Post-transcriptional Regulation of BRCA2 through Interactions with miR-19a and miR-19b.
ABSTRACT: Breast cancer type 2, early onset susceptibility gene (BRCA2) is a major component of the homologous recombination DNA repair pathway. It acts as a tumor suppressor whose function is often lost in cancers. Patients with specific mutations in the BRCA2 gene often display discrete clinical, histopathological, and molecular features. However, a subset of sporadic cancers has wild type BRCA2 and display defects in the homology-directed repair pathway, which is the hallmark of 'BRCAness.' The mechanisms by which BRCAness arises are not well understood but post-transcriptional regulation of BRCA2 gene expression by microRNAs (miRNAs) may contribute to this phenotype. Here, we examine the post-transcriptional effects that some members of the six-miRNA cluster known as the miR-17/92 cluster have on the abundance of BRCA2's messenger RNA (mRNA) and protein. We discuss two interactions involving the miR-19a and miR-19b members of the cluster and the 3'UTR of BRCA2's mRNA. We investigated these miRNA:mRNA interactions in 15 cell lines derived from pancreatic, breast, colon, and kidney tissue. We show that over-expression of these two miRNAs results in a concomitant decrease of BRCA2's mRNA and protein expression in a subset of the tested cell lines. Additionally, using luciferase reporter assays we identified direct interactions between miR-19a/miR-19b and a miRNA response element (MRE) in BRCA2's 3'UTR. Our results suggest that BRCA2 is subject to a complex post-transcriptional regulatory program that has specific dependencies on the genetic and phenotypic background of cell types.
Project description:MicroRNA-19 (miR-19) is identified as the key oncogenic component of the miR-17-92 cluster. When we explored the functions of the dysregulated miR-19 in lung cancer, microarray-based data unexpectedly demonstrated that some immune and inflammatory response genes (i.e., IL32, IFI6 and IFIT1) were generally down-regulated by miR-19 overexpression in A549 cells, which prompted us to fully investigate whether the miR-19 family (i.e., miR-19a and miR-19b-1) was implicated in regulating the expression of immune and inflammatory response genes in cancer cells. In the present study, we observed that miR-19a or miR-19b-1 overexpression by miRNA mimics in the A549, HCC827 and CNE2 cells significantly downregulated the expression of interferon (IFN)-regulated genes (i.e., IRF7, IFI6, IFIT1, IFITM1, IFI27 and IFI44L). Furthermore, the ectopic miR-19a or miR-19b-1 expression in the A549, HCC827, CNE2 and HONE1 cells led to a general downward trend in the expression profile of major histocompatibility complex (MHC) class I genes (such as HLA-B, HLA-E, HLA-F or HLA-G); conversely, miR-19a or miR-19b-1 inhibition by the miRNA inhibitor upregulated the aforementioned MHC Class I gene expression, suggesting that miR-19a or miR-19b-1 negatively modulates MHC Class I gene expression. The miR-19a or miR-19b-1 mimics reduced the expression of interleukin (IL)-related genes (i.e., IL1B, IL11RA and IL6) in the A549, HCC827, CNE2 or HONE1 cells. The ectopic expression of miR-19a or miR-19b-1 downregulated IL32 expression in the A549 and HCC827 cells and upregulated IL32 expression in CNE2 and HONE1 cells. In addition, enforced miR-19a or miR-19b-1 expression suppressed IL-6 production by lung cancer and nasopharyngeal carcinoma (NPC) cells. Taken together, these findings demonstrate, for the first time, that miR-19 can modulate the expression of IFN-induced genes and MHC class I genes in human cancer cells, suggesting a novel role of miR-19 in linking inflammation and cancer, which remains to be fully characterized.
Project description:The primary cause of heart failure is the loss of cardiomyocytes in the diseased adult heart. Previously, we reported that the miR-17-92 cluster plays a key role in cardiomyocyte proliferation. Here, we report that expression of miR-19a/19b, members of the miR-17-92 cluster, is induced in heart failure patients. We show that intra-cardiac injection of miR-19a/19b mimics enhances cardiomyocyte proliferation and stimulates cardiac regeneration in response to myocardial infarction (MI) injury. miR-19a/19b protected the adult heart in two distinctive phases: an early phase immediately after MI and long-term protection. Genome-wide transcriptome analysis demonstrates that genes related to the immune response are repressed by miR-19a/19b. Using an adeno-associated virus approach, we validate that miR-19a/19b reduces MI-induced cardiac damage and protects cardiac function. Finally, we confirm the therapeutic potential of miR-19a/19b in protecting cardiac function by systemically delivering miR-19a/19b into mice post-MI. Our study establishes miR-19a/19b as potential therapeutic targets to treat heart failure.
Project description:Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma, which shows high aggressiveness and lacks biomarkers. RhoB acts as a tumor suppressor that inhibits the progression of ccRCC. In the present study, we examined the effects of oncogenic microRNAs, miR-19a and miR-19b, on RhoB expression in ccRCC cells. The results showed that both miR-19a and miR-19b could directly target the 3'untranslated region (3'UTR) of RhoB, resulting in the reduced expression of RhoB. With RT-PCR analysis, we detected the increased expression of miR-19a and miR-19b in ccRCC tissues compared to adjacent non-tumor renal tissues. These data also demonstrated an exclusive negative correlation between miR-19a/19b and RhoB expression in ccRCC specimens and cell lines. In addition, the knockdown of RhoB or overexpression of miR-19a and miR-19b in ccRCC cells could promote cell proliferation, migration and invasion. These data demonstrate the direct roles of miR-19a and miR-19b on the repression of RhoB and its consequences on tumorigenesis, cancer cell proliferation and invasiveness. These results suggest the potential clinical impact of miR-19a and miR-19b as molecular targets for ccRCC.
Project description:The synergism between c-MYC and miR-17-19b, a truncated version of the miR-17-92 cluster, is well-documented during tumor initiation. However, little is known about miR-17-19b function in established cancers. Here we investigate the role of miR-17-19b in c-MYC-driven lymphomas by integrating SILAC-based quantitative proteomics, transcriptomics and 3' untranslated region (UTR) analysis upon miR-17-19b overexpression. We identify over one hundred miR-17-19b targets, of which 40% are co-regulated by c-MYC. Downregulation of a new miR-17/20 target, checkpoint kinase 2 (Chek2), increases the recruitment of HuR to c-MYC transcripts, resulting in the inhibition of c-MYC translation and thus interfering with in vivo tumor growth. Hence, in established lymphomas, miR-17-19b fine-tunes c-MYC activity through a tight control of its function and expression, ultimately ensuring cancer cell homeostasis. Our data highlight the plasticity of miRNA function, reflecting changes in the mRNA landscape and 3' UTR shortening at different stages of tumorigenesis.
Project description:The synergism between c-MYC and miR-17-19b, a truncated version of the miR-17-92 cluster, is well documented during tumor initiation. However, little is known about miR-17-19b function in established cancers. Here we investigate the role of miR-17-19b in c-MYC-driven lymphomas by integrating SILAC-based quantitative proteomics, transcriptomics and 3’ UTR analysis upon miR-17-19b overexpression. We identify over one hundred novel miR-17-19b targets, of which 40% are co-regulated by c-MYC. Down-regulation of a new miR-17/20 target Chek2 increases the recruitment of HuR to c-MYC transcripts, resulting in the inhibition of c-MYC translation and thus interfering with in vivo tumor growth. Hence, in established lymphomas, miR-17-19b fine-tunes c-MYC activity through a tight control of its function and expression, ultimately ensuring cancer cell homeostasis. Our data highlight the plasticity of miRNA function, reflecting changes in the mRNA landscape and 3’ UTR shortening at different stages of tumorigenesis.
Project description:Several members from microRNA 17-92 cluster, i.e. miR-19a, miR-19b and miR-20a, were found up-regulated in human epidermal keratinocytes at wound-edges compared to the intact skin; however their biological role in keratinocytes during wound repair has not been studied. To study the genes regulated by miR-19a, miR-19b and miR-20a, we transfected miRNA specific mimics, i.e. pre-miR-19a, pre-miR-19b or pre-miR-20a into human primary epidermal keratinocytes to overexpress them. We performed a global transcriptome analysis of keratinocytes upon overexpression of miR-19a or miR-19b or miR-20a using Affymetrix arrays. Overall design: Expression profiling of primary human epidermal keratinocytes transfected with 20nM miR-19a mimics (pre-miR-19a) or 20nM miR-19b mimics (pre-miR-19b) or 20nM miR-20a mimics (pre-miR-20a) or 20nM control oligos (pre-miR-Ctrl) for 24 hours (biological triplicates in each group) was performed using Affymetrix GeneTitan system.
Project description:Lung fibroblasts play a pivotal role in pulmonary fibrosis, a devastating lung disease, by producing extracellular matrix. MicroRNAs (miRNAs) suppress numerous genes post-transcriptionally; however, the roles of miRNAs in activated fibroblasts in fibrotic lungs remain poorly understood. To elucidate these roles, we performed global miRNA-expression profiling of fibroblasts from bleomycin- and silica-induced fibrotic lungs and investigated the functions of miRNAs in activated lung fibroblasts. Clustering analysis of global miRNA-expression data identified miRNA signatures exhibiting increased expression during fibrosis progression. Among these signatures, we found that a miR-19a-19b-20a sub-cluster suppressed TGF-?-induced activation of fibroblasts in vitro. Moreover, to elucidate whether fibroblast-specific intervention against the sub-cluster modulates pathogenic activation of fibroblasts in fibrotic lungs, we intratracheally transferred the sub-cluster-overexpressing fibroblasts into bleomycin-treated lungs. Global transcriptome analysis of the intratracheally transferred fibroblasts revealed that the sub-cluster not only downregulated expression of TGF-?-associated pro-fibrotic genes, including Acta2, Col1a1, Ctgf, and Serpine1, but also upregulated expression of the anti-fibrotic genes Dcn, Igfbp5, and Mmp3 in activated lung fibroblasts. Collectively, these findings indicated that upregulation of the miR-19a-19b-20a sub-cluster expression in lung fibroblasts counteracted TGF-?-associated pathogenic activation of fibroblasts in murine pulmonary fibrosis.
Project description:Recently, rapid advances in bioinformatics analysis have expanded our understanding of the transcriptome to a genome-wide level. miRNA-mRNA-lncRNA interactions have been shown to play critical regulatory role in cancer biology. In this study, we discussed the use of an integrated systematic approach to explore new facets of the oestrogen receptor (ER)-regulated transcriptome. The identification of RNAs that are related to the expression status of the ER may be useful in clinical therapy and prognosis. We used a network modelling strategy. First, microarray expression profiling of mRNA, lncRNA and miRNA was performed in MCF-7 (ER-positive) and MDA-MB-231 cells (ER- negative). A co-expression network was then built using co-expression relationships of the differentially expressed mRNAs and lncRNAs. Finally, the selected miRNA-mRNA network was added to the network. The key miRNA-mRNA-lncRNA interaction can be inferred from the network. The mRNA and non-coding RNA expression profiles of the cells with different ER phenotypes were distinct. Among the aberrantly expressed miRNAs, the expression levels of miR-19a-3p, miR-19b-3p and miR-130a-3p were much lower in the MCF-7 cells, whereas that of miR-148b-3p was much higher. In a cluster of miR-17-92, the expression levels of six of seven miRNAs were lower in the MCF-7 cells, in addition to miR-20b in the miR-106a-363 cluster. However, the levels of all the miRNAs in the miR-106a-25 cluster were higher in the MCF-7 cells. In the co-expression networking, CD74 and FMNL2 gene which is involved in the immune response and metastasis, respectively, had a stronger correlation with ER. Among the aberrantly expressed lncRNAs, lncRNA-DLEU1 was highly expressed in the MCF-7 cells. A statistical analysis revealed that there was a co-expression relationship between ESR1 and lncRNA-DLEU1. In addition, miR-19a and lncRNA-DLEU1 are both located on the human chromosome 13q. We speculate that miR-19a might be co-expressed with lncRNA-DLEU1 to co-regulate the expression of ESR1, which influences the occurrence and development of breast cancer cells with different levels of ER expression. Our findings reveal that the status of ER is mainly due to the differences in the mRNA and ncRNA profile between the breast cancer cell lines, and highlight the importance of studying the miRNA-mRNA-lncRNA interactions to completely illustrate the intricate transcriptome.
Project description:BACKGROUND:Lung cancer is one of the most malignant cancers threatening human health. The miR-17-92 gene cluster is a highly conserved oncogene cluster encoding 6 miRNAs: miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a and miR-92a. This study explored whether these miRNAs can be used as diagnostic markers for non-small-cell lung cancer (NSCLC). METHODS:Serum samples were collected from healthy subjects (n = 23) and NSCLC patients at various stages (n = 74). Serum RNA was extracted by the TRIzol-glycogen method, and cDNA libraries were constructed by reverse transcription. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to detect the expression levels of the 6 miRNAs. RESULTS:The expression levels of the 6 miRNAs varied in different stages of NSCLC. Thus, 2 receiver operating characteristic (ROC) curves, that is, normal subjects and stage I-III patients and normal subjects and stage IV patients, of each miRNA were established to determine the interval of normal ?Ct values. The 2 areas under the curve (AUCs) of each miRNA were investigated (miR-17: 0.8097 and 1.000; miR-18a: 0.7388 and 0.9907; miR-19a/19b: 0.8451 and 0.5104; miR-20a: 0.8975 and 1.000; miR-92a: 0.8097 and 0.8342). In addition, a high positive correlation was discovered between miR-17 and miR-20a expression. Combining these 2 miRNAs can improve the screening effect of NSCLC. CONCLUSION:The miR-17-92 gene cluster can likely serve as a diagnostic marker in NSCLC.
Project description:The precise control of miR-17?92 microRNA (miRNA) is essential for normal development, and overexpression of certain miRNAs from this cluster is oncogenic. Here, we find that the relative expression of the six miRNAs processed from the primary (pri-miR-17?92) transcript is dynamically regulated during embryonic stem cell (ESC) differentiation. Pri-miR-17?92 is processed to a biogenesis intermediate, termed "progenitor-miRNA" (pro-miRNA). Pro-miRNA is an efficient substrate for Microprocessor and is required to selectively license production of pre-miR-17, pre-miR-18a, pre-miR-19a, pre-miR-20a, and pre-miR-19b from this cluster. Two complementary cis-regulatory repression domains within pri-miR-17?92 are required for the blockade of miRNA processing through the formation of an autoinhibitory RNA conformation. The endonuclease CPSF3 (CPSF73) and the spliceosome-associated ISY1 are responsible for pro-miRNA biogenesis and expression of all miRNAs within the cluster except miR-92. Thus, developmentally regulated pro-miRNA processing is a key step controlling miRNA expression and explains the posttranscriptional control of miR-17?92 expression in development.