Coordination of insulin and Notch pathway activities by microRNA miR-305 mediates adaptive homeostasis in the intestinal stem cells of the Drosophila gut

Homeostasis of the intestine is maintained by dynamic regulation of a pool of intestinal stem cells. The balance between stem cell self-renewal and differentiation is regulated by the Notch and insulin signaling pathways. Dependence on the insulin pathway places the stem cell pool under nutritional control, allowing gut homeostasis to adapt to environmental conditions. Here we present evidence that miR-305 is required for adaptive homeostasis of the gut. miR-305 regulates the Notch and insulin pathways in the intestinal stem cells. Notably, miR-305 expression in the stem cells is itself under nutritional control via the insulin pathway. This link places regulation of Notch pathway activity under nutritional control. These findings provide a mechanism through which the insulin pathway controls the balance between stem cell self-renewal and differentiation that is required for adaptive homeostasis in the gut in response to changing environmental conditions.     

MicroRNA-29抑制胰岛素刺激的大鼠L6细胞葡萄糖吸收

 目的 研究microRNA-29（miR-29）在糖尿病模型大鼠 (GK)骨骼肌组织中的表达,并探讨其对大鼠骨骼肌细胞L6葡萄糖吸收的影响。方法 利用real-time PCR方法检测miR-29家族,miR-29a、miR-29b及miR-29c在GK大鼠骨骼肌组织中的表达特征。L6骨骼肌细胞诱导分化为肌管细胞,经葡萄糖与胰岛素处理后,利用real-time PCR与Northern blot分析miR-29家族的表达情况。选择腺病毒表达系统在L6分化的肌管细胞中过表达或抑制内源miR-29的功能,利用葡萄糖吸收实验检测胰岛素刺激的葡萄糖吸收情况。结果 miR-29a、miR-29b及miR-29c在GK大鼠骨骼肌组织中表达上调。L6分化的肌管细胞经葡萄糖与胰岛素处理,miR-29b和miR-29c的表达上调,而miR-29a表达无明显变化。在L6分化的肌管细胞中过表达miR-29可以明显抑制胰岛素刺激的葡萄糖吸收。结论 miR-29参与了GK大鼠骨骼肌细胞葡萄糖耐受及胰岛素抵抗的产生,抑制其表达可能是治疗II型糖尿病的一种新策略。     

基于HepG2细胞胰岛素抵抗模型探讨miR-7-5p对Itch基因的靶向作用

目的:通过体外建立Hep G2细胞胰岛素抵抗模型,检测胰岛素抵抗状态下微小RNA-7-5p(miR-7-5p)及其预测靶基因Itch的差异表达,初步探讨miR-7-5p对Itch基因的靶向作用及其与胰岛素抵抗的关系。方法:采用适宜浓度的软脂酸诱导Hep G2细胞,建立体外胰岛素抵抗模型;基于生物信息学分析预测miR-7-5p可能作用的靶基因及其富集的相关信号通路;运用RT-q PCR和Western blot技术检测在胰岛素抵抗状态下miR-7-5p和Itch的表达变化。结果:0.25 mmol/L的软脂酸作用于Hep G2细胞24 h可诱导细胞产生胰岛素抵抗,RT-q PCR检测表明,与阴性对照组相比,胰岛素抵抗组的miR-7-5p表达下调(P0.01)。生物信息学分析结果表明,miR-7-5p有相当数量的靶基因富集于泛素-蛋白酶体系统,其中E3泛素连接酶Itch基因是与胰岛素抵抗最为相关的靶基因;Western blot结果揭示,在胰岛素抵抗状态下,Hep G2细胞中Itch蛋白表达上调(P0.01)。结论:miR-7-5p可能参与了胰岛素抵抗的病理生理过程,其机制可能通过靶向调控Itch基因的表达,进而直接或间接影响胰岛素信号通路的正常转导。     

miR-143和miR-21在妊娠期糖尿病患者胎盘组织中的表达及与新生儿出生体重的相关性

目的探讨妊娠期糖尿病(GDM)患者胎盘组织和外周血清中miR-143和miR-21定量表达水平与母体胰岛素抵抗程度和新生儿出生体重的相关性,为GDM的发生机制及早期评估疾病风险提供客观的检测指标。方法选择2018年5月至2019年5月入我院诊断GDM患者共80例(观察组),其中轻度50例,中重度30例,另选择30例健康孕妇作为对照组;采用RT-PCR法检测分娩前1日外周血清和胎盘娩出后胎盘组织中miR-143和miR-21水平,并检测分娩前1日空腹血糖和胰岛素水平,评估胰岛素抵抗指数(HOMA-IR),记录新生儿出生体重。结果miR-143和miR-21不仅在观察组患者的外周血清,而且在胎盘组织中表达水平均明显低于对照组,对观察组进行亚组分析发现,中重度患者的血清和胎盘组织miR-143和miR-21水平也明显低于轻度患者(P<0.05)。采用定量资料Pearson检验发现,miR-143和miR-21水平与HOMA-IR呈负相关(r=-0.653和-0.678,P均<0.05)。两组比较新生儿出生体重发现,观察组平均值明显低于对照组,观察组进一步比较也得出,中重度孕妇的新生儿出生体重低于轻度孕妇(P<0.05)。母体血清和胎盘组织miR-143和miR-21水平与新生儿出生体重呈正相关(P<0.05)。结论miR-143和miR-21表达下调可能参与GDM的发生,与母体胰岛素抵抗增加和新生儿出生体重降低有关。     

miR-3960对成骨细胞分化的影响**☆

背景：成骨细胞的分化成熟过程涉及多种激素和细胞因子对成骨细胞分化相关基因表达的调控,近来发现多个微小RNAs也参与了这一调控过程。 目的：观察miR-3960在小鼠骨髓基质细胞向成骨细胞分化过程中的作用。 方法：将miR-3960表达载体pSilencer4.1-miR-3960转染骨髓基质细胞,构建miR-3960过表达细胞模型,随后予300 μg/L骨形态发生蛋白2诱导分化,观察成骨细胞分化指标变化。 结果与结论：转染pSilencer4.1-miR-3960能够在细胞中稳定地高表达miR-3960。miR-3960过表达促进骨髓基质细胞向成骨细胞分化过程中的碱性磷酸酶活性增高和骨钙素分泌,增加细胞中的钙沉积量。抑制miR-3960降低骨髓基质细胞向成骨细胞分化过程中的碱性磷酸酶活性,减少骨钙素分泌,降低钙沉积量。表明miR-3960可以促进成骨细胞分化。     

利拉鲁肽通过调节microRNA-375对胰岛细胞凋亡的影响

目的:观察利拉鲁肽通过微小RNA-375(microRNA-375,miR-375)对db/db小鼠胰岛细胞凋亡的影响,探讨其可能作用机制,为临床应用提供药效学依据。方法:20只8周龄雄性db/m小鼠为正常对照组,皮下注射等量的生理盐水。40只8周龄雄性db/db小鼠随机分为2组,每组20只:糖尿病对照组的db/db小鼠皮下注射等量生理盐水;利拉鲁肽组的db/db小鼠皮下注射利拉鲁肽300μg·kg~(-1)·d~(-1)。给药8周后,检测各组小鼠体重(BW)、空腹血糖(FBG)、空腹胰岛素(FINS)、甘油三酯(TG)、总胆固醇(TC)及低密度脂蛋白胆固醇(LDL-C)含量,并进行腹腔注射葡萄糖耐量实验(IPGTT)和胰岛素耐量实验(ITT);苏木精-伊红(HE)染色检测胰岛组织病理学变化;原位末端转移酶标记技术(TUNEL)检测胰岛凋亡情况;Western blot法检测胰岛凋亡相关蛋白caspase-3、Bcl-2及Bax的蛋白水平;实时荧光定量PCR检测胰岛miR-375的表达水平。小鼠胰岛β细胞系MIN-6分为对照组(等量溶媒孵育)、miRNA-375 mimic组和miRNA-375 mimic+利拉鲁肽组,MTT实验检测细胞活力,Western blot法检测各组细胞caspase-3、Bcl-2及Bax的蛋白水平。结果:整体实验中,与对照组相比,利拉鲁肽组BW、FBG、FINS、TC、TG及LDL-C含量明显降低(P0.05);胰岛数量较模型组增多,体积较大,细胞结构明显改善;胰岛细胞凋亡减少;利拉鲁肽组的Bcl-2表达明显增高,caspase-3和Bax的表达显著下降(P0.05);胰岛组织miR-375的水平显著降低(P0.01)。细胞实验中,经miRNA-375 mimic处理24 h后,MIN-6细胞活力显著降低,Bcl-2表达减少,而caspase-3和Bax的蛋白水平显著增加(P0.05),给予利拉鲁肽组治疗后,MIN-6细胞活力明显上升,Bcl-2表达明显增高,caspase-3和Bax的蛋白水平显著下降(P0.05)。结论:利拉鲁肽可以抑制糖尿病胰岛β细胞的凋亡,其机制可能与调控胰岛组织中miR-375的表达有关。     

microRNA-155 acts as an oncogene by targeting the tumor protein 53-induced nuclear protein 1 in esophageal squamous cell carcinoma

MicroRNA-155 (miR-155) is overexpressed in many human cancers; however, the function of miR-155 is largely unknown in esophageal squamous cell carcinoma (ESCC). In the present study, we found that miR-155 is dramatically increased in ESCC tissues compared with the paired adjacent normal tissues, which suggested that miR-155 acts as an oncogene in ESCC. We predicted that tumor protein p53-induced nuclear protein 1 (TP53INP1) is a candidate target gene of miR-155 given that miR-155 expression decreased mRNA and protein levels of TP53INP1 as determined by RT-PCR and Western blot analysis. In addition, miR-155 and TP53INP1 showed a negative relation in ESCC tissues. Dual luciferase-based reporter assay indicated direct regulation of TP53INP1 by miR-155. Furthermore, we demonstrated that RNA interference of TP53INP1 increased the proliferation and colonies formation of EC-1 cells. Up-regulation of TP53INP1 abrogated miR-155 induced growth in EC-1 cells and mutation of TP53INP1 in 3’-UTR restored the effects when co-transfected with miR-155. We also indicated that overexpression of miR-155 significantly promoted the proliferation of EC-1 cells in vitro and the development of tumors in nude mice. Taken together, our study reveals that miR-155 acts as an oncogene by targeting TP53INP1 in ESCC.     

miR-29 is a major regulator of genes associated with pulmonary fibrosis

MicroRNAs (miRNA) are small regulatory RNAs that control gene expression by translational suppression and destabilization of target mRNAs. There is increasing evidence that miRNAs regulate genes associated with fibrosis in organs, such as the heart, kidney, liver, and the lung. In a large-scale screening for miRNAs potentially involved in bleomycin-induced fibrosis, we found expression of miR-29 family members significantly reduced in fibrotic lungs. Analysis of normal lungs showed the presence of miR-29 in subsets of interstitial cells of the alveolar wall, pleura, and at the entrance of the alveolar duct, known sites of pulmonary fibrosis. miR-29 levels inversely correlated with the expression levels of profibrotic target genes and the severity of the fibrosis. To study the impact of miR-29 down-regulation in the lung interstitium, we characterized gene expression profiles of human fetal lung fibroblast IMR-90 cells in which endogenous miR-29 was knocked down. This confirmed the derepression of reported miR-29 targets, including several collagens, but also revealed up-regulation of a large number of previously unrecognized extracellular matrix-associated and remodeling genes. Moreover, we found that miR-29 is suppressed by transforming growth factor (TGF)-β1 in these cells, and that many fibrosis-associated genes up-regulated by TGF-β1 are derepressed by miR-29 knockdown. Interestingly, a comparison of TGF-β1 and miR-29 targets revealed that miR-29 controls an additional subset of fibrosis-related genes, including laminins and integrins, independent of TGF-β1. Together, these strongly suggest a role of miR-29 in the pathogenesis of pulmonary fibrosis. miR-29 may be a potential new therapeutic target for this disease.     

MicroRNA-34a inhibits tumor invasion and metastasis in gastric cancer by targeting Tgif2

To study how miR-34a acts as a tumor suppressor in inhibiting the invasion and metastasis of the gastric cancer cells. First, real-time polymerase chain reaction (PCR) and western blot analysis were used to analyze the expression of miR-34a and Tgif2 in gastric cancer tissues and the adjacent normal tissues. Next, gastric cancer cells were transfected with miR-34a mimic and Tgif2 siRNA, respectively. After transfection, real-time PCR and western blot analysis were used to detect the relative Tgif2 expression level. Cell proliferation was monitored by the colorimetric water-soluble tetrazolium salt and apoptosis analysis was performed with Annexin-V-FITC Apoptosis Detection Kit I. The expression of miR-34a in the adjacent non-tumor tissues was higher than that in gastric cancer tissues, but Tgif2 was opposite. In gastric cancer cells transfected with miR-34a mimic/Tgif siRNA, Tgif2 expression was remarkably down-regulated. Cells transfected with miR-34a mimic/Tgif2 siRNA grew more slowly than the control groups. The percentage of apoptotic cells in gastric cancer cells transfected with miR-34a mimic/Tgif siRNA was much higher compared to the controls. Therefore, we concluded that miR-34a could inhibit tumor invasion and metastasis in gastric cancer by targeting Tgif2 and may be a novel therapeutic candidate for gastric cancer.     

Exosomal miR-155-5p promotes proliferation and migration of gastric cancer cells by inhibiting TP53INP1 expression

Exosomal microRNA (miRNA) secreted by tumor cells plays an important biological role in tumorigenesis and development. We aimed to explore the effects of exosomal miR-155-5p in gastric cancer (GC) and understand its mechanism of action in GC progression. We isolated exosomes from the human gastric mucosal epithelial cell line GES-1 and gastric cancer cell line AGS, and then identified them according to their surface markers by flow cytometry. Later, we detected the miR-155-5p expression levels in tissues and isolated exosomes using RT-qPCR. Bioinformatics analysis showed that miR-155-5p directly binds to the 3' untranslated region (3'-UTR) of tumor protein p53-induced nuclear protein 1 (TP53INP1) mRNA. We also investigated whether the miR-155-5p-rich exosomes caused changes in cell cycle, proliferation, and migration in AGS cells. In this study, we found that the levels of miR-155-5p were significantly increased in GC tissues and AGS cells, and that the TP53INP1 protein level was downregulated in GC tissues using IHC and IFC. TP53INP1 was found to be directly regulated by miR-155-5p following a dual luciferase-based reporter assay. After co-culturing with the isolated miR-155-5p-rich exosomes, the proliferation and migration capabilities of AGS cells were enhanced. Thus, our results reveal that exosomal miR-155-5p acts as an oncogene by targeting TP53INP1 mRNA in human gastric cancer.     

miR-200a通过调节PTEN影响小鼠肝细胞中AKT/GSK信号通路活性

目的 肝胰岛素抵抗能够导致严重的糖脂代谢紊乱,同2型糖尿病的发病密切相关.miR-200a属于miR-200家族,广泛表达于各个组织中,在许多癌症细胞中高表达.此研究探讨miR-200a通过下游靶基因PTEN调节AKT/GSK信号通路活性的机制,从microRNA角度阐明肝胰岛素抵抗的机制,为胰岛素抵抗的防治提供新的思路.方法 ① 脂质体在小鼠肝脏细胞株HEP1-6中转染miR-200a mimic和miR-200a inhibitor,用Real-time PCR检测细胞中miR-200a水平,并且检测AKT/GSK信号通路;② 用生物信息学方法预测miR-200a的下游靶基因;以双荧光素酶报告分析和Western印迹明确miR-200a的下游基因PTEN.③ 在HEP1-6细胞中共同转染miR-200a和si-PTEN,验证miR-200a通过PTEN调节AKT/GSK信号通路活性.结果 ① 用miR-200a mimic转染HEP1-6细胞,miR-200a水平升高,AKT/GSK信号通路活性增强;用miR-200a inhibitor转染HEP1-6细胞,miR-200a水平降低,AKT/GSK信号通路活性受到抑制;② 双荧光素酶报告分析和Western印迹结果表明,miR-200a能够直接同PTEN3′-UTR结合,抑制PTEN蛋白表达;③ 在HEP1-6细胞中沉默PTEN促进AKT/GSK信号通路活性;同时转染miR-200a和si-PTEN,能够逆转miR-200a inhibitor对AKT/GSK信号通路的抑制作用.结论 在HPE1-6细胞中miR-200a通过调节下游靶基因PTEN影响AKT/GSK信号通路活性.     

miR-375对人类诱导多能干细胞向胰岛素分泌细胞分化的影响

目的:探讨微小RNA-375(miR-375)在调控人类诱导多能干细胞(hiPSCs)分化为胰岛素分泌细胞(IPCs)过程中的作用及可能机制。方法:构建miR-375过表达的慢病毒载体,转染hiPSCs获得miR-375稳定表达的miR-375-hiPSCs,体外诱导其定向分化为IPCs,采用real-time PCR、流式细胞术及ELISA等方法对其标志性基因、分化效率、胰岛素和C肽释放量等进行检测;生物信息学结合萤光素酶报告基因实验预测并验证miR-375的靶基因,real-time PCR及Western blot检测靶基因的表达。结果:过表达miR-375可上调胰岛β细胞标志性转录因子HNF4α、MafA、Pdx1、Pax6、Nkx6.1、glucokinase和insulin的表达,分化效率由对照组的22.3%提高至38.6%;高糖刺激后胰岛素释放量由成年胰岛细胞分泌量的1/8提高到1/5;过表达miR-375可影响靶基因REST和WNT5A的蛋白翻译水平。结论:miR-375可通过干扰靶基因REST和WNT5A的翻译水平,开启胰岛素分泌相关特异性基因的表达,从而促进hiPSCs向IPCs定向分化。     

外泌体miR-27a/b与单纯性肥胖儿童胰岛素抵抗、血脂代谢和生长发育的关系

目的 检测循环血外泌体miR-27a、miR-27b表达与单纯性肥胖儿童胰岛素抵抗、血脂代谢和生长发育的关系.方法 共纳入了120例年龄在6～15岁之间在我院儿保科体检的儿童,包括60例诊断为单纯性肥胖者作为肥胖组和60例非肥胖儿童作为正常对照组.进行人体测量(包括身高、体重、腰围等),采集血样,提取血清外泌体,采用实...     

miR-145过表达对宫颈癌细胞辐射敏感性的影响

目的:探讨微小RNA(miR)-145过表达增强宫颈癌细胞放疗敏感性的分子机制。方法:运用Lipofectamine 2000将miR-145-mimic转染4株宫颈癌细胞系He La、Ca Ski、C33A和Si Ha,以NC-mimic为阴性对照,并以real-time PCR检测子宫内膜基质细胞(ESC)和4株宫颈癌细胞中miR-145的表达水平;转染的细胞经电离辐射照射后于不同时点运用MTT法和Annexin V-FITC/PI染色法联合流式细胞术分别测定细胞活力和细胞凋亡率;运用免疫荧光检测组蛋白γH2AX的表达水平;Western blot检测细胞中螺旋酶样转录因子(HLTF)的蛋白表达水平。结果:miR-145在ESC中高表达,而在4株宫颈癌细胞中均呈低表达,转染miR-145-mimic后4株宫项癌细胞中miR-145的表达水平显著高于NC-mimic组细胞(P0.05)。相同条件下,辐照后miR-145过表达的宫颈癌细胞的活力显著低于NC-mimic组细胞,72 h的细胞凋亡率明显增加(P0.05)。免疫荧光观察结果显示miR-145过表达显著促进电离辐射诱导的γH2AX激活;Western blot结果显示,miR-145过表达显著抑制HLTF的表达,与NC-mimic比较差异有统计学意义(P0.05)。结论:miR-145在正常子宫内膜上皮细胞中高表达,而在宫颈癌细胞系中低表达;miR-145过表达可显著抑制宫颈癌细胞的活力,促进电离辐射诱导的细胞凋亡。miR-145过表达增强宫颈癌细胞的辐射敏感性,其机制可能与下调HLTF表达、抑制DNA损伤修复、促进细胞凋亡有关。     

MicroRNA-155 通过调节CXCR4 / PI3K / AKT 途径影响滋养细胞的侵袭与迁移

目的:以人绒毛膜滋养层细胞系JEG-3 细胞为研究对象,结合该细胞侵袭和迁移能力的变化情况,研究转染miR-155 mimics 和miR-155 inhibitor 之后CXCR4 的表达变化及其对下游PI3K/ AKT 信号通路的影响作用,从而探讨miR-155参与子痫前期发生发展的分子机制。方法:设计miR-155mimics 和miR-155 inhibitor,对JEG-3 进行转染,通过Transwell 侵袭实验、划痕实验,观察转染后细胞的侵袭和迁移能力的变化;利用Real-time PCR 检测CXCR4 mRNA 的表达;利用Western blot检测CXCR4 及下游p鄄AKT 蛋白的表达水平。结果:Real-time PCR 结果显示,miR-155 mimics 转染组CXCR4 mRNA 相对表达量(0.589依0.096)明显低于空白对照组(1.503依0.090)和阴性对照组(1.146依0.153),差异有统计学意义(P<0.05);miR-155 inhibitor 转染组CXCR4 mRNA 相对表达量(1.739依0.083)与两组对照组相比差异也有统计学意义(P<0.05)。Western blot 结果显示miR-155 mimics 转染组CXCR4 蛋白和下游p-AKT 蛋白表达水平均明显降低,而miR-155 inhibitor 转染组CXCR4 和p-AKT 蛋白水平则升高,与空白对照组和阴性对照组相比差异均有统计学意义(P<0.05)。Transwell 侵袭实验结果显示,与空白对照组(63郾46依2郾37)和阴性对照组(49.29依5.81)侵袭细胞数相比,miR-155 mimics 转染组侵袭细胞数(22.89依9.42)明显减少,差异有统计学意义(P<0.05);同时,miR-155 inhibitor 转染组侵袭细胞数(81.50依11.25)明显升高,差异有统计学意义(P<0.05)。划痕实验结果显示,转染miR-155 mimics 后,JEG-3 细胞相对迁移距离(0.159依0.058)低于空白对照组(1.080依0.045)和阴性对照组(0.823依0.201),差异有统计学意义(P<0.05);而miR-155 inhibitor 转染组,JEG-3 细胞相对迁移距离(1.640依0.078)明显增加,差异有统计学意义(P<0.05)。结论:miR-155 可能通过抑制CXCR4 的表达进而抑制其下游PI3K/AKT 信号通路的活化,从而影响滋养细胞的侵袭及迁移能力,最终导致子痫前期的发生发展。