDNA3.1组相比,miR-520e-mimic+AEG-1-pcDNA3.1组的AEG-1的mRNA和蛋白表达水平升高,OD值升高,细胞凋亡率降低,迁移和侵袭细胞数增加,MMP2和MMP9的蛋白表达水平及p65的磷酸化水平均增加(P<0.001)。结论:miR-520e在结直肠癌中表达降低,可通过靶向抑制AEG-1来发挥抗结直肠癌特性,其抗癌机制可能通过NF-κB信号通路介导。

母血microRNA-520α水平与胎儿心脏缺陷关系的研究

目的探讨母血microRNA-520α水平对胎儿心脏缺陷的早期诊断价值及其临床意义。方法选取2016年5月~2018年4月怀有心脏缺陷胎儿孕妇23例(心脏缺陷组),同期产检的神经管畸形胎儿孕妇23例(神经管畸形组)和怀有正常胎儿孕妇30例(正常对照组)作为本文的主要研究对象,检测各组孕妇血液中的microRNA-520α水平,并通过ROC曲线分析评估母血microRNA-520α水平诊断胎儿心脏缺陷关系的价值。结果经分娩/引产证实胎儿心脏缺陷例数为22例,准确率为95.65%,误诊1例,漏诊率为4.35%。心脏畸形组的microRNA-520α水平(1.29±0.75)ng/ml,低于神经管畸形组的(1.81±0.95)ng/ml,显著低于正常对照组的(2.65±1.26)ng/ml,差异有统计学意义(P<0.05)。进一步的ROC曲线分析得出母血microRNA-520α水平诊断胎儿心脏缺陷的曲线下面积(area under curve,AUC)为0.854,在最佳临界值点对应的灵敏度、特异度分别为87%、71.7%,证明microRNA-520α对胎儿心脏缺陷的诊断效能较好。结论母血microRNA-520α水平对胎儿心脏缺陷具有诊断价值,且诊断准确性较好,可为胎儿心脏缺陷早期辅助评估提供客观参考。

血瘀证相关的microRNA-520b对白介素-8的影响及丹参酮Ⅱ_A的干预作用

目的:研究血瘀证相关的micorRNA-520b(miR-520b)对白介素-8(IL-8)的影响及丹参酮ⅡA(TanⅡA)的干预作用。方法:采用脂质体介导的转染方法将microRNA-520b模拟物(50 nmol·L-1)或抑制物(100 nmol·L-1)转染进入含1.6×104cells/well人脐静脉血管内皮细胞(HUVECs)的6孔板中24,36,48 h,采用5,10,20,40 mg·L-1TanⅡA干预24,48 h。荧光显微镜观察转染情况,MTT法检测细胞活性(n=5),硝酸还原酶法、酶联免疫法(ELISA)测定培养液中一氧化氮(NO),内皮素(ET),蛋白C受体(EPCR),血管内假性血友病因子(v WF)和血栓调节蛋白(TM)含量(n=3)。半定量反转录-聚合酶链反应(RT-PCR),蛋白质免疫印迹(Western blotting)检测细胞IL-8信使核糖核酸(mRNA)和蛋白表达水平(n=3)。结果:与对照组比较,模型组(miR-520b组)细胞有荧光,48 h荧光最强。细胞活性降低(P<0.01)且48 h最显著,NO含量下降(P<0.01),EPCR,v WF,ET含量升高(P<0.01),转染48 h后IL-8 mRNA和蛋白质表达下调(P<0.01)。与模型(miR-520b)组比较,给药组(TanⅡA组)细胞活性升高(P<0.01),TanⅡA(10 mg·L-1,48 h)干预效果最佳;NO含量升高(P<0.01);EPCR,v WF,TM,ET含量降低(P<0.01),IL-8 mRNA和蛋白质表达上调(P<0.01)。结论:miR-520b调节IL-8在血瘀证中发挥作用,可能为TanⅡA作用靶点。

Upregulation of miR-345-5p suppresses cell growth of lung adenocarcinoma by regulating ras homolog family member A(RhoA)and Rho/Rho associated protein kinase(Rho/ROCK)pathway

Background:Microribose nucleic acids(miRNAs)are implicated in the progression of lung adenocarcinoma.MicroRNA-345-5p(miR-345-5p)is a recently identified anti-oncogene in some human cancers,but its functional role and possible molecular mechanism in lung adenocarcinoma remain unknown.This study aimed to identify the biological function and underlying mechanism of miR-345-5p in lung adenocarcinoma cells.Methods:In this study,lung adenocarcinoma tissues and adjacent tissues were collected in the First Affiliated Hospital of Anhui Medical University between April 2016 and February 2017.The expression of miR-345-5p and ras homolog family member A(RhoA)in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines(A549,H1650,PC-9,and H441)was detected by reverse transcription quantitative polymerase chain reaction analysis.Functional assays including colony formation,flow cytometry analysis,wound healing,and transwell assays were performed to assess the proliferation,apoptosis,migration,and invasion of lung adenocarcinoma cells.In addition,RNA pulldown and luciferase reporter assays were conducted to evaluate the relationship between miR-345-5p and RhoA.Difference between the two groups was analyzed with Student’st test,while that among multiple groups was analyzed with one-way analysis of variance.Results:MiR-345-5p expression displayed lower level in lung adenocarcinoma tissues(0.241±0.095vs.1.000±0.233,t=19.247,P<0.001)and cell lines(F=56.992,P<0.001)than control tissues and cells.Functional experiments demonstrated that upregulation of miR-345-5p inhibited the malignant phenotypes of lung adenocarcinoma cells via suppressing cell proliferation,migration,invasion,and facilitating cell apoptosis.Additionally,RhoA was verified to be the downstream target of miR-345-5p.Expression of RhoA was downregulated by overexpression of miR-345-5p in PC-9(0.321±0.047vs.1.000±0.127,t=8.536,P<0.001)and H1650(0.398±0.054vs.1.000±0.156,t=4.429,P=0.011)cells.Rescue assays revealed that overexpression of RhoA rescued the suppressive effects of miR-345-5p upregulation on proliferation,migration,and invasion of lung adenocarcinoma cells.Further,miR-345-5p was found to regulate the Rho/Rho-associated protein kinase(ROCK)signaling pathway by downregulation of RhoA in lung adenocarcinoma cells.Conclusions:MiR-345-5p plays a tumor suppressor role in lung adenocarcinoma cells by downregulating RhoA to inactivate the Rho/ROCK pathway.

Effects of moxibustion on miRNA-133b,Pitx3/TH,and neurotransmitters in the midbrain of rats with diarrhea-predominant irritable bowel syndrome

Objective:To investigate the mechanism of moxibustion in the treatment of diarrhea-predominant irritable bowel syndrome(IBS-D),by observing the effects of moxibustion at Tianshu(ST25)and Shangjuxu(ST37)on microRNA-133b(miRNA-133b),pituitary homeobox family factor 3(Pitx3)/tyrosine hydroxylase(TH),and neurotransmitters in the brain tissue of IBS-D rats.Methods:Healthy Sprague-Dawley rats were randomly divided into a normal group,a model group,a moxibustion group,and a Western medicine group,with 12 rats in each group.Except for the normal group,the IBS-D rat model was established by mother-offspring separation and acetic acid enema combined with restraint stress stimulation in all the other groups.No intervention was performed in the normal and model groups.Mild moxibustion was applied to both Tianshu(ST25)and Shangjuxu(ST37)in the moxibustion group.Rifaximin was given by gavage in the Western medicine group.The physical status of rats in each group was observed at different periods.After the intervention,hematoxylineosin staining was performed to observe the histopathological morphology of rat colon;enzyme-linked immunosorbent assay was used to measure the levels of dopamine(DA),noradrenaline(NE),and 5-hydroxytryptamine(5-HT)in plasma,colon,and midbrain tissue of rats;the relative expression levels of miRNA-133b,Pitx3 mRNA,and TH mRNA in the midbrain tissue were measured by real-time fluorescence quantitative polymerase chain reaction,and the relative expression levels of Pitx3 and TH proteins in the midbrain tissue were measured by Western blotting and immunofluorescence.Results:The body weights of rats among groups and at different time points were statistically different(P<0.01).The body weight of the normal group was higher than that of the other groups over time(P<0.01).After modeling,the minimum volume threshold of abdominal withdrawal reflex(AWR)was significantly lower(P<0.01)and the loose stool rate was significantly higher(P<0.01)in the model,moxibustion,and Western medicine groups compared with the normal group;the miRNA-133b expression in the midbrain tissue was significantly lower(P<0.01),the expression levels of Pitx3 and TH in the midbrain tissue were significantly higher(P<0.01),and the levels of DA,NE,and 5-HT in plasma,colon and midbrain tissue were significantly higher(P<0.01).After the intervention,the minimum volume threshold of AWR was significantly higher(P<0.01),the loose stool rate was significantly lower(P<0.01),the miRNA-133b expression was significantly increased(P<0.01 or P<0.05)and the expression levels of Pitx3 and TH were significantly decreased(P<0.01)in the midbrain tissue,the levels of DA,NE,and 5-HT in plasma,colon,and midbrain tissue were significantly reduced(P<0.01)in the moxibustion and Western medicine groups compared with the model group;the levels of 5-HT in the colon and midbrain tissue of the moxibustion group were significantly lower than those in the Western medicine group(P<0.05),and there was no statistical difference compared with the remaining groups(P>0.05).Linear correlation analysis showed that miRNA-133b was negatively correlated with Pitx3(r<0,P<0.01);Pitx3 with TH,TH with DA,and NE with 5-HT were positively correlated(r>0,P<0.01).Conclusion:Moxibustion at Tianshu(ST25)and Shangjuxu(ST37)improves diarrhea symptoms and visceral hypersensitivity in IBS-D rats.The mechanism may be related to up-regulating miRNA-133b,inhibiting Pitx3/TH,and reducing neurotransmitter expression levels in the midbrain tissue.