Altered microRNA expression in cisplatin-resistant ovarian cancer cells and upregulation of miR-130a associated with MDR1/P-glycoprotein-mediated drug resistance

microRNAs (miRNAs) are short non-coding RNA molecules which are involved in the regulation of various biological processes. Drug resistance has become a major obstacle to successful chemotherapy of ovarian cancer. The aim of this study was to investigate microRNA expression profiles in cisplatin-resistant ovarian cancer cells and the role of miR-130a in regulating drug resistance. Analysis of differentially expressed miRNAs between SKOV3 and SKOV3/CIS cells was assessed by miRNA microarrays. Target prediction of miRNAs was determined with the help of PicTar or TargetScan. Among these miRNAs, the expression of miR?130a was verified using qRT-PCR. The expression of MDR1 mRNA and P-glycoprotein (P-gp) after cellular transfection was examined using qRT-PCR and western blotting, respectively. Cisplatin sensitivity was detected by the MTT assay. We indentified 35 downregulated and 54 upregulated miRNAs in SKOV3/CIS compared to those in SKOV3. We found that miR-130a was upregulated in SKOV3/CIS compared to the parental SKOV3 cells, and PTEN was predicted to be the potential target of miR-130a. Moreover, downregulation of miR-130a could inhibit MDR1 mRNA and P-gp expression and overcome the cisplatin resistance in SKOV3/CIS cells, which indicated that miR-130a may be associated with MDR1/P-gp-mediated drug resistance and plays the role of an intermediate in drug-resistance pathways of PI3K/Akt/PTEN/mTOR and ABC superfamily drug transporters in SKOV3/CIS cells. This study provides important information for the development of targeted gene therapy for reversing cisplatin resistance in ovarian cancer.     

Systematic analysis of microRNAs targeting the androgen receptor in prostate cancer cells

Androgen receptor (AR) is expressed in all stages of prostate cancer progression, including in castration-resistant tumors. Eliminating AR function continues to represent a focus of therapeutic investigation, but AR regulatory mechanisms remain poorly understood. To systematically characterize mechanisms involving microRNAs (miRNAs), we conducted a gain-of function screen of 1129 miRNA molecules in a panel of human prostate cancer cell lines and quantified changes in AR protein content using protein lysate microarrays. In this way, we defined 71 unique miRNAs that influenced the level of AR in human prostate cancer cells. RNA sequencing data revealed that the 3'UTR of AR (and other genes) is much longer than currently used in miRNA target prediction programs. Our own analyses predicted that most of the miRNA regulation of AR would target an extended 6 kb 3'UTR. 3'UTR-binding assays validated 13 miRNAs that are able to regulate this long AR 3'UTR (miR-135b, miR-185, miR-297, miR-299-3p, miR-34a, miR-34c, miR-371-3p, miR-421, miR-449a, miR-449b, miR-634, miR-654-5p, and miR-9). Fifteen AR downregulating miRNAs decreased androgen-induced proliferation of prostate cancer cells. In particular, analysis of clinical prostate cancers confirmed a negative correlation of miR-34a and miR-34c expression with AR levels. Our findings establish that miRNAs interacting with the long 3'UTR of the AR gene are important regulators of AR protein levels, with implications for developing new therapeutic strategies to inhibit AR function and androgen-dependent cell growth.     

MicroRNA expression profiling involved in doxorubicin-induced cardiotoxicity using high-throughput deep-sequencing analysis

MicroRNAs (miRNAs/miRs) are sensitive biomarkers and endogenous repressors of gene expression by decreasing mRNA stability and interfering with mRNA translation. Despite a number of investigations revealing the dysregulation of miRNA expression associated with cardiotoxicity induced by doxorubicin (Dox), perturbation of miRNAs directly resulting from Dox at early stage in cardiomyocytes and the target gene interaction remain largely unknown. In the present study, high-throughput deep-sequencing was used to analyze changes in global miRNA expression in H9c2 cardiomyocytes exposed to 5 µg/ml Dox for 0, 12 or 24 h. Compared with the 0-h time point, the expression levels of 386 unique miRNAs were altered. Based on miRNA expression and fold-change, the target genes of 76 selected miRNAs were further analyzed using gene interaction networks and pathway enrichment analysis. These miRNAs were involved in the regulation of different pathways, whose functions included apoptosis, cell proliferation, extracellular matrix remodeling, oxidative stress and lipid metabolism. These differentially expressed miRNAs included let-7 family, miR-29b-3p, miR-378-3/5p, miR-351-3p, miR-664-3p, miR-455-3p, miR-298-3p, miR-702-5p, miR-128-1-5p, miR-671 and miR-421-5p. The present data indicated that global wide miRNA profiling in Dox-induced cardiomyocytes may provide a novel mechanistic insight into understanding Dox-induced heart failure and cardiotoxicity, as well as novel biomarkers and therapeutic targets.     

miR-10a-5p基因甲基化对卵巢癌铂类耐药的影响

目的 卵巢癌是死亡率最高的妇科肿瘤,较强的化疗耐药性是其预后差的主要原因之一,为了阐明卵巢癌对铂类药物的耐药机制,本研究探讨miRNA基因的甲基化水平对卵巢癌铂类耐药的影响.方法 将卵巢癌组织分为敏感组和耐药组,每组各3例;采用基因芯片技术,对比分析了两组微RNA(microRNA,miRNA)的表达差异;采用实时荧光定量PCR,分别在6例敏感和3例耐药组织、铂类药物敏感(CoC1)和耐药的卵巢癌细胞系(CoC1/DDP),检测了候选miRNA的表达差异;应用Massarray技术,检测敏感组织(15例)与耐药组织(6例)中miRNA基因启动子的甲基化差异;应用生物信息学分析,鉴定目标miRNA的潜在靶基因.结果 以铂类药物敏感的卵巢癌组织样本为对照,利用基因芯片筛选,鉴定了6条在耐药组织样本中出现表达上调的miRNA(miR-493-3p、miR-10a-5 p、miR-16-2-3p、miR-1248、miR-451a、miR-628-3p)和6条表达下调的miRNA(miR-509-3p、miR-1197、miR-376a-3p、miR-1273a、miR-550a-3p、miR-19b-3p).组织验证发现,miR-509-3p、miR-493-3p、miR-10a-5p、miR-16-2-3p和miR-451a,与芯片结果一致;培养细胞研究发现,4条miRNA的表达调控方式与组织芯片结果一致,miR-10a-5p、miR-16-2-3p、miR-1248和miR-628-3p在耐药细胞系中高表达.进一步研究发现,与敏感肿瘤组织相比,耐药组织中miR-10a-5p基因启动子的甲基化水平出现显著降低,P=0.04.结合生物信息学预测HOXA1和USF2为miR-10a-5p与耐药相关的靶基因.结论 与敏感组相比,耐药组miR-10a-5p基因启动子甲基化水平显著降低,miR-10a-5p表达升高,通过抑制 HOXA1和USF2,抑制细胞凋亡,导致铂类化疗药物耐受.     

HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer

miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA–target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA–mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.     

miR-212／132簇生物学功能研究进展

目的：总结miR-132和miR-212对肿瘤发生发展的作用及其在神经免疫等领域的生物学功能。方法：应用PubMed及万方数据库检索系统,以“miRNA、miR-212／132、miR-212及miR-132”为检索词,检索1990-2013年相关文献。共检索到中文文献305条,英文文献228条。纳入标准：1）miRNA的作用;2）miR-212／132基因簇结构;3）miR-212／132基因簇的生物学功能。根据纳入标准符合分析的文献55篇。结果：microRNAs是一类具有转录后调控功能的非编码小RNA,参与细胞增殖、凋亡和分化。miR-132和miR-212在脊椎动物中有着相似并且高度保守的序列,位于人染色体17p13．3,在基因组中呈现串联排列,称为miR-212／132基因簇。miR-132和miR212由miR-212／132基因簇共同转录剪切形成。miR-212／132簇不但与肿瘤的发生发展相关,而且在神经元的发育、成熟及功能中起到重要作用。它们还参与了生物钟形成并与免疫调节相关。结论：miR-212／132基因簇在肿瘤、神经及免疫等领域均发挥重要作用,因此对于miR-212／miR-132簇的深入研究必将给多种疾病的治疗带来希望。     

Roles for microRNAs, miR-93 and miR-130b, and tumor protein 53-induced nuclear protein 1 tumor suppressor in cell growth dysregulation by human T-cell lymphotrophic virus 1

A role for microRNAs (miRNA) in human T-cell leukemia virus 1 (HTLV-1)-mediated cellular transformation has not been described. Here, we profiled miRNA expression in HTLV-1-transformed human T-cell lines and primary peripheral blood mononuclear cells from adult T-cell leukemia patients. Analyses of 11 different profiles revealed six miRNAs that were consistently up-regulated. Two of the up-regulated miRNAs (miR-93 and miR-130b) target the 3' untranslated region (3'UTR) of the mRNA for a tumor suppressor protein, tumor protein 53-induced nuclear protein 1 (TP53INP1). A low expression level of TP53INP1 protein was found in HTLV-1-transformed cells. Additionally, when antagomirs were used to knock down miR-93 and miR-130b in these cells, the expression of TP53INP1 was increased, suggesting that the latter is regulated inside cells by the former. A role for TP53INP1 in regulating cell growth was established by experiments that showed that enhanced TP53INP1 expression increased apoptosis. Collectively, the findings implicate a miR-93/miR-130b-TP53INP1 axis that affects the proliferation and survival of HTLV-1-infected/transformed cells.     

miR-195, miR-455-3p and miR-10a are implicated in acquired temozolomide resistance in glioblastoma multiforme cells

To identify microRNAs (miRNAs) specifically involved in the acquisition of temozolomide (TMZ) resistance in glioblastoma multiforme (GBM), we first established a resistant variant, U251R cells from TMZ-sensitive GBM cell line, U251MG. We then performed a comprehensive analysis of miRNA expressions in U251R and parental cells using miRNA microarrays. miR-195, miR-455-3p and miR-10a^∗ were the three most up-regulated miRNAs in the resistant cells. To investigate the functional role of these miRNAs in TMZ resistance, U251R cells were transfected with miRNA inhibitors consisting of DNA/LNA hybrid oligonucleotides. Suppression of miR-455-3p or miR-10a^∗ had no effect on cell growth, but showed modest cell killing effect in the presence of TMZ. On the other hand, knockdown of miR-195 alone displayed moderate cell killing effect, and combination with TMZ strongly enhanced the effect. In addition, using in silico analysis combined with cDNA microarray experiment, we present possible mRNA targets of these miRNAs. In conclusion, our findings suggest that those miRNAs may play a role in acquired TMZ resistance and could be a novel target for recurrent GBM treatment.