mir-17-5p、mir-92a、let-7b表达水平与非小细胞肺癌顺铂耐药的关系

目的探讨mir-17-5p、mir-92a、let-7b表达水平与非小细胞肺癌顺铂耐药关系。 方法以人非小细胞肺癌细胞系A549及其耐药株A549/DDP为研究对象,采用RT-PCR法检测mir-17-5p、mir-92a及let-7b在细胞中的表达水平,采用cck8检测其细胞存活情况,采用细胞克隆平台方法,检测转染前后细胞的增殖情况,采用流式细胞仪检测转染前后细胞的凋亡情况。 结果（1） A549/DDP细胞mir-17-5p的表达水平是A549细胞的2.11±0.25倍（P＜0.05）;A549/DDP细胞mir-92a的表达水平是A549细胞的 7.40 ± 1.05 倍（P＜0.05）;而A549/DDP 细胞let-7b 的表达水平是A549 细胞的（26.54 ± 2.90）%（P＜0.05）;（2）A549 转染mir-17-5pmimic,mir-92a mimic 以及let-7b inhibitor 后对顺铂的敏感性下降（P＜0.05）;A549/ddp 转染mir-17-5p inhibitor, mir-92a inhibitor以及let-7b mimic后对顺铂的敏感性增加（P＜0.05）;（3）A549转染mir-17-5p mimic,mir-92a mimic以及let-7b inhibitor 后,细胞形成克隆集落数量数量多于对照组（P＜0.05）;而A549/ddp 转染mir-17-5p inhibitor,mir-92a inhibitor 以及 let-7b mimic 后,细胞形成克隆集落数量数量少于对照组（P＜0.05）;（4）A549 转染mir-17-5p mimic,mir-92a mimic 以及let-7b inhibitor后,细胞凋亡率明显低于对照组（P＜0.05）;而a549/ddp转染mir-17-5p inhibitor,mir-92a inhibitor以及let-7b mimic后, 细胞凋亡率明显高于对照组（P＜0.05）。结论Mir-17-5p、mir-92a表达水平升高,let-7b表达水平下降,可以促进肺癌细胞增殖, 抑制其凋亡以及使肺癌细胞对顺铂敏感性下降。      

MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors

MicroRNAs (miRNAs) regulate gene expression. It has been suggested that obtaining miRNA expression profiles can improve classification, diagnostic, and prognostic information in oncology. Here, we sought to comprehensively identify the miRNAs that are overexpressed in lung cancer by conducting miRNA microarray expression profiling on normal lung versus adjacent lung cancers from transgenic mice. We found that miR-136, miR-376a, and miR-31 were each prominently overexpressed in murine lung cancers. Real-time RT-PCR and in situ hybridization (ISH) assays confirmed these miRNA expression profiles in paired normal-malignant lung tissues from mice and humans. Engineered knockdown of miR-31, but not other highlighted miRNAs, substantially repressed lung cancer cell growth and tumorigenicity in a dose-dependent manner. Using a bioinformatics approach, we identified miR-31 target mRNAs and independently confirmed them as direct targets in human and mouse lung cancer cell lines. These targets included the tumor-suppressive genes large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A), and expression of each was augmented by miR-31 knockdown. Their engineered repression antagonized miR-31–mediated growth inhibition. Notably, miR-31 and these target mRNAs were inversely expressed in mouse and human lung cancers, underscoring their biologic relevance. The clinical relevance of miR-31 expression was further independently and comprehensively validated using an array containing normal and malignant human lung tissues. Together, these findings revealed that miR-31 acts as an oncogenic miRNA (oncomir) in lung cancer by targeting specific tumor suppressors for repression.     

人淋巴瘤Raji细胞系中的边缘细胞：淋巴瘤干细胞存在之可能

背景：已有研究提示急性白血病、多发性骨髓瘤中肿瘤干细胞富集于SP细胞中,且免疫表型及生物学特性相比于主群细胞存在明显的异质性。而关于淋巴瘤淋巴瘤干细胞的研究报道甚少。目的：检测人淋巴瘤Raji细胞系中是否存在SP细胞,分选该群细胞后观察其与非SP细胞在分化抗原和P-糖蛋白表达以及细胞周期方面的差异,探讨淋巴瘤干细胞存在的可能性。方法：Hoechst-33342染色细胞后运用带紫外激发光的流式细胞仪进行Raji细胞系SP细胞的检测及分选。观察不同质量浓度维拉帕米对SP细胞抑制的程度,选择最佳抑制浓度。分选后分别检测SP和非SP细胞P-糖蛋白、分化抗原CD34,CD19,CD20,CD5表达以及细胞周期,并进行对比。结果与结论：Raji细胞系中存在SP细胞,比例在2%左右,当维拉帕米质量浓度为50mg/L时能被完全抑制。SP和非SP细胞CD20阳性表达率分别为18.2%和93.6%,CD5阳性表达率分别为78.0%和22.2%;P-糖蛋白表达差异无显著性意义,但SP中阳性细胞的平均荧光强度强于非SP细胞。提示Raji细胞系中SP与非SP细胞在分化程度、多药耐药性方面具有明显的差异,SP细胞是具有明显＂异质性＂的细胞亚群。据此可初步推测,Raji细胞系的构成也类似肿瘤干细胞分级组成模式,而淋巴瘤干细胞则有可能富集于SP亚群中。     

Systemic Delivery of Tumor Suppressor microRNA Mimics Using a Neutral Lipid Emulsion Inhibits Lung Tumors in Mice

MicroRNAs (miRNAs) are emerging as potential cancer therapeutics, but effective delivery mechanisms to tumor sites are a roadblock to utility. Here we show that systemically delivered, synthetic miRNA mimics in complex with a novel neutral lipid emulsion are preferentially targeted to lung tumors and show therapeutic benefit in mouse models of lung cancer. Therapeutic delivery was demonstrated using mimics of the tumor suppressors, microRNA-34a (miR-34a) and let-7, both of which are often down regulated or lost in lung cancer. Systemic treatment of a Kras-activated autochthonous mouse model of non-small cell lung cancer (NSCLC) led to a significant decrease in tumor burden. Specifically, mice treated with miR-34a displayed a 60% reduction in tumor area compared to mice treated with a miRNA control. Similar results were obtained with the let-7 mimic. These findings provide direct evidence that synthetic miRNA mimics can be systemically delivered to the mammalian lung and support the promise of miRNAs as a future targeted therapy for lung cancer.     

miRNA Sponge介导的miR-17和miR-20a基因沉默的抗白血病作用机制

本研究旨在检测急性白血病患者中miR-17和miR-20a前体的表达水平,并探讨miRNA Sponge介导的miR-17和miR-20a沉默的抗白血病作用机制.采用荧光实时定量PCR方法检测初治急性白血病患者及8种白血病细胞株中miR-17和miR-20a前体的表达水平,分析其在各型白血病中的表达情况.利用前期构建的针对miR-17和miR-20a基因的miRNA Sponge慢病毒表达载体,感染高表达miR-17和miR-20a的Jurkat细胞株;用CCK-8方法及流式细胞术分别检测miR-17和miR-20a沉默对Jurkat细胞增殖能力及细胞周期的影响.结果表明：初治急性白血病患者中miR-17和miR-20a前体的表达明显高于正常对照（P＜0.05）;且与急性髓系白血病患者相比,急性淋巴细胞白血病患者中的表达量更高;然而二者表达量与患者外周血高白细胞计数无明显相关性（P＞0.05）.miR-17和miR-20a沉默能抑制Jurkat细胞的增殖,使细胞周期阻滞于G1—S期,同时促进细胞的凋亡.结论：急性白血病患者中miR-17和miR-20a呈过表达,可能参与白血病的发生、发展;高表达的miR-17和miR-20a可通过在转录后抑制P21和E2F1表达,从而促进细胞增殖及细胞周期G1 —S期转换,抑制细胞凋亡.     

Let-7a mimic attenuates CCL18 induced breast cancer cell metastasis through Lin 28 pathway

BackgroundMicroRNAs are believed to influence breast cancer cell tumorgenicity by interacting with the production of tumor associated macrophages. At this stage, this hypothesis lacks sufficient empirical evidence. Our study is an investigation of the effects of let-7a on the function of human breast cancer cell lines that had undergone chemokine ligand 18 (CCL18) stimulation.MethodsTwo breast cancer cell lines MDA-MB-231 and MCF-7 were transfected with let-7a mimics with or without CCL18 simulation. The expression level of let-7a was evaluated with qRT-PCR. Our study examined cell proliferation, migration and cell cycles following let-7a treatment. The predicted target of let-7a was identified and confirmed in vitro by a dual luciferase reporter system. The associations between let-7a, CCL18 and target gene expression were evaluated using RT-PCR and the Western blotting method.ResultsThe downregulated expression level of let-7a was observed in both breast cancer cell lines. When compared to the control and CCL18 stimulation groups, cell proliferation and migration in MDA-MB-231 and MCF-7 cells were significantly inhibited by let-7a. Furthermore, the cell cycle was dramatically blocked at the G2/M phase. The luciferase reporter identified Lin28 as the direct binding target of let-7a in both breast cancer cell lines.ConclusionUpregulation of let-7a carries the potential to reverse CCL18 induced cell proliferation and migration alteration in breast cancer cells by regulating Lin28 expression. Our results provided evidence which suggests the use of let-7a as a therapeutic agent in the treatment of breast cancer.     

实时定量PCR检测白血病相关miRNA方法的建立

本研究建立实时定量PCR（RQ-PCR）技术检测白血病相关miRNA的方法,探讨此方法在定量检测miRNA中的应用价值。通过提取82例慢性淋巴细胞白血病（CLL）、70例急性白血病（AL）患者骨髓或外周血标本总RNA,以cDNA为模板进行10倍梯度系列稀释,分别作miRNA及内参U6RNA的标准曲线并计算扩增效率;运用ABF7300定量PCR仪进行定量检测;采用SYBRGreen荧光染料法、以U6RNA作为内参照、循环阈值（ct）比较法进行miRNA表达相对定量分析,以检测CLL患者miR-15a、miR-16-1、miR-29b、miR-181b及AL患者miR．128．1、miR-223、let-7b、miR-155、miR-181a的表达。方法学考核参数包括特异性、线性范围、精密度和重复性。结果表明：RQ—PCR检测miRNA仅需10—50ng总RNA样本,检测下限为0．05ng,miRNA及u6的Ct值均在15—30之间,10倍系列稀释法制作的标准曲线呈现良好的线性关系（R^2〉0．980）,扩增效率均在0．9以上,溶解曲线均为单峰,PCR产物经琼脂糖电泳均显示较亮的目的条带,批内差和批间差分别小于4．8％和6．3％。结论：RQ-PCR技术检测白血病相关miRNA敏感、快速,高通量,节约成本,定量范围宽,极大的方便了对miRNA的定量研究。     

microRNA在非小细胞肺癌组织中的表达及其临床应用研究

摘要:目的：探讨microRNA（miRNA）在非小细胞肺癌（NSCLC）组织中的表达及其临床意义。 方法：用实时荧光定量PCR技术检测40例NSCLC患者癌组织与癌旁组织中miR-21、miR-20a、miR-31、miR-222、miR-223、miR-145、miR-126的表达变化。 结果：NSCLC患者癌组织中miR-21、miR-20a、miR-31、miR-222较癌旁组织呈高表达（P<0.05）,而miR-223、miR-145、miR-126呈低表达（P<0.05）。随着病情进展,miR-21、miR-222表达升高,miR-126表达下降。 结论：miR-21、miR-20a、miR-31、miR-222、miR-145、miR-126、miR-223在NSCLC癌组织中表达存在差异,且miR-21、miR-222和miR-126与NSCLC病情的恶化发展相关,有助于预后判断。     

miRNA在胰腺癌患者血浆中的表达及临床意义

目的探讨miRNA在胰腺癌患者血浆中的表达及临床意义。方法比较40例胰腺癌患者（观察组）及40例正常志愿者（对照组）血浆中的4种miRNA（miR-190、miR-196a、miR-221和miR-222）水平,并分析其与患者年龄、性别、肿瘤部位、分化程度及CA199水平的关系。结果胰腺癌患者血浆中4种miRNA表达量的水平较对照组均明显升高,差异有统计学意义（P〈0.05）。胰腺癌患者血浆中miR-196a的高表达与胰腺癌的分期呈正相关（P〈0.05）,与其他因素无关。其他3种miRNA的表达与以上因素均无关,而且4种miRNA的表达量与血清中CA199无明显相关性（P〉0.05）。结论 miRNA作为敏感而特异的胰腺癌肿瘤标志值得进一步研究。     

局部注射胆固醇包裹let-7a miRNA模拟物下调人3种Ras表达抑制裸鼠皮下肝癌移植瘤的生长和转移

  目的  研究胆固醇包裹let-7a模拟物(mimics)是否能通过下调人3种Ras抑制肝癌发展, 寻找肝癌的潜在治疗策略  目的  采用MTT增殖分析、PI单染和Annexin V/FITC双染方法检测let-7a mimics在体外对肝癌细胞的作用。使用裸鼠皮下移植瘤模型和通过肿瘤局部注射方法观察let-7a mimics对体内肝癌的作用。采用实时定量PCR和Western blot方法检测let-7a及其靶点Ras的表达  结果  与阴性对照组相比, let-7a mimics转染的肝癌细胞let-7a水平升高, 细胞增殖缓慢(P均 < 0.05);let-7a mimics阻滞更多肝癌细胞停留在G0-G1期且促进肝癌细胞凋亡(P均 < 0.05)。经let-7a mimics治疗的裸鼠体内肿瘤体积较阴性对照组减小, 是阴性对照组的54.97%(P=0.039);局部浸润和肝脏转移较阴性对照组明显减轻。肝癌细胞和移植瘤组织内let-7a上调的同时, 人K-Ras、H-Rras和N-Ras mRNA和蛋白的表达降低(P均 < 0.05)  结论  let-7a mimics下调人3种Ras mRNA和蛋白的表达, 影响细胞周期, 进而抑制细胞增殖和促进细胞凋亡。瘤周多点注射胆固醇包裹的let-7a mimics能抑制移植瘤的生长、浸润和转移。提示let-7阻断Ras可成为肝癌治疗的研究策略。     

lncRNA HCG11-201对肾癌细胞增殖和侵袭的影响

目的探讨长链非编码RNA(lncRNA)HCG11-201在肾癌组织中的表达及影响癌细胞增殖和侵袭的分子机制。方法生物信息学方法预测HCG11-201表达与肾癌患者预后的相关性。实时荧光定量PCR(qPCR)检测肾癌组织和癌旁正常组织、肾癌细胞系和正常肾小管上皮细胞中HCG11-201的表达,选择表达最少的肾癌细胞系转染,分别将HCG11-201质粒(实验组)或阴性对照质粒(对照组)转至肾癌细胞。qPCR检测HCG11-201质粒转染效率。MTT法检测转染后细胞增殖活性,Transwell小室实验检测转染后细胞侵袭能力。生物信息学方法预测HCG11-201可吸附结合的微小RNA(miRNA)及miRNA下游基因。qPCR和Western blot检测miRNA和下游基因表达。结果GEPIA数据库显示,HCG11-201相对表达水平越高,患者总生存期越长(P<0.01)。肾癌组织HCG11-201相对表达水平显著低于癌旁正常组织(P<0.01)。肾癌细胞HCG11-201相对表达水平显著低于正常肾小管上皮细胞(P<0.01),其中Caki-1细胞相对表达水平最低(P<0.01)。与对照组比较,实验组Caki-1细胞HCG11-201相对表达水平显著升高(P<0.01),细胞增殖活性显著降低(P<0.05),细胞的侵袭能力显著降低(P<0.05)。HCG11-201可互补结合miR-522,miR-522可互补结合线粒体融合蛋白2(mitofusion-2)。与对照组比较,实验组Caki-1细胞miR-522的相对表达水平显著降低(P<0.01),mitofusion-2的mRNA相对表达水平和蛋白水平显著升高(P<0.01)。结论lncRNA HCG11-201在肾癌组织和细胞系低表达,与肾癌患者预后相关。过表达HCG11-201可明显抑制肾癌细胞增殖和侵袭,其分子机制可能为HCG11-201吸附miR-522进而上调mitofusion-2基因的表达。     

The antidiabetic drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo

Recent studies suggest that metformin, which is commonly used as an oral anti-hyperglycemic agent of the biguanide family, may reduce cancer risk and improve prognosis, but the mechanisms by which metformin affects various cancers, including gastric cancer, remains unknown. The goal of the present study was to evaluate the effects of metformin on human gastric cancer cell proliferation in vitro and in vivo and to study microRNAs (miRNA) associated with antitumor effect of metformin. We used MKN1, MKN45, and MKN74 human gastric cancer cell lines to study the effects of metformin on human gastric cancer cells. Athymic nude mice bearing xenograft tumors were treated with or without metformin. Tumor growth was recorded after 4 weeks, and the expression of cell-cycle-related proteins was determined. In addition, we used miRNA array tips to explore the differences among miRNAs in MKN74 cells bearing xenograft tumors treated with or without metformin in vitro and in vivo. Metformin inhibited the proliferation of MKN1, MKN45, and MKN74 in vitro. Metformin blocked the cell cycle in G(0)-G(1)in vitro and in vivo. This blockade was accompanied by a strong decrease of G(1) cyclins, especially in cyclin D1, cyclin-dependent kinase (Cdk) 4, Cdk6 and by a decrease in retinoblastoma protein (Rb) phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor and insulin-like growth factor-1 receptor in vitro and in vivo. The miRNA expression was markedly altered with the treatment of metformin in vitro and in vivo. Various miRNAs altered by metformin also may contribute to tumor growth in vitro and in vivo.     

伊马替尼耐药K562/G的microRNA表达谱分析及与FOXO3/Bcl-6信号通路关系的研究

目的:探讨CML耐药细胞株K562/G耐药相关的FOXO3/Bcl-6信号通路及相关microRNA(miRNA)机制。方法:采用MTT法检测伊马替尼对K562/G的耐药性;采用Western blot法检测耐药株和敏感株细胞中FOXO3和Bcl-6蛋白的表达情况,实时荧光定量PCR检测FOXO3及Bcl-6 mRNA的表达。运用miRNA芯片技术筛查K562与K562/G细胞之间差异表达的miRNA,进一步筛选FOXO3/Bcl-6信号通路的靶向miRNA。结果:K562/G较K562细胞株的FOXO3及Bcl-6蛋白表达均明显升高(P<0.01);Bcl-6 mRNA的表达水平无明显差异,而K562/G细胞中FOXO3 mRNA表达明显升高(P<0.05)。miRNA芯片结果显示,K562/G与K562细胞之间有109条miRNA存在显著的差异,其中上调miRNA为81个,下调miRNA有28个。经生物信息学反向预测,其中miR-6718-5p、miR-5195-5p、miR-4711-3p、miR-4763-5p、miR-4664-5p和miR-3176与该信号通路相关。结论:伊马替尼耐药与FOXO3/Bcl-6信号通路相关,并且K562/G与K562细胞存在miRNA表达显著差异。     

肺鳞癌患者血清miRNA-181a的表达及其对肺癌细胞功能的影响

【目的】探讨肺鳞癌患者血清中microRNA-181a（miR-181a）水平的变化及其对人肺鳞癌细胞生物学功能的影响。【方法】选择56例肺鳞癌患者和50名健康成人,采用stem-[oop定量PCR的方法检测其血清中miR-181a的表达变化;体外实验采用CCK8和Transwell小室实验检测miR-181a对人肺鳞癌细胞株NCI-H226增殖、侵袭和迁移能力的影响。【结果】miR-181a在肺鳞癌患者血清中表达较健康成人显著降低（P〈0．05）;miR181a可明显抑制肺鳞癌细胞NCI—H226的增殖、侵袭和迁移。【结论】在肺鳞癌患者miR-181a是一种保护性的微小RNA,具有较好的诊断和治疗前景。     

双氢青蒿素对人肺腺癌细胞株A549抑制作用的实验研究

目的观察双氢青蒿素对人肺腺癌细胞株A549的作用。方法采用四唑氮蓝（MTT）法测定不同浓度和时间双氢青蒿素对A549细胞的生长抑制作用;应用流式细胞术检测双氢青蒿素对A549细胞增殖和细胞周期的影响;应用透射电镜分析法检测双氢青蒿素对A549细胞凋亡的影响。结果双氢青蒿素对A549细胞有明显的体外增殖抑制作用,72 h的IC50值为580 nmol/L,且有明显的时间和剂量依赖关系;双氢青蒿素作用后G0/G1期细胞数增加;双氢青蒿素作用A549细胞后可以出现凋亡的形态学改变。结论双氢青蒿素对人肺腺癌A549细胞有生长抑制作用,其机制可能与诱导A549细胞周期阻滞和凋亡有关。     

MicroRNA-199a-3p is downregulated in human osteosarcoma and regulates cell proliferation and migration

microRNAs (miRNA, miR) play an important role in cancer cell growth and migration; however, the potential roles of miRNAs in osteosarcoma remain largely uncharacterized. By applying a miRNA microarray platform and unsupervised hierarchical clustering analysis, we found that several miRNAs have altered expression levels in osteosarcoma cell lines and tumor tissues when compared with normal human osteoblasts. Three miRNAs, miR-199a-3p, miR-127-3p, and miR-376c, were significantly decreased in osteosarcoma cell lines, whereas miR-151-3p and miR-191 were increased in osteosarcoma cell lines in comparison with osteoblasts. Transfection of precursor miR-199a-3p into osteosarcoma cell lines significantly decreased cell growth and migration, thus indicating that the inhibition effect is associated with an increase in the G(1)-phase and a decrease of the S-phase cell population. In addition, we observed decreased mTOR and Stat3 expression in miR-199a-3p transfected cells. This study provides new insights for miRNAs in osteosarcoma and suggests that miR-199a-3p may play a functional role in osteosarcoma cell growth and proliferation. Restoring miR-199a-3p's function may provide therapeutic benefits in osteosarcoma.