17-701 Sigma-AldrichEZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit

Aberrant Hedgehog signaling and excessive activation of the Gli family of transcriptional activators are key drivers of medulloblastoma (MB), the most common human pediatric brain malignancy. MB originates mainly from cerebellar granule neuron progenitors (CGNP), but the mechanisms underlying CGNP transformation remain largely obscure. In this study, we found that suppression of the noncoding RNA Nkx2-2as promoted Sonic Hedgehog (Shh)-potentiated MB development. Nkx2-2as functioned as a competing endogenous RNA against miR-103 and miR-107, sequestering them and thereby derepressing their tumor suppressive targets BTG2 and LATS1 and impeding cell division and migration. We also found that Nkx2-2as tethered miR-548m and abrogated its LATS2 targeting activity. Shh signaling impaired Nkx2-2as expression by upregulating the transcriptional repressor FoxD1. In clinical specimens of Shh-subgroup MB, we validated coordinated expression of the aforementioned proteins. Notably, exogenous expression of Nkx2-2as suppressed tumorigenesis and prolonged animal survival in MB mouse models. Our findings illuminate the role of noncoding RNAs in Hedgehog signaling and MB occurrence, with implications for identifying candidate therapeutic targets for MB treatment.Significance: These findings illuminate the role of noncoding RNAs in Hedgehog signaling and an interplay between the Hedgehog and Hippo pathways in medulloblastoma pathogenesis. Cancer Res; 78(4); 962-73. ©2017 AACR.

This study was aimed to investigate the potential regulatory mechanism of high-content hydrogen water (HHW) in non-alcoholic fatty liver disease (NAFLD). A high-fat diet (HFD)-induced NAFLD mice model and cellular model were prepared. The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total cholesterol (TCH) and triglycerides (TG) were measured. The expression levels of representative five microRNA (miRNAs) (miR-103, miR-488, miR-136, miR-505 and miR-148a) in liver tissues were determined by quantitative real-time PCR (qRT-PCR). The target of miR-136 was validated by RNA immunoprecipitation (RIP) and pull-down assay. MiR-136, MEG3 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression levels following cell treatment were detected in hepatocytes using qRT-PCR and Western blotting. Moreover, cell viability and TG content were conducted. MiR-136 was downregulated, MEG3 as well as Nrf2 was upregulated and serum lipid level was reduced in NAFLD mice model after HHW treatment, which exerted the same effect in cellular model. RIP and RNA pull-down assay confirmed that MEG2 was a downstream target of miR-136. What's more, HHW ameliorated lipid accumulation by regulating miR-136/MEG3/Nrf2 axis in vitro and in vivo. Hence, HHW alleviated NAFLD by downregulation of miR-136 through mediating Nrf2 via targeting MEG3.

Emerging evidence indicates that long non-coding RNAs (lncRNAs) play a vital role in cardiovascular physiology and pathology. Although the lncRNA TUG1 is implicated in atherosclerosis, its function in calcific aortic valve disease (CAVD) remains unknown.In this study, we found that TUG1 was highly expressed in human aortic valves and primary valve interstitial cells (VICs). Moreover, TUG1 knockdown induced inhibition of osteoblast differentiation in CAVD both in vitro and in vivo. Mechanistically, silencing of TUG1 increased the expression of miR-204-5p and subsequently inhibited Runx2 expression at the post-transcriptional level. Importantly, TUG1 directly interacted with miR-204-5p and downregulation of miR-204-5p efficiently reversed the suppression of Runx2 induced by TUG1 short hairpin RNA (shRNA). Thus, TUG1 positively regulated the expression of Runx2, through sponging miR-204-5p, and promoted osteogenic differentiation in CAVD.All together, the evidence generated by our study elucidates the role of lncRNA TUG1 as a miRNA sponge in CAVD, and sheds new light on lncRNA-directed diagnostics and therapeutics in CAVD.

LncRNA colon-cancer-associated transcript-1 (CCAT1) was proved to be a potential prognostic biomarker for breast cancer progression. However, the role of CCAT1 in regulating radiosensitivity of breast cancer and its underlying mechanism have not been investigated. The present study showed that CCAT1 was up-regulated and miR-148b was down-regulated in radioresistant breast cancer tissues compared with radiosensitive breast cancer tissues. Radiation treatment triggered a significant increase in CCAT1 and an obvious decrease in miR-148b. CCAT1 down-regulation reduced colony formation rates and caspase3 activity in breast cancer cells under irradiation. Moreover, CCAT1 could negatively regulate miR-148b expression. Furthermore, overexpression of miR-148b suppressed colony survival fraction and caspase3 expression under irradiation in breast cancer cells, which was exacerbated by CCAT1 knockdown. Taken together, this study demonstrated that CCAT1 down-regulation improved radiosensitivity of breast cancer cells via negatively regulating miR-148b expression, providing a crucial clue for lncRNA-miRNA interaction in the mechanism of radiosensitivity of breast cancer.

Colorectal cancer (CRC) is one of the most common cancers and the third leading cause of cancer-related deaths due to its rapid progression and poor prognosis. 5-Fluorouracil (5-FU)-based chemotherapies are the standard treatment for locally advanced CRC. However, a considerable percentage of CRCs have inherent or acquired 5-FU resistance, which critically impedes clinical outcomes. In the present study, we reported that the expression level ENST00000547547 was downregulated in 5-FU-resistant CRC cells in comparison with the parental cells, While rising with the treatment of 5-FU in parental cells. Overexpression of ENST00000547547 promoted 5-FU-induced cell apoptosis and reduced the chemoresistance of 5-FU in vitro. Moreover, we found that ENST00000547547 was a target of miR-31, as confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. Notably, miR-31 was upregulated in 5-FU-resistant CRC cells, and knockdown of miR-31 increased the chemosensitivity of 5-FU-resistant CRC cells. Furthermore, we demonstrated that ENST00000547547 reduced the chemoresistance of 5-FU via competitive binding to miR-31 in 5-FU-resistant CRC cell lines. Collectively, our findings revealed that ENST00000547547 reduced chemoresistance in 5-FU of 5-FU-resistant CRC cells through competitive binding to miR-31 and has the potential to serve as a therapeutic target in CRC patients.

In the context of diabetes, obesity, and metabolic syndrome, the inflammatory signaling has critical roles in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), but the underlying mechanisms remain poorly delineated. Herein, early and persistently elevated, proinflammatory cytokine HMGB1 expression was detected in a high-fat diet (HFD)-induced NAFLD model in C57BL/6 mice. The expression and extracellular release of HMGB1 was rapidly and dramatically induced by saturated palmitic acid in vitro. HFD-induced inflammatory response and liver function impairment were both mitigated after the inhibition of endogenous HMGB1 by neutralizing antibody in vivo. The up-regulation of HMGB1 was thought to be modified by dual channels: in the transcriptional level, it was regulated by JNK1/JNK2-ATF2 axis; post-transcriptionally, it was regulated by the microRNA (miR)-200 family, especially miR-429. miR-429 liver conditional knockout mice (miR-429Δhep), fed either a normal diet or an HFD, showed severe liver inflammation and dysfunction, accompanied by greater expression of HMGB1. Intriguingly, the up-regulation and release of HMGB1 could in turn self-activate TLR4-JNK1/JNK2-ATF2 signaling, thus forming a positive feedback. Our findings reveal a novel mechanism by which HMGB1 expression was regulated by both the JNK1/2-ATF2 axis and the miR-200 family, which provides a potential new approach for the treatment of NAFLD.-Chen, X., Ling, Y., Wei, Y., Tang, J., Ren, Y., Zhang, B., Jiang, F., Li, H., Wang, R., Wen, W., Lv, G., Wu, M., Chen, L., Li, L., Wang, H. Dual regulation of HMGB1 by combined JNK1/2-ATF2 axis with miR-200 family in nonalcoholic steatohepatitis in mice.

Ischemic stroke is related to a variety of physiological and pathological processes including autophagy and apoptosis. Growth arrest-specific 5 (GAS5), a long non-coding RNA (lncRNA), is known to negatively regulate cell survival and plays a key role in the pathogenesis of numerous diseases. However, the function and molecular mechanism of lncRNA GAS5 in ischemic stroke have not been reported. Real-time PCR was used to detect GAS5 and microRNA-137 (miR-137) expression in the brain tissues of mice underwent middle cerebral artery occlusion (MCAO) surgery and oxygen-glucose deprivation (OGD)-treated mouse primary brain neurons. Gain- or loss-of-function approaches were used to manipulate GAS5, miR-137, and Notch1. The mechanism of GAS5 in ischemic stroke was evaluated both in vivo and in vitro via bioinformatics analysis, MTT, flow cytometry, luciferase assay, RNA immunoprecipitation, and Western blot. GAS5 level was up-regulated and negatively correlated with miR-137 expression in MACO-injured brain and in OGR-stimulated primary brain neurons. GAS5 siRNA notably increased the cell viability, suppressed the activation of caspase-3 and cell apoptosis in neurons subjected to OGD. Furthermore, we also found that GAS5 functioned as a competing endogenous RNA (ceRNA) for miR-137 to regulate the de-repression of its endogenous target Notch1 and decrease neuron survival through inactivation of the Notch1 signaling pathway. Taken together, these findings indicate that GAS5 may promote the progression of ischemic stroke through acting as a ceRNA for miR-137 to mediate the Notch1 signaling pathway, which contributes to an extensive understanding of ischemic stroke and may provide novel therapeutic options for this disease.

A growing number of long non-coding RNAs (lncRNAs) have been found to be involved in diverse biological processes such as cell cycle regulation, embryonic development, and cell differentiation. However, limited knowledge is available concerning the underlying mechanisms of lncRNA functions. In this study, we found down-regulation of TCONS_00041960 during adipogenic and osteogenic differentiation of glucocorticoid-treated bone marrow mesenchymal stem cells (BMSCs). Furthermore, up-regulation of TCONS_00041960 promoted expression of osteogenic genes Runx2, osterix, and osteocalcin, and anti-adipogenic gene glucocorticoid-induced leucine zipper (GILZ). Conversely, expression of adipocyte-specific markers was decreased in the presence of over-expressed TCONS_00041960. Mechanistically, we determined that TCONS_00041960 as a competing endogenous RNA interacted with miR-204-5p and miR-125a-3p to regulate Runx2 and GILZ, respectively. Overall, we identified a new TCONS_00041960-miR-204-5p/miR-125a-3p-Runx2/GILZ axis involved in regulation of adipogenic and osteogenic differentiation of glucocorticoid-treated BMSCs.

The prognosis for bladder cancer patients with lymph node (LN) metastasis is dismal and only minimally improved by current treatment modalities. Elucidation of the molecular mechanisms that underlie LN metastasis may provide clinical therapeutic strategies for LN-metastatic bladder cancer. Here, we report that a long noncoding RNA LINC00958, which we have termed bladder cancer-associated transcript 2 (BLACAT2), was markedly upregulated in LN-metastatic bladder cancer and correlated with LN metastasis. Overexpression of BLACAT2 promoted bladder cancer-associated lymphangiogenesis and lymphatic metastasis in both cultured bladder cancer cell lines and mouse models. Furthermore, we demonstrate that BLACAT2 epigenetically upregulated VEGF-C expression by directly associating with WDR5, a core subunit of human H3K4 methyltransferase complexes. Importantly, administration of an anti-VEGF-C antibody inhibited LN metastasis in BLACAT2-overexpressing bladder cancer. Taken together, these findings uncover a molecular mechanism in the lymphatic metastasis of bladder cancer and indicate that BLACAT2 may represent a target for clinical intervention in LN-metastatic bladder cancer.

Studies have shown that long noncoding ribonucleic acids (lncRNAs) play critical roles in multiple biologic processes. However, the Small Nucleolar RNA Host Gene 1 (SNHG1) function and underlying molecular mechanisms in ischemic stroke have not yet been reported. In the present study, we found that SNHG1 expression was remarkably increased both in isolated cerebral micro-vessels of a middle cerebral artery occlusion (MCAO) mice model, and in oxygen-glucose deprivation (OGD)-cultured mice brain micro-vascular endothelial cells (BMECs), meanwhile, the SNHG1 level was negatively correlated with miR-18a in MCAO mice. Mechanistically, SNHG1 inhibition presents larger brain infarct size and worsens neurological scores in MCAO mice. Consistent with the in vivo findings, SNHG1 inhibition also significantly increased caspase-3 activity and cell apoptosis in OGD-cultured BMECs. Furthermore, we found that SNHG1 functions as a competing endogenous RNA (ceRNA) for miR-18a, thereby regulating the de-repression of its endogenous target HIF-1α and promoting BMEC survival through HIF-1α/VEGF signaling. This study found a neuroprotective effect of SNHG1 mediated by HIF-1α/VEGF signaling through acting as a ceRNA for miR-18a. These findings reveal a novel function of SNHG1, which contributes to an extensive understanding of ischemic stroke and provides novel therapeutic options for this disease.