NNT-AS1 enhances bladder cancer cell growth by targeting miR-1301-3p/PODXL axis and activating Wnt pathway

As a tumor involved in the urinary system, bladder cancer (BC) seriously threatens human health. Emerging as crucial biomarkers, long noncoding RNAs (lncRNAs) play an important role in the regulation of many cancers. lncRNA NNT-AS1 has been studied in a series of cancers, whereas its role and potential molecular mechanism was poorly understood in BC. Here, we found that NNT-AS1 was upregulated in BC cells. Functionally, the silencing of NNT-AS1 inhibited cell proliferation, migration, invasion, and endothelial-mesenchymal transition. Furthermore, the apoptosis of BC cells was induced upon NNT-AS1 knockdown. Later, miR-1301-3p, the downstream gene of NNT-AS1, was found at a low level in BC cells. In addition, we found that miR-1301-3p targeted to PODXL. PODXL expression downregulated in NNT-AS1-silenced cells was restored by miR-1301-3p inhibition. Importantly, NNT-AS1 was discovered to activate Wnt pathway, and the treatment of LiCl recovered the repressive role of NNT-AS1 silencing in BC cell growth. Through restoration assays, we observed that PODXL overexpressing countervailed NNT-AS1 depletion-mediated suppression on BC cell growth and Wnt pathway. These data suggested that NNT-AS1 enhances BC cell growth and activates Wnt pathway by targeting miR-1301-3p/PODXL axis.     

miR-187-5p 对骨髓间充质干细胞成骨分化能力的调控作用

目的利用小鼠骨髓间充质干细胞(BMSCs)的成骨分化模型,探讨miR-187-5p在成骨分化中的表达趋势及调控作用。方法通过切除雌性小鼠双侧卵巢构建小鼠骨质疏松模型;应用qRT-PCR技术检测组织和细胞中miR-187-5p的表达;应用基因转染技术观察过表达或敲减miR-187-5p对BMSCs向成骨分化的影响;应用茜素红和碱性磷酸酶染色检测BMSCs中矿化结节的数量和矿化区域的染色面积。结果 qRT-PCR结果显示miR-187-5p在骨质疏松模型小鼠的骨组织及BMSCs中均表达下降。过表达miR-187-5p可提高ALP、Collagen-1、Runx2、BMP4、OCN和OPN等成骨分化相关基因mRNA的表达,促进BMSCs向成骨分化;而敲减miR-187-5p降低ALP等成骨分化相关基因mRNA的表达,抑制BMSCs向成骨分化。在体实验同样证实,过表达miR-187-5p可以显著促进骨质疏松小鼠BMSCs向成骨分化,改善小鼠的骨质疏松表型。结论过表达miR-187-5p促进BMSCs向成骨分化,敲减miR-187-5p抑制BMSCs向成骨分化。     

miR-155 modulates TNF-α-inhibited osteogenic differentiation by targeting SOCS1 expression

Bone morphogenetic proteins (BMPs) can induce ectopic bone formation, which is negatively regulated by inflammatory cytokines, such as tumor necrosis factor (TNF)-α. Recently, miR-155 has been reported to regulate the transforming growth factor (TGF)-β signaling pathway and inflammatory responses. However, whether and how miR-155 modulates TNF-α-regulated osteogenic differentiation have not been explored. In this study, we demonstrated that miR-155 was involved in TNF-α-mediated inhibition of osteogenic differentiation. Knockdown of miR-155 partially mitigated the inhibition of TNF-α on BMP-2-induced osteogenic differentiation. Bioinformatic analysis identified the candidate target site in the 3' untranslated region (3'UTR) of SOCS1. Knockdown of miR-155 increased SOCS1 protein expression during TNF-α stimulation in MC3T3-E1 cells. And transfection with miR-155 inhibited the wild-type, but not the mutant, 3'UTR of SOCS1-regulated luciferase activity, indicating that SOCS1 is a direct target of miR-155 in osteoblast cells. Furthermore, miR-155 expression could be induced by TNF-α through the JNK pathway. As the result of increased SOCS1 expression, knockdown of miR-155 significantly reduced the JNK/c-Jun activation. In addition, transfection of SOCS1 siRNA or overexpression of SOCS1 coding region could narrow the differences of alkaline phosphatase (ALP) and osteocalcin (OSC) expression between the control and miR-155 inhibitor transfected cells. These data indicated that miR-155 modulates TNF-α-regulated osteogenic differentiation by targeting SOCS1, at least partially through the SAPK/JNK pathway. These findings may provide new insights into understanding the regulatory role of miR-155 in the process of osteogenic differentiation in inflammatory condition.     

High levels of β‐catenin signaling reduce osteogenic differentiation of stem cells in inflammatory microenvironments through inhibition of the noncanonical Wnt pathway

Periodontal ligament stem cells (PDLSCs), a new population of mesenchymal stem cells (MSCs), have been isolated from the periodontal ligament (PDL). The capacity of multipotency and self‐renewal makes them an excellent cell source for bone regeneration and repair. However, their bone‐regeneration ability could be awakened in inflammatory microenvironments, which may be the result of changes in their differentiation potential. Recently, genetic evidences has shown that the Wnt pathway plays an important role in bone homeostasis. In this study we have determined the specific role of β‐catenin in osteogenic differentiation of PDLSCs obtained from inflammatory microenvironments (P‐PDLSCs). The inflammatory microenvironment, while inhibiting osteogenic differentiation potential, promotes proliferation of MSCs. A higher the level of β‐catenin in P‐PDLSCs than in H‐PDLSCs (PDLSCs obtained from a healthy microenvironment) resulted in the same disparity in canonical Wnt signaling pathway activation between each cell type. Here we show that activation of β‐catenin suppresses the noncanonical Wnt/Ca²⁺ pathway, leading to increased proliferation but reduced osteogenic differentiation of P‐PDLSCs. Downregulation of the levels of β‐catenin by treatment with dickkopf‐1 (DKK‐1) leads to activation of the noncanonical Wnt/Ca²⁺ pathway, which, in turn, results in the promotion of osteogenic differentiation in P‐PDLSCs. Interestingly, β‐catenin can affect both the canonical Wnt/β‐catenin pathway and the noncanonical Wnt/Ca²⁺ pathway. Our data indicate that β‐catenin plays a central role in regulating osteogenic differentiation of MSCs in inflammatory microenvironments. Given the important role of Wnt signaling in osteogenic differentiation, it is possible that agents that can modify this pathway may be of value in bone regeneration by MSCs in chronic inflammatory microenvironments. © 2011 American Society for Bone and Mineral Research     

MiR-122-5p promotes peritoneal fibrosis in a rat model of peritoneal dialysis by targeting Smad5 to activate Wnt/β-catenin pathway

Peritoneal fibrosis (PF) is the main reason leading to declining efficiency and ultrafiltration failure of peritoneum, which restricts the application of peritoneal dialysis (PD). We aimed to investigate the effects and mechanisms of miR-122-5p on the PF. Sprague-Dawley (SD) rats were infused with glucose-based standard PD fluid to establish PF model. HE staining was performed to evaluate the extent of PF. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and fluorescence in situ hybridization (FISH) were performed to measure the expression level of miR-122-5p. Western blot was used to test the expression of transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF)-A, Fibronectin 1 (FN1), extracellular matrix protein 1 (ECM1), Smad5, α-smooth muscle actin (SMA), collagen type 1(COL-1), Vimentin, E-Cadherin, Wnt1, β-catenin, p-β-catenin, c-Myc, c-Jun, and Cyclin D1. Immunohistochemistry (IHC) staining was used to detect type I collagen alpha 1 (Col1α1), α-SMA, and E-Cadherin expression. We found PF was glucose concentration-dependently enhanced in peritoneum of PD rat. The PD rats showed increased miR-122-5p and decreased Smad5 expression. MiR-122-5p silencing improved PF and epithelial–mesenchymal transition (EMT) process in PD rats. MiR-122-5p silencing attenuated the activity of the Wnt/β-catenin signaling pathway. Importantly, dual-luciferase reporter assay showed Smad5 was a target gene of miR-122-5p. Smad5 overexpression significantly reversed the increases of PF and EMT progression induced by miR-122-5p overexpression. Moreover, miR-122-5p mimic activated Wnt/β-catenin activity, which was blocked by Smad5 overexpression. Overall, present results demonstrated that miR-122-5p overexpression showed a deterioration effect on PD-related PF by targeting Smad5 to activate Wnt/β-catenin pathway.     

Stretch‐induced inhibition of Wnt/β‐catenin signaling in mineralizing osteoblasts

Wnt signaling is important for bone formation and osteoblastic differentiation. Recent findings indicate a stimulating role of Wnt signaling in bone mechanotransduction. However, negative effects of Wnt signaling on osteoblast differentiation and mineralization have been described as well. We conducted in vitro stretch experiments using human pre‐osteoblasts to study short‐ and long‐term effects of mechanical loading on Wnt/β‐catenin signaling. As the extracellular regulated kinase (ERK) pathway is known to be involved in mechanotransduction in osteoblasts, we also evaluated its role in Wnt/β‐catenin signaling. Stretch experiments up to 21 days (using stretch episodes of 15 min, alternated with 90 min rest) resulted in higher mineralization compared to static control cultures. We found that 15 min of stretch initially increased nuclear β‐catenin, but ultimately resulted in significant decrease at 12 and 40 h after stretch. Downregulation of Wnt‐responsive element activity 16 h after stretch, using a luciferase construct, further supported these findings. The presence of the ERK inhibitor U0126 did not alter the stretch‐induced decrease of β‐catenin levels. Our data indicate a biphasic effect of mechanical loading on β‐catenin in mineralizing human differentiating osteoblasts, which is independent of the ERK pathway. The osteogenic potential of our loading regime was confirmed by an increase in osteogenic differentiation markers such as alkaline phosphatase activity and calcium deposition after 3 weeks of culture. We conjecture that the biphasic aspect of Wnt/β‐catenin signaling with a strong decrease up to 40 h after the stretch induction, is important for the anabolic effects of mechanical stretch on bone. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:390–396, 2010     

Wnt signaling inhibits osteogenic differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) from the bone marrow represent a potential source of pluripotent cells for autologous bone tissue engineering. We previously discovered that over activation of the Wnt signal transduction pathway by either lithium or Wnt3A stimulates hMSC proliferation while retaining pluripotency. Release of Wnt3A or lithium from porous calcium phosphate scaffolds, which we use for bone tissue engineering, could provide a mitogenic stimulus to implanted hMSCs. To define the proper release profile, we first assessed the effect of Wnt over activation on osteogenic differentiation of hMSCs. Here, we report that both lithium and Wnt3A strongly inhibit dexamethasone-induced expression of the osteogenic marker alkaline phosphatase (ALP). Moreover, lithium partly inhibited mineralization of hMSCs whereas Wnt3A completely blocked it. Time course analysis during osteogenic differentiation revealed that 4 days of Wnt3A exposure before the onset of mineralization is sufficient to block mineralization completely. Gene expression profiling in Wnt3A and lithium-exposed hMSCs showed that many osteogenic and chondrogenic markers, normally expressed in proliferating hMSCs, are downregulated upon Wnt stimulation. We conclude that Wnt signaling inhibits dexamethasone-induced osteogenesis in hMSCs. In future studies, we will try to limit release of lithium or Wnt3A from calcium phosphate scaffolds to the proliferative phase of osteogenesis.     

Mechanical stretching induces osteoprotegerin in differentiating C2C12 precursor cells through noncanonical Wnt Pathways

Mechanical loading is known to be important for maintaining the formation and resorption rates of bone. To study the mechanisms by which mechanical loading regulates osteogenesis, we investigated the role of the Wnt pathway in C2C12 cells committed to osteogenic differentiation in response to cyclic mechanical stretching. Osteoprotegerin (OPG) acts as a decoy receptor for RANKL to inhibit osteoclastogenesis and resorption of bone. Our results demonstrate that stretching leads to a sustained increase in OPG expression in C2C12 cells. The expression of osteogenic marker genes, such as osteocalcin and alkaline phosphatase, was transiently decreased by stretching at 24 hours and returned to control levels at 48 hours. The addition of inhibitors of the canonical Wnt/β‐catenin pathways, such as the secreted FZD‐related peptide sRFP2, as well as siRNA‐mediated knockdown, did not inhibit the effect of stretching on OPG expression. In contrast, treatment with inhibitors of noncanonical Wnt signaling, including KN93, and siRNA for Nemo‐like kinase (NLK) blocked most of the mechanical inductive effect on OPG. Furthermore, stretching‐induced OPG production in the culture medium was able to inhibit the osteoclast formation of bone marrow macrophages. These results suggest that mechanical stretching may play an important role in bone remodeling through the upregulation of OPG and that the mechanical signaling leading to OPG induction involves the noncanonical Wnt pathway. © 2010 American Society for Bone and Mineral Research     

力学刺激下Wnt信号通路在促进成骨分化中作用的研究进展

Wnt信号通路在骨形成过程中发挥重要作用,主要表现为对骨组织中成骨分化等功能的调节。研究表明,激活Wnt信号可增强成骨特异性基因的表达,促进骨形成;机械力学刺激在促进骨骼的发育及维持良好的骨骼形态方面具有关键作用。本文就Wnt信号通路参与力学刺激下骨向分化的调节进行综述。     

FAK Promotes Osteoblast Progenitor Cell Proliferation and Differentiation by Enhancing Wnt Signaling

Decreased bone formation is often associated with increased bone marrow adiposity. The molecular mechanisms that are accountable for the negative correlation between bone mass and bone marrow adiposity are incompletely understood. Focal adhesion kinase (FAK) has critical functions in proliferation and differentiation of many cell types; however, its roles in osteoblast lineage cells are largely unknown. We show herein that mice lacking FAK in Osterix‐expressing cells exhibited decreased osteoblast number and low bone mass as well as increased bone marrow adiposity. The decreased bone mass in FAK‐deficient mice was accounted for by decreased proliferation, compromised osteogenic differentiation, and increased adipogenic differentiation of bone marrow Osterix‐expressing cells resulting from downregulation of Wnt/β‐catenin signaling due to the reduced expression of canonical Wnt ligands. In contrast, FAK loss in calvarial preosteoblasts had no adverse effect on their proliferation and osteogenic differentiation and these cells had intact Wnt/β‐catenin signaling. © 2016 American Society for Bone and Mineral Research.     

The role of proton transporters in epithelial Wnt signaling pathways

The planar cell polarity (PCP) pathway polarizes epithelia in the plane of a tissue. It regulates form and function of tissues and manifests itself by the polarized formation of cellular appendages such as epidermal hairs and cilia. Defects in the pathway are often associated with organ malformation and disease. In the kidney, the molecular events leading to cyst formation in polycystic kidney disease involve the PCP pathway. PCP is, however, best understood in Drosophila where genetic screens have identified a group of PCP core proteins including the Wnt receptor Frizzled (Fz), Dishevelled (Dsh), and Flamingo (Fmi). These proteins can localize to opposite parts of the plasma membrane in response to a poorly understood symmetry breaking event. Recent evidence suggests that proton transporters may play a role in regulating the asymmetric localization of PCP core proteins. Several papers have reported that the (pro)renin receptor, which is an associated subunit of the proton pumping V-ATPase, is required for PCP, but also for canonical Wnt signaling. Here, we discuss the implications of these findings for diverse developmental settings.     

血管内皮生长因子对脂肪干细胞成骨分化基因转录水平的调节

目的探讨内源性血管内皮生长因子（VEGF）体外诱导脂肪干细胞（ADSCs）分化成骨的转录活化机制。方法脂肪干细胞来自大白鼠腹股沟处脂肪,脂肪内干细胞的分离根据JMGimblel所描述的方法获取,培养鉴定后以BMP-2修饰组作为阳性对照组,提取细胞总RNA,采用RT-PCR法及琼脂糖凝胶电泳方法分析在不同诱导条件下Cbfal和Osterix表达的差异。结果BMP-2重组蛋白组的Cbfal基因转录水平明显高于VEGF诱导组（P〈0．01）,两者联合应用未见Cbfal基因转录增强;在以VEGF为诱导因子的ADSCs组,Osterix的mRNA水平明显低于BMP-2组（P〈0．01）,与Cbfal基因转录模式不同,VEGF联合BMP-2共同诱导ADSCs成骨分化组,Osterix基因的mRNA水平有增高的趋势。结论通过Cbfal和Osterix在ADSC成骨分化的不同时间的表达差异,本研究认为在ADSCs诱导成骨分化过程中,Cbfal启动和激活Osterix转录,仍是主要的信号传导途径。VEGF具有一定的诱导ADSCs成骨分化能力,但其作用主要是非依赖性Cbfal通路调控的Osterix表达。     

Wnt信号传导途径与骨质疏松

过去20年,在分子生物学和基础医学领域对骨质疏松症的研究取得了长足进展。Wnt信号通路在胚胎发育及生命体成长过程中起着关键的调控作用。2001年,基因学研究证实标准的Wnt信号通路在骨骼形成过程中起到关键的调控作用,最近,有报道非经典的Wnt信号通路在骨骼形成过程中有调控作用。由于目前应用调控合成代谢途径预防和治疗骨质疏松症尚无有效的报道。因此,本文主要从Wnt信号通路与骨质疏松症的发病机制及治疗进展的角度展开综述。     

Wnt3a联合骨形态发生蛋白2对骨髓间充质干细胞增殖及成骨分化的影响

目的 观察经典Wnt/β-catenin以及骨形态发生蛋白(BMP)信号通路对大鼠骨髓来源的间充质干细胞(BMSCs)体外增殖以及向成骨方向分化的影响.方法 应用密度梯度离心联合贴壁筛选法分离培养骨髓间充质干细胞并分4组:对照组、Wnt组、BMP组、Wnt3a和BMP-2联合组,在不同时间点用细胞计数试剂盒-8(CCK-8)法检测细胞增殖活性,碱性磷酸酶(ALP)活性定量测定、Yon Kossa染色观察细胞向成骨分化和基质矿化程度.逆转录-聚合酶链反应(RT-PCR)检测成骨特异性标志物表达.结果 培养第7天,CCK-8测吸光度值联合组为0.845,Wnt组为0.738,明显高于对照组0.409(P＜0.05).培养第6天和第12天,ALP活性吸光度值联合组为63.8和144.3,BMP-2组为40.8和104.1,均明显高于对照组7.3和18.9(P ＜0.05).培养14 d后,联合组骨钙素mRNA表达量高于对照组,而Runx2及Osterix表达量与其他组比较增高不显著.培养3周后,Yon Kossa染色显示联合组钙结节数量及大小均高于对照组.结论 Wnt3a因子和骨形态发生蛋白-2联合诱导能有效地促进骨髓间充质干细胞增殖及向成骨方向分化.     

miR-129-5p inhibits gemcitabine resistance and promotes cell apoptosis of bladder cancer cells by targeting Wnt5a

Objectives

Gemcitabine resistance is a major obstacle for effective treatment of bladder cancer. This study was aimed to investigate the potential role of miR-129-5p in the development of gemcitabine resistance in bladder cancer cells and its underlying mechanism.

Methods

The IC50 for gemcitabine in 20 bladder cancer cells was first profiled from Genomics of Drug Sensitivity in Cancer. miR-129-5p level and gene mRNA expression were detected using quantitative real-time PCR (qRT-PCR). Cell viability, apoptosis, and gene protein level were assessed by MTT, flow cytometry, and Western blot, respectively. Regulatory relationship between Wnt5a and miR-129-5p was determined using luciferase reporter assay.

Results

We found that down-regulated miR-129-5p level contributed to gemcitabine resistance in bladder cancer cells and tissues. We also observed restoration of miR-129-5p could significantly increase cell sensitivity to gemcitabine and promote cell apoptosis. Mechanism analysis revealed that Wnt5a is a direct target gene of miR-129-5p and knock-down of Wnt5a reversed gemcitabine resistance.

Conclusions

Taken together, our findings indicate that miR-129-5p and Wnt5a may be novel therapeutic targets for overcoming gemcitabine resistance in bladder cancer treatment.

     

Activation of Wnt Planar cell polarity (PCP) signaling promotes growth plate column formation in vitro

Disrupting the Wnt Planar Cell Polarity (PCP) signaling pathway in vivo results in loss of columnar growth plate architecture, but it is unknown whether activation of this pathway in vitro is sufficient to promote column formation. We hypothesized that activation of the Wnt PCP pathway in growth plate chondrocyte cell pellets would promote columnar organization in these cells that are normally oriented randomly in culture. Rat growth plate chondrocytes were transfected with plasmids encoding the Fzd7 cell‐surface Wnt receptor, a Fzd7 deletion mutant lacking the Wnt‐binding domain, or Wnt receptor‐associated proteins Ror2 or Vangl2, and then cultured as three‐dimensional cell pellets in the presence of recombinant Wnt5a or Wnt5b for 21 days. Cellular morphology was evaluated using histomorphometric measurements. Activation of Wnt PCP signaling components promoted the initiation of columnar morphogenesis in the chondrocyte pellet culture model, as measured by histomorphometric analysis of the column index (ANOVA p = 0.01). Activation of noncanonical Wnt signaling through overexpression of both the cell‐surface Wnt receptor Fzd7 and receptor‐associated protein Ror2 with addition of recombinant Wnt5a promotes the initiation of columnar architecture of growth plate chondrocytes in vitro, representing an important step toward growth plate regeneration. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1906–1914, 2012