miR-125b-5p促进分枝杆菌在宿主细胞和小鼠体内存活的研究

通过观察miR-125b-5p对分枝杆菌在宿主细胞和小鼠体内存活情况的影响,探究其在抗结核免疫过程中的作用。采用不同培养基对分枝杆菌进行培养并计数;以1640培养基加10%胎牛血清培养所有实验用细胞。将终浓度50 nmol/L的miR-125b-5p 模拟物、miR-125b-5p 抑制剂及磷酸盐缓冲液(PBS)对照加入细胞后,在不同时间点收集细胞。用分枝杆菌分别感染宿主细胞(A549、THP-1和RAW264.7)以及C57BL/6小鼠。采用定量聚合酶链反应检测miR-125b-5p的表达量。结果miR-125b-5p在分枝杆菌感染的多种宿主细胞及小鼠中都显著上调表达,其中小鼠肺部的表达量提高了约15倍。分别转染模拟物和抑制剂后,再用分枝杆菌感染细胞,结果发现miR-125b-5p可促进分枝杆菌在宿主细胞内的生长。当miR-125b-5p抑制剂注射到卡介苗(BCG)感染的小鼠体内时,小鼠体内的细菌载量显著降低(P<0.05)。本研究证明miR-125b-5p可调控分枝杆菌在宿主细胞及小鼠体内的生长,在抗结核免疫过程中发挥了重要作用。进一步对其作用机制的深入研究将为临床结核病的治疗提供理论指导。     

miR-199b-5p在糖尿病肾病大鼠肾脏纤维化中作用机制

[目的]探索miR-199b-5p在糖尿病肾病大鼠肾脏纤维化中的作用机制。[方法]选择92只雄性大鼠,随机分为空白对照组、DN组、慢病毒对照组、miR-199b-5p敲低组,造模结束后第12 w处死大鼠。对比上述4组大鼠在肾组织病理学、肾功能指标,Klotho、Wnt、β-catenin等相关蛋白表达差异。[结果] DN组、慢病毒对照组的miR-199b-5p表达高于空白对照组和miR-199b-5p敲低组,miR-199b-5p敲低组高于空白对照组(1.08±0.24、3.27±0.12、3.22±0.53、1.62±0.15;P<0.05);DN组、慢病毒对照组的Scr、BUN、β-catenin、α-SMA、MMP-7均显著高于空白对照组和miR-199b-5p敲低组,miR-199b-5p敲低组高于空白对照组(78.66±2.12 vs 61.14±3.78;14.04±1.09 vs 9.26±1.73;32.33±2.57 vs 27.23±1.78;0.97±0.02 vs 0.34±0.35;3.49±0.28 vs 1.43±0.23;P<0.05);而D...     

miR-125b-5p对人血管瘤内皮细胞HemES增殖和凋亡的影响及其机制

目的:研究miR-125b-5p对人血管瘤内皮细胞HemECs增殖、凋亡的影响.方法:RT-qPCR检测人血管瘤内皮细胞HemECs及其旁系组织细胞中miR-125b-5p与MCL-1 mRNA的表达;选取HemECs细胞分为对照组、miR-NC 组、miR-125b-5p mimic 组、miR-125b-5p in...     

白藜芦醇抑制肠癌细胞焦亡的作用*

目的: 研究白藜芦醇(Res)对肠癌细胞焦亡的影响。方法: ①葡聚糖硫酸钠(DSS)诱发小鼠结肠癌(CRC)实验:30只C57BL/6小鼠随机分为正常对照组(Contro组),氧化偶氮甲烷(AOM)组,AOM/DSS组,AOM/DSS+Res组和Res组,每组6只,造模周期共70 d。第1周第1日对AOM组、AOM/DSS组和AOM/DSS+Res组小鼠AOM(10 mg/kg)腹腔注射一次,无菌水饮水,1% DSS水供AOM/DSS组和AOM/DSS+Res组饮用,对AOM/DSS+Res组和Res组小鼠灌胃给予Res(50 mg/kg),造模结束后,取小鼠结肠组织固定、包埋、切片; IHC和Western blot检测小鼠结肠组织NLRP3、Caspase-1、IL-18蛋白表达情况。②离体实验:HCT 116细胞给予Res(2.4 μg/L)以及转染miR-31,加Res实验分为4组,分别标记0 h、12 h、24 h、48 h组;细胞转染分组为5组,即control组、miR-31 mimic组、miR-31 mimic+Res组、miR-31inhibitor组、miR-31inhibitor+Res组,48 h后收集细胞,每组设置三个复孔,并通过Western blot检测细胞NLRP3、Caspase-1、GSDMD-N、IL-18和IL-1β蛋白表达情况。结果: 动物实验:与control组相比较,AOM/DSS组NLRP3、Caspase-1、IL-18蛋白表达显著升高(P<0.01),AOM/DSS+Res组NLRP3、Caspase-1、IL-18蛋白表达水平相较于AOM/DSS组有显著下降(P<0.01);细胞实验:与control组相比,miR-31 mimic组NLRP3(P<0.01)、GSDMD-N(P<0.05)、IL-18(P<0.01)蛋白表达显著升高, miR-31 inhibitor组NLRP3、GSDMD-N、IL-18蛋白表达显著降低(P<0.05)。结论: Res可通过细胞焦亡抑制结肠癌。     

血清miR-34b-5p、miR-155表达与早产儿急性呼吸窘迫综合征炎症因子和预后的关系分析

摘要 目的：探讨血清微小核糖核酸（miR）-34b-5p、miR-155表达与早产儿急性呼吸窘迫综合征（ARDS）炎症因子和预后的关系。方法：选择2019年2月至2021年3月我院收治的92例ARDS早产儿,根据ARDS病情严重程度将其分为轻度组（31例）、中度组（43例）和重度组（18例）,追踪患儿临床结局,根据院内死亡情况将其分为存活组（51例）和死亡组（41例）。检测所有患儿的血清miR-34b-5p、miR-155表达水平以及白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）水平,比较各组间上述指标差异,分析ARDS早产儿血清miR-34b-5p、miR-155表达与炎症因子的相关性以及miR-34b-5p、miR-155预测ARDS早产儿预后的价值。结果：重度组miR-155表达水平及IL-1β、TNF-α、IL-6水平均高于中度组和轻度组,且中度组高于轻度组（P＜0.05）,重度组miR-34b-5p表达水平低于中度组和轻度组,且中度组低于轻度组（P＜0.05）。死亡组miR-155表达水平及IL-1β、TNF-α、IL-6水平高于存活组（P＜0.05）,死亡组miR-34b-5p表达水平低于存活组（P＜0.05）。ARDS早产儿miR-155表达水平与IL-1β、TNF-α、IL-6水平均呈正相关,而miR-34b-5p表达水平与IL-1β、TNF-α、IL-6水平均呈负相关（P＜0.05）。联合miR-34b-5p、miR-155预测ARDS早产儿死亡的曲线下面积（AUC）为0.853,高于两指标单独预测的0.688、0.649。结论：ARDS早产儿血清miR-34b-5p、miR-155表达水平与患儿血清炎症因子水平以及预后有关,可作为ARDS早产儿病情评估以及预后预测的潜在指标。     

miR-125b-5p对创伤性脑损伤诱发大鼠认知功能障碍的干预作用及其机制

目的:研究miR-125b-5p对创伤性脑损伤(TBI)引起的大鼠认知功能障碍的影响及其分子机制。方法:将大鼠随机分为Control组、TBI组(模型组)、NC agomir组(假阴性组)、miR-125b-5p agomir组(高表达组),每组5只。假阴性组和高表达组分别脑室注射NC agomir和miR-125b-5p agomir,除Control组外各组均用改良Feeney法建立脑损伤模型。通过行为学实验对大鼠的运动协调、学习记忆、神经损伤程度等进行评估。通过ELISA和Western blot对各组大鼠海马组织中miR-125b-5p、炎性因子,以及神经相关因子的表达水平进行检测。最后使用生物信息学结合RT-PCR和Western blot对miR-125b-5p的下游靶基因进行预测和验证。结果:与对照组相比,模型组和假阴性组大鼠miR-125b-5p的表达水平显著下调、mNSS评分显著升高、运动协调能力及学习记忆能力显著下降、白介素(IL-1β、IL-6)及肿瘤坏死因子(TNF-α)表达水平显著升高、BDNF和NGF的表达水平显著下降、GFAP的表达水平显著升高(P<...     

miR-99b-5p对紫杉醇致大鼠神经病理性疼痛的缓解作用及对神经细胞焦亡和凋亡的影响

目的:研究miR-99b-5p(非编码RNA)通过抑制NLRP3炎性小体活化缓解紫杉醇化疗后病理性神经疼痛的干预作用,以及对神经细胞焦亡和凋亡的影响。方法:SD大鼠随机分为空白组(Blank)、模型组(Model)、agomiR-99b-5p治疗组和agomiR-NC对照组,每组6只。空白组接受生理盐水治疗作为对照,模型组通过紫杉醇诱导建立疼痛模型,agomiR-99b-5p组和agomiR-NC组为在模型组的基础上注射agomiR-99b-5p和agomiR-NC进行干预。RT-qPCR检测空白组、模型组、agomiR-99b-5p治疗组和agomiR-NC组大鼠背根神经节中miR-99b-5p的表达差异;vonFrey纤维丝检测空白组、模型组与治疗组机械缩足阈值(MWT);TUNEL法测定背根神经节细胞凋亡情况;试剂盒检测大鼠背根神经节中ROS、MDA和SOD;免疫荧光染色检测炎症小体NLRP3、Caspase-1和IL-1β蛋白的表达。结果:与空白组比较,模型组miR-99b-5p含量较低;与模型组比较,agomiR-99b-5p治疗组可以显著增加大鼠背根神经节miR-99b-5...     

miR-181b-5p靶向调控Nr6a1抑制GC-1spg细胞的增殖与迁移

Micro RNA(mi RNA)是一段长约为22 nt的内源性非编码单链RNA,研究表明mi RNA对正常精子发生和雄性发育必不可少。为了探究mi R-181b-5p在雄性生殖细胞发育中的功能以及寻找其靶基因,首先采用流式细胞术、噻唑蓝检测以及划痕实验分析了mi R-181b-5p在小鼠GC-1spg精原细胞系中的生物学效应,随后利用Targetscan和GO数据库等生物信息学软件预测发现生殖核受体Nr6a1可能为其靶基因,进而通过双荧光素酶报告基因实验以及q PCR证实了mi R-181b-5p与Nr6a1的靶向识别关系,最后采用细胞功能回补实验明确了mi R-181b-5p通过靶向调控Nr6a1来抑制GC-1spg细胞的增殖和迁移。结果表明mi R-181b-5p通过其靶基因Nr6a1在精子发生中起到负调控的作用。     

miR-216b-5p靶向调控BTN3A2促进胶质瘤细胞LN-229的迁移以及侵袭能力

大量证据表明microRNA（miRNA）通过靶向调控靶基因的表达从而在肿瘤侵袭与转移中发挥重要作用。然而关于microRNA-216b-5p (miR-216b-5p )通过靶向嗜乳脂蛋白第3亚家族膜蛋白A2（butyrophilin subfamily 3 member A2,BTN3A2）促进胶质瘤侵袭与转移的机制尚不明确。本研究通过GSE15824与GSE4290差异表达分析筛选出同时在2个芯片中表达上调的BTN3A2（P＜0.05）。生存曲线结果显示,高表达BTN3A2病人总生存期明显下降（P＜0.001）。表达量分析结果显示,BTN3A2表达随WHO分级升高而升高（P＜0.05）,同时1p/19q未联合缺失与IDH突变型病人BTN3A2表达升高（P＜0.001）。基因集富集分析（gene set enrichment analysis,GSEA）结果显示,BTN3A2与众多癌症相关通路有关（P＜0.05）;Western印迹结果显示,BTN3A2在7例胶质瘤组织和胶质瘤细胞系U87、U251和LN-229中表达上调,过表达miR-216b-5p （miR-216b-5p mimics）后BTN3A2蛋白表达水平降低;Transwell结果显示,转染BTN3A2干扰质粒（si-BTN3A2）和miR-216b-5p mimics后可以抑制LN 229细胞体外迁移与侵袭能力（P＜0.05）;在线预测网站证实,miR-216b-5p 为BTN3A2潜在靶基因;生存曲线结果显示,与低表达miR-216b-5p 病人相比,高表达病人生存率明显上调（P=0.025）;荧光定量RT PCR结果显示,miR-216b-5p 在胶质瘤U87、U251和LN-229细胞中表达下降（P＜0.05）;双荧光素酶结果显示,BTN3A2存在与miR-216b-5p 的结合靶点(P<005);综上所述,BTN3A2可能通过结合miR-216b-5p 促进胶质瘤细胞LN 229的迁移以及侵袭。     

长链非编码RNAIFNG-AS1靶向miR-19b-1-5p调控oxLDL诱导的血管内皮细胞增殖、凋亡的机制研究

为了探讨长链非编码RNA干扰素活化基因的反义核糖核酸(lncRNA IFNG-AS1)对氧化型低密度脂蛋白(oxLDL)诱导的人脐静脉血管内皮细胞EVC-304增殖、凋亡的影响和调控机制,该研究采用100 μg/mL的oxLDL分别处理转染si-IFNG-AS1、miR-19b-1-5p mimics或共转染si-IF...     

Downregulation of lysyl oxidase-like 4 LOXL4 by miR-135a-5p promotes lung cancer progression in vitro and in vivo

Aberrant microRNAs are widely identified in multiple cancers, including lung cancer. miR-135a-5p can function as a significant tumor regulator in diverse cancers via impacting multiple genes in oncogenic pathways. Nevertheless, the biological role of miR-135a-5p in lung cancer is poorly known. Here, we investigated its function in lung cancer. As exhibited, miR-135a-5p was elevated in lung cancer cells in contrast to BEAS-2B cells. Then, we inhibited miR-135a-5p expression by transfecting LV-anti-miR-135a-5p into lung cancer cells. As displayed, miR-135a-5p was obviously reduced in A549 and H1299 cells. Knockdown of miR-135a-5p repressed lung cancer cell growth and cell proliferation. Meanwhile, cell colony formation capacity was depressed, cell apoptosis was enhanced and cell cycle progression was blocked in G1 phase by inhibition of miR-135a-5p in vitro. Additionally, the migration and invasion of A549 and H1299 cells was strongly depressed by LV-anti-miR-135a-5p. For another, by using informatics analysis, lysyl oxidase-like 4 (LOXL4) was speculated as the downstream target of miR-135a-5p. We validated their direct correlation and moreover, overexpression of miR-135a-5p restrained LOXL4 levels in lung cancer cells. Subsequently, we proved that miR-135a-5p promoted lung cancer development via targeting LOXL4 by carrying out the in vivo assays. Taken these together, our study revealed miR-135a-5p might be indicated as a perspective for lung cancer via targeting LOXL4.     

Long non-coding RNA ZFAS1 alleviates sepsis-induced myocardial injury via target miR-34b-5p/SIRT1

Long non-coding RNA ZFAS1 is down-regulated in sepsis. However, whether ZFAS1 participates in sepsis-induced cardiomyopathy (SIC) remains largely unknown. LPS injection to rats was used to establish an in vivo sepsis model, while LPS stimulation with H9C2 cell was used to mimic an in vitro sepsis-induced myocardial injury model. Western blots and quantitative RT-PCR were performed to evaluate protein and mRNA levels, respectively. ELISA was conducted to determine cytokine levels in supernatant. Flow cytometry was used to test apoptosis. Dual-luciferase assay was performed to validate binding between ZFAS1 and miR-34b-5p, miR-34b-5p and SIRT1. Our data revealed that ZFAS1 and SIRT1 were down-regulated, while miR-34b-5p was up-regulated in LPS-induced H9C2 cells. Inhibition of miR-34b-5p or overexpression of ZFAS1 alleviated inflammatory response and cell apoptosis in LPS-stimulated H9C2 cells. A mechanism study revealed that ZFAS1 sponged miR-34b-5p and thus elevated expression of SIRT1, which was prohibited by miR-34b-5p. ZFAS1 alleviated inflammatory response and cell apoptosis in LPS-stimulated H9C2 cells via the miR-34b-5p/SIRT1 axis, providing novel potential therapeutic targets for SIC.     

Circulating miR-3613-5p but not miR-125b-5p,miR-199a-3p,and miR-451a are biomarkers of endometriosis

ObjectiveThis study aimed to assess the utility of circulating miR-125b-5p, miR-199a-3p, miR-451a, and miR-3613-5p as biomarkers of endometriosis.Study designPatients with stage III or IV of endometriosis according to the revised American Society of Reproductive Medicine (rASRM) staging classification, as well as control women, were recruited. We created a prospective study conducted on a group of 48 patients (n = 25 controls, n = 24 endometriosis) who had laparoscopic surgery. Blood samples were taken and plasma miRNA levels were measured by quantitative real-time polymerase chain reaction (RT-qPCR) and assessed with AUC and ROC curves.ResultsMiR-451a and miR-3613-5p were significantly decreased in the plasma of endometriosis patients. miR-451a had a receiver-operating characteristic (ROC) area under the curve 0.8283 and miR-3613-5p had a ROC area under the curve 0.7617. The concentration of circulating miR-125b-5p and miR-199-3p did not differ between endometriosis patients and controls. Plasma miRNA levels did not change with BMI, smoking status, fertility problems, or menstrual pain according to the VAS scale (p > 0.05).ConclusionCirculating miR-451a and miR-3613-5p levels significantly differed between endometriosis and controls. However, the levels of miR-451a were discordant with previous studies. Therefore, miR-3613-5p may have better potential as the endometriosis biomarker. Circulating miR-125b-5p and miR-199a-3p cannot be used as reliable markers of endometriosis.     

lncRNA ZFPM2-AS1 promotes proliferation via miR-18b-5p/VMA21 axis in lung adenocarcinoma

Lung cancer has been proved to be one of the most common kinds of cancers around the globe. Meanwhile, as the predominant type of lung cancer, lung adenocarcinoma (LUAD) has received increasing attention in cancer research. Long noncoding RNAs (lncRNAs) are known to be associated with oncogenesis and progression of various cancers. However, many lncRNAs have not been thoroughly detected in LUAD. In this study, through bioinformatics analysis we found that zinc finger protein multitype 2 antisense RNA 1 (ZFPM2-AS1) was associated with poor prognosis of LUAD patients. Also, ZFPM2-AS1 was detected to be overexpressed in LUAD tissues and cells. Furthermore, ZFPM2-AS1 could promote the proliferation of LUAD cells. Next, miR-18b-5p was found to bind with and negatively regulated by ZFPM2-AS1. VMA21, target gene of miR-18b-5p, could bind with and be negatively regulated by miR-18b-5p. More importantly, both ZFPM2-AS1 and VMA21 were found to be attached to the RNA-induced silencing complex constructed from miR-18b-5p and Ago2. Also, ZFPM2-AS1 could regulate the expression of VMA21. Therefore, ZFPM2-AS1 were confirmed to regulate VMA21 by competitively binding with miR-18b-5p. Finally, rescue assays confirmed that ZFPM2-AS1 could regulate LUAD cell proliferation via miR-18b-5p/VMA21 axis.     

miR-181a/b-5p ameliorates inflammatory response in monocrotaline-induced pulmonary arterial hypertension by targeting endocan

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by vascular remodeling, endothelial cell (EC) dysfunction, and inflammation. The roles of microRNAs have received much critical attention. Thus, this study was attempted to show the biological function of miR-181a/b-5p (miR-181a/b) in monocrotaline (MCT)-induced PAH. Here, rats injected with MCT were used as PAH models. The expression of miR-181a/b and its effect on PAH pathologies were examined using miR-181a/b overexpression lentivirus. A luciferase reporter analysis was performed to measure the relationships between miR-181a/b and endocan. Additionally, primary rat pulmonary arterial endothelial cells (rPAECs) treated with tumor necrosis factor-α (TNF-α) were employed to further validate the regulatory mechanism of miR-181a/b in vitro. Our results showed that miR-181a/b expression was reduced in PAH, and its upregulation significantly attenuated the short survival period, right ventricular systolic pressure and mean pulmonary artery pressure increments, right ventricular remodeling, and lung injury. Furthermore, the increase of intercellular cell adhesion molecule-1 (ICAM1) and vascular cell adhesion molecule-1 (VCAM1) in PAH rats was inhibited by miR-181a/b overexpression. Similarly, our in vitro results showed that inducing miR-181a/b suppressed TNF-α-stimulated increase of ICAM1 and VCAM1 in rPAECs. Importantly, the increased expression of endocan in PAH model or TNF-α-treated rPAECs was restored by miR-181a/b upregulation. Further analysis validated the direct targeting relationships between miR-181a/b and endocan. Collectively, this study suggests that miR-181a/b targets endocan to ameliorate PAH symptoms by inhibiting inflammatory states, shedding new lights on the prevention and treatment of PAH.