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.     

Human microRNA similarity in breast cancer

MicroRNAs (miRNAs) play important roles in a variety of human diseases, including breast cancer. A number of miRNAs are up- and down-regulated in breast cancer. However, little is known about miRNA similarity and similarity network in breast cancer. Here, a collection of 272 breast cancer-associated miRNA precursors (pre-miRNAs) were utilized to calculate similarities of sequences, target genes, pathways and functions and construct a combined similarity network. Well-characterized miRNAs and their similarity network were highlighted. Interestingly, miRNA sequence-dependent similarity networks were not identified in spite of sequence–target gene association. Similarity networks with minimum and maximum number of miRNAs originate from pathway and mature sequence, respectively. The breast cancer-associated miRNAs were divided into seven functional classes (classes I–VII) followed by disease enrichment analysis and novel miRNA-based disease similarities were found. The finding would provide insight into miRNA similarity, similarity network and disease heterogeneity in breast cancer.     

Discovery of novel human breast cancer microRNAs from deep sequencing data by analysis of pri-microRNA secondary structures

MicroRNAs (miRNAs) are key regulators of gene expression and contribute to a variety of biological processes. Abnormal miRNA expression has been reported in various diseases including pathophysiology of breast cancer, where they regulate protumorigenic processes including vascular invasiveness, estrogen receptor status, chemotherapy resistance, invasion and metastasis. The miRBase sequence database, a public repository for newly discovered miRNAs, has grown rapidly with approximately >10,000 entries to date. Despite this rapid growth, many miRNAs have not yet been validated, and several others are yet to be identified. A lack of a full complement of miRNAs has imposed limitations on recognizing their important roles in cancer, including breast cancer. Using deep sequencing technology, we have identified 189 candidate novel microRNAs in human breast cancer cell lines with diverse tumorigenic potential. We further show that analysis of 500-nucleotide pri-microRNA secondary structure constitutes a reliable method to predict bona fide miRNAs as judged by experimental validation. Candidate novel breast cancer miRNAs with stem lengths of greater than 30 bp resulted in the generation of precursor and mature sequences in vivo. On the other hand, candidates with stem length less than 30 bp were less efficient in producing mature miRNA. This approach may be used to predict which candidate novel miRNA would qualify as bona fide miRNAs from deep sequencing data with approximately 90% accuracy.     

Blockade of invasion and metastasis of breast cancer cells via targeting CXCR4 with an artificial microRNA

miRNAs have been shown to function as regulatory molecules and to play an important role in cancer progression. Very little is currently known about the increasing invasion and metastasis of breast cancer due to the loss of expressive levels of certain miRNAs in breast tumor cells. In order to determine whether the CXCR4/SDF-1 pathway is regulated by expression of miRNAs, we designed and synthesized pre-miRNA against CXCR4. This double-stranded miRNA gene was ligated with a miR-155-based Block-iT Pol II miR RNAi Expression Vector (Invitrogen). Expression levels of CXCR4 in CXCR4-miRNA-transfected breast tumor cells had significantly declined. These cells exhibited reduced migration and invasion in vitro. Furthermore, they formed fewer lung metastases in vivo compared to ctrl-miRNA-transfected cells. These data support the conclusion that miRNA against CXCR4 can serve as an alterative means of therapy to lower CXCR4 expression and to block the invasion and metastasis of breast cancer cells.     

microRNAs as oncogenes and tumor suppressors

microRNAs (miRNAs) are a new class of non-protein-coding, endogenous, small RNAs. They are important regulatory molecules in animals and plants. miRNA regulates gene expression by translational repression, mRNA cleavage, and mRNA decay initiated by miRNA-guided rapid deadenylation. Recent studies show that some miRNAs regulate cell proliferation and apoptosis processes that are important in cancer formation. By using multiple molecular techniques, which include Northern blot analysis, real-time PCR, miRNA microarray, up- or down-expression of specific miRNAs, it was found that several miRNAs were directly involved in human cancers, including lung, breast, brain, liver, colon cancer, and leukemia. In addition, some miRNAs may function as oncogenes or tumor suppressors. More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites, suggesting that miRNAs may play a more important role in the pathogenesis of a limited range of human cancers than previously thought. Overexpressed miRNAs in cancers, such as mir-17-92, may function as oncogenes and promote cancer development by negatively regulating tumor suppressor genes and/or genes that control cell differentiation or apoptosis. Underexpressed miRNAs in cancers, such as let-7, function as tumor suppressor genes and may inhibit cancers by regulating oncogenes and/or genes that control cell differentiation or apoptosis. miRNA expression profiles may become useful biomarkers for cancer diagnostics. In addition, miRNA therapy could be a powerful tool for cancer prevention and therapeutics.     

Impact of miRNA Sequence on miRNA Expression and Correlation between miRNA Expression and Cell Cycle Regulation in Breast Cancer Cells

The miRNAs regulate cell functions by inhibiting expression of proteins. Research on miRNAs had usually focused on identifying targets by base pairing between miRNAs and their targets. Instead of identifying targets, this paper proposed an innovative approach, namely impact significance analysis, to study the correlation between mature sequence, expression across patient samples or time and global function on cell cycle signaling of miRNAs. With three distinct types of data: The Cancer Genome Atlas miRNA expression data for 354 human breast cancer specimens, microarray of 266 miRNAs in mouse Embryonic Stem cells (ESCs), and Reverse Phase Protein Array (RPPA) transfected by 776 miRNAs in MDA-MB-231 cell line, we linked the expression and function of miRNAs by their mature sequence and discovered systematically that the similarity of miRNA expression enhances the similarity of miRNA function, which indicates the miRNA expression can be used as a supplementary factor to predict miRNA function. The results also show that both seed region and 3'' portion are associated with miRNA expression levels across human breast cancer specimens and in ESCs; miRNAs with similar seed tend to have similar 3'' portion. And we discussed that the impact of 3'' portion, including nucleotides , is not significant for miRNA function. These results provide novel insights to understand the correlation between miRNA sequence, expression and function. They can be applied to improve the prediction algorithm and the impact significance analysis can also be implemented to similar analysis for other small RNAs such as siRNAs.     

TDP-43 regulates cancer-associated microRNAs

Aberrant regulation of miRNA genes contributes to pathogenesis of a wide range of human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a RNA/DNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs regulated by TDP-43 using RNA-Seq coupled with an siRNA-mediated knockdown approach. TDP-43 knockdown affected the expression of a number of miRNAs. In addition, TDP-43 down-regulation led to alterations in the patterns of different isoforms of miRNAs (isomiRs) and miRNA arm selection, suggesting a previously unknown role of TDP-43 in miRNA processing. A number of TDP-43 associated miRNAs, and their candidate target genes, are associated with human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p. In contrast, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Reduced expression of miR-500a-3p is associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Cancer-associated genes LIF and PAPPA are possible targets of miR-500a-3p. Our work suggests that TDP-43-regulated miRNAs may play multifaceted roles in the pathogenesis of cancer.     

Evaluation of exosomal miR-9 and miR-155 targeting PTEN and DUSP14 in highly metastatic breast cancer and their effect on low metastatic cells

Breast cancer is one of the most prevalent cancers in women. Triple-negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA-MB-231 and MCF-7 cells and characterized. Overexpression of the miRNAs of MDA-MB-231 cells and their exosomes were analyzed using quantitative Real-time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF-7 cells were treated with MDA-MB-231 cells’ exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR-9 and miR-155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and DUSP14 tumor suppressor genes. Quantitative Real-time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF-7 cells with MDA-MB-231 cells’ exosomes resulted in target genes downregulation in MCF-7 cells. We found that miR-9 and miR-155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.     

Genome-wide screen for aberrantly expressed miRNAs reveals miRNA profile signature in breast cancer

Dysregulation in the expression of miRNAs contributes to the occurrence and development of many human cancers. We herein attempted to obtain the potential association between miRNA expression profile and breast cancer by applying high-throughput sequencing technology. Small RNAs from seven paired tumor and adjacent normal tissue samples were sequenced. To determine the miRNA expression profiles in tissues and sera, another five equally pooled serum samples from 20 patients and 30 normal women were sequenced. Despite a similar number in abundantly expressed miRNAs across samples, we detected varying miRNA expression profiles. Some miRNAs showed inconsistent or opposite dysregulation trends across different tumor tissues, including some abundantly expressed miRNA gene clusters and gene families. Wilcoxon sign-rank test for paired samples analysis revealed that abnormal miRNAs showed a higher level of variation across the seven tumor samples. We also completely surveyed abnormal miRNAs expressed in tumor and serum tissues in the mixed datasets based on the relative expression levels. Most of these miRNAs were significantly down-regulated in tumor samples, but nine abnormal miRNAs (miR-18a, 19a, 20a, 30a, 103b, 126, 126*, 192, 1287) were consistently expressed in tumor tissues and serum samples. Based on experimentally validated target mRNAs, functional enrichment analysis indicated that these abnormal miRNAs and miRNA groups (miRNA gene clusters and gene families) have important roles in multiple biological processes. Dynamic miRNA expression profiles, various abnormal miRNA profiles and complexity of the miRNA regulatory network reveal that the miRNA expression profile is a potential biomarker for classifying or detecting human disease.     

microRNA层面上癌症的公共机制

microRNAs(miRNA)是一类内源性的非编码小RNA。已有研究表明miRNAs的靶基因中有不少癌症的相关基因。为了全面研究miRNA与癌症的关系,作者将19种癌症的相关基因集合分别富集到494个miRNAs靶基因集合上,得到各类癌症所富集的miRNAs。结果发现19种癌症仅集中地富集在144个miRNAs上,由此验证了癌症在miRNAs上的公共机制。在此基础上,作者对癌症富集较多的8个miRNAs做了进一步研究,结果发现这8个miRNAs均为高度保守的miRNAs,且它们的靶基因集合一致富集在基因本体论(gene ontology,GO)的基本生物学过程上,并与转录因子活性以及蛋白激酶活性相关。另一方面,在基于miRNA构建的癌症网络中,前列腺癌与乳腺癌,结肠癌与乳腺癌之间共享较多的miRNAs,表明了这些癌症在miRNA层面上存在密切的关系。     

The comparison of miRNAs that respond to anti-breast cancer drugs and usnic acid for the treatment of breast cancer

This study was designed to compare usnic acid with anti-breast cancer drug molecules (A-BCDM) routinely used in the treatment of breast cancer. The miRNA information of 17 anti-breast cancer drug used in breast cancer treatment was obtained from the Small Molecule-miRNA Network-Based Inferance (SMIR-NBI) tool. We had been determined common and different expressed miRNAs between 17 A-BCDM & usnic acid and were classified according to the common miRNAs to reveal molecular similarity. As a result of the bioinformatic analyzes, 20 common miRNAs were determined between 17 A-BCDM and usnic acid. The common miRNAs were analyzed with bioinformatic tolls for determining pathways and targets. The most common miRNAs for 6 of 17 A-BCDM and usnic acid were determined as miR-374a-5p and miR-26a-5p. We compared the anti-proliferative effect of usnic acid and one of the 17 A-BCDM that tamoxifen on MDA-MB-231 triple negative breast cancer cell with real-time cell analysis system. The real time PCR assay was carried out with miR-26a-5p for evaluate to expression level of MDA-MB-231 breast cancer cell and MCF-12A non-cancerous epithelial breast cell. As a result of study, usnic acid as novel candidate drug molecule showed high similarity ratio with 5-Fluorouracil, Sulindac Sulfide, Curcumin and Cisplatin A-BCDM used in treatment of breast cancer. miR-26a-5p as common response miRNA of usnic acid and tamoxifen was showed a decreased level of expression by validated qRT-PCR assay. The obtained from study, in addition to 17 A-BCDM, usnic acid has also the potential to be used as a candidate molecule in the treatment of breast cancer. Moreover, miR-26a-5p might be used as a biomarker in the treatment of breast cancer but further analysis is required.