hsa-miR-125a-5p通过上调Rock-1表达促进肺癌细胞侵袭

背景与目的 MicroRNAs(miRNAs)是广泛存在于真核生物体中的内源性非编码的小分子RNA,通过与靶mRNA不完全互补连接的方式转录后调控靶基因的表达,影响了生物的许多复杂的生命过程.本研究探讨了hsa-miR-125a-5p促进肺癌细胞侵袭的作用机制.方法 应用microRNA.org靶基因预测资源预测hsa-miR-125a-5p的靶基因及其结合位点,并进一步根据预测结果用RT-PCR和Western blot的方法榆测了Rock-1的mRNA及蛋白的表达情况.用Rock-1抗体阻断的方法,检测转染正义的hsa-miR-125a-5p 2'-O-甲基寡核苷酸后A549细胞侵袭能力的变化.结果 转染正义的hsa-miR-125a-5p 2'-O-甲基寡核苷酸36h后,A549细胞中Rocb-1 mRNA和蛋白表达均增加,在转染反义的hsa-miR-125a-5p 2'-O-甲基寡核苷酸的A549细胞中则减少.Transwell小室的结果显示,与未阻断组相比较,阻断Rock-1后,转染正义的hsa-miR-125a-5p 2'-O-甲基寡核廿酸的A549细胞侵袭能力减弱.结论 hsa-miR-125a-5p能够上调Rock-1的表达,并促进肺癌细胞侵袭.     

整合素连接激酶在非小细胞肺癌中过度表达及促进肺癌细胞的侵袭和迁移

目的:探讨整合素连接激酶(ILK)在非小细胞肺癌(NSCLC)中的表达及在侵袭和迁移中的作用和相关分子机制。方法:免疫组化法检测ILK蛋白在NSCLC患者中的表达,细胞转染、siRNA干扰、细胞划痕试验、实时定量PCR、Westernblot方法探讨ILK在肺癌A549细胞中的表达及分子机制。结果:ILK蛋白在原发性NSCLC组织中过度表达30.6%(33/108)并且和TNM分期(P=0.001)、淋巴结转移(P=0.033)相关。ILK在A549细胞中过度表达并且通过下调E-cadherin,上调波形蛋白、纤维连接蛋白、Snail、Slug导致上皮-间质转化(EMT)。此外,NF-κB抑制剂BAY11-7028和小干扰靶RNA(siRNA)NF-p65可诱导E-cadher in的表达下调。结论:ILK在原发性NSCLC组织中高表达并与TNM分期和淋巴结转移相关,其促进肺癌细胞的侵袭和迁徙机制可能是经NF-κB信号通路诱导EMT所致。     

整合素连接激酶在非小细胞肺癌中过度表达及促进肺癌细胞的侵袭和迁移

目的:探讨整合素连接激酶（ILK）在非小细胞肺癌（NSCLC）中的表达及在侵袭和迁移中的作用和相关分子机制。方法:免疫组化法检测ILK蛋白在NSCLC患者中的表达,细胞转染、siRNA干扰、细胞划痕试验、实时定量PCR、Western blot方法探讨ILK在肺癌A549细胞中的表达及分子机制。结果:ILK蛋白在原发性NSCLC组织中过度表达30.6％（33/108）并且和TNM分期（P=0.001）、淋巴结转移（P=0.033）相关。ILK在A549细胞中过度表达并且通过下调E-cadherin,上调波形蛋白、纤维连接蛋白、Snail、Slug导致上皮-间质转化（EMT）。此外,NF-κB抑制剂BAY 11-7028和小干扰靶RNA（siRNA）NF-p65可诱导E-cadherin的表达下调。结论:ILK在原发性NSCLC组织中高表达并与TNM分期和淋巴结转移相关,其促进肺癌细胞的侵袭和迁徙机制可能是经NF-κB信号通路诱导EMT所致。     

miR-216a-5p通过下调MMP16表达抑制肺癌细胞的侵袭

背景与目的：微小RNA(microRNA,miRNA)是一类存在于真核生物体内只有19~39 bp大小的内源性非编码RNA,它能在转录和翻译水平调控基因的表达,在细胞的增殖分化、新陈代谢、免疫调控和凋亡等方面起着重要的作用。本研究检测miR-216a-5p在肺癌组织和肺癌细胞系的表达并探讨其对肺癌细胞侵袭能力的影响及其调控机制。方法：使用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测55例肺癌患者的肺癌组织和7种肺癌细胞系中miR-216a-5p的表达情况;miR-216a-5p瞬时转染A549、95D和H460 3种肺癌细胞系,使用Transwell侵袭实验检测miR-216a-5p对肺癌细胞系侵袭能力的影响;预测并构建miR-216a-5p的候选靶基因基质金属蛋白酶16(matrix metalloproteinase 16,MMP16)基因的双荧光素酶报告基因表达质粒,使用qRT-PCR和蛋白［质］印迹法(Western blot)检测miR-216a-5p对靶基因MMP16的mRNA和蛋白表达的影响;小干扰RNA(siRNA)干扰MMP16与上调miR-216a-5p对比检测其对肺癌细胞侵袭能力的影响。结果：90.91%(50/55)的肺癌患者肿瘤组织中miR-216a-5p表达明显低于对应的癌旁组织(P<0.05)。7种肺癌细胞系中miR-216a-5p的表达量仅为对照组的7.00%~32.00%(P<0.05)。上调miR-216a-5p的表达能够抑制肺癌细胞的侵袭;siRNA干扰MMP16与转染上调miR-216a-5p都能够抑制肺癌细胞中MMP16的表达,并抑制肺癌细胞侵袭。结论：miR-216a-5p可以作为临床肺癌诊断的候选标志物之一,并且其能够通过下调MMP16的表达从而抑制肺癌细胞的侵袭。     

转染microRNA-330-5p对肺癌细胞侵袭和迁移能力的影响

摘 要：［目的］ 探讨miR-330-5p表达对肺癌细胞增殖、侵袭和迁移能力等生物学行为的影响,揭示miR-330-5p的作用机制。［方法］ 分析非小细胞肺癌（NSCLC）细胞系95C和95D的 miRNA表达谱芯片中miR-330-5p的表达情况并用靶标软件预测其靶基因;转染miR-330-5p类似物（mimics）至95D、转染miR-330-5p 抑制剂(inhibitor)至95C中,应用CCK-8法、transwell小室法检测miR-330-5p对肺癌细胞增殖、侵袭迁移等生物学行为的影响;应用Western blot法检测miR-330-5p表达对哺乳动物雷帕霉素靶蛋白（mTOR）表达水平的影响。［结果］ miRNA表达谱芯片显示miR-330-5p在95D中表达明显低于95C;靶标预测软件预测mTOR mRNA可能是miR-330-5p的靶基因;转染miR-330-5p mimics后95D细胞增殖能力显著降低,其24h侵袭穿膜细胞数明显低于对照组,mTOR蛋白表达明显下调(P<0.01);而转染miR-330-5p inhibitor后95C细胞的增殖能力增强,其24h侵袭穿膜细胞数较对照组明显增加,mTOR蛋白表达上调(P<0.01)。［结论］ 提高miR-330-5p表达能够明显抑制肺癌细胞的增殖、侵袭与迁移能力,mTOR mRNA可能是其重要的靶基因。     

miR-625-5p通过靶向调控PRKACA促进肺腺癌细胞的增殖和侵袭

背景 与目的:肺腺癌是非小细胞肺癌的一种亚型,虽在诊断和治疗方面已经取得了很大进展,但晚期肺腺癌临床预后和总生存仍较差.近年来多项研究表明,miRNA在多种癌症中发挥作用,并在细胞增殖、转移、炎症等生物学过程中发挥重要作用.探究miR-625-5p对肺腺癌细胞增殖和侵袭能力的影响及分子机制,旨在为后续肺腺癌的诊断和治疗...     

PAGE5对非小细胞肺癌细胞生长、凋亡和侵袭能力的影响

目的：探讨前列腺相关基因5（PAGE5）在非小细胞肺癌（NSCLC）中的表达及其对肺癌细胞生物学行为的影响。方法：采用实时定量PCR和Western blot方法检测37例NSCLC及其癌旁组织中PAGE5的表达;采用MTT法、流式细胞术、Transwell以及Western blot法检测转染PAGE5 siRNA对A549和H1299细胞生长、凋亡、侵袭以及Bax、Bcl－2和MMP－2表达的影响。结果：在37．84％（14／37）的肺癌组织样本中PAGE5基因表达上调。转染PAGE5 siRNA的A549和H1299细胞生长无明显变化、细胞凋亡显著增加、侵袭能力显著下降（P＜0．05）,Bax蛋白表达显著上调,Bcl－2和MMP－2蛋白表达显著下调（P＜0．05）。结论：PAGE5可能通过调控Bax、Bcl－2及MMP－2蛋白的表达影响NSCLC细胞的生长、凋亡与侵袭,有望成为肺癌诊断标志物及潜在的治疗靶点。     

miR-218-1-3p对非小细胞肺癌细胞侵袭和迁移影响

目的 非小细胞肺癌(non-small cell lung cancer,NSCLC)因其侵袭性而导致预后不良,miR-218可对多种肿瘤的进展起到抑制作用.本研究探讨miR-218-1-3p对NSCLC A549细胞侵袭和迁移影响.方法 使用LipofectamineTM 2000 Reagent将miR-218-1-3p mimic转染入NSCLC A549细胞中,实时定量聚合酶链反应(real-time PCR,RT-qPCR)检测各组细胞中miR-218-1-3p的表达,Transwell小室法测定其侵袭及迁移能力,荧光定量PCR检测细胞迁移侵袭相关指标基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)、MMP-7、MMP-9、Rho A、Rho B和Rho C的变化.结果 同对照组相比,上调mir-218-1-3p明显抑制了A549细胞的侵袭(t=4.028,P=0.016)和迁移(t=8.911,P=0.001),其侵袭和迁移的抑制率分别为37.8%和53.6%.MMP-7降低至对照组的(0.68±0.19)倍,t=2.931,P=0.043;MMP-9降低至对照组的(0.58±0.15)倍,t=4.875,P=0.080.结论 miR-218-1-3p能够抑制NSCLC A549细胞的侵袭和迁移.     

miR-125a-5p通过下调BAG4表达抑制胃癌细胞迁移和侵袭

目的:探讨miR-125a-5p通过调控Bcl-2相关永生基因4(Bcl-2-associated athanogene 4,BAG4)的表达抑制胃癌细胞迁移和侵袭的分子机制.方法:选用2014年1月至2015年12月兰州大学第一医院手术切除的82例胃癌组织标本及配对的癌旁组织以及人胃癌细胞系MGC803、BGC823...     

miR-424对非小细胞肺癌A549细胞生长和侵袭的影响及分子机制

背景与目的已有的研究表明miR-424可抑制肾癌细胞增殖,抑制宫颈鳞癌细胞的迁移和侵袭能力,而其对非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞的影响目前尚无系统研究。本研究探讨miR-424对NSCLC A549细胞生长和侵袭迁移能力的影响并进一步研讨其分子机制。方法应用CCK8检测过表达及抑制miR-424的表达对A549细胞增殖的影响。应用Transwell检测过表达及抑制miR-424的表达对A549细胞侵袭能力的影响。应用Western blot检测过表达及抑制miR-424的表达对A549细胞中MMP9和MMP2蛋白水平的影响。构建E2F63’UTR区的荧光素酶报告载体,验证miR-424对E2F6的靶向作用。采用Western blot检测过表达及抑制miR-424的表达后,A549细胞中E2F6的表达。结果过表达miR-424后,A549的生长和侵袭能力显著降低。过表达miR-424后,A549细胞的MMP-2和MMP-9表达下降。荧光素酶活性检测表明miR-424能够抑制E2F6的荧光素酶活性。过表达miR-424后,细胞内E2F6的表达降低。结论 miR-424能够通过调控E2F6而抑制A549的生长和侵袭能力。     

hsa-miR-23a-3p在肺腺癌组织中的表达及意义

[目的]探讨hsa-miR-23a-3p在肺腺癌织中的表达水平及临床意义。[方法]收集我院呼吸科就诊的42例肺腺癌患者的癌组织及癌旁组织样本,同时选取37例非肺腺癌患者的肺穿刺样本为对照组。采用荧光定量PCR法检测各组患者肺组织中hsa-miR-23a-3p相对表达水平,分析hsa-miR-23a-3p在肺腺癌组织中的表达及与患者临床特征的关系;以患者血清癌胚抗原(CEA)指标为参考,利用受试者工作特征曲线(ROC)评估肺腺癌患者癌组织中hsa-miR-23a-3的表达临床应用价值。[结果]对照组与癌旁组织中hsa-miR-23a-3p的表达水平差异无统计学意义(4.32±0.34 vs 4.16±0.41,P>0.05),与对照组与癌旁组织比,肺腺癌组织hsa-miR-23a-3p表达较高(6.08±0.47 vs 4.32±0.34,6.08±0.47 vs 4.16±0.41,P<0.01)。不同性别(6.16±0.82 vs 5.89±0.73)、分化程度(6.20±0.84 vs 5.97±0.67)癌组织的hsa-miR-23a-3p表达差异无统计学意义(P>0.05),TNM分期越高(5.13±0.49 vs 6.26±0.51)、肿瘤越大(6.47±0.65 vs 5.94±0.71)、淋巴结转移(6.32±0.53 vs 5.83±0.46)及CEA阳性(6.47±0.44 vs 5.79±0.41)癌组织的hsa-miR-23a-3p表达显著性增高(P<0.001)。ROC曲线分析显示,与CEA比,hsa-miR-23a-3p的曲线下面积高(0.779vs 0.683)。[结论]肺腺癌组织hsa-miR-23a-3p呈高表达,与淋巴结转移、肿瘤大小及TNM分期相关,可用于辅助诊断肺腺癌。     

Up-Regulation of MiRNA-125a-5p Inhibits Cell Proliferation and Increases EGFR-TKI Induced Apoptosis in Lung Cancer Cells

Background: Despite the dramatic efficacy of erlotinib, an EGFR tyrosine kinase inhibitor (TKI), most of non-small cell lung cancer (NSCLC) patients ultimately acquire resistance to this agent. Different studies indicated that miRNA-125a-5p is down-regulated in human lung cancer cells and may function as a tumor suppressor by targeting EGFR. However, the biological function of miRNA-125a-5p in NSCLC resistance to EGFR-TKIs is not fully understood. In this study the effect of miRNA-125a-5p on cell proliferation, apoptosis and sensitivity of the A549 lung cancer cells to erlotinib was investigated. Methods: After miRNA-125a-5p transfection, the expression levels of EGFR mRNA were measured by QRT-PCR. Trypan blue assays were performed to evaluate the proliferation of the A549 lung cancer cells. The cytotoxic effects of miRNA-125a-5p and erlotinib, alone and in combination, were determined using MTT assay. Combination index study was performed using the method of Chou-Talalay. Apoptosis was assessed using an ELISA cell death assay kit. Results: MiRNA-125a-5p clearly reduced the expression of EGFR mRNA in a time dependent manner, causing marked cell proliferation inhibition and spontaneous apoptosis (p<0.05, relative to control). Pretreatment with miRNA-125a-5p synergistically increased the cytotoxic effect of erlotinib and decreased its IC50. Furthermore, miRNA-125a-5p significantly enhanced the apoptotic effect of erlotinib. Negative control miRNA had no significant effect on biological parameter of the tumor cells. Conclusions: Our data suggest that suppression of EGFR by miRNA-125a-5p can effectively trigger apoptosis and overcome EGFR-TKs resistance of lung cancer cells. Therefore, miRNA-125a-5p may be a potential therapeutic adjuvant in patients with lung cancer.     

Circulating exosomal miR-125a-5p as a novel biomarker for cervical cancer

Exosomal microRNAs (miRs/miRNAs) have been reported to be associated with cervical cancer. The aim of the present study was to investigate circulating exosomal miRNA as a biomarker for cervical cancer diagnosis. In the present study, samples from 6 patients with cervical cancer and 6 healthy control subjects were retrieved for exosomal RNA-sequencing. The results revealed that a total of 39 miRNAs were differentially expressed between patients with cervical cancer and healthy controls (P<0.001; fold-change >2.0). Exosomal miR-125a-5p was further quantified in plasma from 60 subjects, which included 22 healthy individuals and 38 patients with cervical cancer. miR-16a-5p served as the reference miRNA for quantitative PCR analysis of exosomal miR-125a-5p in patients with cervical cancer and healthy individuals. The results revealed that exosomal miR-125a-5p expression levels in the patients with cervical cancer were significantly lower than those in the healthy controls (P<0.001). Receiver operating characteristic (ROC) curve analyses were performed and the results revealed that the level of plasma exosomal miR-125a-5p was a potential marker for differentiating between non-cervical cancer and cervical cancer, with an ROC area under the curve of 0.7129. At the cut-off value of 2.537 for miR-125a-5p, cervical cancer diagnostic sensitivities and specificities were 59.1 and 84.2%, respectively. The present study provides confirmation that exosomal miR-125a-5p could potentially serve as a biomarker for cervical cancer diagnosis. The present study involved only a small number of clinical samples; more samples are required to support the conclusions of the present study.     

miR-125a-5p is a prognostic biomarker that targets HDAC4 to suppress breast tumorigenesis

Identifying stably expressed tumor markers that can be used easily to detect cancer is currently an important area of cancer research. By using miRNA microarray, we identified 20 differentially expressed miRNAs in serum samples of breast cancer patients. Expression of miR-125a-5p was relatively lower in patients with shorter survival compared to long-term survivors. In a cohort of breast cancer patients (N = 300), serum expression of miR-125a-5p was negatively and significantly correlated with tumor grade (P = 0.004), lymph-node status (P = 0.004), and tumor size (P < 0.001). Low miR-125a-5p expression was an independent prognostic marker (OR = 0.421; 95% CI = 0.184 to 0.961; P = 0.04) associated with poor survival rates (P = 0.0062). We show that miR-125a-5p directly inhibits expression of the HDAC4 gene, resulting in tumor suppression in vitro and in vivo. Together these results demonstrate that serum miR-125a-5p level in breast cancer may be a useful prognostic biomarker and offer a novel therapeutic avenue by targeting HDAC4 in breast cancer.     

miR-199a-3p对前列腺癌细胞迁移及侵袭能力的影响

目的从miR-199a-3p在前列腺癌高、低转移潜能细胞中的表达差异出发,研究其在高转移前列腺癌细胞株1E8细胞运动转移中的作用及可能的分子机制。方法运用Realtime PCR法检测 miR-199a-3p在前列腺癌高、低转移潜能配对细胞系中的表达差异。通过划痕实验及transwell实验观察1E8细胞及转染 miR-199a -3p mimics及mimics NC后该细胞运动迁移能力的变化。结果（1）Realtime PCR结果显示高转移潜能1E8细胞中miR-199a-3p表达水平明显低于低转移潜能2B4细胞。（2）上调miR-199a-3p会减弱1E8细胞的运动转移能力。结论miR-199a-3p的上调可以抑制前列腺癌细胞的运动迁移能力。     

人支气管上皮hsa-miR-148a-3p 低表达细胞株的建立

目的： 建立稳定的hsa-miR-148a-3p低表达人支气管上皮细胞株(16HBE)。方法：根据miRBase中提供的序列信息设计hsa-miR-148a-3p tough decoy RNA(TuD RNA),并将其连接到慢病毒载体pLKO.1-puro上。将重组慢病毒载体转染至293FT细胞并包装为慢病毒后,收集病毒上清,感染正常16HBE细胞。用嘌呤霉素筛选出has-miR-148a-3p低表达的16HBE细胞株,通过荧光定量PCR对其进行鉴定,然后对筛选出的细胞分别采用荧光定量PCR和Western blot检测DNA甲基转移酶1(DNMT1) mRNA和蛋白的表达水平。结果：测序结果表明含hsa-miR-148a-3p TuD RNA的重组慢病毒载体构建成功;荧光定量PCR检测显示has-miR-148a-3p低表达的16HBE细胞株has-miR-148a-3p的表达量比正常16HBE细胞低44%(P<0.01),其作用靶基因DNMT1的mRNA和蛋白表达水平分别为正常细胞的3.4倍和2.0倍(P均<0.01)。结论：成功建立hsa-miR-148a-3p低表达的16HBE细胞,hsa-miR-148a-3p的低表达能提高DNMT1 mRNA和蛋白的表达水平。     

CircularRNA circPARP4 promotes glioblastoma progression through sponging miR-125a-5p and regulating FUT4

Circular RNA (circRNA) is a widely expressed non-coding RNA element characterized by a covalently closed continuous loop. Emerging evidence suggests important roles of circRNAs in the pathogenesis of human cancers. However, the functions and underlying mechanisms of circRNAs in glioma remain largely unclear. Previously, our studies uncovered a batch of abnormally expressed circRNAs in glioma tissue, among which circPARP4 was significantly upregulated with the top fold change. Here, we focused on the functional investigation toward circPARP4 in glioblastoma progression and looked for insight into its underlying mechanisms. The results confirmed the elevated expression of circPARP4 in glioma and found its association with glioma pathological grade. Gain- and loss-of-function strategies showed that circPARP4 could obviously promote glioma cell proliferation, migration, invasion, and epithelial-mesenchymal transition. Mechanistically, in vivo and in vitro studies demonstrated that circPARP4, as a miRNA sponge, directly interacted with miR-125a-5p, which then regulated FUT4 to exert the oncogenic effect on glioma behavior. Our findings illustrate functions of circPARP4 in modulating glioma progression through miR-125a-5p/FUT4 pathway, which provides a novel and potential target for glioma therapy.