miR-221/222在肝癌细胞抵抗内质网应激凋亡中的作用

目的 研究内质网应激对miR-221/222的调控及其在肝癌细胞抵抗内质网应激诱导细胞凋亡中作用.方法 采用miR-221/222抑制物和miR-221/222类似物分别阻断或模拟内源性miR221/222的功能,并利用Western blot和流式细胞技术分析内质网应激条件下miR-221/222对肝癌细胞周期和凋亡的调控作用.结果 内质网应激诱导miR-221/222表达下调,miR-221/222类似物和抑制物分别抑制和促进内质网应激诱导的p27Kip1表达上调,干扰p27Kip1不仅抑制了内质网应激诱导的肝癌细胞G0/G1期阻滞,也促进了内质网应激介导的肝癌细胞凋亡.结论 内质网应激诱导miR-221/222下调能够通过促进p27Kipl表达对内质网应激条件下肝癌细胞周期和凋亡起重要调控作用.

Abstract：

Objective To investigate the role of miR-221/222 in inhibiting endoplasmic reticulum stress-induced human hepatocarcinoma cells apoptosis. Method miR-221/222 mimics and inhibitors were used to mimic or block the function of endogenous miR-221/222 respectively. Western blot and flow cytometry were used to test the effects of miR-221/222 on cell cycle and apoptosis under endoplasmic reticulum stress in human hepatocellular carcinoma cells. Results Endoplasmic reticulum stress resulted in miR-221/222down-regulation in human hepatocellular carcinoma cells. miR-221/222 mimics and inhibitors inhibited and promoted respectively endoplasmic reticulum stress-mediated p27Kip1 induction. Moreover, p27Kip1 suppression not only resulted in reduction in the fraction of G1 phase cells, but also promoted the endoplasmic reticulum stress-mediated apoptosis in human hepatocellular carcinoma cells. Conclusions miR-221/222 were downregulated by endoplasmic reticulum stress in human hepatocellular carcinoma cells, which subsequently protected human hepatocellular carcinoma cells against endoplasmic reticulum stress-induced apoptosis through p27KiP1 regulation.     

病毒性脑炎患儿脑脊液外泌体中miR-221/222检测及其临床意义

目的检测病毒性脑炎患儿脑脊液外泌体中微小RNA-221/222(miR-221/222)水平,并探讨miR-221、miR-222与病毒性脑炎发生及预后的关系。方法选取2018年3月–2019年6月于本院住院治疗的79例病毒性脑炎患儿为病脑组,并根据预后评定将患儿分为预后良好组50例,预后不良组29例。选取同期68例以头痛为主诉而脑脊液正常患儿作为对照组。采用实时荧光定量PCR(qRT-PCR)法检测脑脊液外泌体中miR-221、miR-222表达水平;分析病毒性脑炎患儿脑脊液外泌体中miR-221与miR-222的相关性;分析脑脊液外泌体中miR-221、miR-222检测对病毒性脑炎的诊断价值及其对病毒性脑炎患儿预后的影响。结果透射电镜观察病脑组和对照组患儿脑脊液中均存在中间较周围淡染、边界较清晰的椭圆形或类圆形囊泡,且囊泡内可见致密低电子影;病脑组患儿脑脊液外泌体中miR-221、miR-222相对表达量分别为1.02±0.27和1.03±0.26,对照组分别为1.49±0.33和1.60±0.43,差异均有统计学意义(t值分别为9.354和9.531,均P0.01),且miR-221与miR-222水平呈正相关(r=0.540,P0.05)。脑脊液外泌体miR-221、miR-222诊断病毒性脑炎的AUC分别为0.846、0.879,截断值分别为1.393、1.436,特异性分别为87.9%、74.9%,敏感度分别为65.6%、85.1%;二者联合诊断的AUC为0.914,特异性为83.5%,敏感度为86.8%。预后不良组病脑患儿脑脊液外泌体中miR-221、miR-222相对表达量分别为1.59±0.41和1.79±0.49,预后良好组分别为1.59±0.41和1.79±0.49,差异均有统计学意义(t值分别为4.314和3.846,均P0.01)。多因素Logistic回归分析miR-222是影响病毒性脑炎患儿预后不良的独立危险因素(P0.05)。结论病毒性脑炎患儿脑脊液外泌体中miR-221、miR-222水平显著升高,两者对诊断病毒性脑炎及其预后评估可能有重要价值。     

阿托伐他汀对兔急性心肌梗死后心肌miR-221/miR-222表达及血管新生的影响

目的:探讨阿托伐他汀对兔急性心肌梗死后梗死心肌组织miR-221/miR-222表达以及血管新生的影响。方法:40只新西兰大白兔随机选取8只设为假手术组,只在左冠状动脉前降支下穿线不结扎,剩余32只兔建立急性心肌梗死模型后随机均分为2组:模型组和他汀组。他汀组给予阿托伐他汀钙片1mg·kg-1·d-1,连续灌胃4周;假手术组和模型组给予等量助溶剂连续灌胃4周。4周后处死,取梗死心肌组织,采用免疫组织化学法检测梗死区心肌组织微血管密度(MVD),采用RT-PCR法检测miR-221/miR-222的表达水平。结果:与模型组MVD(3.450±1.036)相比,他汀组(7.640±1.804)明显增加(P0.05)。与假手术组miR-221/miR-222表达水平(0.716±0.083/0.692±0.069)比较,模型组miR-221/miR-222表达水平(1.010±0.043/1.009±0.042)明显升高,他汀组(0.431±0.111/0.374±0.118)明显降低(P0.05)。他汀组梗死组织MVD和miR-221/miR-222的表达水平呈显著负相关(r=-0.755,P0.01;r=-0.804,P0.01)。结论:阿托伐他汀可能通过抑制miR-221/miR-222的表达促进心肌梗死后梗死心肌组织的血管新生。     

前列腺癌组织miR-221和miR-222的表达及临床意义

目的探讨前列腺癌(PCa)组织中miR-221和miR-222的表达及其临床意义。方法采用茎环RT-PCR的方法检测8例正常前列腺组织、74例PCa组织及其对应的癌旁组织中miR-221和miR-222的表达情况,并结合临床病理资料进行统计学分析。结果 miR-221和miR-222在PCa组织中的表达显著高于其对应癌旁组织(P<0.05)及正常前列腺组织(P<0.05),其中miR-221的水平还与癌组织分化程度(P<0.05)以及远端转移情况(P<0.05)相关。结论 miR-221和miR-222在PCa组织中表达量升高,其中miR-221的水平还与癌组织分化程度与转移相关,可能作为PCa诊断和预后的指标。     

MiR-221/222在动脉粥样硬化相关性血管重塑中的作用

正近年来,微小RNA(microRNA、miRNA)对血管生理和病理性结构改变的研究越来越备受关注。其中,miR-221/222已被证实参与调节多个重要的血管生物学行为,如血管新生、内膜增生、伤口愈合及血管老化等[1]。血管重塑是血管新生和损伤修复,维持血管功能和结构完整,防止血栓形成的关键机制。然而,血管重塑在动脉粥样硬化血管中则是斑块增长的一种适应性反应。研究发现,血管重塑与miR-221/222的     

退行性心脏瓣膜病患者血清微小RNA-222和白细胞介素6水平变化及其临床意义

目的探讨退行性心脏瓣膜病患者血清微小RNA-222(miR-222)、白细胞介素6(interleukin-6,IL-6)水平变化及其临床意义。方法选择2016年1~11月徐州医科大学附属淮安医院老年科住院的退行性心脏瓣膜病患者100例,其中单瓣膜病变组48例,多瓣膜病变组52例;健康体检者30例作为对照组。比较3组血清miR-222和IL-6水平。直线相关性分析退行性心脏瓣膜病患者血清miR-222与IL-6的相关性。结果单瓣膜病变组和多瓣膜病变组血清miR-222和IL-6水平明显高于对照组[0.136±0.013、0.205±0.018 vs 0.029±0.005,(3.814±0.108)ng/L、(4.479±0.104)ng/L vs(2.351±0.116)ng/L,P0.01],且多瓣膜病变组血清miR-222和IL-6水平明显高于单瓣膜病变组,差异有统计学意义(P0.01)。退行性心脏瓣膜病患者血清miR-222与IL-6呈正相关(r=0.586,P0.01)。结论血清miR-222和IL-6可能参与了退行性心脏瓣膜病的发生、发展。     

微小RNA-200c在胰腺癌中的研究进展

微小RNA-200c(miR-200c)是一类非编码小分子RNA,参与基因转录后水平调控,与肿瘤的发生发展密切相关。本文介绍了miR-200c对胰腺癌侵袭和转移的抑制作用,及其在胰腺癌的早期诊断、预后判断等方面的临床应用价值,提示miR-200c可作为抑癌基因及有效分子标志物和未来临床研究重点。     

miR-221通过细胞周期蛋白D1介导同型半胱氨酸诱导人冠状动脉内皮细胞损伤

目的研究miR-221通过细胞周期蛋白D1介导同型半胱氨酸(Hcy)诱导人冠状动脉内皮细胞(HCAEC)损伤。方法培养HCAEC并分为4组,对照组用无血清培养基处理,Hcy组用含有1 mmol/L Hcy的培养基处理,Hcy+NC(阴性对照)组转染NC抑制物后用含有1 mmol/L的培养基处理,Hcy+miR-221组转染miR-221抑制物后用含有1 mmol/L的培养基处理。采用荧光定量PCR检测miR-221的表达水平,Western blot检测细胞周期蛋白D1的表达水平,MTS检测细胞活力OD_(490 nm)水平,流式细胞术检测细胞周期,双荧光素酶报告基因实验验证miR-221靶向细胞周期蛋白D1。结果与对照组比较,Hcy组HCAEC的miR-221表达水平、细胞G0/G1期比例明显增加,OD_(490 nm)水平、S期及G2/M期比例、细胞周期蛋白D1表达水平明显降低。与Hcy组及Hcy+NC组比较,Hcy+miR-221组的miR-221表达水平、G0/G1期比例明显降低,OD_(490 nm)水平、S期及G2/M期比例、细胞周期蛋白D1表达水平明显增加。细胞周期蛋白D1基因mRNA 3′UTR第1224-1231碱基是miR-221的结合位点,miR-221能够降低野生型细胞周期蛋白D1双荧光素酶报告基因的荧光活力。结论 miR-221在Hcy诱导HCAEC损伤过程中表达增加,抑制miR-221表达能够减轻Hcy诱导的HCAEC损伤,靶向细胞周期蛋白D1是可能的分子机制。     

微小RNA-221对HepG2细胞增殖及其凋亡的影响

目的 探讨微小RNA-221(miR-221)对肝癌细胞株HepG2细胞增殖与凋亡的影响.方法 将miR-221阻遏物及模拟物转染至HepG2后,用实时荧光定量RT-PCR检测miR-221的表达水平;用细胞增殖试剂盒、Hoechst 33342及碘化丙啶(PI)双重染色、流式细胞仪及caspase3/7活性试剂盒检测HepG2细胞增殖与凋亡情况.对数据进行多样本的单因素方差分析,两两比较采用LSD法;相关性比较用Pearson检验.结果 RT-PCR结果显示,转染miR-221阻遏物后其表达受到抑制,而转染miR-221模拟物可促进其表达.细胞增殖试剂盒及Hoechst 33342/PI染色结果显示,48 h后miR-221阻遏物明显抑制HepG2细胞增殖(P＜0.05),而miR-221模拟物促进HepG2细胞增殖(P＜0.05),两种方法结果呈正相关(r=0.993,P＜0.01).流式细胞仪检测细胞周期显示,miR-221模拟物组G1期细胞比例(47.6%±1.53%)明显低于空白对照组(59.00%±1.00%)及阴性对照组(58.00%±1.00%,F=81.77,P＜0.01); S期细胞比例(20.33%±1.15%)明显高于空白对照组(11.00%±1.00%)及阴性对照组(12.00%±1.00%,F=70.90,P＜0.01).Hoechst 33342/PI染色、流式细胞仪膜联蛋白V凋亡试剂盒检测均显示,转染miR-221阻遏物48 h后,细胞凋亡和坏死增加(P＜0.05),差异有统计学意义.Caspase-3/7试剂盒检测caspase-3/7活性变化结果显示,转染miR-221阻遏物24 h后,细胞caspase3/7活性明显增高(P＜0.05),差异有统计学意义.结论 miR-221可促进肝癌细胞生长增殖,抑制miR-221表达可诱导细胞凋亡.miR-221有望成为治疗肝癌新的分子靶点之一.     

MicroRNA和动脉粥样硬化的关系

MicroRNA是一组生物进化过程中高度保守的非编码的小RNA,在转录后水平调节基因表达.研究显示MircoRNA在心血管系统中高度表达,在动脉粥样硬化中起重要的作用.MicroRNA126,MicroRNA221/222,MicroRNA92-a参与调节内皮细胞功能与血管生成, MicroRNA217促进内皮细胞的衰老,MicroRNA221/222,MicroRNA34对内皮祖细胞有调节作用,MicroRNA145/143,MicroRNA221/222参与调节血管平滑肌细胞的表型转化,增殖和迁移.MicroRNA-125a-5p参与巨噬细胞对致动脉粥样硬化脂质的炎症应答.本文简要介绍近年有关MicroRNA与动脉粥样硬化关系研究的部分进展.     

Role of gga-miR-221 and gga-miR-222 during Tumour Formation in Chickens Infected by Subgroup J Avian Leukosis Virus

Subgroup J avian leukosis virus (ALV-J) causes a neoplastic disease in infected chickens. Differential expression patterns of microRNAs (miRNAs) are closely related to the formation and growth of tumors. (1) Background: This study was undertaken to understand how miRNAs might be related to tumor growth during ALV-J infection. We chose to characterize the effects of miR-221 and miR-222 on cell proliferation, migration, and apoptosis based on previous microarray data. (2) Methods: In vivo, the expression levels of miR-221 and miR-222 were significantly increased in the liver of ALV-J infected chickens (p < 0.01). Over-expression of gga-miR-221 and gga-miR-222 promoted the proliferation, migration, and growth of DF-1 cells, and decreased the expression of BCL-2 modifying factor (BMF) making cells more resistant to apoptosis. (3) Results: Our results suggest that gga-miR-221 and gga-miR-222 may be tumour formation relevant gene in chicken that promote proliferation, migration, and growth of cancer cells, and inhibit apoptosis. BMF expression was significantly reduced in vivo 70 days after ALV-J infection. They may also play a pivotal role in tumorigenesis during ALV-J infection.     

MicroRNAs (miR)-221 and miR-222, both overexpressed in human thyroid papillary carcinomas, regulate p27Kip1 protein levels and cell cycle

We have recently reported that MicroRNAs (miR)-221 and miR-222 were up-regulated in human thyroid papillary carcinomas in comparison with the normal thyroid tissue. Bioinformatic analysis proposed the p27(Kip1) protein, a key regulator of cell cycle, as a candidate target for the miR-221/222 cluster. Here, we report that the enforced expression of miR-221 and miR-222 was able to reduce p27(Kip1) protein levels in thyroid carcinoma and HeLa cells in the absence of significant changes in specific p27(Kip1) mRNA levels. This effect is direct as miR-221 and miR-222 negatively regulate the expression of the 3'-untranslated region-based reporter construct from the p27(Kip1) gene, and is dependent on two target sites in this region. Consistent with these results, an enforced expression of the miR-221 and miR-222 induced the thyroid papillary carcinoma cell line (TPC-1) to progress to the S phase of the cell cycle. It is likely that the negative regulation of p27(Kip1) by miR-221 and miR-222 might also have a role in vivo since we report an inverse correlation between miR-221 and miR-222 up-regulation and down-regulation of the p27(Kip1) protein levels in human thyroid papillary carcinomas. Therefore, the data reported here demonstrate that miR-221 and miR-222 are endogenous regulators of p27(Kip1) protein expression, and thereby, the cell cycle.     

Cell-specific effects of miR-221/222 in vessels: molecular mechanism and therapeutic application

MicroRNAs (miRNAs) are noncoding RNAs that impact almost every aspect of biology and disease. Until now, the cell-specific effects of miRNAs in cardiovascular system have not been established. In the current study, the cellular functions of miR-221 and miR-222 (miR-221/222) in vascular smooth muscle cells (VSMCs) and vascular endothelial cells (ECs) were compared. In cultured cells, we identified that the effects of miR-221/222 on proliferation, migration, and apoptosis are opposite between VSMCs and ECs. In VSMCs, miR-221/222 had effects of pro-proliferation, pro-migration, and anti-apoptosis. In contrast, miR-221/222 had effects of anti-proliferation, anti-migration, and pro-apoptosis in ECs. The different expression profiles of their target genes, p27(Kip1), p57(kip2), and c-kit between the two cell types might be related to the opposite effects. Finally, the opposite cellular effects of miR-221/222 were verified in vivo in balloon-injured rat carotid artery as demonstrated by different consequences in neointimal growth and re-endothelialization. The results suggest that the biological functions of miR-221/222 in vascular walls are cell-specific. The opposite cellular effects of miR-221/222 on VSMCs and ECs may have important therapeutic applications in many vascular diseases such as atherosclerosis and restenosis after angioplasty.     

Human polynucleotide phosphorylase selectively and preferentially degrades microRNA-221 in human melanoma cells

MicroRNAs (miRNA), small noncoding RNAs, affect a broad range of biological processes, including tumorigenesis, by targeting gene products that directly regulate cell growth. Human polynucleotide phosphorylase (hPNPase^old-35), a type I IFN-inducible 3′-5′ exoribonuclease, degrades specific mRNAs and small noncoding RNAs. The present study examined the effect of this enzyme on miRNA expression in human melanoma cells. miRNA microarray analysis of human melanoma cells infected with empty adenovirus or with an adenovirus expressing hPNPase^old-35 identified miRNAs differentially and specifically regulated by hPNPase^old-35. One of these, miR-221, a regulator of the cyclin-dependent kinase inhibitor p27^kip1, displayed robust down-regulation with ensuing up-regulation of p27^kip1 by expression of hPNPase^old-35, which also occurred in multiple human melanoma cells upon IFN-β treatment. Using both in vivo immunoprecipitation followed by Northern blotting and RNA degradation assays, we confirm that mature miR-221 is the target of hPNPase^old-35. Inhibition of hPNPase^old-35 by shRNA or stable overexpression of miR-221 protected melanoma cells from IFN-β–mediated growth inhibition, accentuating the importance of hPNPase^old-35 induction and miR-221 down-regulation in mediating IFN-β action. Moreover, we now uncover a mechanism of miRNA regulation involving selective enzymatic degradation. Targeted overexpression of hPNPase^old-35 might provide an effective therapeutic strategy for miR-221–overexpressing and IFN-resistant tumors, such as melanoma.     

Liver tumorigenicity promoted by microRNA-221 in a mouse transgenic model

MicroRNA-221 (miR-221) is one of the most frequently and consistently up-regulated microRNAs (miRNAs) in human cancer. It has been hypothesized that miR-221 may act as a tumor promoter. To demonstrate this, we developed a transgenic (TG) mouse model that exhibits an inappropriate overexpression of miR-221 in the liver. Immunoblotting and immunostaining confirmed a concomitant down-regulation of miR-221 target proteins. This TG model is characterized by the emergence of spontaneous nodular liver lesions in approximately 50% of male mice and by a strong acceleration of tumor development in 100% of mice treated with diethylnitrosamine. Similarly to human hepatocellular carcinoma, tumors are characterized by a further increase in miR-221 expression and a concomitant inhibition of its target protein-coding genes (i.e., cyclin-dependent kinase inhibitor [Cdkn]1b/p27, Cdkn1c/p57, and B-cell lymphoma 2-modifying factor). To validate the tumor-promoting effect of miR-221, we showed that in vivo delivery of anti-miR-221 oligonucleotides leads to a significant reduction of the number and size of tumor nodules. Conclusions: This study not only establishes that miR-221 can promote liver tumorigenicity, but it also establishes a valuable animal model to perform preclinical investigations for the use of anti-miRNA approaches aimed at liver cancer therapy. (HEPATOLOGY 2012;56:1025-1033).     

The manipulation of miRNA-gene regulatory networks by KSHV induces endothelial cell motility

miRNAs have emerged as master regulators of cancer-related events. miRNA dysregulation also occurs in Kaposi sarcoma (KS). Exploring the roles of KS-associated miRNAs should help to identify novel angiogenesis and lymphangiogenesis pathways. In the present study, we show that Kaposi sarcoma-associated herpesvirus (KSHV), the etiological agent of KS, induces global miRNA changes in lymphatic endothelial cells (LECs). Specifically, the miR-221/miR-222 cluster is down-regulated, whereas miR-31 is up-regulated. Both latent nuclear antigen (LANA) and Kaposin B repress the expression of the miR-221/miR-222 cluster, which results in an increase of endothelial cell (EC) migration. In contrast, miR-31 stimulates EC migration, so depletion of miR-31 in KSHV-transformed ECs reduces cell motility. Analysis of the putative miRNA targets among KSHV-affected genes showed that ETS2 and ETS1 are the downstream targets of miR-221 and miR-222, respectively. FAT4 is one of the direct targets of miR-31. Overexpression of ETS1 or ETS2 alone is sufficient to induce EC migration, whereas a reduction in FAT4 enhances EC motility. Our results show that KSHV regulates multiple miRNA-mRNA networks to enhance EC motility, which eventually contributes to KS progression by promoting the spread of malignant KS progenitor cells. Targeting KSHV-regulated miRNAs or genes might allow the development of novel therapeutic strategies that induce angiogenesis or allow the treatment of pathogenic (lymph)angiogenesis.