汉黄芩素调控miRNA-135b-3p靶向SIX4抑制A549细胞上皮间质转化的分子机制

目的 探究汉黄芩素对人非小细胞肺癌A549细胞侵袭转移的影响及其对EMT的作用和分子机制。 方法 （1）研究汉黄芩素对A549增殖、侵袭转移的影响。MTT实验检测汉黄芩素对A549细胞的毒性作用，HE染色观察汉黄芩素处理前后形态学变化，划痕实验、Transwell小室实验检测侵袭转移能力，Western Blot和qRT-PCR实验测定EMT相关标志物表达情况。（2）研究A549细胞EMT相关miRNA及调控机制。利用高通量测序技术，检测汉黄芩素处理TGF-β1诱导的A549后差异表达的miRNA，进行qRT-PCR验证；划痕实验、Transwell小室实验分析细胞转染miRNA mimic、inhibitor和NC后A549细胞侵袭转移能力，Western Blot和qRT-PCR实验测定EMT相关标志物的表达情况。运用GO和KEGG数据库分析，找出目标miRNA作用的靶基因；Western Blot实验和qRT-PCR实验验证miRNA mimic、inhibitor和NC转染后靶基因及蛋白的表达情况；双荧光素报告酶基因实验证明该miRNA对靶基因翻译的调控作用。 结果 （1）汉黄芩素可抑制A549细胞的生长增殖，上调TGF-β1诱导的A549细胞E-cadherin及下调Vimentin的表达，降低细胞迁移的能力，逆转细胞的形态变化。（2）高通量测序得到差异表达的miRNA，其中汉黄芩素可逆转miRNA-135b-3p在A549细胞EMT过程中的过表达，抑制A549细胞的侵袭和转移。 pathway分析及miRNA数据库靶基因预测miRNA-135b-3p可调控SIX4的表达。双荧光素报告基因实验显示miRNA-135b-3p可结合于SIX4基因的3’-UTR产生调控，过表达miRNA-135b-3p的A549细胞SIX4表达降低，敲低miRNA-135b-3p后可恢复。 结论 汉黄芩素调控miRNA-135b-3p靶向SIX4抑制A549细胞的侵袭转移和EMT过程。     

山楂酸与顺铂合用对肺癌A549细胞增殖、凋亡的影响

目的探讨山楂酸与顺铂合用对肺癌细胞A549增殖及凋亡产生的影响。方法将人的肺癌细胞系A549经过药物干预(山楂酸、顺铂),并将干预后的肺癌细胞通过MTT实验,分析细胞增殖能力变化,通过流式细胞技术,分析细胞的凋亡水平变化。通过Western blot分析细胞中XIAP、Survivin的表达水平变化。结果山楂酸和顺铂合用后,A549细胞的增殖率显著降低,凋亡水平显著提高;荧光电子显微镜下观察到凋亡细胞明显增加,核碎裂明显,细胞大片脱落;Western blot结果显示,山楂酸和顺铂合用,显著下调XIAP、Survivin的蛋白表达量。结论山楂酸和顺铂合用抑制肺癌细胞的增殖,促进肺癌细胞的凋亡,并下调细胞中凋亡抑制相关指标XIAP、Survivin的表达水平。     

山楂酸对肺癌95D细胞增殖、凋亡的影响

目的探讨山楂酸对肺癌细胞95D增殖及凋亡产生的影响。方法将山楂酸作用于人的肺癌细胞系95D,通过四甲基偶氮唑蓝(MTT)法检测山楂酸对细胞增殖的影响;通过流式细胞仪Annexin V/PI双染法检测细胞凋亡;通过荧光显微镜检测细胞核的形态变化;采用Western blot方法检测凋亡相关蛋白的影响。结果随着山楂酸浓度增大,95D细胞的增殖率明显降低,早期及晚期凋亡率逐渐增加,与对照组比较差异有统计学意义;荧光电子显微镜观察山楂酸作用后的细胞,可见染色体边集、核碎裂等典型的凋亡改变,而对照组没有相应的变化;Western blot显示,随着山楂酸浓度的增加,Bcl-2蛋白表达逐渐降低,Bax蛋白表达逐渐升高。结论山楂酸可以抑制肺癌95D细胞增殖,且具有浓度依赖性,山楂酸抑制肺癌95D细胞增殖是通过诱导细胞凋亡实现的。     

尿多酸肽对人非小细胞肺癌细胞生长与凋亡的影响

目的:初步探讨尿多酸肽(CDA-2)体外诱导非小细胞肺癌细胞凋亡作用及其可能的分子机制。方法:采用四甲基偶氮唑盐(MTT)试验检测CDA-2对正常人呼吸道上皮细胞HBE细胞及肺癌细胞系A549和H157细胞增殖的抑制效应;采用流式细胞仪检测细胞凋亡;采用Western印迹法检测凋亡相关蛋白Bcl-2和Bax表达。结果:CDA-2显著抑制A549和H157细胞增殖,且具有浓度依赖性,其对正常人呼吸道上皮HBE细胞无影响。流式细胞DNA分布宽度检测显示,CDA-2处理组A549细胞亚G1期凋亡峰明显高于对照组(P<0.01);而HBE细胞给药前后比较无统计学意义(P>0.05);流式细胞仪An-nexinV和PI检测结果显示,CDA-2处理组A549细胞凋亡率和死亡率均明显高于对照组(P<0.01),而HBE细胞给药前后比较无统计学意义(P>0.05)。Western印迹分析显示,CDA-2处理组A549细胞抗凋亡蛋白Bcl-2表达强而快速的下调而促凋亡蛋白Bax表达显著上调,Bcl-2/Bax蛋白比率下降。结论:CDA-2可抑制非小细胞肺癌A549和H157细胞增殖和诱导细胞凋亡,其机制可能是改变Bcl-2/Bax比率,通过线粒体途径诱导肺癌细胞凋亡。     

阿司匹林下调lncRNA CCAT1增加肺癌细胞顺铂敏感性的研究

目的研究阿司匹林通过调控长链非编码RNA(lnc RNA)结肠癌相关转录因子1(CCAT1)对人非小细胞肺癌耐顺铂细胞A549(A549/DDP)的顺铂敏感性的影响。方法阿司匹林以不同浓度(1、3、6和9 mmol/L)作用于A549/DDP细胞,采用噻唑蓝(MTT)法检测细胞存活率;用流式细胞术和蛋白质印迹法(Western blot)检测阿司匹林处理A549/DDP细胞的凋亡率和凋亡相关蛋白Bax和Bcl-2的表达;实时荧光定量PCR(qRT-PCR)检测多种肺癌细胞中CCAT1的表达差异,并分析阿司匹林作用A549/DDP细胞后CCAT1的表达情况;运用双酶切法构建pcDNA3.1-CCAT1表达载体;流式细胞仪和Western blot分别检测阿司匹林作用于CCAT1过表达的A549/DDP细胞后,细胞的凋亡率和凋亡相关蛋白Bax和Bcl-2的表达。结果阿司匹林可明显抑制A549/DDP细胞增殖(P0.05),具有剂量依耐性;且可通过调节Bax等相关蛋白表达增强A549/DDP细胞凋亡效应(P0.05);A459/DDP细胞中CCAT1较非耐药细胞株明显高表达(P0.05);阿司匹林作用后,CCAT1表达明显下调(P0.05);成功构建了pcDNA3.1-CCAT1表达载体并成功转染;证实CCAT1过表达逆转阿司匹林对A549/DDP细胞敏感性的影响。结论阿司匹林通过下调lncRNA CCAT1的表达,提高A549/DDP细胞对顺铂的敏感性。     

基于生物信息学分析关键miRNAs在肺鳞癌发生发展中的功能及意义

目的 应用生物信息学方法探究关键miRNAs在肺鳞癌发生发展中的功能及临床意义。方法 利用GEO2R在线工具分析GSE17681数据集中肺癌样本和对照样本的差异表达miRNA。利用miRTarBase数据库预测排在前3位的上调和下调miRNA的靶基因。利用DAVID数据库对靶基因进行GO和KEGG富集分析。通过STRING数据库构建靶基因间的蛋白互作网络及miRNA-基因互作网络，然后利用Cytoscape软件的cytoHubba插件分析关键miRNA及其调控的Hub靶基因。利用UALCAN数据库分析关键miRNA及其调控的Hub靶基因在肺鳞癌中的表达。最后利用Kaplan-Meier Plotter工具分析关键miRNA对肺鳞癌生存状况的影响。结果 共筛选出116个差异表达的miRNA,包括93个上调miRNA,23个下调miRNA。预测了前3位上调和下调miRNA的1282个靶基因，参与了肺鳞癌的相关通路，如癌症途径、MAPK信号通路等。通过miRNA-基因互作网络筛选到上调和下调的关键miRNA为hsa-miR-19a和hsa-miR-126,其调控的Hub靶基因分别为TNF、P...     

缺氧预处理诱导大鼠心肌细胞差异表达microRNA的芯片筛选与生物信息学分析

目的筛选缺氧预处理(hypoxia preconditioning,HPC)诱导成年大鼠心肌细胞差异表达的microRNA(miRNA),并预测分析miRNA调控的靶基因与功能。方法体外分离培养成年大鼠心室肌细胞,分为对照组(CON)和缺氧预处理组(HPC),CON组细胞正常培养,HPC组细胞经历10 min缺氧和30 min再给氧,提取心肌细胞总RNA,行miRNA芯片筛选,qRT-PCR验证芯片结果。利用软件预测差异表达miRNA调控的靶基因,分析靶基因富集的基因功能(gene ontology,GO)和信号通路(pathway)。结果与CON组相比,HPC可诱导大鼠心肌细胞miRNA表达谱发生明显改变,共有12个miRNA上调,14个miRNA下调(P<0.01,FDR<0.05);选择其中荧光信号值>500的7个miRNA,用于生物信息学分析。qRT-PCR检测miR-133b-5p、miR-664-1-5p、miR-6216水平,变化趋势与芯片结果一致。生物信息学分析显示差异表达miRNA所调控的靶基因功能明显富集于27个GO和6条信号通路。结论 HPC可诱导成年大鼠心肌细胞miRNA表达谱明显改变,这些差异表达miRNA可能通过调控靶基因参与HPC介导的心肌细胞保护作用。     

Mir-181a通过靶向c-fos基因抑制肺癌细胞迁移

目的:利用双荧光蛋白报告基因分析系统验证miR-181a的直接靶基因c-fos,探讨miR-181a促进肺癌细胞迁移的可能机制。方法:Transwell小室检测miR-181a对肺癌细胞迁移的影响;应用生物信息学预测软件TargetScan对miR-181a靶基因进行预测,并通过双荧光素酶报告基因检测miR-181a对靶基因c-fos的3’UTR区的调控作用;通过Westernblot方法检测miR-181a对c-fos蛋白表达的影响;结果:A549细胞转染miR-181a后,细胞的迁移能力降低;生物信息学预测c-fos是miR-181a的靶基因,构建融合c-fos的3’UTR区表达质粒pMIR-c-fos,在此基础上对c-fos的3’UTR"种子区"进行定点突变。双荧光素酶报告基因分析表明miR-181a能够作用于c-fos的3’UTR。Western blot方法进一步证实c-fos是miR-181a的靶基因。结论:Mir-181a可能通过靶向c-fos基因,抑制肺癌细胞迁移。     

miR-552通过PTEN/AKT信号通路促进非小细胞肺癌A549细胞的恶性生物学行为

目的 探讨miR-552通过调控PTEN/AKT信号通路促进人非小细胞肺癌A549细胞的恶性生物学行为及其相关机制。方法 通过qRT-PCR检测人肺癌组织样本和人非小细胞肺癌A549细胞系中miR-552、PTEN mRNA的表达情况;通过Western blotting检测PTEN、AKT、p-AKT蛋白的表达情况;通过CCK-8检测miR-552表达对A549细胞增殖能力的影响;通过Transwell小室检测miR-552表达对A549细胞迁移和侵袭能力的影响;通过流式细胞术检测miR-552表达对A549细胞凋亡能力的影响。结果 miR-552在肺癌组织和A549细胞中的表达显著上调。过表达miR-552可显著促进A549细胞的增殖、迁移和侵袭,并抑制细胞凋亡,而抑制其表达则结果相反。与癌旁组织相比,肺癌组织中PTEN表达显著下调。过表达miR-552可下调PTEN蛋白表达,上调p-AKT蛋白表达,对AKT蛋白无影响,而抑制其表达则结果相反。结论 miR-552可能通过PTEN/AKT信号通路促进人非小细胞肺癌A549细胞的恶性生物学行为。     

Lipocalin-2过表达对人肺癌 A549细胞增殖、凋亡及侵袭力的影响

目的：采用基因过表达方法上调人肺癌 A549细胞 Lipocalin-2表达,观察 Lipocalin-2对细胞增殖、凋亡、细胞周期调控及侵袭力的影响。方法构建 Lipocalin-2过表达慢病毒载体,转染人肺癌 A549细胞。Real-time PCR 和 Western blot 检测转染后人肺癌 A549细胞中 Lipocalin-2表达水平。MTT 实验检测细胞增殖。流式细胞术检测细胞周期及凋亡情况。Transwell 小室实验检测细胞侵袭力。结果与阴性对照组和空载体对照组比较,转染组 Li-pocalin-2 mRNA 和蛋白表达水平均显著增加,差异有统计学意义（ P ﹤0.05）。Lipocalin-2过表达慢病毒载体转染后,细胞增殖率和侵袭力显著增加,同时细胞凋亡率及 G0/ G1期细胞比例显著降低,S 期细胞比例显著提高,差异有统计学意义（ P ﹤0.05）。结论 Lipocalin-2过表达通过调控人肺癌 A549细胞周期、抑制凋亡促进细胞的增殖,还明显提高细胞的侵袭力。     

筛选三阴型乳腺癌差异表达 microRNA 的临床转化研究

目的：应用 microRNAs（miRNAs）芯片技术筛选成球培养法及贴壁培养法培养细胞系 MDA-MB-231差异表达的 miRNA,发现三阴型乳腺癌（TNBC）相关的 miRNA。方法用干细胞培养基成球培养法筛选 TNBC 干细胞,应用 miRNAs 芯片技术筛选成球培养法及贴壁培养法培养细胞系 MDA-MB-231差异表达的 miRNA,同时选取5例新鲜 TNBC 癌组织及其癌旁组织运用荧光定量 RT-PCR 对差异表达的 miRNAs 进行验证。运用靶基因预测软件预测差异表达 miRNAs 可能的调控靶基因。结果通过 miRNAs 芯片的检测及 SAM 软件分析,选取细胞培养数据中筛选得到 TNBC 干细胞较 TNBC 细胞表达上调的5个 miRNA（ hsa-miR-297、hsa-miR-8063、hsa-miR-6768-5p、hsa-miR-1231、hsa-miR-489-3p）,下调的4个 miRNA（ hsa-miR-30c-5p、hsa-miR-25-3p、hsa-miR-18a-3p、U25）。荧光定量 RT-PCR 结果是 hsa-miR-297、hsa-miR-8063、hsa-miR-6768-5p、hsa-miR-18a-3p、U25表达上调,hsa-miR-30c-5p、hsa-miR-25-3p、hsa-miR-489-3p、hsa-miR-1231表达下调。芯片检测结果及荧光定量 RT-PCR 结果一致的 miRNA 为 hsa-miR-297、hsa-miR-8063、hsa-miR-6768-5p、hsa-miR-30c-5p、hsa-miR-25-3p,并对其进行生物信息学分析;分析出 MicroRNA-297所对应靶基因为 SLC7A6、SLC7A5、SLC25A44、7A5、AAK1、SMYD1、NDE1、PDE3B、STC1、SUSD1。结论 MicroRNA-297及其靶基因SLC7A5可能在乳腺癌发生发展及干性形成过程中发挥重要作用。     

Inhibitory effects of anti-miRNA oligonucleotides (AMOs) on A549 cell growth

MicroRNAs (miRNAs) are a new class of non-protein-coding, endogenous small RNA molecules of 18–24 nt in size. miRNAs can specifically down-regulate gene expression involved in proliferation, apoptosis, and differentiation in cancer cells. Our purpose was to identify several functional miRNAs as potential drug targets by using specific antisense-microRNA oligonucleotides (AMOs), and to study the inhibitory effects of these AMOs on A549 cell growth. miR-16, miR-21, miR-214, and miR-181a were selected as target candidates, based on which specific AMOs were designed, synthesized, and transfected into A549 cells. The viable cells were counted by using trypan blue dye exclusion assay. Apoptosis of A549 cells were determined flowcytometrically, and miR-21 expression levels in A549 cells were determined by real-time PCR. The results showed that AMO-miR-21, AMO-miR-16, and AMO-miR-181a inhibited A549 cell growth by inducing apoptosis and S-phase arrest. These inhibitory effects increased with dose and time. It was found that AMO-miR-21 down-regulated miR-21 expression in A549 cells. We conclude that miR-21, miR-16, and miR-181a are potential targets for lung cancer therapy, and specific AMOs can be a powerful technique for miRNA inhibition.     

MicroRNA delivery by cationic lipoplexes for lung cancer therapy

Lung cancer is the leading cause of cancer deaths in western countries and carries a poor overall five year survival rate. Several studies demonstrate that microRNAs (miRNAs or miRs) are actively involved in tumor development by serving as tumor suppressors, oncogenes or both. In lung cancer, miRNAs may serve as both diagnostic and prognostic biomarkers as well as regulate in vitro and in vivo tumor progression. However, miRNA-based therapy is faced with several challenges including lack of tissue specificity, lack of optimal delivery systems, poor cellular uptake and risk of systemic toxicity. Here, we report a cationic lipid based miRNA delivery system to address some of these challenges. Among many lung cancer related miRNAs, miR-133b, a tumor suppressor, was selected as a therapeutic target because it directly targets the prosurvival gene MCL-1 thus regulating cell survival and sensitivity of lung cancer cells to chemotherapeutic agents. The efficacy of pre-miR-133b containing lipoplexes was evaluated in A549 non-small cell lung cancer (NSCLC) cells. Compared with siPORT NeoFX transfection agent, lipoplexes delivered pre-miR-133b in a more efficient manner with ~2.3-fold increase in mature miR-133b expression and ~1.8-fold difference in MCL-1 protein downregulation in vitro. In the in vivo biodistribution study, lipoplexes achieved ~30% accumulation in lung tissue, which was ~50-fold higher than siPORT NeoFX transfection agent. Mice treated with pre-miR-133b containing lipoplexes had mature miR-133b expression in lung ~52-fold higher than untreated mice. Our results demonstrated that cationic lipoplexes are a promising carrier system for the development of miRNA-based therapeutics in lung cancer treatment.     

Aberrant expression of miR-638 contributes to benzo(a)pyrene-induced human cell transformation

Identification of aberrant microRNA (miRNA) expression during chemical carcinogen-induced cell transformation will lead to a better understanding of the substantial role of miRNAs in cancer development. To explore whether aberrant miRNAs expression can be used as biomarkers of chemical exposure in risk assessment of chemical carcinogenesis, we analyzed miRNA expression profiles of human bronchial epithelial cells expressing an oncogenic allele of H-Ras (HBER) at different stages of transformation induced by benzo(a)pyrene (BaP) by miRNA array. It revealed 12 miRNAs differentially expressed in HBER cells at both pretransformed and transformed stages. Differentially expressed miRNAs were confirmed in transformed cells and examined in 50 pairs of primary human non-small-cell lung cancer (NSCLC) tissues using real-time PCR. Among these miRNAs, downregulation of miR-638 was found in 68% (34/50) of NSCLC tissues. However, the expression of miR-638 in HBER cells increased upon treatment of BaP in a dose-dependent manner. The expression of miR-638 was also examined in peripheral lymphocytes from 86 polycyclic aromatic hydrocarbons (PAHs)-exposed (PE) workers. We found that the average expression level of miR-638 in peripheral lymphocytes from 86 PE workers increased by 72% compared with control group. The levels of miR-638 were correlated with the concentration of urinary 1-hydroxypyrene (1-OHP) and external levels of PAHs. Overexpression of miR-638 aggravated cell DNA damage induced by BaP, which might be mediated by suppression of breast cancer 1 (BRCA1), one of the target genes of miR-638. In summary, we suggest that miR-638 is involved in the BaP-induced carcinogenesis by targeting BRCA1.     

全反式维甲酸联合阿司匹林对体外抑制肺腺癌细胞株A549增殖和促凋亡的作用及其机制

目的：观察全反式维甲酸（ATRA）联合阿司匹林（ASA）体外抑制肺癌A549细胞增殖,促凋亡作用和机制。方法：体外培养人肺腺癌A549细胞,5,10 μmol·L^-1的ATRA单独及分别联合5,10 μmol·L^-1的ASA进行干预48 h、72 h后,MTT法检测细胞增殖抑制率;TUNEL法观测药物作用48 h后细胞凋亡状态;RT-PCR法检测药物作用后肺腺癌A549细胞COX-2 mRNA的表达;Western blot法检测凋亡相关因子Bax、Bcl-2、COX-2和caspase-3蛋白的表达。结果： ATRA与ASA联用对A549增殖具有协同作用;TUNEL法结果显示ATRA可诱导A549细胞凋亡,与ASA联用凋亡率显著升高（P<0.05）;RT-PCR显示A579细胞中COX-2 mRNA呈现高表达状态,ASA可下调COX-2表达,ATRA无此作用;Western blot结果显示ATRA和ASA协同作用使A549细胞中Bcl-2、COX-2蛋白表达受到抑制,Bax蛋白表达增加,Bcl-2/Bax值降低,对凋亡通路的影响大于单药作用（P<0.05）。结论： ASA联合ATRA可协同抑制肺腺癌细胞A549的增殖及以及促进其发生凋亡,其作用机制可能是ASA通过抑制COX-2蛋白表达,与ATRA共同通过Bcl/caspase通路诱导肿瘤凋亡实现的。     

let-7 microRNA functions as a potential growth suppressor in human colon cancer cells

MicroRNAs (miRNAs) are endogenously expressed RNAs, 18-25 nucleotides in length, that repress protein translation through binding to target mRNAs. miRNAs have been implicated in many cellular processes including cell proliferation, differentiation, and death. Recently, let-7 miRNAs were found to regulate human RAS oncogene expression and to be often down-regulated in human lung tumors. In this study, we examined the expression of let-7 miRNAs in human colon cancer tumors and cell lines, with the result that 2 of 6 cases and 1 of 3 cell lines showed reduced expression of let-7. When let-7 low-expressing DLD-1 human colon cancer cells were transfected with let-7a-1 precursor miRNA, which is located at chromosome 9q22.3, the cells underwent significant growth suppression. At that time, the levels of RAS and c-myc proteins were lowered after the transfection, whereas the levels of both of their mRNAs remained almost unchanged. These findings suggest the involvement of let-7 miRNA in the growth of colon cancer cells. Thus, miRNAs might provide a basis for novel RNA anti-cancer agents.