基于生物信息学分析研究miR-182-5p通过靶向调控EP300促进乳腺癌细胞的侵袭和转移

近期研究表明,miR-182-5p对多种癌症的侵袭和转移具有重要作用,但其在乳腺癌侵袭转移中的研究相对较少。本研究通过网上在线microRNA分析工具下载乳腺癌组织及正常乳腺组织表达比较的数据集,分析发现在GSE4589、GSE38167、GSE61438等3个数据库中,在乳腺癌组织中存在26个相同的microRNA,其中8个上调,而我们实验验证发现hsa-miR-182在8例病理组织中的表达上调差异最显著(P=0.001),选定目的基因hsa-miR-182;qRT-PCR检测细胞中miR-182-5p的表达,结果显示,与MCF-10A相比,miR-182-5p在MDA-MB-231、T47D、MDA-MB-453、MCF-7中表达上调(P<0.05);转染miR-182-5p干扰质粒,qRT-PCR检测细胞中miR-182-5p的表达情况。结果显示,miR-182-5p表达显著降低(P=0.003),提示转染成功;Transwell侵袭结果显示,MDAMB-231细胞敲低miR-182-5p,与对照组相比,体外侵袭能力明显降低(P=0.002);Western印迹检测转染miR-182-5p干扰质粒时,MDA-MB-231中上皮-间质转化(epithelial-mesenchymal transition,EMT)相关标志物的表达情况,结果显示,与对照组相比,敲低miR-182-5p使细胞中上皮-钙黏着蛋白(E-cadherin)表达上调,神经-钙黏着蛋白(N-cadherin)、波形蛋白(vimentin)表达下调。为研究探讨miR-182-5p的靶蛋白,采用在线预测软件预测可能与miR-182-5p结合的靶蛋白,cytoscape构建蛋白质互作网络图并筛选出hub基因;双荧光素酶结果证实,miR-182-5p可与EP300靶向结合(P=0.001);采用qRT-PCR、Western印迹检测转染miR-182-5p干扰质粒后EP300在mRNA及蛋白质水平的表达,结果显示,与对照组相比,在敲低miR-182-5p组中EP300在mRNA及蛋白质的表达上调(P=0.001)。综上所述,miR-182-5p可靶向调节EP300,促进乳腺癌细胞的侵袭与转移。     

MiR-150-5p通过靶向调控Rab1A抑制乳腺癌细胞MDA-231的上皮-间质转化

先前的研究表明,miR-150-5p发挥抑癌基因的作用,调控肿瘤细胞的侵袭与转移。然而,关于其在乳腺癌细胞侵袭与转移中的机制尚不明确。本实验旨在研究miR-150-5p负向调控Rab1A在乳腺癌细胞上皮-间质转化（epithelial-mesenchymal transition,EMT）中的作用。双荧光素酶的结果显示,miR-150-5p可负向调控Rab1A。荧光定量PCR (qRT-PCR) 结果显示,miR-150-5p在乳腺癌细胞MCF-7及MDA-MB-231（MDA-231）中的表达水平明显低于正常乳腺上皮细胞MCF-10A; 在MDA-231中过表达miR-150-5p后,qRT-PCR结果显示,Rab1A mRNA的表达水平明显降低。Western印迹结果显示,过表达miR-150-5p后,miR-150-5p组细胞中的Rab1A、波形蛋白(vimentin)及N-钙黏着蛋白(N-cadherin)的表达水平相对于对照组（NC）细胞明显降低,而E-钙黏着蛋白(E-cadherin)的表达水平明显增加。Transwell侵袭和划痕实验显示,与miR-150-5p+Con组细胞相比,miR-150-5p+Rab1A组细胞的侵袭和迁移能力明显增加。qRT-PCR结果显示,miR-150-5p+Rab1A组细胞的Rab1A mRNA表达水平明显增加。Western印迹结果显示,miR-150-5p+Rab1A组细胞中的波形蛋白、N-钙黏着蛋白表达水平明显增加, 而E-钙黏着蛋白表达明显降低,过表达Rab1A后显著逆转了miR-150-5p对EMT的影响。综上所述,miR-150-5p可以通过负向调控Rab1A抑制EMT,进而抑制乳腺癌细胞的侵袭和迁移。     

miR-222 and miR-29a contribute to the drug-resistance of breast cancer cells

Adriamycin (Adr) and docetaxel (Doc) are two chemotherapeutic agents commonly used in the treatment of breast cancer. However, patients with breast cancer who are treated by the drugs often develop resistance to them and some other drugs. Recently studies have shown that microRNAs (miRNAs, miRs) play an important role in drug-resistance. In present study, miRNA expression profiles of MCF-7/S and its two resistant variant MCF-7/Adr and MCF-7/Doc cells were analyzed using microarray and the results were confirmed by real-time quantitative polymerase chain reaction. Here, 183 differentially expressed miRNAs were identified in the two resistant sublines compared to MCF-7/S. Then, five up-regulated miRNAs (miR-100, miR-29a, miR-196a, miR-222 and miR-30a) in both MCF-7/Adr and MCF-7/Doc were selected to explore their roles in acquisition of drug-resistance using transfection experiment. The results showed that miR-222 and miR-29a mimics and inhibitors had partially changed the drug-resistance of breast cancer cells, which was also confirmed by apoptosis assay. Western blot results suggested that miR-222 and -29a could regulate the expression of PTEN, maybe through which the two miRNAs conferred Adr and Doc resistance in MCF-7 cells. Finally, pathway mapping tools were employed to further analyze signaling pathways affected by the two miRNAs. In summary, this study demonstrates that altered miRNA expression pattern is involved in acquiring resistance to Adr and Doc in breast cancer MCF-7 cells, and that there are some miRNAs who displayed consistent up- or down-regulated expression changes in the two resistant sublines. The most importance is that we identify two miRNAs (miR-222 and miR-29a) involved in drug-resistance, at least in part via targeting PTEN.     

Down-regulation of MicroRNAs (MiRs) 203, 887, 3619 and 182 Prevents Vimentin-triggered,Phospholipase D (PLD)-mediated Cancer Cell Invasion

Breast cancer is a leading cause of morbidity and mortality among women. Metastasis is initiated after epithelial-mesenchymal-transition (EMT). We have found a connection between EMT markers and the expression of four microRNAs (miRs) mediated by the signaling enzyme phospholipase D (PLD). Low aggressive MCF-7 breast cancer cells have low endogenous PLD enzymatic activity and cell invasion, concomitant with high expression of miR-203, -887, and -3619 (that decrease PLD2 translation and a luciferase reporter) and miR-182 (targeting PLD1) that are, therefore, “tumor-suppressor-like” miRs. The combination miR-887+miR-3619 abolished >90% of PLD enzymatic activity. Conversely, post-EMT MDA-MB-231 cells have low miR expression, high levels of PLD1/2, and high aggressiveness. The latter was reversed by ectopically transfecting the miRs, which was negated by silencing miRs with specific siRNAs. We determined that the molecular mechanism is that E-cadherin triggers expression of the miRs in pre-EMT cells, whereas vimentin dampens expression of the miRs in post-EMT invasive cells. This novel work identifies for the first time a set of miRs that are activated by a major pre-EMT marker and deactivated by a post-EMT marker, boosting the transition from low invasion to high invasion, as mediated by the key phospholipid metabolism enzyme PLD.     

miR-100 Induces Epithelial-Mesenchymal Transition but Suppresses Tumorigenesis,Migration and Invasion

Whether epithelial-mesenchymal transition (EMT) is always linked to increased tumorigenicity is controversial. Through microRNA (miRNA) expression profiling of mammary epithelial cells overexpressing Twist, Snail or ZEB1, we identified miR-100 as a novel EMT inducer. Surprisingly, miR-100 inhibits the tumorigenicity, motility and invasiveness of mammary tumor cells, and is commonly downregulated in human breast cancer due to hypermethylation of its host gene MIR100HG. The EMT-inducing and tumor-suppressing effects of miR-100 are mediated by distinct targets. While miR-100 downregulates E-cadherin by targeting SMARCA5, a regulator of CDH1 promoter methylation, this miRNA suppresses tumorigenesis, cell movement and invasion in vitro and in vivo through direct targeting of HOXA1, a gene that is both oncogenic and pro-invasive, leading to repression of multiple HOXA1 downstream targets involved in oncogenesis and invasiveness. These findings provide a proof-of-principle that EMT and tumorigenicity are not always associated and that certain EMT inducers can inhibit tumorigenesis, migration and invasion.     

Long noncoding RNA SNHG15 promotes human breast cancer proliferation,migration and invasion by sponging miR-211-3p

Long non-coding RNAs (lncRNA) have been demonstrated to act as essential regulators in the development and progression of breast cancer. In our study, we found that long noncoding RNA SNHG15 was highly expressed in breast cancer tissues and cell lines. And the expression of SNHG15 was correlated with TNM stage, lymphnode metastasis and survival in breast cancer patients. SNHG15 knockdown significantly inhibited the proliferation and induced apoptosis in breast cancer cells in vitro and in vivo. Besides, SNHG15 downregulation suppressed cell migration and invasion in MCF-7 and BT-20 cells, and inhibited epithelial-mesenchymal transition (EMT). In mechanism, we found that SNHG15 acted as a competing endogenous RNA to sponge miR-211-3p, which was downregulated in breast cancers and inhibited cell proliferation and migration. Our results showed that there was a negative correlation between SNHG15 and miR-211-3p expression in breast cancer patients. Collectively, we, for the first time, revealed the functions of SNHG15 and miR-211-3p in breast cancer.     

miR-219-5p targets TBXT and inhibits breast cancer cell EMT and cell migration and invasion

miR-219-5p has been reported to act as either a tumor suppressor or a tumor promoter in different cancers by targeting different genes. In the present study, we demonstrated that miR-219-5p negatively regulated the expression of TBXT, a known epithelial–mesenchymal transition (EMT) inducer, by directly binding to TBXT 3′-untranslated region. As a result of its inhibition on TBXT expression, miR-219-5p suppressed EMT and cell migration and invasion in breast cancer cells. The re-introduction of TBXT in miR-219-5p overexpressing cells decreased the inhibitory effects of miR-219 on EMT and cell migration and invasion. Moreover, miR-219-5p decreased breast cancer stem cell (CSC) marker genes expression and reduced the mammosphere forming capability of cells. Overall, our study highlighted that TBXT is a novel target of miR-219-5p. By suppressing TBXT, miR-219-5p plays an important role in EMT and cell migration and invasion of breast cancer cells.     

RP4 通过miR-183-5p.1上调FOXO1抑制乳腺癌细胞增殖

近年来,越来越多的证据表明,长非编码RNAs在肿瘤发生发展中发挥重要作用。位于12号染色体的长非编码RNA RP4-816N1.7（简称RP4）在乳腺癌细胞中的作用未见报道。我们通过实时荧光定量PCR证实,RP4在乳腺癌细胞中的表达量普遍低于其在正常乳腺上皮细胞MCF-10A中的表达量。RP4在MCF-7和MDA-MB-231中表达量分别比其在MCF-10A中的表达量下调21.57%和91.33%。过表达RP4可明显抑制乳腺癌细胞增殖。敲低RP4可显著增加乳腺癌细胞的增殖能力。生物信息学预测,RP4可能与miR-183-5p.1结合,且叉头蛋白O1(FOXO1)可能是miR-183-5p.1的潜在靶标。实时荧光定量PCR结果提示,RP4可下调miR-183-5p.1,而miR-183-5p.1也可下调RP4和FOXO1的表达。双荧光素酶报告基因结果证实,miR-183-5p.1可与RP4结合,下调其表达,也能与FOXO1 3′UTR结合,抑制其mRNA和蛋白质水平的表达量。最后,本文通过BrdU实验证实,RP4通过FOXO1抑制乳腺癌细胞的增殖。总之,RP4通过内源性结合miR-183-5p.1,上调FOXO1表达,进而抑制乳腺癌细胞增殖。     

Re-expression of miR-21 contributes to migration and invasion by inducing epithelial-mesenchymal transition consistent with cancer stem cell characteristics in MCF-7 cells

MiR-21 is known to play an important role in the development and progression, including migration and invasion, of many malignancies including breast cancer. Accumulating evidence suggest that the induction of epithelial-mesenchymal transition (EMT) phenotype and acquisition of cancer stem cell (CSC) characteristics are highly interrelated, and contribute to tumorigenesis, tumor progression, metastasis, and relapse. The molecular mechanisms underlying EMT and CSC characteristics during miR-21 contributes to cell migration and invasion of breast cancer are poorly understood. Therefore, we established miR-21 re-expressing breast cancer MCF-7 (MCF-7/miR-21) cells, which showed increasing cell growth, migration and invasion, self-renewal and clonogenicity. Our data showed that re-expression of miR-21 induced the acquisition of EMT phenotype by activation of mesenchymal cell markers (N-cadherin, Vimentin, α-SMA) and inhibition of epithelial cell marker (E-cadherin) in MCF-7/miR-21 cells, which consistent with increased cell subpopulation expressing CSC surface markers (ALDH1(+) and CD44(+)/CD24(-/low)) and the capacity of sphereforming (mammospheres). Our results demonstrated that re-expression of miR-21 is responsible for migration and invasion by activating the EMT process and enhancing the characteristics of CSCs in MCF-7 cells.     

Synthetic miRNA-Mowers Targeting miR-183-96-182 Cluster or miR-210 Inhibit Growth and Migration and Induce Apoptosis in Bladder Cancer Cells

Background

MicroRNAs (miRNAs) function as endogenous regulators of biological behaviors of human cancers. Several natural non-coding RNAs are reported to inhibit miRNAs by base-pairing interactions. These phenomena raise questions about the ability of artificial device to regulate miRNAs. The purpose of this study is to create synthetic devices that target a single miRNA or a miRNA cluster and to ascertain their therapeutic effects on the phenotypes of bladder cancer cells.

Methodology/Principal Findings

Tandem bulged miRNA binding sites were inserted into the 3′ untranslated region (UTR) of the SV-40 promoter-driven Renilla luciferase gene to construct two “miRNA-mowers” for suppression of miR-183-96-182 cluster or miR-210. A third device with tandem repeat sequences not complementary to any known miRNA was generated as an untargeted-control. In functional analyses, bladder cancer T24 and UM-UC-3 cells were transfected with each of the three devices, followed by assays for detection of their impacts. Luciferase assays indicated that the activities of the luciferase reporters in the miRNA-mowers were decreased to 30–50% of the untargeted-control. Using Real-Time qPCR, the expression levels of the target miRNAs were shown to be reduced 2-3-fold by the corresponding miRNA-mower. Cell growth, apoptosis, and migration were tested by MTT assay, flow cytometry assay, and in vitro scratch assay, respectively. Cell growth inhibition, increased apoptosis, and decreased motility were observed in miRNA-mowers-transfected bladder cancer cells.

Conclusions/Significance

Not only a single target miRNA but also the whole members of a target miRNA cluster can be blocked using this modular design strategy. Anti-cancer effects are induced by the synthetic miRNA-mowers in the bladder cancer cell lines. miR-183/96/182 cluster and miR-210 are shown to play oncogenic roles in bladder cancer. A potentially useful synthetic biology platform for miRNA loss-of-function study and cancer treatment has been established in this work.     

miR-17-5p promotes proliferation and epithelial-mesenchymal transition in human osteosarcoma cells by targeting SRC kinase signaling inhibitor 1

MicroRNA-17-5p (miR-17-5p) and epithelial-mesenchymal transition (EMT) have been reported to participate in the development and progression of multiple cancers. However, the relationship between the miR-17-5p and EMT in osteosarcoma (OS) is still poorly understood. This study was to investigate the effects of the miR-17-5p and its potential mechanism in regulating proliferation, apoptosis, and EMT of human OS. Quantitative real-time PCR was used to detect the miR-17-5p and SRC kinase signaling inhibitor 1 (SRCIN1) messenger RNA expression in OS specimens and cell lines. After transfection with miR-17-5p inhibitors, proliferation, apoptosis, migration, and invasion of OS cells were assessed by using the Cell Counting Kit-8, the annexin V-FITC apoptosis, wound-healing, and transwell assays. The SRCIN1 was validated as a target of the miR-17-5p through bioinformatics algorithms and luciferase reporter assay. Moreover, the expression of EMT markers, E-cadherin, N-cadherin, and Snail was identified by the Western blot analysis. MiR-17-5p was significantly upregulated in OS tumor samples and cell lines. It inhibited proliferation and EMT, and promoted apoptosis in OS. The SRCIN1 was identified as a direct target of the miR-17-5p. Silenced miR-17-5p could change the expression of EMT markers, such as upregulating the expression of E-cadherin, and downregulating the expression of N-cadherin and Snail through targeting the antioncogenic SRCIN1. These findings suggest that the miR-17-5p promotes cell proliferation, and EMT in human OS by directly targeting the SRCIN1, and reveal a branch of the miR-17-5p/SRCIN1/EMT signaling pathway involved in the progression of OS.     

miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

目的：通过敲低微小RNA （microRNA,miRNA）-449a的方法研究miR-449a对人乳腺癌细胞MCF-7的增殖和迁移能力的影响。方法：采用miRNA芯片在乳腺癌细胞MCF-7和人正常乳腺细胞MCF-10A筛选具有表达差异的miRNA;化学合成法制备miR-449a的抑制剂（inhibitor）,转染后经real-time PCR验证表达的变化;细胞增殖CCK-8实验对转染后细胞增殖能力进行检测;划痕实验检测细胞转移能力,transwell小室实验检测细胞侵袭的改变;蛋白免疫印迹法（Western blot）实验对MCF-7细胞增殖和迁移相关的β-catenin和E-cadherin蛋白进行检测;通过生物信息学软件预测miR-449a潜在靶基因为Notch 1,荧光素酶实验检测Notch 1是miR-449a的靶基因。结果：分别收集MCF-7和MCF-10A细胞,芯片结果显示miR-449a在MCF-7细胞的表达水平显著高于MCF-10A;本研究将细胞分为未处理组（Mock组）,阴性对照组（negative control组,NC组）和处理组,通过收集不同组MCF-7细胞进行试验,CCK-8结果显示miR-449a下调后MCF-7细胞增殖能力显著降低;划痕实验结果显示miR-449a表达降低导致MCF-7细胞转移能力降低;transwell实验结果显示MCF-7细胞侵袭受到抑制;Western blot结果发现miR-449a敲低后β-catenin表达降低,E-cadherin表达增加;荧光素酶试验结果显示,miR-449a能够显著降低Notch 1-3'-UTR质粒的荧光素活性（P<0.01）。结论：在乳腺癌细胞MCF-7中敲低miR-449a能够显著抑制癌细胞增殖和迁移,而这一变化可能通过降低Notch 1蛋白表达实现的。     

miR-183-96-182 cluster is overexpressed in prostate tissue and regulates zinc homeostasis in prostate cells

Decreased zinc levels are a hallmark of prostate cancer tumors as zinc uniquely concentrates in healthy prostate tissue. Increased dietary zinc correlates with decreased risk of advanced prostate cancer and decreased mortality from prostate cancer. The mechanisms of prostatic zinc homeostasis are not known. Lower zinc levels in the tumor are correlated directly with decreased expression of the zinc transporter hZIP1. We report identification of a microRNA cluster that regulates multiple zinc transporters, including hZIP1. Screening in laser capture microdissected prostate cancer tumors identified miR-182 as a potential regulator of hZIP1. Regulation of hZIP1 by miR-182 via two binding sites was confirmed in primary prostate cell cultures. miR-96 and miR-183 are expressed as a cluster with miR-182 and share similar sequences. Array profiling of tissue showed that miR-183, -96, and -182 are higher in prostate cancer tissue compared with normal prostate. Overexpression of the entire miR-183-96-182 cluster suppressed five additional zinc transporters. Overexpression of miR-183, -96, and -182 individually or as a cluster diminished labile zinc pools and reduced zinc uptake, demonstrating this miR cluster as a regulator of zinc homeostasis. We observed regulation of zinc homeostasis by this cluster in prostate cells and HEK-293 cells, suggesting a universal mechanism that is not prostate-specific. To our knowledge, this is the first report of a miR cluster targeting a family of metal transport proteins. Individually or as a cluster, miR-183, -96, and -182 are overexpressed in other cancers too, implicating this miR cluster in carcinogenesis.     

Role of Deregulated microRNAs in Breast Cancer Progression Using FFPE Tissue

MicroRNAs (miRNAs) contribute to cancer initiation and progression by silencing the expression of their target genes, causing either mRNA molecule degradation or translational inhibition. Intraductal epithelial proliferations of the breast are histologically and clinically classified into normal, atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). To better understand the progression of ductal breast cancer development, we attempt to identify deregulated miRNAs in this process using Formalin-Fixed, Paraffin-Embedded (FFPE) tissues from breast cancer patients. Following tissue microdissection, we obtained 8 normal, 4 ADH, 6 DCIS and 7 IDC samples, which were subject to RNA isolation and miRNA expression profiling analysis. We found that miR-21, miR-200b/c, miR-141, and miR-183 were consistently up-regulated in ADH, DCIS and IDC compared to normal, while miR-557 was uniquely down-regulated in DCIS. Interestingly, the most significant miRNA deregulations occurred during the transition from normal to ADH. However, the data did not reveal a step-wise miRNA alteration among discrete steps along tumor progression, which is in accordance with previous reports of mRNA profiling of different stages of breast cancer. Furthermore, the expression of MSH2 and SMAD7, two important molecules involving TGF-β pathway, was restored following miR-21 knockdown in both MCF-7 and Hs578T breast cancer cells. In this study, we have not only identified a number of potential candidate miRNAs for breast cancer, but also found that deregulation of miRNA expression during breast tumorigenesis might be an early event since it occurred significantly during normal to ADH transition. Consequently, we have demonstrated the feasibility of miRNA expression profiling analysis using archived FFPE tissues, typically with rich clinical information, as a means of miRNA biomarker discovery.