Roles of microRNA-1 in hypoxia-induced apoptotic insults to neuronal cells

Hypoxia is a common occurrence in brain tumors and traumatic brain injury. microRNA (miR)-1 participates in the regulation of brain development and neuronal function. Interestingly, miR-1 can mediate ischemia-induced injury to cardiomyocytes. This study was designed to evaluate the roles of miR-1 in hypoxia-induced insults to neurons and the possible mechanisms. Exposure of neuro-2a cells to oxygen/glucose deprivation (OGD) or cobalt chloride decreased cell viability and induced cell apoptosis in time-dependent manners. In parallel, OGD caused augmentation of cellular Bax and cytochrome c levels, a reduction in the mitochondrial membrane potential (MMP), activation of caspase-3, and fragmentation of DNA. miR-1 was induced in neuro-2a cells by OGD. Knocking down miR-1 expression using specific antisense inhibitors significantly alleviated OGD-induced neuronal death. Administration of OGD to neuro-2a cells induced heat-shock protein (HSP)-70 messenger (m)RNA and protein expressions. A bioinformatic search revealed that miR-1-specific binding elements exist in the 3′-untranslated region of HSP-70 mRNA. Overexpression of miR-1 simultaneously attenuated OGD-induced HSP-70 mRNA and protein expressions. In comparison, knocking down miR-1 expression synergistically enhanced OGD-induced HSP-70 mRNA. As to the mechanism, reducing miR-1 expression lowered OGD-induced alterations in the MMP, caspase-3 activation, DNA fragmentation, and cell apoptosis. Taken together, this study shows that miR-1 can target HSP-70 expression and consequently mediate hypoxia-induced apoptotic insults to neuro-2a cells via an intrinsic Bax–mitochondrion–caspase protease pathway.     

七氟烷对肺腺癌细胞增殖和迁移能力的影响

目的 研究七氟烷对肺腺癌细胞增殖和迁移能力的影响及相关机制.方法 体外培养对数生长期肺腺癌细胞系H1299,将H1299细胞传代并分为对照组及实验组,对照组用含5％CO2常规气体培养,实验组用2.5％七氟烷及5％CO2气体培养,处理细胞6 h.以TRIzol法收集2组细胞总RNA,以逆转录实时荧光定量聚合酶链反应检测2...     

乙酰葛根素对氧糖剥离诱导海马神经元凋亡的影响

目的研究新型异黄酮类化合物乙酰葛根素对氧糖剥离神经元的保护作用及其机制。方法采用氧糖剥离建立细胞模型。DAPI法观察神经元凋亡率,RT-PCR法检测NF-κBp65、HIF-1α及p53mRNA的表达,Western blot法测定Hsp70蛋白的含量及乙酰葛根素对其的影响。结果与模型组相比较,乙酰葛根素可以减少凋亡细胞数;降低NF-κBp65、HIF-1α及p53mRNA表达;乙酰葛根素(低,中和高浓度组)增加Hsp70蛋白的表达。结论乙酰葛根素降低NF-κBp65、HIF-1α、p53的表达和升高Hsp70蛋白表达。     

Inhibition of microRNA-199a-5p ameliorates oxygen-glucose deprivation/reoxygenation-induced apoptosis and oxidative stress in HT22 neurons by targeting Brg1 to activate Nrf2/HO-1 signalling

MicroRNAs (miRNAs) have emerged as critical regulators of neuronal survival during cerebral ischaemia/reperfusion injury. Accumulating evidence has shown that miR-199a-5p plays a crucial role in regulating apoptosis and survival in various cell types. However, whether miR-199a is involved in regulating neuronal survival during cerebral ischaemia/reperfusion injury remains unknown. In this study, we aimed to explore the biological role of miR-199a-5p in regulating neuronal injury induced by oxygen-glucose deprivation/reoxygenation (OGD/R), an in vitro cellular model of cerebral ischaemia and reperfusion injury. We found that miR-199a-5p expression was significantly altered in neurons in response to OGD/R treatment. Overexpression of miR-199a-5p facilitated OGD/R-induced apoptosis and reactive oxygen species (ROS) production, whereas miR-199a-5p inhibition alleviated OGD/R-induced apoptosis and ROS production. Notably, our results identified Brahma-related gene 1 (Brg1) as a target gene of miR-199a-5p. Moreover, inhibition of miR-199a-5p promoted the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signalling via targeting Brg1. However, silencing of Brg1 markedly reversed the miR-199a-5p inhibition-mediated neuroprotective effect. Taken together, our results suggest that downregulation of miR-199a-5p protects neurons from OGD/R-induced neuronal injury through upregulating Brg1 to activate Nrf2/HO-1 signalling. The miR-199a-5p/Brg1/Nrf2/HO-1 regulation axis may play an important role in regulating neuronal survival during cerebral ischaemic/reperfusion injury in vivo.     

Inhibition of microRNA-148b-3p alleviates oxygen-glucose deprivation/reoxygenation-induced apoptosis and oxidative stress in HT22 hippocampal neuron via reinforcing Sestrin2/Nrf2 signalling

MicroRNAs (miRNAs) have emerged as crucial regulators of neuronal injury during cerebral ischaemia/reperfusion injury. Various miRNAs are dysregulated during this pathological process; however, the precise role of these miRNAs in regulating neuronal injury remains largely unknown. In the current study, we explored the potential function of microRNA-148b-3p (miR-148b-3p) in regulating neuronal injury induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro, a cellular model for mimicking cerebral ischaemia/reperfusion injury. We found that miR-148b-3p expression was significantly decreased in neurons in response to OGD/R exposure. Importantly, miR-148b-3p overexpression decreased cell viability and exacerbated apoptosis and reactive oxygen species (ROS) production in OGD/R-exposed neurons. By contrast, miR-148b-3p inhibition improved cell viability and decreased apoptosis and ROS production in OGD/R-exposed neurons. Notably, Sestrin2, a cytoprotective gene, was identified as a miR-148b-3p target gene. miR-148b-3p inhibition markedly increased Sestrin2 expression as well as the activation of nuclear factor erythroid-2-related factor 2 (Nrf2) antioxidant signalling. Moreover, silencing of Sestrin2 or Nrf2 significantly reversed the miR-148-3p-inhibition-mediated protective effect in OGD/R-injured neurons. Overall, these results demonstrate that miR-148b-3p inhibition protects neurons from OGD/R-induced apoptosis and ROS production through reinforcing Nrf2 antioxidant signalling via upregulation of Sestrin2. Our study indicates that the miR-148b-3p/Sestrin2/Nrf2 axis plays an important role in regulating neuronal injury and may serve as a potential therapeutic target for providing neuroprotection during cerebral ischaemia/reperfusion injury.     

表没食子儿茶素没食子酸酯对缺血再灌注损伤诱导的大鼠神经细胞凋亡的影响

目的 探讨表没食子儿茶素没食子酸酯(EGCG)对缺血再灌注损伤诱导的大鼠神经细胞凋亡、氧化应激的影响及其对长链非编码RNA MALAT1(lncRNA MALAT1)/微小RNA-2115-3p(miR-2115-3p)分子轴的调控作用.方法 体外培养大鼠神经元细胞HT22,建立氧糖剥夺/复氧损伤(OGD/R)神经细胞...     

MicroRNA-665-3p attenuates oxygen-glucose deprivation-evoked microglial cell apoptosis and inflammatory response by inhibiting NF-κB signaling via targeting TRIM8

Microglial inflammation induced by ischemic stroke aggravates brain damage. MicroRNAs (miRNAs) have emerged as pivotal regulators in ischemic stroke-induced inflammation in microglial cells. miR-665-3p has been reported as a critical inflammation-associated miRNA. However, whether miR-665-3p participates in regulating microglial inflammation during ischemic stroke is underdetermined. This study investigated the potential role of miR-665-3p in stroke-induced inflammation in microglial cells using a cellular model of oxygen-glucose deprivation (OGD)-stimulated microglial cells in vitro. We found that miR-665-3p expression was decreased in microglial cells exposed to OGD treatment. Functional experiments demonstrated that the overexpression of miR-665-3p attenuated OGD-induced apoptosis and inflammation in microglial cells. Notably, tripartite motif 8 (TRIM8) was identified as a target gene of miR-665-3p. TRIM8 expression was induced by OGD treatment in microglial cells and the knockdown of TRIM8 protected microglial cells from OGD -induced cytotoxicity and inflammation. Moreover, TRIM8 knockdown or miR-665-3p overexpression blocked OGD-induced activation of nuclear factor (NF)-κB signaling in microglial cells. In addition, TRIM8 overexpression partially reversed the miR-665-3p overexpression-mediated inhibitory effect on OGD-induced inflammation in microglial cells. Taken together, these results indicate that miR-665-3p up-regulation protects microglial cells from OGD-induced apoptosis and inflammatory response by targeting TRIM8 to inhibit NF-κB signaling.     

Alisol A 24-acetate protects oxygen–glucose deprivation-induced brain microvascular endothelial cells against apoptosis through miR-92a-3p inhibition by targeting the B-cell lymphoma-2 gene

ContextAlisol A 24-acetate has been used to treat vascular diseases. However, the underlying mechanisms still remain unclear.ObjectiveThe present study evaluated the antiapoptotic effect of alisol A 24-acetate on brain microvascular endothelial cells (BMECs) and explored the underlying mechanisms.Materials and methodsBMECs were injured through oxygen -glucose deprivation (OGD) after alisol A 24-acetate treatment. Cell viability and half-maximal inhibitory concentration (IC₅₀) were measured using CCK-8, whereas inflammatory factors and oxidative stress indicators were measured using enzyme linked immunosorbent assay. Cell invasion and wound healing assays were detected. Cell apoptosis was assessed using flow cytometry. B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X (Bax) expression were analyzed using Western blotting. Dual-luciferase assay was applied to detect target genes of miR-92a-3p.ResultAlisol A 24-acetate had an IC₅₀ of 98.53 mg/L and inhibited cell viability at concentrations over 50mg/L. OGD induced apoptosis and promoted miR-92a-3p overexpression in BMECs. However, alisol A 24-acetate treatment suppressed inflammation, improved migration and invasion abilities, increased Bcl-2 expression, inhibited Bax expression, and repressed apoptosis and miR92a-3p overexpression in OGD-induced BMECs. MiR-92a-3p overexpression promoted cell apoptosis and suppressed Bcl-2 expression, whereas its inhibitor reversed the tendency. Alisol A 24-acetate treatment relieved the effects of miR-92a-3p overexpression. Dual-luciferase assay confirmed that miR-92a-3p negatively regulated the Bcl-2 expression.ConclusionsThese findings suggest that alisol A 24-acetate exerts antiapoptotic effects on OGD-induced BMECs through miR-92a-3p inhibition by targeting the Bcl-2 gene, indicating its potential for BMECs protection and as a novel therapeutic agent for the treatment of cerebrovascular disease.     

hsa_circ_0054345靶向miR-206对TGF-β1诱导的大鼠肝星状细胞增殖、活化及凋亡的影响

目的 探讨环状RNA _0054345(hsa_circ_0054345)是否通过靶向微小RNA-206(miR-206)调控转化生长因子β1(TGF-β1)诱导的大鼠肝星状细胞增殖、活化及凋亡.方法 体外培养大鼠肝星状细胞HSC-T6,使用TGF-β1处理细胞建立模型,采用实时荧光定量聚合酶链反应(qRT-PCR)检...     

MicroRNA-1对大鼠心肌细胞凋亡的影响

摘要 目的 探讨microRNA-1(miR-1) 对大鼠心肌细胞凋亡的影响。方法 应用转染的方法使培养的H9C2大鼠心肌细胞高表达miR-1（miR-1组）,荧光定量PCR的方法确定miR-1上调后,采用MTT法和流式细胞术检测各组细胞活力和凋亡情况、用荧光定量PCR和Western Blot的方法检测凋亡抑制因子Bcl-2的mRNA和蛋白表达情况。正常细胞和转染随机合成的miRNA阴性对照片段分别为正常对照组和阴性对照组。结果 与正常对照组相比,阴性对照组各个指标的变化均没有统计学差异。miR-1组的miR-1 mRNA表达水平显著升高,细胞活力下降,凋亡率增加,Bcl-2的mRNA和蛋白表达水平均明显降低。 结论 转染的miR-1 mimic能够上调细胞内的miR-1mRNA表达水平,抑制心肌细胞增殖,促进细胞凋亡。     

lncRNA NEAT1通过靶向抑制miR-29a促进低氧低糖诱导的肺纤维化进程的机制研究

目的 探究lncRNA NEAT1通过负调控miR-29a促进低氧低糖诱导的肺纤维化进程的机制.方法 收集健康者和缺血再灌注引起急性肺损伤患者的血清样本,通过qRT-PCR检测血清样本中NEAT1与miR-29a的表达.构建肺纤维化细胞模型和动物模型,进行离体和在体的RNA干扰,通过显微镜观察细胞的形态学改变,Hoec...     

Ischemic insult induced apoptotic changes in PC12 cells: protection by trans resveratrol

In this study, we determined the protective potential of trans resveratrol against oxygen-glucose deprivation (OGD) induced reactive oxygen species mediated apoptotic damages in PC12 cells. In vitro model of ischemic cerebral stroke was created by keeping cells in an OGD condition for 6h followed by 24h reoxygenation. Cells received biologically safe doses (5, 10, and 25 μM) of trans resveratrol in the following schedules for 24h prior to OGD; during 6h of OGD; for 24h post OGD and whole treatment group which starts from 24h before OGD and lasted to 24h post OGD. Anti-ischemic potential of trans resveratrol was assessed by measuring the regulation of lipid peroxidation, reactive oxygen species production, glutathione content, and expression (mRNA and protein) of apoptotic markers such as Bax, Bcl(2) and Caspase-3. Hypoxia inducible factor-1α (HIF-1α) was also assessed to correlate the changes with ischemic injuries. Significant (P<0.05) restoration in lipid peroxidation, reactive oxygen species, and glutathione content were observed following the treatment of trans resveratrol in cells receiving OGD and re-oxygenation. Changes induced by trans resveratrol could be correlated well with alterations in the expression of Bax, Bcl(2), Caspase-3 and HIF-1α. These results indicate that trans resveratrol administration attenuates free radical formation and mitochondria mediated apoptosis perhaps by regulating the expressions of Bax, Bcl(2,) and Caspase-3 in PC12 cells receiving OGD and re-oxygenation insult.     

Dehydrocostuslactone protects against oxygen-glucose deprivation/reperfusion-induced injury by inhibiting autophagy and apoptosis in PC12 cells

OBJECTIVE TO investigate the neural protection of dehydrocostus lactone(DHL) against neuronal injury induced by oxygen and glucose deprivation/reperfusion(OGD/R) in differentiated PC12 cells. METHODS We used a cellular model of 2 h of OGD and 24 h of reperfusion to mimic cerebral ischemia-reperfusion injury. Cell viability was used to reflect the degree of OGD/R-induced injury. Cells were treated with DHL during the reperfusion phase. Cell Counting Kit(CCK-8) and LDH assays were performed to determine the optimal dose of DHL and cel viability. Flow cytometry analysis and Monodansylcadaverine(MDC) staining were then conducted to detect apoptosis rate and autophagosome formation after OGD/R in PC12 cells. Immunofluorescence and Western blotting analyses were used to detect the expression of proteins associated with autophagy and apoptosis. RESULTS OGD/R significantly decreased cell viability and increased apoptosis rate. The expression levels of autophagy-related proteins, namely, LC3 and Beclin-1, and apoptosisrelated proteins, namely, Bax and caspase-3 increased, but that of the anti-apoptosis Bcl-2 protein decreased. However,DHL attenuated OGD/R-induced neuronal injury through inhibition of apoptosis and autophagy properties by modulating autophagy-associated proteins(LC3 and Beclin-1) and apoptosis-modulating proteins(caspase-3 and Bcl-2/Bax). CONCLUSION Our data provide an evidence for the neuroprotective effect of DHL against ischemic neuronal injury. Hence, DHL could be a promising candidate for treatment of ischemic stroke.     

miR-34a通过调控雄激素受体基因抑制前列腺癌细胞系LNCaP增殖

目的:探究miR-34a对雄激素受体(AR)基因的调控作用及对前列腺癌(PCa)LNCaP细胞增殖、凋亡的影响。方法:收集2016年10月至2019年9月本院泌尿外科行前列腺穿刺活检确诊为PCa患者组织标本36例,另取同期行手术治疗切除的良性前列腺增生(BPH)组织标本41例。体外培养LNCaP细胞,分别转染miR-34a mimics(mimics组),miR-34a mimic NC(NC组),设正常生长细胞为空白对照组(BC组),另在mimics组基础上转染AR过表达(AR过表达组)载体及其对照(AR对照组),采用CCK-8试剂盒检测细胞活性,Annexin V-FITC/PI双染色流式细胞凋亡检测试剂盒检测细胞凋亡率,实时定量PCR(RT-qPCR)检测miR-34a及AR mRNA表达水平,免疫印迹法检测AR蛋白、细胞周期蛋白D1(cyclinD1)及原癌基因产物(c-Mcy)、细胞凋亡相关蛋白(Bcl-2、Bax、capase-3)的表达水平。结果:与BPH组织比较,PCa组织中miR-34a表达水平显著降低(P<0.05),AR mRNA及蛋白表达水平均显著增加(P<0.05);与BC、NC组比较,mimics组LNCap细胞miR-34a表达水平、细胞凋亡率、Bax蛋白表达水平显著增加(P<0.05),AR、Cyclin D1、Bcl-2、Bax蛋白表达水平显著降低(P<0.05),同一时间LNCap细胞活力均显著降低(P<0.05);双荧光素酶实验结果显示,miR-34a与AR可能存在一定的调控关系,过表达AR基因可逆转miR-34a mimics对LNCaP细胞增殖抑制,促进凋亡作用。结论:miR-34a可能通过调控AR抑制前列腺癌LNCaP细胞增殖,促进其凋亡。     

芒柄花素对体外培养成骨细胞缺氧损伤的保护作用

目的研究芒柄花素在缺氧培养条件下对成骨细胞增殖、细胞周期、凋亡和成骨分化等的影响。方法采用酶消化法分离培养SD大鼠颅骨成骨细胞,并用三气培养箱建立缺氧模型。将细胞分为常氧对照组、缺氧对照组和缺氧加药组,其中缺氧加药组进一步分为10-6、10-5和10-4 mol·L-1组,分别加入10-6、10-5和10-4 mol·L-1芒柄花素。于缺氧处理36 h后分析各组的细胞存活率、LDH漏出率、细胞凋亡、细胞周期等,并用RT-PCR法检测缺氧诱导因子-1α、Bcl-2及Caspase-3的基因表达情况。缺氧处理48 h后检测ALP活性、钙化结节面积等成骨分化指标。结果与缺氧对照组比较,芒柄花素可提高细胞存活率,降低LDH漏出率,减少细胞凋亡并提高G1期细胞百分比,提高HIF-1α和Bcl-2mRNA的表达水平,抑制Caspase-3 mRNA的表达水平,对ALP活性和钙化结节面积等也有提高作用,且均呈现出剂量依赖性特点。结论芒柄花素对成骨细胞缺氧损伤具有明显保护作用。     

过表达微小 RNA-877-5p通过靶向叉头框转录因子 M1调控胃癌细胞活力和凋亡

目的探讨微小 RNA-877-5p(miR-877-5p)对胃癌细胞活力、凋亡的影响及其分子机制。方法本研究时间为 2020年 1—7月。胃癌和正常胃黏膜上皮细胞株购自美国典型培养物保藏中心。实时定量基因扩增荧光检测( qPCR)检测胃癌细胞株 HGC-27、SUN-1、AGS和正常胃黏膜上皮细胞株 GES-1中 miR-877-5p和叉头框转录因子 M1(FOXM1)信使核糖核酸( mRNA)表达。建立 miR-877-5p过表达或抑制 FOXM1表达细胞株,观察其在 HGC-27细胞的活力、凋亡中的作用。 MTT法检测细胞的活力,流式细胞术检测细胞凋亡,蛋白质印迹法( Western blotting)检测 FOXM1、细胞周期蛋白 D1(cyclinD1)、细胞周期依赖性激酶抑制因子 p21、p27、B细胞淋巴瘤 /白血病 -2(Bcl-2)、 Bcl-2相关 X蛋白( Bax)、活化的含半胱氨酸的天冬氨酸蛋白水解酶 3(cleaved-caspase 3)蛋白表达。 TargetScan预测结合双荧光素酶报告实验分析 miR-877-5p和 FOXM1的靶向关系。共转染 miR-877-5p模拟物和 FOXM1过表达载体( pcDNA-FOXM1),研究 FOXM1过表达对 miR-877-5p过表达诱导的 HGC-27细胞增殖和凋亡的影响。结果与正常胃黏膜上皮细胞 GES-1比较,胃癌细胞 HGC-27、SUN-1、AGS中的 miR-877-5p表达下调( 1.00±0.08比 0.34±0.03,0.51±0.05,0.44±0.04,P<0.05)FOXM1 mRNA和蛋白表达上调( 1.00±0.09比 2.41±0.23,2.58±0.24,2.26±0.23,P< 0.05)。 miR-877-5p过表达显著降低 HGC-27细,胞 48 h、72 h的细胞活力( P<0.05),明显提高细胞凋亡率、 p21、p27、Bax、cleaved-caspase3蛋白的水平( P<0.05)显著减少 cyclinD1、Bcl-2蛋白表达量( P<0.05)。抑制 FOXM1表达显著降低 48 h、72 h的细胞活力( P<0.05)提高细胞凋亡率、 p,21、Bax蛋白水平( P<0.05),减少 cyclinD1、Bcl-2蛋白表达量( P<0.05)。 miR-877-5p靶向调控 FOXM1的表达,。FOXM1过表达后, miR-877-5p过表达对 HGC-27细胞增殖、 cyclinD1、Bcl-2蛋白表达的抑制作用被逆转,其对细胞凋亡、 p21、Bax蛋白表达的促进作用也被逆转。结论过表达 miR-877-5p通过靶向调控 FOXM1表达抑制胃癌细胞的活     

Epirubicin-mediated expression of miR-302b is involved in osteosarcoma apoptosis and cell cycle regulation

Epirubicin is widely used in osteosarcoma chemotherapy. Growing evidence indicates that the microRNA (miRNA) expression levels which are induced by chemotherapeutic agents play an important role in osteosarcoma development and progression. In this study we investigate the alterations of miRNA expression in the osteosarcoma cells after epirubicin treatment and whether miRNAs can enhance its anti-osteosarcoma effect. After epirubicin exposure, microarray shows 40 miRNAs are differentially expressed in osteosarcoma cells including 24 down-regulated miRNAs. Notably, miR-302b, which is stably low-expressed in osteosarcoma, could be induced by the epirubicin. Furthermore, we find that miR-302b can inhibit the osteosarcoma cell proliferation, promote cell apoptosis and cell cycle arrest MiR-302b can activate caspase-3 and regulate the Akt/pAkt, Bcl-2, Bim expression to increase the cell apoptosis. Meanwhile, miR-302b also attenuates cyclin D1 and CDKs expression to induce cell cycle arrest. Therefore, our results suggest miR-302b can play an essential role in osteosarcoma treatment as a potential tumor suppressor.     

Metformin protects PC12 cells against oxygen-glucose deprivation/reperfusion injury

Oxidative stress has a causative role in ischemic reperfusion-induced cell death. Evidence has shown that metformin is capable to reduce ischemic reperfusion injuries. The current study investigated the effect of metformin on ischemia/reperfusion-induced apoptosis in PC12 cells by evaluation of Bcl-2 family proteins expression. Cells were exposed to a time-dependent in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) injury and then treated with metformin. The intracellular reactive oxygen species (ROS) levels were measured. Western blotting was used to examine the expression of anti- and pro-apoptotic proteins. Moreover, the number of apoptotic cell death was evaluated by TUNEL assay. Our results showed that metformin attenuated ROS generation, downregulated pro-apoptotic BAX expression, and upregulated expression of the Bcl-2 protein in the PC12 cells. Moreover, metformin reduced cell death under OGD/R condition which was confirmed by lower apoptotic cell death in the TUNEL assay. These findings suggest that neuroprotective effect of metformin on OGD/R-induced cell death is possibly mediated by inhibition of ROS-induced apoptosis pathway.