microRNA-199a-5p suppresses glioma progression by inhibiting MAGT1

microRNAs (miRNAs) can function as a tumor suppressor or oncogenic genes in human cancers. Alternation expression of miR-199a-5p has been revealed in several human cancers. However, its expression pattern and biological roles in glioma remain unclear. Expression levels of miR-199a-5p in glioma were evaluated at first. The effects of miR-199a-5p expression on cell proliferation, migration, and invasion were investigated using the MTT assay, wound-healing assay, and transwell invasion assay. The expression of miR-199a-5p was found to be reduced in glioma cell lines. Overexpression of miR-199a-5p inhibits glioma cell proliferation, migration, and invasion in vitro. Furthermore, the target of miR-199a-5p was predicted by TargetScan and validated by luciferase activity reporter assay. We found magnesium transporter 1 (MAGT1) was a direct target of miR-199a-5p. Overexpression of MAGT1 reversed the effects of miR-199a-5p on glioma cell behaviors. Taken together, our study revealed that miR-199a-5p and MAGT1 have the potential to be used as a biomarker for glioma.     

Helvolic acid attenuates osteoclast formation and function via suppressing RANKL-induced NFATc1 activation

Excessive osteoclast formation and function are considered as the main causes of bone lytic disorders such as osteoporosis and osteolysis. Therefore, the osteoclast is a potential therapeutic target for the treatment of osteoporosis or other osteoclast-related diseases. Helvolic acid (HA), a mycotoxin originally isolated from Aspergillus fumigatus , has been discovered as an effective broad-spectrum antibacterial agent and has a wide range of pharmacological properties. Herein, for the first time, HA was demonstrated to be capable of significantly inhibiting receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption in vitro by suppressing nuclear factor of activated T cells 1 (NFATc1) activation. This inhibition was followed by the dramatically decreased expression of NFATc1-targeted genes including Ctr (encoding calcitonin receptor), Acp5 (encoding tartrate-resistant acid phosphatase [TRAcP]), Ctsk (encoding cathepsin K), Atp6v0d2 (encoding the vacuolar H+ ATPase V0 subunit d2 [V-ATPase-d2]) and Mmp9 (encoding matrix metallopeptidase 9) which are osteoclastic-specific genes required for osteoclast formation and function. Mechanistically, HA was shown to greatly attenuate multiple upstream pathways including extracellular signal-regulated kinase (ERK) phosphorylation, c-Fos signaling, and intracellular Ca ²⁺ oscillation, but had little effect on nuclear factor-κB (NF-κB) activation. In addition, HA also diminished the RANKL-induced generation of intracellular reactive oxygen species. Taken together, our study indicated HA effectively suppressed RANKL-induced osteoclast formation and function. Thus, we propose that HA can be potentially used in the development of a novel drug for osteoclast-related bone diseases.     

Diosmetin inhibits osteoclast formation and differentiation and prevents LPS-induced osteolysis in mice

Osteolytic bone diseases are closely linked to the over-activation of osteoclasts and enhancement of bone resorption. It has become a major health issue in orthopedic practice worldwide. Inhibition of osteoclasts is proposed to be the main treatment for osteolytic disorders. Diosmetin (DIO) is a natural flavonoid with properties of antioxidant, anti-infection, and antishock. The effect of DIO on osteoclast differentiation is poorly understood. In this study project, we found that DIO could inhibit osteoclastic formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in a dose-dependent manner. The expression of the osteoclast differentiation marker genes, cathepsin K, nuclear factor of activated T-cells 1 (NFATc1), Acp5, Ctr, Atp6v0d2, and Mmp9 were also decreased by the treatment of DIO. In addition, DIO attenuated the formation of actin ring and the ability of bone resorption. Further, the western blotting showed that DIO inhibits the phosphorylation of the mitogen-activated protein kinases signaling pathway induced by RANKL, accompanied by the downregulation of NFATc1 and c-Fos expression. We also found that DIO could reduce the accumulation of reactive oxygen species (ROS) induced by RANKL. In vivo, the study revealed that DIO can significantly reduce LPS-induced osteolysis in mice. Collectively, our study shows that DIO can inhibit osteoclast formation and activation, and could serve as a potential therapeutic drug for osteolytic bone diseases.     

LncRNA TMPO-AS1 serves as a ceRNA to promote osteosarcoma tumorigenesis by regulating miR-199a-5p/WNT7B axis

Osteosarcoma (OS) is a common kind of aggressive tumor in bone which was mostly identified in children and adolescents with extremely high risk of death. Accumulating research works have displayed that long noncoding RNAs (lncRNAs) exert an essential role in the development of multiple cancers. It has been reported that TMPO-AS1 is an oncogene in cancers; nonetheless, its molecular mechanism in OS is totally unclear. Our present study elucidated that a remarkable overexpression of TMPO-AS1 was found in OS tissues and cells. Moreover, TMPO-AS1 depletion restrained Wnt/β-catenin pathway and cell proliferation as well as facilitated cell apoptosis. Further molecular mechanism investigations showed that TMPO-AS1 can sponge to miR-199a-5p. Moreover, miR-199a-5p was at a low level at OS cells. Importantly, miR-199a-5p's overexpression was associated with the OS cells' decreased proliferation and increased apoptosis. In addition, WNT7B was confirmed as a downstream gene of miR-199a-5p. Also the WNT7B expression was reversely modulated by miR-199a-5p and positively modulated by TMPO-AS1. Rescue experiments suggested that downregulated WNT7B rescued miR-199a-5p inhibitor-mediated repression on OS progression, but the treatment of LiCl counteracted the effect of WNT7B downregulation. In a word, TMPO-AS1 serves as a competing endogenous RNA to boost osteosarcoma tumorigenesis by regulating miR-199a-5p/WNT7B axis, which provided an underlying therapeutic target for patients with OS.     

FZD6 expression is negatively regulated by miR-199a-5p in human colorectal cancer

Colorectal cancer (CRC), the third most common cancer worldwide, also has the highest rate of cancer-related morbidity and mortality. WNT signaling is initiated by binding of WNT to various receptors, including frizzleds (FZDs), and plays a critical role in CRC and other tumor development by regulating proliferation, differentiation, migration, apoptosis, and polarity. Among the members of the FZD family, FZD6 is broadly expressed in various tissues, and its overexpression has been reported in several cancers, suggesting an important role in cancer development. In this study, we investigated the expression of FZD6 in patients with CRC and found it to be increased in tumors, as compared to paired adjacent non-tumor tissues. Additionally, we found that FZD6 expression was negatively regulated by miR199a5p in CRC cells. These results suggest that overexpression of FZD6, mediated by reduced expression of miR-199a-5p, may play an important role in the development of CRC. [BMB Reports 2015; 48(6): 360-366]     

The MicroRNA miR-199a-5p Down-regulation Switches on Wound Angiogenesis by Derepressing the v-ets Erythroblastosis Virus E26 Oncogene Homolog 1-Matrix Metalloproteinase-1 Pathway

miR-199a-5p plays a critical role in controlling cardiomyocyte survival. However, its significance in endothelial cell biology remains ambiguous. Here, we report the first evidence that miR-199a-5p negatively regulates angiogenic responses by directly targeting v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1). Induction of miR-199a-5p in human dermal microvascular endothelial cells (HMECs) blocked angiogenic response in Matrigel® culture, whereas miR-199a-5p-deprived cells exhibited enhanced angiogenesis in vitro. Bioinformatics prediction and miR target reporter assay recognized Ets-1 as a novel direct target of miR-199a-5p. Delivery of miR-199a-5p blocked Ets-1 expression in HMECs, whereas knockdown endogenous miR-199a-5p induced Ets-1 expression. Matrix metalloproteinase 1 (MMP-1), one of the Ets-1 downstream mediators, was negatively regulated by miR-199a-5p. Overexpression of Ets-1 not only rescued miR-199a-5p-dependent anti-angiogenic effects but also reversed miR-199a-5p-induced loss of MMP-1 expression. Similarly, Ets-1 knockdown blunted angiogenic response and induction of MMP-1 in miR-199a-5p-deprived HMECs. Examination of cutaneous wound dermal tissue revealed a significant down-regulation of miR-199a-5p expression, which was associated with induction of Ets-1 and MMP-1. Mice carrying homozygous deletions in the Ets-1 gene exhibited blunted wound blood flow and reduced abundance of endothelial cells. Impaired wound angiogenesis was associated with compromised wound closure, insufficient granulation tissue formation, and blunted induction of MMP-1. Thus, down-regulation of miR-199a-5p is involved in the induction of wound angiogenesis through derepressing of the Ets-1-MMP1 pathway.     

Silencing H19 regulated proliferation,invasion, and autophagy in the placenta by targeting miR-18a-5p

Fetal growth restriction (FGR) is a serious pregnancy complication associated with increased perinatal mortality and morbidity. It may lead to neurodevelopmental impairment and adulthood onset disorders. Recently, long noncoding RNAs (lncRNAs) were found to be associated with the pathogenesis of FGR. Here we report that the lncRNAH19 is significantly decreased in placentae from pregnancies with FGR. Downregulation of H19 leads to reduced proliferation and invasion of extravillous trophoblast cells. This is identified with reduced trophoblast invasion, which has been discovered in FGR. Autophagy is exaggerated in FGR. Downregulation of H19 promotes autophagy via the PI3K/AKT/mTOR and MAPK/ERK/mTOR pathways of extravillous trophoblast cells in FGR. We also found that the expression level of microRNAs miR-18a-5p was negatively correlated with that of H19. H19 can act as an endogenous sponge by directly binding to miR-18a-5p, which targets IRF2. The expression of miR-18a-5p was upregulated, but IRF2 expression was downregulated after the H19 knockdown. In conclusion, our study revealed that H19 downexpressed could inhibit proliferation and invasion, and promote autophagy by targeting miR-18a-5pin HTR8 and JEG3 cells. We propose that aberrant regulation of H19/miR-18a-5p-mediated regulatory pathway may contribute to the molecular mechanism of FGR. We indicated that H19 may be a potential predictive, diagnostic, and therapeutic modality for FGR.     

MiR-125a-5p抑制3T3-L1前体脂肪细胞分化

MicroRNAs(miRNAs) 是一类在脂肪组织发育中发挥重要作用的小非编码RNA. 为探明miR-125a-5p在3T3-L1前体脂肪细胞中的作用,采用实时qPCR检测了miR-125a-5p在小鼠各组织及3T3-L1前体脂肪细胞分化过程中的表达|使用经化学修饰的miR-125a-5p模拟物agomir及抑制剂antagomir转染3T3-L1前体脂肪细胞,采用实时qPCR 和 Western印迹检测成脂标志基因Pparγ和aP2的表达,油红O染色观察脂肪细胞脂质积累. 结果显示,miR-125-5p在小鼠脂肪组织中高丰度表达,在3T3-L1前体脂肪细胞分化过程中表达下降.过表达miR-125a-5p,与对照组相比,成脂标志基因Pparγ和aP2在mRNA和蛋白质水平均明显下降|油红O染色及定量结果显示脂质积累减少. 抑制剂处理结果显示,Pparγ和aP2在mRNA和蛋白质水平均有不同程度上升,但油红O染色及定量结果差异不显著. 以上结果表明,miR-125a-5p在脂肪细胞分化中发挥负调控作用.     

MiR-99a-5p inhibits bladder cancer cell proliferation by directly targeting mammalian target of rapamycin and predicts patient survival

Bladder cancer is a common malignancy and miR-99a-5p has been reported to be downregulated in bladder cancer, but its function and the underlying mechanism in bladder cancer development remains largely unclear. Here, we report that miR-99a-5p expression was decreased in bladder cancer compared with the adjacent normal tissues. Receiver operating characteristic curve revealed that miR-99a-5p expression signature had area under curve value of 0.7989 in differing bladder cancer from the adjacent normal tissues. Bladder cancer patients with low expression of miR-99a-5p had a poor survival rate. Gain-of-function and loss-of-function approaches demonstrated that miR-99a-5p inhibited bladder cell proliferation and cell cycle. Furthermore, we identified that mammalian target of rapamycin (mTOR) was a direct target of miR-99a-5p and mTOR restore could rescue the proliferative ability of bladder cancer cells. Moreover, miR-99a-5p/mTOR axis regulated S6K1 phosphorylation. These suggested that miR-99a-5p/mTOR axis might be a therapeutic target for bladder cancer.     

miR-125a-5p通过靶向调控GAB2抑制乳腺癌细胞的迁移能力

miR-125a-5p可负性调节GAB2表达,抑制胶质瘤细胞的侵袭和转移。本研究旨在证明miR-125a-5p抑癌作用的普遍性,即miR-125a-5p是否可通过靶向抑制GAB2抑制乳腺癌细胞的迁移。荧光素酶实验结果显示,miR-125a-5p可特异识别GAB2的3′-UTR,抑制报告酶的表达。荧光定量PCR结果揭示,与正常乳腺上皮细胞MCF-10A比较,miR-125a-5p在乳腺癌细胞MDA231和MCF-7中的表达明显降低;与迁移能力相对较低的MCF-7细胞比较,miR-125a-5p在迁移能力较高的MDA231细胞中的表达量更低。Western 印迹结果证明,与空载体（对照）和anti-miR125a 5p转染细胞比较,转染miR-125a-5p明显抑制GAB2蛋白在乳腺癌细胞中的表达。Transwell结果显示,与空载体转染的对照细胞比较,转染miR-125a-5p的乳腺癌细胞穿过基质胶的细胞数明显减少;相反,转染anti-miR125a-5p的细胞穿过基质胶的细胞数却明显增多。上述结果提示,miR-125a-5p在正常的乳腺细胞中高表达,而在乳腺癌细胞中低表达,其表达水平与癌细胞的迁移能力和GAB2表达呈反向关系。本研究结果还提示,miR-125a-5p通过靶向负调控GAB2抑制乳腺癌细胞的迁移能力。总之,本研究证明,miR-125a-5p在肿瘤中发挥抑癌作用。     

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.     

Receptor activator of nuclear factor‐kappa B ligand induces osteoclast formation in RAW 264.7 macrophage cells via augmented production of macrophage–colony‐stimulating factor

RAW 264.7 macrophage cells differentiate into osteoclast‐like cells in the presence of RANKL. Participation of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells was examined. TRAP‐positive osteoclast‐like cells appeared in RAW 264.7 cells cultured in the presence of RANKL. RANKL‐induced osteoclast formation was markedly inhibited by anti‐M‐CSF antibody. RANKL augmented M‐CSF mRNA expression and M‐CSF production in RAW 264.7 cells. Further, anti‐M‐CSF antibody inhibited the expression of RANK, c‐fms, c‐fos and TRAP mRNA in RANKL‐stimulated RAW 264.7 cells. However, anti‐M‐CSF antibody did not affect the expression of DC‐STAMP in the stimulated cells. Therefore, RANKL was suggested to induce osteoclast formation in RAW 264.7 cells via augmented production of M‐CSF. The putative role of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells is discussed.     

MiR‐199b‐5p inhibits osteogenic differentiation in ligamentum flavum cells by targeting JAG1 and modulating the Notch signalling pathway

Ossification of the ligamentum flavum (OLF) is a pathology almost only reported in East Asian countries. The leading cause of OLF is thoracic spinal canal stenosis and myelopathy. In this study, the role of miR‐199b‐5p and jagged 1 (JAG1) in primary ligamentum flavum cell osteogenesis was examined. MiR‐199b‐5p was found to be down‐regulated during osteogenic differentiation in ligamentum flavum cells, while miR‐199b‐5p overexpression inhibited osteogenic differentiation. In addition, JAG1 was found to be up‐regulated during osteogenic differentiation in ligamentum flavum cells, while JAG1 knockdown via RNA interference caused an inhibition of Notch signalling and osteogenic differentiation. Moreover, target prediction analysis and dual luciferase reporter assays supported the notion that JAG1 was a direct target of miR‐199b‐5p, with miR‐199b‐5p found to down‐regulate both JAG1 and Notch. Further, JAG1 knockdown was demonstrated to block the effect of miR‐199b‐5p inhibition. These findings imply that miR‐199b‐5p performs an inhibitory role in osteogenic differentiation in ligamentum flavum cells by potentially targeting JAG1 and influencing the Notch signalling pathway.     

PC-PLC is involved in osteoclastogenesis induced by TNF-α through upregulating IP3R1 expression

The precise mechanism of how TNF-α promotes osteoclast formation is not clear. Previous reports show TNF-α targets molecules that regulate calcium signaling. Inositol-1,4,5-trisphosphate receptors (IP3Rs) are important calcium channel responsible for evoking intracellular calcium oscillation. We found that TNF-α increased the expression of IP3R1 and promoted osteoclastogenesis in RANKL-induced mouse BMMs. Phosphatidylcholine-specific phospholipase C (PC-PLC) specific inhibitor D609 eliminated the upregulation of IP3R1 by TNF-α, and decreased the autoamplification of nuclear factor of activated T-cells 1 (NFATc1), thus resulted in less osteoclasts formation. However, D609 did not inhibit RANKL-induced osteoclastogenesis. Our data suggest TNF-α promotes RANKL-induced osteoclastogenesis, at least partially, through PC-PLC/IP3R1/NFATc1 pathway.