MicroRNA-199b-5p impairs cancer stem cells through negative regulation of HES1 in medulloblastoma.
ABSTRACT: Through negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3-4 years and 8-9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies.In a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept.Despite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.
Project description:Micro-RNA (miR) 199b-5p targets Hes1 in medulloblastoma, one of the downstream effectors of both the canonical Notch and noncanonical Sonic Hedgehog pathways. In medulloblastoma patients, expression of miR-199b-5p is significantly decreased in metastatic cases, thus suggesting a downregulation mechanism. We studied this mechanism, which is mediated mostly by Hes1 and epigenetic promoter modifications. The miR-199b-5p promoter region was characterized, which identified a Hes1 binding site, thus demonstrating a negative feedback loop of regulation. MiR-199b-5p was shown to be downregulated in several medulloblastoma cell lines and in tumors by epigenetic methylation of a cytosine-phosphate-guanine island upstream of the miR-199b-5p promoter. Furthermore, the cluster of differention (CD) carbohydrate antigen CD15, a marker of medulloblastoma tumor-propagating cells, is an additional direct target of miR-199b-5p. Most importantly, regulation of miR-199b-5p expression in these CD15+/CD133+ tumor-propagating cells was influenced by only Hes1 expression and not by any epigenetic mechanism of regulation. Moreover, reverse-phase protein array analysis showed both the Akt and extracellular-signal-regulated kinase pathways as being mainly negatively regulated by miR-199b-5p expression in several medulloblastoma cell lines and in primary cell cultures. We present here the finely tuned regulation of miR-199b-5p in medulloblastoma, underlining its crucial role by its additional targeting of CD15.
Project description:Epithelial ovarian cancer is a highly lethal and aggressive gynecological malignancy. The high mortality rate is due in part to the fact that many advanced cancer patients become refractory to current chemotherapeutic agents, leading to tumor recurrence and death. However, the underlying mechanisms leading to chemoresistance remain obscure. Here, we report that the loss of miR-199b-5p due to progressive epigenetic silencing leads to the activation of the JAG1-mediated Notch1 signaling cascade, thereby leading to the development of acquired chemoresistance in ovarian cancer. Using miRCURY LNA™ microRNA array and Q-PCR analyses of two pairs of cisplatin-sensitive and -resistant ovarian cancer cell lines, we identified miR-199b-5p as significantly down-regulated in cisplatin-resistant ovarian cancer cells and confirmed that miR-199b-5p is clinically associated with advanced and poor survival ovarian cancers. Interestingly, the loss of miR-199b-5p could be restored by 5-Aza-dC-mediated demethylation, and methylated specific PCR (MS-PCR), bisulfite-sequencing and pyrosequencing revealed that the promoter region of miR-199b-5p was hypermethylated. Computational and mechanistic analyses identified JAG1 as a primary target of miR-199b-5p. Notably, the reduced expression of miR-199b-5p was found to be inversely correlated with the increased expression of JAG1 using an ovarian cancer tissue array. Enforced expression of miR-199b-5p sensitized ovarian cancer cells to cisplatin-induced cytotoxicity both in vitro and in vivo. Conversely, re-expression of miR-199b-5p and siRNA-mediated JAG1 knockdown or treatment with Notch specific inhibitor ?-secretase (GSI) attenuated JAG1-Notch1 signaling activity, thereby enhancing cisplatin-mediated cell cytotoxicity. Taken together, our study suggests that the epigenetic silencing of miR-199b-5p during tumor progression is significantly associated with acquired chemoresistance in ovarian cancer through the activation of JAG1-Notch1 signaling.
Project description:<h4>Background</h4>Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies.<h4>Methodology/principal findings</h4>In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133(+)/CD15(+) tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1(+/-) p53(-/-)), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo.<h4>Conclusions/significance</h4>Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.
Project description:Atrasentan is a promising therapy for treating diabetic nephropathy (DN). Here we evaluated whether atrasentan down-regulated the miR-199b-5p expression, thereby increasing klotho and preventing renal tubular injury in DN. One-hundred patients with type 2 diabetes mellitus (T2DM) and 40 healthy subjects were included. A DN mice model was established by an injection of streptozotocin (STZ). Human renal proximal tubular epithelial HK-2 cells were exposed to high glucose (20?mmol/L). Treated the mice and HK-2 cells with atrasentan, and we then investigated whether and how miR-199b-5p and Klotho were involved in preventing renal tubular injury in DN. In patients, the serum miR-199b-5p level increased and the klotho concentration decreased in accordance with elevated albuminuria. Atrasentan down-regulated miR-199b-5p and up-regulated klotho of the DN mice and HK-2 cells exposed to high glucose. High glucose promoted the binding of histone H3 to the miR-199b-5p promoter, and atrasentan canceled this effect. MiR-199b-5p targeted the 3'?UTR of klotho. Overexpression of miR-199b-5p canceled the effects of atrasentan on klotho expression and apoptosis of renal tubular cells in both in vivo and in vitro. The increased serum klotho, mediated by miR-199b-5p, is a possible mechanism by which atrasentan prevents renal tubular injury in DN.
Project description:<h4>Aims</h4>Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalized medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation toward ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration.<h4>Methods and results</h4>In this study, we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in vascular endothelial growth factor (VEGF) transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation toward smooth muscle markers. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis.<h4>Conclusions</h4>This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses. Stem Cells 2015;33:1405-1418.
Project description:Angiogenesis is a crucial event during cancer progression that regulates tumor growth and metastasis. Activin receptor-like kinase 1 (ALK1), predominantly expressed in endothelial cells, plays a key role in the organization of neo-angiogenic vessels. Therapeutic targeting of ALK1 has been proposed as a promising strategy for cancer treatment, and microRNAs (miRNAs) are increasingly being explored as modulators of angiogenesis. However, the regulation of ALK1 by miRNAs is unclear. In this study, we identified that ALK1 is directly targeted by miR-199b-5p, which was able to inhibit angiogenesis in vitro and in vivo. Moreover, it was found that miR-199b-5p was repressed in breast cancer cells and its expression was decreased during the VEGF-induced angiogenesis process of human umbilical vein endothelial cells (HUVECs). Overexpression of miR-199b-5p inhibited the formation of capillary-like tubular structures and migration of HUVECs. Furthermore, overexpression of miR-199b-5p inhibited the mRNA and protein expression of ALK1 in HUVECs by directly binding to its 3'UTR. Additionally, overexpression of miR-199b-5p attenuated the induction of ALK1/Smad/Id1 pathway by BMP9 in HUVECs. Finally, overexpression of miR-199b-5p reduced tumor growth and angiogenesis in in vivo. Taken together, these findings demonstrate the anti-angiogenic role of miR-199b-5p, which directly targets ALK1, suggesting that miR-199b-5p might be a potential anti-angiogenic target for cancer therapy.
Project description:Accumulating evidence indicates that N-cadherin is a cell adhesion molecule that has critical roles in tumour progression. However, the role of N-cadherin in hepatocellular carcinoma (HCC) remains controversial.This study aims to investigate the expression status of N-cadherin and its molecular mechanisms in HCC.The expression of N-cadherin was markedly overexpressed in HCC tissues and cell lines. We identified that miR-199b-5p binds to the 3'-UTR of N-cadherin mRNA, thus decreasing N-cadherin expression in HCC cells. We also found the downregulation of miR-199b-5p in HCC specimens, which was inversely correlated with N-cadherin upregulation, predicted poor clinical outcomes in HCC patients. Next, we determined that miR-199b-5p overexpression promoted cell aggregation, suppressed cell migration and invasion in HCC cells, and inhibited xenografts tumour metastasis in nude mice. Moreover, we demonstrated that miR-199b-5p attenuated TGF-?1 induced epithelial-mesenchymal transition (EMT) -associated traits, while its effects could be partially reversed by N-cadherin restoration. Finally, we examined that N-cadherin downregulation or miR-199b-5p overexpression suppressed TGF-?1-induced Akt phosphorylation, and inhibition of PI3K/Akt pathway blocked TGF-?1-induced N-cadherin overexpression in HCC cells.Our data demonstrate that N-Cadherin was markedly overexpressed and miR-199b-5p was significantly downregulated in HCC. MiR-199b-5p exerts inhibitory effects on EMT, and directly targets N-cadherin in HCC, supporting the potential utility of miR-199b-5p as a promising strategy to treat HCC. Also, a positive regulatory loop exists between N-cadherin and Akt signalling represents a novel mechanism of TGF-?1-mediated EMT in HCC cells.
Project description:Almost all cervical cancers are associated with human papillomavirus (HPV); however, the majority of women infected with this virus do not develop cervical cancer. Therefore, new markers are needed for reliable screening of cervical cancer, especially in relation to HPV infection. We aimed to identify potential microRNAs that may serve as diagnostic markers for cervical cancer development in high-risk HPV-positive patients. We evaluated the microRNA expression profiles in 12 cervical tissues using the hybridization method and verified them by quantitative polymerase chain reaction (qPCR). Finally, we evaluated the effects of HPV16 oncoproteins on the expression of selected microRNAs using cervical cancer cells (CaSki and SiHa) and RNA interference. With the hybridization method, eight microRNAs (miR-9-5p, miR-136-5p, miR-148a-3p, miR-190a-5p, miR-199b-5p, miR-382-5p, miR-597-5p, and miR-655-3p) were found to be expressed differently in the HPV16-positive cervical cancer group and HPV16-positive normal group (fold change ? 2). The results of qPCR showed that miR-148a-3p, miR-190a-5p, miR-199b-5p, and miR-655-3p levels significantly decreased in the cancer group compared with the normal group. Upon silencing of HPV16 E5 and E6/E7, miR-148a-3p levels increased in both cell lines. Silencing of E6/E7 in SiHa cells led to the increase in miR-199b-5p and miR-190a-5p levels. Three HPV16 oncoproteins (E5, E6, and E7) downregulate miR-148a-3p, while E6/E7 inhibit miR-199b-5p and miR-190a-5p expression in cervical carcinoma. The three microRNAs, miR-148a-3p, miR-199b-5p, and miR-190a-5p, may be novel diagnostic biomarkers for cervical cancer development in high-risk HPV-positive patients.
Project description:This study screened microRNAs (miRNAs) that are abnormally expressed in papillary thyroid carcinoma (PTC) tissues to identify PTC and nodular goiter and the degree of PTC malignancy. A total of 51 thyroid tumor tissue specimens paired with adjacent normal thyroid tissues were obtained from the Department of Surgical Oncology of Hangzhou First People's Hospital from June-December 2011. miRNA expression profiles were examined by microarrays and validated by quantitative real-time PCR (qRT-PCR). Expression levels of the miRNAs were analyzed to assess if they were associated with selected clinicopathological features. Eleven miRNAs were significantly differentially expressed between nodular goiter and PTC and between highly invasive and low invasive PTC. miR-199b-5p and miR-30a-3p were significantly differentially expressed among the three groups. miR-30a-3p, miR-122-5p, miR-136-5p, miR-146b-5p and miR-199b-5p were selected for further study by qRT-PCR and miR-146b-5p, miR-199b-5p and miR-30a-3p were different between the PTC and nodular goiter groups. miR-199b-5p was over-expressed in PTC patients with extrathyroidal invasion and cervical lymph node metastasis. In conclusion miR-146b-5p, miR-30a-3p, and miR-199b-5p may serve as biomarkers for the diagnosis of PTC and miR-199b-5p is associated with PTC invasiveness.
Project description:Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.