Molecular imaging of a cancer-targeting theragnostics probe using a nucleolin aptamer- and microRNA-221 molecular beacon-conjugated nanoparticle

MicroRNAs (miRNA, miR) have been reported as cancer biomarkers that regulate tumor suppressor genes. Hence, simultaneous detecting and inhibiting of miRNA function will be useful as a cancer theragnostics probe to minimize side effects and invasiveness. In this study, we developed a cancer-targeting therangostics probe in a single system using an AS1411 aptamer - and miRNA-221 molecular beacon (miR-221 MB)-conjugated magnetic fluorescence (MF) nanoparticle (MFAS miR-221 MB) to simultaneously target to cancer tissue, image intracellularly expressed miRNA-221 and treat miRNA-221-involved carcinogenesis. AS1411 aptamer-conjugated MF (MFAS) nanoparticles displayed a great selectivity and delivery into various cancer cell lines. The miR-221 MB detached from the MFAS miR-221 MB in the cytoplasm of C6 cells clearly imaged miRNA-221 biogenesis and simultaneously resulted in antitumor therapeutic effects by inhibiting miRNA function, indicating a successful astrocytoma-targeting theragnostics. MFAS miRNA MB can be easily applied to other cancers by simply changing a targeted miRNA highly expressed in cancers.     

MicroRNA-139-5p inhibits cell proliferation and invasion by targeting insulin-like growth factor 1 receptor in human non-small cell lung cancer

Introduction: Increasing evidence suggested that microRNAs (miRNAs) play a critical role in tumorigenesis. Decreased expression of miRNA-139-5p has been observed in various types of cancers. However, the biological function of miRNA-139-5p in non-small cell lung cancer (NSCLC) is still largely unknown. Methods: Quantitative real-time PCR (qRT-PCR) was used to explore the expression level of miRNA-139-5p in NSCLC tissues and cell lines. Then, we investigated the role of miRNA-139-5p to determine its potential roles on lung cancer cell proliferation, migration and invasion in vitro. A luciferase reporter assay was performed to confirm the target gene of miRNA-139-5p and the results were validated in renal cancer cells. Results: miRNA-139-5p was significantly decreased in NSCLC tissues and cell lines. Over-expression of miRNA-139-5p could inhibit lung cancer cell proliferation, migration, and invasion in vitro. Furthermore, we identified insulin-like growth factor 1 receptor (IGF1R) as a target of miR-139-5p and miR-139-5p function as a tumor suppressor via targeting IGF1R in NSCLC. Conclusions: Our results indicated that miR-139-5p acts as a tumor suppressor in NSCLC partially via down-regulating IGF1R expression.     

A frequent somatic mutation in CD274 3'-UTR leads to protein over-expression in gastric cancer by disrupting miR-570 binding

Inhibitory costimulatory molecule CD274 expresses in various cancers and contributes to cancer immune evasion by inhibiting T cell activation and proliferation, yet the regulatory mechanisms for CD274 overexpression in cancers are poorly understood. In this study, we discovered a novel mechanism of CD274 expression regulated by miR-570. A guanine-to-cytosine mutation at the 3'-UTR of CD274 mRNA led to CD274 overexpression by disrupting the miR-570 binding. The mutations were widely observed in cancers by sequencing of 276 gastrointestinal cancers (esophageal, gastric, colorectal, hepatocellular, and pancreatic cancers). This mutation was significantly associated with CD274 overexpression in gastric cancer (P = 1.44×10(-10)) and with the pathological features including differentiation grade, depth of tumor invasion, lymph node metastasis, and tumor-node-metastases (TNM) stage. These findings suggest a novel regulatory mechanism for CD274 overexpression in gastric cancer mediated by miR-570 and a somatic mutation in CD274 3'-UTR, and provide a new insight to gastric carcinogenesis.     

miRNA-519a在子宫内膜癌中的诊断及预后价值及机制探寻

目的利用TCGA数据库探寻miRNA-519a在子宫内膜癌中的诊断及预后价值及机制。方法通过对TCGA数据库中UCEC数据进行差异表达和生存分析,挑选出肿瘤组织中显著高表达且高表达提示预后不良的miRNA,并对该miRNA的靶基因进行富集分析,将富集出的关键通路中的mRNA与UCEC组织中低表达的mRNA交集,找出miRNA在UCEC中的靶基因和通路,并进一步利用双荧光素酶报告基因分析检测miRNA与靶基因mRNA的靶向结合进而调控其功能。结果TCGA-UCEC数据库中癌旁组织和癌组织存在不同miRNA差异表达,共148个miRNA上调,75个miRNA下调(|logFC|>2);进一步对差异表达miRNA进行预后分析发现,和正常组织相比,miRNA-519a在UCEC组织中显著高表达,且高表达的患者预后不良;通过mirDB数据库预测miRNA-519a靶基因(共685个),并用DAVID数据库将这些mRNA进行KEGG-PATHWAY富集分析,发现miRNA-519a的靶基因富集于肿瘤相关通路,且该通路相关基因中PDGFRA和TGFBR2在UCEC组织中显著低表达;双荧光素酶报告基因分析证明,miRNA-519a可与PDGFRA和TGFBR2 mRNA的3'-UTR直接结合并抑制两者的表达。结论miRNA-519a能够作为UCEC诊断和预后的生物标志物,miRNA-519a通过调控PDGFRA和TGFBR2的表达进而调控肿瘤的进程。     

Oncolytic adenovirus co-expressing miRNA-34a and IL-24 induces superior antitumor activity in experimental tumor model

It has been demonstrated that numerous microRNAs (miRNAs) have potent tumor-suppressing effects on a variety of cancers, implicating a possible application of miRNA in tumor therapy. Oncolytic adenovirus is a suitable vector to deliver tumor suppressor genes for treatment of cancers. However, it remains unknown whether co-expression of tumor suppressor genes and miRNAs can contribute to a more potent antitumor capacity within an oncolytic adenovirus delivery system. In this study, we found that expression of miRNA-34a was reduced in hepatocellular carcinoma (HCC), and the reduced expression of miRNA-34a was associated with worse outcome of HCC patients. Thus, we developed an oncolytic adenoviral vector, AdCN205, to co-express miRNA-34a and IL-24 driven by an adenovirus endogenous E3 promoter in HCC cells. High levels of miRNA-34a and IL-24 expression were detected in AdCN205-IL-24-miR-34a-infected HCC cells. AdCN205-IL-24-miR-34a significantly induced dramatic antitumor activity, as compared with that induced by AdCN205-IL-24 or AdCN205-miR-34a alone. Transfer of miRNA-34a into HCC cells inhibited the expression of its target genes, Bcl-2 and SIRT1. Treatment of established xenograft HCC tumors with AdCN205-IL-24-miR-34a in a mouse model resulted in complete tumor regression without recurrence. Taken together, our data provide a promising and reasonable delivery strategy of double-aimed cancer therapy, in which miRNAs and tumor-suppressing genes are used simultaneously.     

Knockdown of LncRNA SNHG7 inhibited epithelial-mesenchymal transition in prostate cancer though miR-324-3p/WNT2B axis in vitro

It is identified that long non-coding RNAs (lncRNAs) play important roles in cancer progression and metastasis. LncRNA SNHG7 was reported to play an oncogenic role in the progression of cancers including prostate cancer. However, its potential regulatory mechanism of endothelial-mesenchymal transition in PCa remains unclear. In this study, We found a lncRNA SNHG7 was overexpressed in PCa cell lines and tissues. LncRNA SNHG7 promotes prostate cancer migration and invasion by modulating EMT. Further study indicated that lncRNA SNHG7 acts as a sponge for miRNA-324-3p and positively regulates WNT2B by a sponge effect. Moreover, We confirmed that WNT2B, an important protein in the Wnt signal pathway, promotes the malignant phenotype of PCa cells and mediated the biological effects exerted by lncRNA SNHG7. Overall, our study suggested that lncRNA SNHG7 could promote PCa EMT via miR-324-3p and WNT2B in vitro. The lncRNA SNHG7/miR-324-3p /WNT2B axis regulatory network might provide a potential new therapeutic strategy for PCa treatment.     

MicroRNA-570 promotes lung carcinoma proliferation through targeting tumor suppressor KLF9

Increasing studies have shown that MicroRNAs (miRNAs) play critical roles in the progression of lung carcinoma. In the present study, the expression and functions of miR-570 were investigated. We found that miR-570 was significantly up-regulated in lung cancer tissues, compared with adjacent non-cancerous tissues. In vitro studies further showed that miR-570 mimics could promote, while its antisense oligos inhibit cell proliferation and invasion. At the molecular level, krüppel-like factor 9 (KLF9), a tumor suppressor gene, was identified as a potential target of miR-570 in lung cancer cells. Indeed, miR-570 could negatively regulate protein levels of KLF9 through targeting its 3’-untranslated region. Taken together, our results suggest a previously unknown miR-570/KLF9 molecular network controlling lung carcinoma progression.     

miRNA-101-3p靶向调控EZH2蛋白抑制胃癌细胞增殖和迁移

目的:研究微小RNA-101-3p(miRNA-101-3p)对人胃癌细胞增殖、凋亡和迁移的影响及其可能的调控机制。方法:Real-time PCR检测2种人胃癌细胞和1种胃黏膜细胞中miRNA-101-3p和zeste增强子同源物2(EZH2)的表达水平;采用脂质体瞬时转染技术过表达miRNA-101-3p;流式细胞术检测miRNA-101-3p对胃癌细胞周期和凋亡的影响;Transwell实验、CCK-8法和台盼蓝染色法检测miRNA-101-3p对胃癌细胞迁移和增殖能力的影响;Western blot法检测EZH2的表达。结果:miRNA-101-3p在胃癌细胞的表达水平显著低于胃黏膜细胞(P0.05);过表达miRNA-101-3p后,流式细胞术结果显示胃癌细胞的S期比例减少,G0/G1期比例增加,早期凋亡率增加(P0.05);CCK-8法、台盼蓝染色法及Transwell实验结果显示胃癌细胞的增殖和迁移能力显著降低(P0.05);Western blot结果显示胃癌细胞中EZH2蛋白的表达明显下降(P0.05)。结论:miRNA-101-3p可能通过靶向负调控EZH2蛋白的表达抑制胃癌细胞的增殖和迁移,进而促进胃癌细胞凋亡。     

Differential Expression of miRNAs in Papillary Thyroid Carcinoma Compared to Multinodular Goiter Using Formalin Fixed Paraffin Embedded Tissues

microRNAs (miRNAs) are approximately 22 nt RNAs that negatively regulate target gene expression. Their dysregulation has been implicated in the pathogenesis of a number of human cancers, including papillary thyroid carcinoma (PTC). Whereas previous studies using microarray technologies have largely relied on the ability to procure fresh tissue at the time of surgery to characterize miRNA signatures in PTC, we exploited the ability to procure sufficient miRNA from formalin-fixed paraffin-embedded (FFPE) tissue to describe a series of miRNAs whose expression is dysregulated in PTC compared to benign proliferative multinodular goiter (MNG). We identified 13 miRNAs upregulated and 26 miRNAs downregulated in PTC versus MNG. These include miRNA-21, miRNA-31, miRNA-221, and miRNA-222. Their dysregulation was further validated by real time RT-PCR analysis in an independent set of FFPE tissues. Many of these have previously been described in fresh tissue studies as altered in PTC, confirming the utility of this approach. These results further highlight the applicability of miRNA expression patterns as potential markers of human cancer, and our results suggest that FFPE tissues are suitable resources for such miRNA expression analyses. The ability to utilize FFPE tissue in the molecular characterization of human malignancy will unlock a rich resource for future cancer studies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

miRNA-212在乳腺癌的表达及其与临床病理特征的关系

目的分析miRNA-212在乳腺癌中的表达及其与临床病理特征的关系。方法选择90例乳腺癌患者的手术标本(癌组织及其癌旁组织)提取总RNA,采用TaqMan探针逆转、实时定量PCR检测miRNA-212在乳腺癌及其癌旁正常组织中的表达量,并分析其与临床病理特征关系。结果乳腺癌组织miRNA-212表达量为3.06(0.146-10.274),与癌旁正常组织相比显著上调;miRNA-212的表达与TNM分期相关,TNM分期越高,miRNA-212表达越高;miRNA-212的表达也与淋巴结转移相关,淋巴结转移患者miRNA-212表达增加。结论miRNA-212表达上调可能与乳腺癌的发生及其转移有关。     

miRNA-199 a和 miRNA-214通过负调控MAPK通路因子抑制宫颈癌细胞生长增殖

目的：研究 miRNA-199a和miRNA-214调控宫颈癌细胞生长增殖的分子机制。方法用细胞生长曲线实验和克隆形成实验研究 miRNA-199a和miRNA-214对宫颈癌细胞系 HeLa 细胞生长增殖的抑制作用;利用信息学手段及双荧光素酶报告基因方法筛选鉴定miRNA-199a和miRNA-214的靶基因,用Real-timePCR和Western印迹方法研究它们对靶基因表达量的影响。结果miRNA-199a和miRNA-214显著抑制了HeLa细胞生长增殖（P ＝0.010、0.036）;鉴定了MAPK通路的KRAS和MKK4（JNKK1／MAP 2K4）是miRNA-199a的靶基因,同时 KRAS 也是 miRNA-214的靶基因;miRNA-199a能够抑制HeLa细胞 KRAS 和 MKK 4的表达（P ＝0.231、0.011）,而miRNA-214也能够抑制 KRAS 的表达（P ＝0.029）。结论miRNA-199a和miRNA-214通过调节MAPK信号途径因子的表达,参与了宫颈癌细胞的生长增殖。     

MicroRNA-101 inhibits growth and metastasis of human ovarian cancer cells by targeting PI3K/AKT

IntroductionOvarian cancer is the most frequent cause of gynecological cancer related mortality in woman. This study was designed to investigate the role and therapeutic potential of miRNA-101 in ovarian cancer.Material and methodsExpression analysis was carried out by real-time quantitative polymerase chain reaction. Transfections were performed with the help of Lipofectamine 2000 reagent. AO/EB and annexin V/PI staining was used to detect apoptosis and flow cytometry was used for cell cycle analysis. Western blotting was employed for cell cycle analysis.ResultsIt was found that miRNA-101 was significantly down-regulated in ovarian cancer cells. The over-expression of miRNA-101 causes a significant decrease in the viability of ovarian cancer cells via the initiation of apoptosis and sub-G1 arrest of OVACAR-3 cells. It was indicated that PTEN was the potential target of miRNA-101 in OVACAR-3 cells. There was 4.5-fold up-regulation of PTEN expression in ovarian cancer cell lines and the over-expression of miRNA-101 in OVACAR-3 cells resulted in the down-regulation of PTEN expression. The inhibition of PTEN in the OVACAR-3 cells arrested the proliferation of these cells. The over-expression of miRNA-101 causes significant down-regulation in PI3K and AKT expression of OVACAR-3 cells.ConclusionsIt can be concluded that miRNA-101 acts as a tumor suppressor which may be beneficial in the treatment of ovarian cancer.     

Antagonism of microRNA-99a promotes cell invasion and down-regulates E-cadherin expression in pancreatic cancer cells by regulating mammalian target of rapamycin

MicroRNA-99a (miRNA-99a), a potential tumor suppressor, has been implicated in tumorigenesis of many human malignancies. However, the role of miRNA-99a in pancreatic cancer remains unclear. In the present study, we transfected miRNA-99a antagonism into human pancreatic cancer AsPC-1 cells to inhibit miRNA-99a expression and investigated its influence on cell migration and invasion as well as the underlying possible mechanisms. We found that miRNA-99a antagonism significantly increased proliferation, migration and invasion abilities of AsPC-1 cells, which was accompanied by increased expression of mesenchymal phenotype cell biomarkers (N-cadherin, Vimentin, and α-SMA), and decreased expression of epithelial phenotype cell biomarker (E-cadherin). Interestingly, small interfering RNA (siRNA)-mediated knockdown of mammalian target of rapamycin (mTOR) remarkably restored miRNA-99a antagonism-induced down-regulation of E-cadherin. In conclusion, our data suggest that miRNA-99a is involved in pancreatic cancer migration and invasion by regulating mTOR, and may provide a target for effective therapies against pancreatic cancer.     

Decreased expression of miR-378 correlates with tumor invasiveness and poor prognosis of patients with glioma

microRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in a variety of biological process. It has been reported that dysregulation of miRNA is always associated with cancer progression and development, and miR-378 aberrant expression has been found in some types of cancers. However, the association of miR-378 and glioma has not been evaluated. In this work, we measured the expression of miR-378 in glioma tissues and non-neoplastic brain tissues was measured using real-time PCR, and found that miRNA-378 expression level was significantly lower in glioma tissues compared with non-neoplastic brain tissues. Patients with lower miR-378 expression level had significantly poorer overall survival. Multivariate Cox regression analysis showed that miR-378 expression was an independent prognostic factor for 5-year overall survival. Over-expression of miR-378 inhibits glioma cell migration and invasion. In conclusion, our results indicated that miR-378 may serve as a tumor suppressor and play an important role in inhibiting tumor migration and invasion. Our work implicates the potential effect of miR-378 on the prognosis of glioma.     

miRNA-326调控胃癌细胞活力和凋亡的机制及其在胃癌组织中的表达及临床意义

目的:探讨微小RNA-326(miRNA-326)调控胃癌细胞活力和凋亡的机制及其在胃癌组织中的表达及临床意义。方法:采用RT-qPCR检测miRNA-326在55例胃癌组织中的表达情况,并分析其与临床病理特征的关系。以胃癌细胞BGC-823为研究对象,RT-qPCR检测miRNA-326在细胞中的表达;将BGC-823细胞随机分为正常对照组(未转染)、mimic-NC组(转染mimic阴性对照)和miRNA-326 mimic组(转染miRNA-326 mimic),以脂质体转染法上调miRNA-326表达后,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测基质金属蛋白酶9(MMP-9)、p21、细胞周期蛋白D1(cyclin D1)、Bcl-2和cleaved caspase-3的蛋白水平,RT-qPCR检测cyclin D1的mRNA表达,双萤光素酶报告基因检测法验证CCND1 (cyclin D1的基因)是否是miRNA-326的靶基因。结果:miRNA-326在胃癌组织中的表达明显低于癌旁组织(P 0. 05),其表达与肿瘤大小、淋巴结转移、分化程度及临床分期均有显著相关性(P 0. 05),而与患者的年龄和性别无关;此外,还与患者生存率密切相关(P 0. 05)。miRNA-326在BGC-823细胞中的表达明显低于正常胃黏膜GES-1细胞(P 0. 05)。与正常对照组相比,mimic-NC组细胞中miRNA-326的表达无明显变化,而miRNA-326 mimic组细胞中miRNA-326的表达明显升高(P 0. 05)。与正常对照组相比,miRNA-326 mimic组细胞活力明显减弱,而细胞凋亡增强(P 0. 05);此外,与正常对照组相比,miRNA-326 mimic组细胞中MMP-9、cyclin D1、Bcl-2蛋白及cyclin D1 mRNA表达下降,而p21和cleaved caspase-3的蛋白水平上升(P 0. 05)。然而,mimic-NC组与正常对照组相比,各检测指标的差异均无统计学显著性。与mimic-NC+miR-326 mimics组相比,转染pmiR-CCND1-WT质粒细胞的萤光素酶活性降低(P 0. 05),而转染pmiRCCND1-Mut质粒的萤光素酶活性无改变。结论:miRNA-326在胃癌组织中低表达,可能通过靶向调控CCND1表达而促进细胞活力并抑制细胞凋亡。     

miRNA-146a与肺部疾病的研究进展

MicroRNA（miRNA）是近二十年来分子生物学和遗传学领域的研究热点,随着人们对miRNA的不断研究和认识,发现miRNAs与吸入性肺损伤的发生发展密切相关。miRNA-146a是第一个被发现在免疫系统中具有调节作用的miRNA,是近年来研究较前沿、较广泛的miRNA之一,其下游基因介导参与了炎症、免疫、肿瘤的发生和发展。近年来,对miRNA-146a这种急性时相反应的负调节剂的研究越来越多,被认为是固有免疫及适应性免疫细胞分化及功能的重要调控者。本文首先从miRNA-146a在固有免疫、适应性免疫参与炎症负反馈机制出发,归纳了在Nuclear factorκB（NF-κB）信号通路中路径图,再通过回顾miRNA-146a在肺部疾病中的表达水平,对参与吸入性肺损伤及肺癌病程中分子机制及预后中作用的最新研究进展作一综述。     

miRNA-15a 在前列腺癌中的表达及诊断意义

目的:检测前列腺癌患者miRNA-15a 的表达情况,探讨其在前列腺癌诊断的意义。方法:选择2014 年1 月至2015 年1 月本院泌尿外科收治的前列腺癌患者血清及肿瘤组织36 例,良性前列腺增生患者(Prostatic hyperplasia,BPH)血清40 例,健康对照血清40 例。miRNA鄄15a 表达采用实时定量聚合酶链反应(Real鄄time PCR)技术检测。结果:miRNA-15a 在前列腺癌患者、良性前列腺增生患者和健康对照血清的表达量分别为(0.193±0.081)、(0.359±0.04)和(0.376±0.037),miRNA-15a 在前列腺癌患者血清中的表达量显著低于良性前列腺增生患者和健康对照(P<0.05),在BPH 组和对照组中的表达差异无统计学意义(P>0.05),前列腺癌患者血清miRNA-15a 表达在不同PSA 表达水平、Gleason 评分、临床分期、有无远处转移之间差异有统计学意义(P<0.05),与其他病理因素无关(P>0.05);前列腺癌患者肿瘤组织中的miRNA-15a 的表达与PSA 表达水平、Gleason 评分、临床分期、有无远处转移之间差异有统计学意义(P <0.05),与其他病理因素无关(P >0.05)。结论:miRNA-15a 低表达参与了前列腺癌的发生、发展过程,是前列腺癌辅助诊断及治疗的潜在的指标。     

结直肠癌患者化疗后miRNA-21、Ataxin-3、CD133表达情况及其与预后的关系分析

目的 探讨结直肠癌患者化疗后微小核糖核酸21(miRNA-21)、多聚谷氨酰胺蛋白3(Ataxin-3)、白细胞分化抗原133(CD133)表达情况及其与预后的关系.方法 收集2012年2月至2014年3月医院收治的93例结直肠癌患者的临床资料,按是否接受术前新辅助化疗(NC)分为NC组(n=43)与非NC组(n=50),所有患者均留取病理标本,有完整miRNA-21、Ataxin-3、CD133测定结果,分析化疗后结直肠癌患者癌组织上述因子表达情况及其与患者预后的关系.结果 NC组Ataxin-3阳性表达率、miRNA-21表达水平低于非NC组,CD133阳性表达率高于非NC组(P＜0.05);随化疗反应程度的降低,Ataxin-3阳性率降低,CD133阳性率上升,miRNA-21表达增加(P＜0.05);不同T分期、N分期、TNM分期及不同Ataxin-3、CD133、miRNA-21表达水平结直肠癌患者3年生存率比较有统计学意义(P ＜0.05);Ataxin-3表达水平与结直肠癌3年生存率呈正相关(P＜0.05),CD133、miRNA-21与结直肠癌3年生存率呈负相关(P＜0.05).结论 结直肠癌患者化疗后miRNA-21表达降低,Ataxin-3阳性率降低,CD133阳性率上升,且上述因子表达均与患者化疗反应性及预后有关.