miR-34a Gene Research Articles

Abstract 1960: Implication of miR-34a in radioresistance of bladder cancer .

Abstract Purpose : Investigate resistance of radiation via miR-34a expression in human urothelial carcinoma Methods : Human urothelial carcinoma cell lines derived from well-differentiated superficial bladder tumors as well as from high-grade invasive tumors were screened for their sensitivity to radiation. Growth inhibition was monitored by clonogenic assays. qRT-PCR analysis was used to evaluate expression of miR-34a among the human urothelial carcinoma cell lines. Methylation-specific polymerase chain reaction (MSP) was performed to determine CpG methylation status of miR-34a gene promoter among the cell lines. Association between miR-34a and sensitivity to radiation was also investigated by ectopic expression of miR-34a. Western blot was performed to evaluate protein expression. Results: Remarkable differences in sensitivity to radiation have been shown among those cell lines in vitro. Of interest, miR-34a expression correlates with human bladder cancer cell response to radiation, with resistant cell lines showing lower expression of miR-34a and that is due to CpG methylation of its promoter. Additionally, an inverse correlation was observed between miR-34a and expression of sirtuin1 (SIRT1). Ectopic expression of miR-34a in cell lines resistant to radiation showed a positive effect of miR-34a to the response therapy. Growth inhibition observed after treatment could be explained, in part, by the increased expression of the cell cycle inhibitor p21. Conclusion: Preliminary results suggest that detection of miR-34a expression may potentially be used as a predictor of therapeutic efficacy, and its regulation represent an attractive mechanism for the management of bladder cancer therapy. Citation Format: Jose J. Mansure, Roland Nassim, Wassim Kassouf. Implication of miR-34a in radioresistance of bladder cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1960. doi:10.1158/1538-7445.AM2013-1960

Abstract 3163: Exploration of microRNA-34a effects in human glioma cell lines

Abstract We have previously demonstrated that miR34a targets and downregulates multiple oncogenes, and therefore it may be considered as a potent tumor suppressor factor. In this report we show that miR34a expression is strictly dependent on the ability of major tumor suppressor protein TP53 to bind to miR34a transcriptional regulatory elements. In order to further investigate the effects of miR34a we generated a panel of human glioma cell lines where both miR34a and copGFP gene reporter are expressed in a tet-inducible manner. The constitutive expression of cyan fluorescent protein, the mutant GFP derived from Aequorea Victoria, and tet-inducible expression of copGFP derived from Anthozoa allows co-localizing and tracking both tet-induced and non-induced cells in vivo. We present data indicating that miR34a-caused inhibition of cell proliferation both in vitro and in vivo is due to the down-regulation of cyclin D1 and transcriptional factors E2F3 and E2F5. We show that miR199a, which targets other cyclins and cyclin-dependent kinases and therefore complements the action of miR34a, is up-regulated upon miR34a overexpression. miR199a-5P and miR199a-3P, but not miR34a, target E2F6 and USP7 correspondingly. Overexpression of miR34a using tet-inducible cell lines and lentiviral vectors downregulated E2F6 and USP7 on the 5th day after induction/transduction. However, at 48-72 hours after p53 overexpression accompanied by the modest upregulation of miR34a no changes in miR199a levels were observed. The results indicate that miR34a induces miR199a overexpression indirectly, most probably at the transcriptional level, thus eliciting the synergistic anti-tumor effects. We have also found that overexpression of miR34a in tet-inducible manner significantly inhibits the growth of brain tumors and prolongs the survival of immunodeficient animals. Finally, we show that miR34a targets and downregulates phosphoprotein enriched in astrocytes-15, which has been reported to promote autophagy and contribute to the resistance to radiation therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3163. doi:1538-7445.AM2012-3163

Transcriptional activation of microRNA-34a by NF-kappa B in human esophageal cancer cells

BackgroundmiR-34a functions as an important tumor suppressor during the process of carcinogenesis. However, the mechanism of miR-34a dysregulation in human malignancies has not been well elucidated. Our study aimed to further investigate the regulation mechanism of miR-34a.ResultsWe found that overexpression of NF-kappa B p65 subunit could increase miR-34a levels in EC109, an esophageal squamous cancer cell line, while ectopic expression of DN IkappaB leaded to a significant reduction of miR-34a expression. Bioinformatics analysis suggested three putative KB sites in promoter region of miR-34a gene. Mutation two of these KB sites impaired p65 induced miR-34a transcriptional activity. Chromatin immunoprecipitation and electrophoretic mobility shift assays both showed that NF-kappaB could specifically bind to the third KB site located in miR-34a promoter. In addition, we found that overexpression of NF-kappaB p65 could not successfully induce miR-34a expression in esophageal cancer cell lines with mutant p53 or decreased p53. Reporter assay further showed that NF-kappaB-induced miR-34a transcriptional activity was reduced by p53 impairment. Nevertheless, CHIP analysis suggested binding of NF-kappaB to miR-34a promoter was not affected in cells with mutant p53.ConclusionsOur work indicates a novel mechanism of miR-34a regulation that NF-kappaB could elevate miR-34a expression levels through directly binding to its promoter. And wildtype p53 is responsible for NF-kappaB-mediated miR-34a transcriptional activity but not for NF-kappaB binding. These findings might be helpful in understanding miR-34a abnormality in human malignancies and open new perspectives for the roles of miR-34a and NF-kappaB in tumor progression.

Effect of microRNA‐34a in cell cycle, differentiation, and apoptosis: A review

The tumor suppressor gene p53 was shown to directly regulate the expression of microRNA-34a (miR-34a). miR-34a regulates a plethora of target proteins, which are involved in cell cycle, apoptosis, differentiation, and cellular development.miR-34a resides in the region of chromosome 1p36.23, which is commonly deleted in many tumor types, while it results in the loss expression of miR-34a. The promoters of the miR-34a gene subject to inactivation by CpG methylation also induce the loss expression of miR-34a. Ectopic miR-34a expression induces apoptosis, cell cycle arrest, and differentiation or reduces migration. This review summarizes the progress regarding the role of miR-34a in cell cycle, differentiation, and apoptosis.

MiR-34a as potential fecal biomarker Colitis-induced Colorectal cancer

Colorectal cancer represents one of the most dreaded complications of chronic ulcerative colitis. Typical surveillance approaches (every other year colonoscopy with >30 random or targeted biopsies) have been suboptimal at protection against CRC. Our group has been developing biomarkers of field carcinogenesis for colorectal cancer risk stratification (reviewed in Gastroenterology, 2011). MicroRNAs (miRNAs) are being increasingly recognized as powerful cancer biomarkers given the critical role in regulation of gene expression. In particular miR34a has been shown to be a tumor promotor (oncomiR) involved in both initiation and progression phases of colon carcinogenesis. Our group has developed a novel modality of isolating and analyzing mucus layer fecal colonocytes(MLFC). MLFC are abraded from the normal colonic epithelium by passage of solid stool and therefore represents a potential means of field carcinogenesis. We have recently observed that miR 34a was overexpressed in the microscopically normal mucosa of both animal models (azoxymethane-treated rat) and patients harboring neoplasia elsewhere in the colon. In this study, we assess the potential translatability of fecal miR 34 to colitis-induced CRC. We used an in vivo and in vitro model using citrobacter rodentium as the model of inducing inflammation In the in vitro model, CRC cell line HT-29 was infected with C. rodentium and total RNA was processed for miRNA analysis. For the in vivo C57BL/J6 mice were infected with C. rodentium. Stool was collected with isolation of mucus layer fecal colonocytes (MLFC) and total RNA was extracted and processed for miRNA detection. Total RNA was processed for reverse transcription using miRNA reverse transcription kit (Applied Biosystems) and assayed for specific miRNAs using individual Taqman miRNA assays namely, miR-142-3p, miR-21, miR-34a, miR-146b, miR-148b-3p and miR-494 (Applied Biosystems) using manufacturer's instructions. The six miRNAs tested had been previously demonstrated to be upregulated in colonic field carcinogenesis. Out of the miRNAs tested, miR-34a was found to be significantly induced in HT-29 cells.(4.8 fold induction, p=0.02). We next both in the fecal colonocytes from C. rodentium infected mice and noted a dramatic induction (7.2 fold, p<0.05). To demonstrate the relevance to colon carcinogenesis, we used gold-standard model, the azoxymethane-treated rat (a carcinogen induced model). We noted that at premalignant time points there was a marked increase in mucus layer fecal colonocytes (4.2 to 6.2 fold, p<0.05) We present herein the first data that fecal miR 34a may be a marker for neoplastic risk of in both an inflammation model and a well-validated CRC models. Importantly, miR 34a is regulated by p53, one of the earliest events in colitis-induced CRC (found in flat dysplasia or histologically normal mucosa of patients harboring dysplasia). Thus, while confirmation for IBD dysplasia will be performed with standard DSS or transgenic (IL10 knockouts etc.) models, this work provides a powerful proof of concept that miR34a may serve as a biomarker for colitis-induced CRC. Furthermore, the development of mucus layer fecal colonocytes as a marker of field carcinogenesis may represent significant advance in the surveillance for IBD-related neoplasia.

MiR-34a Replacement As a Novel Therapeutic Approach for Multiple Myeloma: Preclinical In Vitro and In Vivo Evidence

Abstract 2910Multiple myeloma (MM) remains an incurable disease despite important therapeutic advances in the last few years. Small non-coding RNAs (miRNAs) synthetic mimics are a new class of biological agents which have recently demonstrated preclinical activity against a variety of human neoplasms. miRNA antitumor activity has been related to their capacity to interfere with mRNA stability and protein transducing activity. miR-34a has tumor suppressor activity and is transcriptionally regulated by p53. We investigated the in vitro and in vivo therapeutic potential of pre-miR-34a mimics against human MM cells. Transient expression of pre-miR-34a mimics, after electroporation of SKMM1 and RPMI-8226 MM cell lines which display low constitutive miR-34a levels, triggered antiproliferative effects, as demonstrated by MTT and long-term soft-agar colony assays. 48 hours after cell transfection, apoptotic events were detected in both cell lines exposed to miR-34a mimics. In parallel experiments, MM cells stably transduced with miR-34a gene cloned in a lentiviral vector showed significant growth reduction as compared to empty vector-transduced cell colonies, providing additional evidence of miR-34a tumor suppressor activity in MM cells. qRT-PCR analysis of treated MM cells showed that pre-miR-34a mimics induced down-regulation of mRNAs coding for Notch1 and the cell-cycle dependent kinase 6 (CDK6), validated miR-34a targets. Furthermore, decreased anti-apoptotic Bcl-2 and CDK6 proteins was detected after pre-miR-34a mimic expression, evidence by western blotting analysis. The anti-MM activity of pre-miR-34a mimics was also evaluated in vivo using xenografted SCID models of human MM. Intra-tumor delivery of pre-miR-34a was performed by a novel formulation with Neutral Lipid Emulsion (NLE). Following 4 injections (3 days apart) of pre-miR34a formulated in NLE particles, a highly significant inhibition of tumor growth was detected in SKMM1 xenografted SCID mice. At day 13 after the first treatment, tumors in mice treated with formulated pre-miR-34a were significantly smaller than tumors in mice treated with the formulated scrambled sequence (P=0.0002) or vehicle (P=0.0002) or PBS (P=0.0001). Interestingly, at day 21a three mice enrolled in the miR-34a treated group showed complete regression of tumors. Formulated synthetic pre-miR-34a also produced a significant increase of mice survival (P=0.01 versus formulated scrambled sequence). A similar in vivo tumor growth inhibition was observed in mice xenografted with SKMM1 MM cells stably transduced with a miR-34a lentiviral construct, as compared to cells transduced with the empty vector. We here provide the first proof-of-principle demonstrating that replacement of miR-34a produces therapeutic activity against MM cells with low constitutive miR-34a expression. Our findings provide a framework for development of miR-34a-based therapeutic strategies in MM.Supported by AIRC 5 per mille, Molecular Clinical Oncology Program No. 9980 Disclosures:Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Epidermal growth factor-mediated signaling in ovarian cancer and the role of miR-34a and 6p22 gene amplification-based E2F3a control.

e15535 Background: Cell cycle acceleration based on a selective induction of proliferation-promoting transcription factor E2F3a is a crucial step in EGFR mediated mitogenic signalling in ovarian cancer. Herein we report on the molecular background and clinical relevance of two alternative mechanisms of E2F3a control. One is acting epigenetically via promoter methylation of miR-34a, which by its physical interaction with E2F3a transcripts causes their degradation, and the second is based on 6p22 gene locus amplification. Methods: Expression levels of E2F3 isoforms and miR-34a were estimated by RT-PCR, miR-34a promoter methylation status by means of MethyLight-PCR. MiRIDIAN based knockdown or induction of miR-34a was performed in ovarian cancer cell lines and FISH analyses were used to estimate 6p22 amplification status in cell lines and primary tumor specimens. The clinical relevance of miR-34a and 6p22 amplification was evaluated in a large collective of 130 ovarian cancer patients. Results: MiRIDIAN-based knock-down or induction of miR-34a evidenced a direct regulatory link between miR-34a and E2F3a, and the tumor-suppressive character of miR-34a was documented by its inverse correlation with grading (p=0.016), residual disease (p=0.013) and association with improved survival. We further showed that in a significant number of ovarian cancer specimens miR-34a depletion is based on promoter methylation. Although, 6p22 gene locus amplification was detected in a significant number of ovarian cancer specimens and correlated with grading (p=0.04) and FIGO (p=0.007), 6p22 ploidy was not relevant in predicting survival. In the Cox regression analysis E2F3a, but not activated EGFR or miR-34a expression, retained independent prognostic significance (progression-free survival: HR 3.785, p=0.013; overall survival: HR 4.6511; p=0.013). Conclusions: These clinical findings highlight the relevance of E2F3a in the biology of ovarian cancer. Moreover, identification of EGFR-independent mechanisms in E2F3a control can be helpful in explaining non-responsiveness of therapeutic EGFR targeting in ovarian cancer.

Frequent concomitant inactivation of miR-34a and miR-34b/c by CpG methylation in colorectal, pancreatic, mammary, ovarian, urothelial, and renal cell carcinomas and soft tissue sarcomas

The microRNA encoding genes miR-34a and miR-34b/c represent direct p53 target genes and possess tumor suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. We previously reported that the miR-34a gene is subject to epigenetic inactivation by CpG methylation of its promoter region in primary prostate cancer and melanomas, and in 110 different cancer cell lines of diverse origin. Here we analyzed the methylation status of miR-34a and miR-34b/c in additional primary tumors of divergent sites. We found methylation of miR-34a or miR-34b/c in formalin-fixed, paraffin-embedded (FFPE) tumor samples from 178 patients with the following frequencies: colorectal cancer (74% miR-34a, 99% miR-34b/c; n = 114), pancreatic cancer (64%, 100%; n = 11), mammary cancer (60%, 90%; n = 10), ovarian cancer (62%, 69%; n = 13), urothelial cancer (71%, 57%; n = 7), and renal cell cancer (58%, 100%; n = 12). Furthermore, soft tissue sarcomas showed methylation of miR-34 gene promoters in FFPE samples (64%, 45%; n = 11), in explanted, cultured cells (53%, 40%; n = 40), and in frozen tissue samples (75%, 75%, n = 8). In the colorectal cancer samples a statistically significant correlation of miR-34a methylation and the absence of p53 mutation was detected. With the exception of sarcoma cell lines, the inactivation of miR-34a and miR-34b/c was concomitant in most cases. These results show that miR-34 inactivation is a common event in tumor formation, and suggest that CpG methylation of miR-34a and miR-34-b/c may have diagnostic value. The mutual exclusiveness of miR-34a methylation and p53 mutation indicates that miR-34a inactivation may substitute for loss of p53 function in cancer.

A Pathway Involving Farnesoid X Receptor and Small Heterodimer Partner Positively Regulates Hepatic Sirtuin 1 Levels via MicroRNA-34a Inhibition

Sirtuin 1 (SIRT1) is a NAD-dependent deacetylase that is critically involved in diverse cellular processes including metabolic disease, cancer, and possibly aging. Despite extensive studies on SIRT1 function, how SIRT1 levels are regulated remains relatively unknown. Here, we report that the nuclear bile acid receptor farnesoid X receptor (FXR) inhibits microRNA-34a (miR-34a) in the liver, which results in a positive regulation of SIRT1 levels. Activation of FXR by the synthetic agonist GW4064 decreases hepatic miR-34a levels in normal mice, and consistently, hepatic miR-34a levels are elevated in FXR-null mice. FXR induces expression of small heterodimer partner (SHP), an orphan nuclear receptor and transcriptional corepressor, which in turn results in repression of p53, a key activator of the miR-34a gene, by inhibiting p53 occupancy at the promoter. MiR-34a decreased SIRT1 levels by binding to the 3'-untranslated region of SIRT1 mRNA, and adenovirus-mediated overexpression of miR-34a substantially decreased SIRT1 protein levels in mouse liver. Remarkably, miR-34a levels were elevated, and SIRT1 protein levels were reduced in diet-induced obese mice, and FXR activation in these mice reversed the miR-34a and SIRT1 levels, indicating an intriguing link among FXR activation, decreased miR-34a, and subsequently, increased SIRT1 levels. Our study demonstrates an unexpected role of the FXR/SHP pathway in controlling SIRT1 levels via miR-34a inhibition and that elevated miR-34a levels in obese mice contribute to decreased SIRT1 levels. Manipulation of this regulatory network may be useful for treating diseases of aging, such as metabolic disease and cancer.

MicroRNA-34a Inhibits Cell Proliferation by Repressing Mitogen-Activated Protein Kinase Kinase 1 during Megakaryocytic Differentiation of K562 Cells

Phorbol 12-myristate 13-acetate (PMA) induces megakaryocytic differentiation of the human chronic myelocytic leukemia cell line K562. We examined the potential regulatory role of microRNAs (miRNAs) in this process. Genome-wide expression profiling identified 21 miRNAs (miRs) that were induced by the treatment of K562 cells with PMA. Among them, the expression of miR-34a, miR-221, and miR-222 was induced in the early stages and maintained throughout the late stages of differentiation. Cell signaling analysis showed that the activation of extracellular signal-regulated protein kinase (ERK) in response to PMA strongly induced miR-34a expression by transactivation via the activator protein-1 binding site in the upstream region of the miR-34a gene. Reporter gene assays identified mitogen-activated protein kinase kinase 1 (MEK1) as a direct target of miR-34a and c-fos as a direct target of miR-221/222. Although overexpression of the three miRNAs had little effect on cell differentiation, overexpression of miR-34a significantly repressed the proliferation of K562 cells with a concomitant reduction in MEK1 protein expression. Conversely, a locked nucleic acid probe against miR-34a significantly enhanced the proliferation of PMA-treated K562 cells. Taken together, the results show that PMA activates the MEK-ERK pathway and strongly induces miRNA-34a expression, which in turn inhibits cell proliferation by repressing the expression of MEK1. Thus, the results highlight an important regulatory role for miR-34a in the process of megakaryocytic differentiation, especially in the arrest of cell growth, which is a prerequisite for cells to enter differentiation.

Micro-RNA deletion in neuroblastoma: A possible alternative mechanism of MYCN overexpression

9052 Background: Micro-RNAs (miRNAs) are small noncoding RNAs of 18 to 25 nucleotides that regulate protein expression. The biological functions of miRNAs are not yet fully understood. miRNA genes were recently found to be abnormally expressed in several types of cancer. By negatively regulating proto-oncogenes, miRNAs could act as tumor suppressors and conversely, by inhibiting tumor suppressors function as oncogenes. Neuroblastoma (NB) is the most common solid tumor in children under 5 years of age. Major adverse prognostic factors include: age over 1 year, advanced stages, adrenal primary site, MYCN amplification, diploid or tetraploid DNA content (DI), and chromosomal aberrations - including 1p deletions. We studied a possible involvement of miRNAs in neuroblastoma. Methods: The cohort consisted of 72 patients: 65% of them were above 1 year of age, 72% with advanced stages, 33% of the patients progressed. Median follow up was 60 months (ranging 1–221). Genomic DNA from 72 tumors were analyzed for the presence of 7 miRNAs located on chromosome 1p, ranging from 1p36.33 to 1p31.1 by PCR. Results: Deletion of one or more of the miRNAs was identified in 50% of the tumors.The most prevalent deletions were of two miRNAs: miR 30e located on 1p34.2 (29%) and miR 34a on 1p 36.23 (22%), accounting for 40% of the patients with a deletion of one or both of these miRs. Interestingly, miR 30e and miR 34a are known to negatively regulate the oncogene MYCN. MYCN protein expression is currently being evaluated in this cohort. Conclusions: The deletions of these miRNAs in neuroblastoma tumors suggest their involvement as tumor suppressor genes. This implies a new mechanism for the overexpression of MYCN. Discovery of the miRNAs and their targets involved in neuroblastoma has a therapeutic potential, for the development of new targeted therapies. No significant financial relationships to disclose.