miR-574-3p在结直肠癌中的表达和作用机制研究

目的检测并分析结直肠癌组织和细胞系中miR-574-3p的表达情况及其对结直肠癌细胞发生、发展的影响。方法收集武汉市第三医院11对手术切除结直肠癌患者的癌组织和对应癌旁组织,3种结直肠癌细胞系和1种正常结直肠上皮细胞,通过实时荧光定量聚合酶链反应(qRT-PCR)检测临床样本及结直肠癌细胞系中miR-574-3p的表达水平;通过转染miR-574-3p mimic及相应对照mimic NC实现上调结直肠癌细胞(HCT-8、HCT-116)中miR-574-3p的表达水平,采用CCK-8、EdU、克隆形成实验、流式凋亡实验和流式细胞周期实验检测过表达miR-574-3p后对结直肠癌细胞的增殖、凋亡和细胞周期的影响。结果癌旁组织中miR-574-3p的表达差异倍数显著高于对应癌组织(P<0.05),在3种结直肠癌细胞系中miR-574-3p的表达较正常结肠上皮细胞也显著降低(P<0.05)。CCK-8、EdU、克隆形成实验表明,过表达miR-574-3p抑制结直肠癌细胞的增殖能力。流式凋亡实验结果显示,过表达miR-574-3p促进结直肠癌细胞的凋亡能力。流式细胞周期结果显示,过表达miR-574-3p使结直肠癌细胞发生G₀/G₁期阻滞。结论结直肠癌组织和细胞系中的miR-574-3p均低表达,上调结直肠癌细胞(HCT-8、HCT-116)中miR-574-3p可以抑制细胞增殖,促进凋亡并发生G₀/G₁期阻滞。     

miR-206与心血管疾病研究进展

微小RNA(microRNA,miRNA)是一大类短链非编码RNA,可以直接结合靶基因的3′非翻译区,进而影响基因表达,在心血管疾病中发挥关键作用。其中,microRNA-206(miR-206)是心脏发育和生理活动的关键调控因子,不仅可以作为诊断的标志分子,也可作为疾病治疗的定向作用点。本文综述了miR-206调节心肌细胞、内皮细胞、平滑肌细胞、自主神经细胞等细胞的基本功能,以及miR-206在冠状动脉疾病、心肌梗死、心力衰竭、心律失常、肺动脉高压等疾病发生发展中的具体作用和机制。     

Long non-coding RNA TP73-AS1 promotes pancreatic cancer growth and metastasis through miRNA-128-3p/GOLM1 axis

BACKGROUNDPrevious studies have suggested that long non-coding RNAs (lncRNA) TP73-AS1 is significantly upregulated in several cancers. However, the biological role and clinical significance of TP73-AS1 in pancreatic cancer (PC) remain unclear. AIMTo investigate the role of TP73-AS1 in the growth and metastasis of PC.METHODSThe expression of lncRNA TP73-AS1, miR-128-3p, and GOLM1 in PC tissues and cells was detected by quantitative real-time polymerase chain reaction. The bioinformatics prediction software ENCORI was used to predict the putative binding sites of miR-128-3p. The regulatory roles of TP73-AS1 and miR-128-3p in cell proliferation, migration, and invasion abilities were verified by Cell Counting Kit-8, wound-healing, and transwell assays, as well as flow cytometry and Western blot analysis. The interactions among TP73-AS1, miR-128-3p, and GOLM1 were explored by bioinformatics prediction, luciferase assay, and Western blot. RESULTSThe expression of TP73-AS1 and miRNA-128-3p was dysregulated in PC tissues and cells. High TP73-AS1 expression was correlated with a poor prognosis. TP73-AS1 silencing inhibited PC cell proliferation, migration, and invasion in vitro as well as suppressed tumor growth in vivo. Mechanistically, TP73-AS1 was validated to promote PC progression through GOLM1 upregulation by competitively binding to miR-128-3p. CONCLUSIONOur results demonstrated that TP73-AS1 promotes PC progression by regulating the miR-128-3p/GOLM1 axis, which might provide a potential treatment strategy for patients with PC.     

miR-214-5p通过靶向调控PTEN的表达对破骨分化和骨质疏松的影响

 目的 探讨miR-214-5p通过靶向调控PTEN的表达对破骨分化和骨质疏松的影响。方法 选取2019-03至2020-05在空军军医大学唐都医院外科进行脊柱手术的骨质疏松患者20例(骨质疏松组),同时选取非骨质疏松症患者20例为对照组,抽取两组骨髓血。将miR-214-5pmimics(miR-214-5pmimics组)、miR-214-5p空质粒(miR-214-5pvector组)、shZFAS1载体(sh PTEN组)、shZFAS1空载体(sh vector组)转染到骨质疏松患者细胞,对照组细胞不进行任何转染处理。采用RT-qPCR 检测骨髓单个核细胞中的miR-214-5p和PTEN mRNA及 Raw264.7单核巨噬细胞中NFaTc1、MMP9、TRAP、CTSK的mRNA水平;通过TRAP染色检测TRAP染色的阳性率;通过Western blot 检测PTEN、NFATc1、AKT和PI3K蛋白水平。结果 骨质疏松组miR-214-5p水平的表达明显高于对照组(P＜0.001),骨质疏松组PTEN的表达显著低于对照组(P=0.009);miR-214-5pmimics组中TRAP表达阳性率明显高于对照组和miR-214-5p vector组(P＜0.001);sh PTEN组中TRAP表达阳性率明显高于对照组和sh vector组(P＜0.001);miR-214-5p mimics组中的NFaTc1、MMP9、TRAP和CTSK水平显著高于对照组(P<0.001);sh PTEN组中的NFaTc1、MMP9、TRAP和CTSK水平显著高于对照组(P<0.001);与miR-214-5p mimics组相比,miR-214-5pmimics+PTEN组中TRAP表达阳性率显著降低(P＜0.001)。结论 骨质疏松症患者的骨髓单个核细胞中miR-214-5p表达上调,miR-214-5p可以调控PTEN表达,过表达miR-214-5p可能通过靶向PTEN促进破骨细胞分化。     

Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists

On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific.

Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70.

Platelet activation triggered the release of 57–79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (r² > 0.98; p < 0.0001 for all), and with the microRNA content of the parent platelets (r² > 0.98; p < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect.

These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.     

Down-regulation of LncRNA CRNDE aggravates kidney injury via increasing MiR-181a-5p in sepsis

Long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) is reported to be linked to inflammation and cell apoptosis. However its role in sepsis induced kidney injury remains unclear. This study aims to explore the possible mechanism of CRNDE in kidney injury induced by sepsis. In vivo urine-derived sepsis (US) rat model and in vitro LPS-induced HK-2 and HEK293 cells were established. Kidney function was measured in rats from different groups. Relative levels of tumor necrosis factor-α (TNF-α) and interleukin-1β(IL-1β) in kidney tissue were detected via Enzyme-linked immune sorbent assay (ELISA). Then we up- or down-regulated CRNDE and miRNA-181a-5p expression in the cells. The biological influence of CRNDE and miR-181a-5p on cells was studied using CCK-8 assay and Annexin V assay. Interaction between CRNDE and miR-181a-5p was determined by bioinformatics analysis, RT-PCR, and dual luciferase reporter assay. Peroxisome proliferator-activated receptor-α (PPARα) and cell apoptosis related molecules were detected by western blot. We demonstrated that CRNDE was markedly down-regulated while miR-181a-5p was significantly up-regulated in sepsis models. CRNDE interacted with miR-181a-5p, and negatively regulated its expression level. CRNDE knockdown in rats increased the urea nitrogen and serum creatinine in plasma. Knockdown of CRNDE or transfection of miR-181a-5p significantly inhibited proliferation and promoted apoptosis of HK-2 and HEK293 cells, while overexpression of CRNDE and transfection of miR-181a-5p inhibitors had opposite effects. For mechanism, miR-181a-5p directly targeted the 3′ untranslated region of PPARα, and depressed its protein level, and PPARα was regulated indirectly by CRNDE. We concluded that CRNDE protected renal cell from sepsis-induced injury via miR-181a-5p/PPARα pathway.     

Inhibition of miR-429 improves neurological recovery of traumatic brain injury mice and attenuates microglial neuroinflammation

BackgroundNeuroinflammation is a common therapeutic target for traumatic brain injury (TBI) due to its contribution to delayed secondary cell death and has the potential to occur for years after the initial insult. Previous studies demonstrate that miR-429 is up-regulated in the brain lesions of TBI mice, while its role in regulating neuroinflammation and brain injury remains largely unknown.MethodThe expression of miR-429 in LPS-activated microglia and microglia in TBI model was detected by RT-PCR. The effects of miR-429 inhibitors on LPS-activated microglia in vitro as well as neurological recovery and post-traumatic neuroinflammatory response in TBI model mice were detected in vivo.ResultsLPS and TBI significantly induce the up-expression of miR-429, inflammatory cytokines, MAPK-p38 and phosphorylated NF-κB in microglia, which were all inhibited by miR-429 inhibitors. Meanwhile, miR-429 inhibitors also attenuated the neurological impairment in TBI mice. Bioinformatics analysis showed that miR-429 could target and inhibit the expression of dual specificity protein phosphatase 1 (DUSP1), thus inhibiting the expression of MAPK-p38 and phosphorylated NF-κB.ConclusionmiR-429 plays a pro-inflammatory role in activated microglia by targeting DUSP1 signaling pathway. Inhibiting miR-429 can attenuate the inflammatory response of microglia and TBI-mediated brain damage.     

Long noncoding RNA GAS5 promotes microglial inflammatory response in Parkinson's disease by regulating NLRP3 pathway through sponging miR-223-3p

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. Neuroinflammation induced by microglia plays an important role in the pathogenesis of PD. Long noncoding RNA GAS5 was showed to have significant effects on regulating inflammatory response. Here, we aim to investigate the effects of GAS5 on the inflammatory response of PD, and the underlying mechanism. An in vivo model of PD was established in C57BL/6 mice by rotenone and an in vitro cell model was conducted on microglia by lipopolysaccharide (LPS). Our results indicated that GAS5 was upregulated in tissues in a mice model of PD and microglia activated by LPS. Gain- and loss- of functional experiments demonstrated that GAS5 promoted the inflammation of microglia in vitro. Besides, the knockdown of GAS5 repressed the PD progression in vivo. Mechanistically, GAS5 positively regulated the NLRP3 expression via competitively sponging miR-223-3p. Overall, our finding illuminates that GAS5 accelerates PD progression through targeting miR-223-3p/NLRP3 axis.     

circ-KEL在急性髓系白血病中的表达及其对白血病细胞的调控作用和机制

目的探讨circ-KEL在急性髓系白血病（AML）患者中的表达及其对AML细胞的调控作用和机制。方法收集116例AML患者以及40名健康者骨髓标本，分离骨髓单个核细胞，使用实时定量RT-PCR法检测circ-KEL的表达水平并分析其与AML患者临床特征之间的关系。通过生物信息学分析以及双荧光素酶报告基因实验等验证circ-KEL与miR-335-5p/LRG1的靶向关系。运用CCK-8、流式细胞术等方法检测circ-KEL在AML细胞中的生物学作用。结果circ-KEL在AML患者中的表达较正常人群明显增高（−7.117±1.831对−8.669±1.771，P<0.001），circ-KEL高表达患者总生存期（OS）明显短于低表达者（P＝0.037）。AML患者接受化疗后circ-KEL表达水平较初诊时下降。同时，circ-KEL可充当miR-335-5p的“海绵”，靶向调节LRG1。数据库分析显示miR-335-5p高表达患者预后好，且其与LRG1水平呈负相关。LRG1可促进AML细胞的增殖，抑制其凋亡，在AML患者中表达水平较健康人群也显著升高。circ-KEL可以通过miR-335-5p/LRG1轴发挥对白血病细胞的促增殖以及抑制凋亡作用。结论circ-KEL在AML患者中高表达，与AML患者预后高度相关，通过影响miR-335-5p/LRG1在AML疾病进展中发挥重要作用。