miR-21 Induction Research Articles

Abstract 124: Hypoxia-inducible miR-210 is involved in hypoxia-induced cell migration and invasion in human hepatocellular carcinoma

Abstract MicroRNA dysregulation is implicated in many human malignancies, yet little is known regarding the regulatory mechanisms of these small noncoding RNAs. Hypoxia is a prevalent tumor microenvironment in hepatocellular carcinoma (HCC) and plays a key role in modulating tumor aggressiveness. In the present study, we investigated the effects of hypoxia on microRNA expression in human HCC. First, we examined the miRNA expression profiles with TaqMan human microRNA Low-Density Array on HCC cell lines put under normoxic and hypoxic conditions to identify expression changes of microRNA. From the 664 microRNAs examined, miR-210 was overexpressed in SMMC-7721, Huh-7 and MHCC-97L HCC cell lines under 0.1% O2 hypoxic condition. The hypoxic induction of miR-210 was further validated in another two HCC cell lines (Hep3B and PLC/PRF/5) by individual real-time quantitative PCR (qPCR) and the same expression pattern was observed, suggesting that miR-210 is consistently induced in HCC cells under hypoxic conditions. The abundance of mature human miR-210 was also examined by qPCR in a human HCC cohort consisting of 48 pairs of primary HCC tissues and their corresponding non-tumorous livers. We found that miR-210 expression was significantly higher in the HCC tissues as compared with the corresponding non-tumorous livers (P=0.0003). Furthermore, we used the hypoxia marker carbonic anhydrase IX (CAIX) coupled with microdissection technique to isolate tissue samples in the peri-necrotic areas, which are considered pathologically hypoxic, in clinical formalin-fixed paraffin-embedded (FFPE) HCC samples. We extracted the microRNA from the tissues and compared the miR-210 expression with those from histologically preserved HCC areas away from necrotic areas. In line with the in vitro observations, with qPCR, miR-210 was remarkably overexpressed in hypoxic HCC regions. Furthermore, miR-210 overexpression significantly and inversely correlated with the overall survival and disease-free survival rates (P= 0.029 and 0.043, respectively) of HCC patients, indicating that overexpression of miR-210 in HCC was associated with a poorer prognosis. The biological function of the miR-210 induction in HCC was also investigated. Notably, with transwell and cell invasion assays, we demonstrated that the cell migratory and invasive abilities of SMMC-7721 were dramatically enhanced during hypoxia, and this enhancement was abolished upon miR-210 suppression using locked nucleic acid (LNA) inhibitor. Taken together, our present data indicate that miR-210 is a hypoxia-inducible microRNA in HCC, which may enhance HCC aggressiveness under hypoxic conditions. 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 124. doi:1538-7445.AM2012-124

DNA damage‐induced NF‐kB activation promotes breast cancer metastasis via upregulation of microRNA‐21

OBJECTIVETo investigate the role and mechanism of NF‐κB in cancer metastasis.METHODSDNA damage induced NF‐κB activity was determined by EMSA and microRNA‐21(miR‐21) expression was examined by Quantitative PCR separately. In addition, migration of MDA‐MB‐231 cells was investigated by Transwell invasion and wound healing assay in vitro.RESULTSWe found that chemotherapeutic drug‐induced NF‐κB activation promotes breast cancer cell migration and invasion. The increased metastatic potential is dependent on IL‐6 induction mediated by genotoxic NF‐κB activation. Moreover, genotoxic treatment also upregulates oncogenic microRNA‐21 expression through eliciting NF‐κB recruitment to miR‐21 promoter region, where it cooperates with signal transducer and activator of transcription 3 (STAT3) to activate miR‐21 transcription. Induction of miR‐21 may enable cancer cells to elude DNA damage‐induced apoptosis and enhance the metastatic potential of breast cancer cells through repressing expression of PTEN and PDCD4.CONCLUSIONSOur data support a critical role of DNA damage‐induced NF‐κB activation in promoting cancer metastasis following genotoxic treatment, and NF‐κB‐dependent miR‐21 induction may contribute to both therapeutic resistance and metastasis in breast cancer. Financial support: This work was supported by NIH grants ‐ CA149251 (to Z.W.) and CA133322 (to L.M.P)

MicroRNA-210 is upregulated in hypoxic cardiomyocytes through Akt- and p53-dependent pathways and exerts cytoprotective effects

microRNA-210 (miR-210) is upregulated in hypoxia, but its function in cardiomyocytes and its regulation in response to hypoxia are not well characterized. The purpose of this study was to identify upstream regulators of miR-210, as well as to characterize miR-210's function in cardiomyocytes. We first showed miR-210 is upregulated through both hypoxia-inducible factor (HIF)-dependent and -independent pathways, since aryl hydrocarbon nuclear translocator (ARNT) knockout mouse embryonic fibroblasts (MEF), lacking intact HIF signaling, still displayed increased miR-210 levels in hypoxia. To determine the mechanism for HIF-independent regulation of miR-210, we focused on p53 and protein kinase B (Akt). Overexpression of p53 in wild-type MEFs induced miR-210, whereas p53 overexpression in ARNT knockout MEFs did not, suggesting p53 regulates miR-210 in a HIF-dependent mechanism. Akt inhibition reduced miR-210 induction by hypoxia, whereas Akt overexpression increased miR-210 levels in both wild-type and ARNT knockout MEFs, indicating Akt regulation of miR-210 is HIF-independent. We then studied the effects of miR-210 in cardiomyocytes. Overexpression of miR-210 reduced cell death in response to oxidative stress and reduced reactive oxygen species (ROS) production both at baseline and after treatment with antimycin A. Furthermore, downregulation of miR-210 increased ROS after hypoxia-reoxygenation. To determine a mechanism for the cytoprotective effects of miR-210, we focused on the predicted target, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3), known to induce cell death. Although miR-210 reduced AIFM3 levels, overexpression of AIFM3 in the presence of miR-210 overexpression did not reduce cellular viability either at baseline or after hydrogen peroxide treatment, suggesting AIFM3 does not mediate miR-210's cytoprotective effects. Furthermore, HIF-3α, a negative regulator of HIF signaling, is targeted by miR-210, but miR-210 does not modulate HIF activity. In conclusion, we demonstrate a novel role for p53 and Akt in regulating miR-210 and demonstrate that, in cardiomyocytes, miR-210 exerts cytoprotective effects, potentially by reducing mitochondrial ROS production.

Akt2 Regulates All Akt Isoforms and Promotes Resistance to Hypoxia through Induction of miR-21 upon Oxygen Deprivation

The growth and survival of tumor cells in an unfavorable hypoxic environment depend upon their adaptability. Here, we show that both normal and tumor cells expressing the protein kinase Akt2 are more resistant to hypoxia than cells expressing Akt1 or Akt3. This is due to the differential regulation of microRNA (miR) 21, which is upregulated by hypoxia only in Akt2-expressing cells. By upregulating miR-21 upon oxygen deprivation, Akt2 downregulates PTEN and activates all three Akt isoforms. miR-21 also targets PDCD4 and Sprouty 1 (Spry1), and the combined downregulation of these proteins with PTEN is sufficient to confer resistance to hypoxia. Furthermore, the miR-21 induction by Akt2 during hypoxia depends upon the binding of NF-κB, cAMP responsive element-binding protein (CREB), and CBP/p300 to the miR-21 promoter, in addition to the regional acetylation of histone H3K9, all of which are under the control of Akt2. Analysis of the Akt2/miR-21 pathway in hypoxic MMTV-PyMT-induced mouse mammary adenocarcinomas and human ovarian carcinomas confirmed the activity of the pathway in vivo. Taken together, this study identifies a novel Akt2-dependent pathway that is activated by hypoxia and promotes tumor resistance via induction of miR-21.

Abstract 5004: In vivo profiling of hypoxic mRNA and miRNA expression in gliomas and head and neck tumors

Abstract Hypoxia plays a key role in tumor aggressiveness and radiation resistance, yet little is known regarding hypoxic global gene regulation in vivo. Although the regulation of mRNA and miRNA expression by hypoxia has been investigated in isolation in vitro, it has not yet been analyzed directly in tumor samples. We have used the hypoxia marker EF5 coupled with laser capture microdissection (LCM) to isolate RNA from viable hypoxic and normoxic regions of 9L experimental rat gliomas and from human head and neck (H&N) tumor samples. This was followed by microarray analysis of mRNA expression and comparison of this signature with that obtained from treating 9L cells with hypoxia in vitro. Through this, we have identified several mRNAs (including the HIF targets Vegf, Glut-1 and Hsp27) with increased levels under hypoxia compared to normoxia both in vitro and in vivo. We also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. An intriguing finding was the hypoxic-downregulation of a number of immunomodulatory and DNA repair proteins including CXCL9, CD3D and RAD51 in vivo, consistent with a pro-tumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic vs. normoxic tumor regions. Moreover, CD8+ T cells which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo. Global microRNA expression changes have been reported to occur in response to hypoxia in vitro. Hence, our second objective was to identify the cluster of miRNAs differentially expressed in hypoxic vs. normoxic regions of the human tumors using TaqMan® Array MicroRNA Cards. We employed the same technique used with the 9L tumors to analyze miRNA expression patterns in hypoxic vs. normoxic samples from H&N cancer patients who have been administered EF5 prior to surgical removal of the tumor. Consistent with published in vitro studies, miR-210 emerged as a major miRNA robustly induced in the hypoxic regions. We also confirmed miR-210 induction by an individual real time-PCR assay and found it to be upregulated > 2-fold in all hypoxic RNA samples. In addition, we have also found several miRNAs whose levels were upregulated and downregulated in hypoxic vs. normoxic areas. This is the first study to analyze the influence of hypoxia on mRNA and miRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5004. doi:10.1158/1538-7445.AM2011-5004

Ischemic Preconditioning Augments Survival of Stem Cells via miR-210 Expression by Targeting Caspase-8-associated Protein 2

MicroRNAs (miRs) participate in most cellular functions by posttranscriptional regulation of gene expression albeit with little information regarding their role in ischemic preconditioning (IP) of stem cells. We report that IP of bone marrow-derived mesenchymal stem cells (MSCs) with two cycles of 30-min ischemia/reoxygenation (I/R) supported their survival under subsequent longer exposure to anoxia and following engraftment in the infarcted heart. IP significantly reduced apoptosis in MSCs through activation of Akt (Ser(473)) and ERK1/2 (Thr(202)/Tyr(204)) and nuclear translocation of hypoxia-inducible factor-1alpha (HIF-1alpha). We observed concomitant induction of miR-210 in the preconditioned MSCs ((PC)MSCs). Inhibition of HIF-1alpha or of miR-210 abrogated the cytoprotective effects of preconditioning. Extrapolation of these data to in vivo studies in a rat model of acute myocardial infarction predominantly improved stem cell survival after engraftment with a role for miR-210. Notably, multiple I/R cycles more effectively regulated the miR-210 and hence promoted MSC survival compared with single-cycle hypoxia of an equal duration. Real time PCR array for rat apoptotic genes, computational target gene analyses, and luciferase reporter assay identified FLICE-associated huge protein (FLASH)/caspase-8-associated protein-2 (Casp8ap2) in (PC)MSCs as the target gene of miR-210. Induction of FLASH/CASP8AP2 in miR-210 knocked-down (PC)MSCs resulted in increased cell apoptosis. Taken together, these data demonstrated that cytoprotection afforded by IP was regulated by miR-210 induction via FLASH/Casp8ap2 suppression. These results highlighted that IP by multiple short episodes of I/R is a novel strategy to promote stem cell survival.

Emerging Roles of microRNAs in the Molecular Responses to Hypoxia

Recent studies have established that the regulation of microRNAs (miRs) is a feature of the hypoxic response. In this review, we discuss the role of hypoxia-regulated miRs, with an emphasis on miR-210 and miR-373, and anticipate directions for clinical applications. The induction of miR-210 and miR-373 is dependent upon hypoxia inducible factor (HIF), and their up-regulation has been detected in a variety of solid tumors. Both miRs have been associated with adverse prognosis and metastatic potential. The increased expression of miR-210 is linked to an in vivo hypoxic signature. MiR-210 also participates in endothelial and neuronal cells' response to oxygen deprivation and may possess a role in the regulation of angiogenesis. A variety of miR-210 and miR-373 targets that may be relevant to hypoxia have been validated or proposed. Very recently, targets of these miRs that are implicated in DNA repair have been identified, thus establishing an additional link between the hypoxic tumor microenvironment and DNA damage. Extending beyond cancer biology, some of miR-210 targets are likely involved in the regulation of angiogenesis, and neuronal cell survival. Inactivation of miRs affected by hypoxia presents a promising therapeutic strategy in the case of difficult-to-treat cancers, as well as in other non-cancer-related diseases.

Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

To better understand microRNA miR-21 function in carcinogenesis, we analyzed miR-21 expression patterns in different stages of colorectal cancer development using in situ hybridization (ISH). Locked nucleic acid (LNA)/DNA probes and a biotin-free tyramide signal amplification system were used in ISH analyses of miRNA expression. Conditions for specific detection of miR-21 were determined using human cell lines and miR-21-expressing lentiviral vectors. Expression was determined in 39 surgically excised colorectal tumors and 34 endoscopically resected colorectal polyps. In the surgical samples, miR-21 expression was much higher in colorectal cancers than in normal mucosa. Strong miR-21 expression was also observed in cancer-associated stromal fibroblasts, suggesting miR-21 induction by cancer-secreted cytokines. Protein expression of PDCD4, a miR-21 target, was inversely correlated with miR-21 expression, confirming that miR-21 is indeed a negative regulator of PDCD4 in vivo. In the endoscopic samples, miR-21 expression was very high in malignant adenocarcinomas but was not elevated in nontumorigenic polyps. Precancerous adenomas also frequently showed miR-21 up-regulation. Using the LNA-ISH system for miRNA detection, miR-21 was detectable in precancerous adenomas. The frequency and extent of miR-21 expression increased during the transition from precancerous colorectal adenoma to advanced carcinoma. Expression patterns of miR-21 RNA and its target, tumor suppressor protein PDCD4, were mutually exclusive. This pattern may have clinical application as a biomarker for colorectal cancer development and might be emphasized by self-reinforcing regulatory systems integrated with the miR-21 gene, which has been previously shown in cell culture.

Abstract 3829: Endothelial Cell Response to Hypoxia is Modulated by Microrna-210 Direct Targeting of Ephrin-A3

MicroRNAs (miRNAs) are small non-protein-coding RNAs that negatively modulate gene expression. In this study, miRNAs role in endothelial cell response to hypoxia was investigated. It was found that the expression of miR-210 progressively increased upon exposure to hypoxia. Interestingly, HIF1-alpha was necessary and sufficient for miR-210 induction, while HIF2-alpha was not necessary. miR-210 over-expression in normoxic endothelial cells increased the formation of capillary-like structures on Matrigel and VEGF-induced cell migration. Conversely, miR-210 blockade via LNA-anti-miRNA transfection decreased the formation of capillary-like structures stimulated by hypoxia and inhibited cell migration in response to VEGF. miR-210 over-expression did not affect endothelial cell growth in both hypoxia and normoxia. However, anti-miR-210 transfection inhibited cell growth and induced apoptosis, both in normoxia and in hypoxia. We found that one relevant target of miR-210 in hypoxia was Ephrin-A3, since miR-210 was necessary and sufficient to down-regulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences: indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210, prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. A combination of proteomic and transcriptomic approaches has been undertaken, identifying potential direct and indirect targets of miR-210 action. We conclude that miR-210 induction is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration and differentiation.

MiR-210 links hypoxia with cell cycle regulation and is deleted in human epithelial ovarian cancer

Tumor growth results in hypoxia. Understanding the mechanisms of gene expression reprogramming under hypoxia may provide important clues to cancer pathogenesis. We studied miRNA genes that are regulated by hypoxia in ovarian cancer cell lines by TaqMan miRNA assay containing 157 mature miRNAs. MiR-210 was the most prominent miRNA consistently stimulated under hypoxic conditions. We provide evidence for the involvement of the HIF signaling pathway in miR-210 regulation. Biocomputational analysis and in vitro assays demonstrated that e2f transcription factor 3 (e2f3), a key protein in cell cycle, is regulated by miR-210. E2F3 was further confirmed to be downregulated at the protein level upon induction of miR-210. Importantly, we found remarkably high frequency of miR-210 gene copy deletions in ovarian cancer patients (64%, n=114) and that gene copy number correlates with miR-210 expression levels. Taken together, our results indicate that miR-210 plays a crucial role in tumor onset as a key regulator of the hypoxia response and provide evidence for a link between hypoxia and the regulation of cell cycle.

Interleukin-6–dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer

AbstractSignal transducer and activator of transcription 3 (Stat3) is implicated in the pathogenesis of many malignancies and essential for IL-6–dependent survival and growth of multiple myeloma cells. Here, we demonstrate that the gene encoding oncogenic microRNA-21 (miR-21) is controlled by an upstream enhancer containing 2 Stat3 binding sites strictly conserved since the first observed evolutionary appearance of miR-21 and Stat3. MiR-21 induction by IL-6 was strictly Stat3 dependent. Ectopically raising miR-21 expression in myeloma cells in the absence of IL-6 significantly reduced their apoptosis levels. These data provide strong evidence that miR-21 induction contributes to the oncogenic potential of Stat3.