Moderate activation of Wnt/β-catenin signaling promotes the survival of rat nucleus pulposus cells via regulating apoptosis,autophagy, and senescence

This study aimed to investigate the specific role of Wnt/β-catenin signaling in compression-induced apoptosis, autophagy, and senescence in rat nucleus pulposus (NP) cells. Initially, the cells underwent various periods of exposure to 1.0 MPa compression. Wnt/β-catenin signaling associated molecules were assessed in detail, and then 0, 24 and 48 hours exposure periods were selected. The cells were then divided into control, Wnt/β-catenin inhibitor (IWP-2), Wnt/β-catenin activator (LiCl), and β-catenin overexpression groups. After 0, 24, and 48 hours of compression, apoptosis, autophagy, and senescence were evaluated by Western blot analysis and real-time polymerase chain reaction and were visually observed by Hoechst33258, monodansylcadaverine, and SA-β-gal stainings, respectively. Additionally, the regulatory effect of Wnt/β-catenin signaling on cell morphology, viability, cell cycle, death ratio, and ultrastructure was detected to thoroughly evaluate the survival capacity of NP cells. The results established that compression elicited a time-dependent activation of Wnt/β-catenin signaling. The IWP-2 treatment decreased cell survival rate, which corresponded to downregulation of autophagy as well as increases in apoptosis and senescence. LiCl treatment enabled more efficient of cell survival accompanied by increased autophagy and downregulated apoptosis and senescence; however, in contrast to LiCl, overexpression of β-catenin aggravated compression-induced NP cells death. In conclusion, moderate activation of Wnt/β-catenin signaling enables more efficient of NP cells survival via downregulation of apoptosis, senescence, and upregulation of autophagy, and overactivation of Wnt/β-catenin signaling achieved the opposite effect. Treatment strategies that aim to regulate Wnt/β-catenin signaling might be a novel target for improving compression-induced NP cells death and potential treatment of intervertebral disc degeneration.     

CircSEMA4B targets miR-431 modulating IL-1β-induced degradative changes in nucleus pulposus cells in intervertebral disc degeneration via Wnt pathway

Intervertebral disc (IVD) degeneration (IDD), characterized by elevated levels of proinflammatory mediators, increased Aggrecan and collagen degradation, and increased degradation of extracellular matrix (ECM), has been widely regarded as a significant contributor to low back pain. Genetics are significant factors contribute to IDD. Based on previous data, circular RNA SEMA4B (circSEMA4B) is down-regulated in IDD specimens; herein, we demonstrated circSEMA4B overexpression could attenuate the effect of IL-1β on nucleus pulposus cell (NPC) proliferation, senescence, and ECM and Aggrecan degradation in IDD via Wnt signaling. Moreover, miR-431, a direct target of circSEMA4B, could bind to the 3′UTR of SFRP1 or GSK-3β, two inhibitory regulators of Wnt signaling, to inhibit their expression thus playing a role similar to the activator of Wnt signaling in NPCs. The effect of circSEMA4B knockdown on NPCs was partially reversed by miR-431 inhibition; circSEMA4B serves as a miR-431 sponge to compete with SFRP1 or GSK-3β for miR-431 binding, thus inhibiting IL-1β-induced degenerative process in NPCs through Wnt signaling. Rescuing circSEMA4B expression in NPCs in IDD might present a potential strategy for IDD improvement.     

TGM2 interference regulates the angiogenesis and apoptosis of colorectal cancer via Wnt/β-catenin pathway

Angiogenesis and apoptosis are critical for the growth of colorectal cancer (CRC). The study aimed to investigate the effects of TGM2 in CRC. Forty-two patients were recruited and their TGM2 levels were detected by performing Realtime-qPCR (RT-qPCR), Western blot and immunohistochemistry , respectively. Levels of TGM2, MMP-2 and MMP-9 in four CRC cell lines and in normal cells were determined using RT-qPCR and Western blot. TGM2-siRNA was transfected into LoVo and HCT116 cells, respectively. TGM2 levels, cell viability, cell apoptosis, angiogenesis and related factors were determined. the tumorigenesis rates of mice were detected after TGM2-siRNA transfection. TGM2 were upregulated in patients with CRC. High TGM2 level of CRC patients had a lower survival rate. The levels of TGM2, MMP-2 and MMP-9 were upregulated in all detected CRC cell lines. Silencing TGM2 could inhibit cell viabilities, angiogenesis and suppress the expressions of MMP-2, MMP-9, Wnt3a, β-catenin and Cyclin D1 , whereas cell apoptosis and the expressions of Caspase-3 and TIMP-1 were promoted. Tumor weights and volumes were reduced by TGM2-siRNA interference. The effects of TGM2-siRNA interference might be related to Wnt/β-catenin Pathway. This might prove that TGM2 could be used as a molecular target in the treatment of CRC.     

Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/β-catenin pathway

The Wnt/β-catenin signaling pathway is important for tumor initiation and progression. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling and represents a promising anticancer target. Recently, the antihelminthic drug, niclosamide was found to inhibit Wnt/β-catenin signaling, although the mechanism was not well defined. We found that niclosamide was able to suppress LRP6 expression and phosphorylation, block Wnt3A-induced β-catenin accumulation, and inhibit Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of niclosamide on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were conformed in human prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. Moreover, we showed that the mechanism by which niclosamide suppressed LRP6 resulted from increased degradation as evident by a shorter half-life. Finally, we demonstrated that niclosamide was able to induce cancer cell apoptosis, and displayed excellent anticancer activity with IC(50) values less than 1 μM for prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin signaling in prostate and breast cancer cells. Our data indicate that niclosamide is a unique small molecule Wnt/β-catenin signaling inhibitor targeting the Wnt co-receptor LRP6 on the cell surface, and that niclosamide has a potential to be developed a novel chemopreventive or therapeutic agent for human prostate and breast cancer.     

Pinostilbene hydrate suppresses hepatic stellate cell activation via inhibition of miR-17–5p-mediated Wnt/β-catenin pathway

BackgroundIn the development of liver fibrosis, activated hepatic stellate cells (HSCs) contribute to the synthesis and deposition of extracellular matrix (ECM) proteins. HSC activation is considered as a central driver of liver fibrosis. Recently, microRNAs (miRNAs) have been reported to act as key regulators in HSC activation.PurposePinostilbene hydrate (PSH), a methylated derivative of resveratrol, has demonstrated anti-inflammatory, antioxidant and anti-tumour activities. However, the effects of PSH on HSC activation remain unclear.MethodsThe effects of PSH on HSC activation were examined. Moreover, the roles of WNT inhibitory factor 1 (WIF1) and miR-17–5p in the effects of PSH on HSC activation were examined.ResultsPSH induced a significant reduction in HSC proliferation. PSH also effectively inhibited HSC activation, with reduced α-SMA and collagen expression. Notably, it was found that Wnt/β-catenin signalling was involved in the effects of PSH on HSC activation. PSH resulted in Wnt/β-catenin signalling inactivation, with a reduction in TCF activity as well as β-catenin nuclear translocation. Further studies showed that PSH inhibited Wnt/β-catenin signalling via regulation of WIF1 and miR-17–5p. Reduced HSC activation caused by PSH could be restored by loss of WIF1 or miR-17–5p mimics. Luciferase reporter assays further confirmed that WIF1 was a target of miR-17–5p.ConclusionPSH has a significant protective effect against HSC activation. In addition, we demonstrate that PSH enhances WIF1 expression and inhibits Wnt/β-catenin signalling via miR-17–5p, contributing to the suppression of HSC activation.     

miR-612 suppresses the stemness of liver cancer via Wnt/β-catenin signaling

Previous research showed that microRNA-612 (miR-612) has inhibitory effects on cell proliferation, migration, invasion, and metastasis of hepatocellular carcinoma (HCC). AKT2 was confirmed to be a direct target of miR-612, through which the epithelial–mesenchymal transition (EMT) and metastasis of HCC were inhibited. Our present findings reveal that miR-612 is able to suppress the stemness of HCC by reducing the number and size of tumorspheres as well as clone formation in soft agar, and to relieve drug resistance to cisplatin and 5-fluorouracil. In addition, miR-612 hampered the capacity of tumorigenesis in NOD/SCID mice and redistributed the tumor invasive frontier of miR-612-modulating cells. Finally, our findings suggest that Wnt/β-catenin signaling is required in the regulation of EMT-associated stem cell-like traits by miR-612.     

miR-218 promoted the apoptosis of human ovarian carcinoma cells via suppression of the WNT/β-catenin signaling pathway

MicroRNA-218 (miR-218) is a short, noncoding RNA, with multiple biological functions. In this study, we aimed to investigate the potential effects of miR-218 on the apoptosis of human ovarian carcinoma cells and the underlying mechanisms by which miR-218 exerted its actions. After over-expressing miR-218 in human ovarian carcinoma (OVCAR3) cells, cell viability was determined by MTT method, cell apoptosis was observed by flow cytometry (FCM), mRNA expression of miR-218, Bcl2, Bax was measured by RT-PCR and protein expression levels of Wnt, tankyrase and β-catenin were quantified by Western blots. Over-expression of miR-218 potently suppressed cell viability and promoted the apoptosis of human ovarian carcinoma cells in a time-dependent manner. In addition, the down-regulation of tankyrase expression level was detected in miR-218-over-expressed cells. Following the block of the Wnt/β-catenin signaling pathway using the inhibitor XAV-939, the effects of miR-218 on the proliferation and apoptosis of human ovarian carcinoma cells were significantly suppressed. Augmenting expression of miR-218 and/or miRNA-218 mimicking therapeutics may provide viable avenue for the treatment of ovarian cancer.     

LncRNA CRNDE promotes the epithelial-mesenchymal transition of hepatocellular carcinoma cells via enhancing the Wnt/β-catenin signaling pathway

Colorectal neoplasia differentially expressed (CRNDE) is a significantly upregulated long noncoding RNA in hepatocellular carcinoma (HCC). CRNDE could promote cell proliferation, migration, and invasion, while its molecular mechanisms were still largely unclear. In this study, we investigated the expression and function of CRNDE. CRNDE was significantly upregulated in tumor tissues compared with adjacent normal tissues. In vitro, we revealed that knockdown of CRNDE inhibited cell proliferation, migration, and cell invasion capacities in HCC. Animal studies indicated that CRNDE knockdown represses both growth and metastasis of HCC tumors in vivo. Moreover, knockdown of CRNDE suppressed the cell epithelial-mesenchymal transition (EMT) process by increasing the expression of E-cadherin and ZO-1, whereas, decreasing the expression of N-cadherin, slug, twist, and vimentin in HCC cells. We also revealed that knockdown of CRNDE suppressed the Wnt/β-catenin signaling in HCC. Thus, CRNDE could modulate EMT of HCC cells and knockdown of CRNDE impaired the mesenchymal properties. CRNDE increased invasion of HCC cells might be through activating the Wnt/β-catenin signaling pathway.     

MiR-125b regulates the proliferation and metastasis of triple negative breast cancer cells via the Wnt/β-catenin pathway and EMT

Background/aim: MiR-125b plays an important role in breast cancer. The current study was to explore the expression and function of miR-125b in triple negative breast cancer cells. Materials and methods: The expression of miR-125b in human TNBC samples and cell lines were examined by qRT-PCR. MTT, scratch assays and transwell assays were utilized to observe the proliferation, migration and invasion ability. MiR-125b’s target gene and downstream signaling pathways were investigated by Luciferase Reporter Assays, qRT-PCR, immunofluorescence assays and western bolt. Results: MiR-125b was highly expressed in human TNBC tissues and cell lines. Inhibiting miR-125b expression suppressed the proliferation, cell migration and invasion. The three-prime untranslated region (3´-UTR) of adenomatous polyposis coli (APC) mRNA contains miR-125b binding sites, and inhibiting miR-125b expression suppressed the activity of the intracellular Wnt/β-catenin pathways and EMT. Conclusion: Inhibiting miR-125b regulates the Wnt/β-catenin pathway and EMT to suppress the proliferation and migration of MDA-MB-468 TNBC cells.     

MicroRNA-15b silencing inhibits IL-1β-induced extracellular matrix degradation by targeting SMAD3 in human nucleus pulposus cells

Objectives

To determine the role of microRNA-15b (miR-15b) in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation in the nucleus pulposus (NP).

Results

MiR-15b was up-regulated in degenerative NP tissues and in IL-1β-stimulated NP cells, as compared to the levels in normal controls (normal tissue specimens from patients with idiopathic scoliosis). Bioinformatics and luciferase activity analyses showed that mothers against decapentaplegic homolog 3 (SMAD3), a key mediator of the transforming growth factor-β signaling pathway, was directly targeted by miR-15b. Functional analysis demonstrated that miR-15b overexpression aggravated IL-1β-induced ECM degradation in NP cells, while miR-15b inhibition had the opposite effects. Prevention of IL-1β-induced NP ECM degeneration by the miR-15b inhibitor was attenuated by small-interfering-RNA-mediated knockdown of SMAD3. In addition, activation of MAP kinase and nuclear factor-κB up-regulated miR-15b expression and down-regulated SMAD3 expression in IL-1β-stimulated NP cells.

Conclusions

MiR-15b contributes to ECM degradation in intervertebral disc degeneration (IDD) via targeting of SMAD3, thus providing a novel therapeutic target for IDD treatment.

     

TMPyP4-regulated cell proliferation and apoptosis through the Wnt/β-catenin signaling pathway in SW480 cells

Background: The aim of this study was to investigate the potential effects of the 5, 10, 15, 20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of SW480 cells and the underlying mechanisms by which TMPyP4 exerted its actions. Methods: After treated with different doses of TMPyP4, cell viability was determined by MTT method, the apoptosis was observed by flow cytometry (FCM) and the expression of Wnt, GSK-3β, β-catenin and cyclinD1 was measured by RT-PCR and Western blot analysis. Results: The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of SW480 cells in a dose-dependent manner. In addition, the downregulation of Wnt, β-catenin and cyclinD1 expression levels was detected in TMPyP4-treated SW480 cells. However, followed by the block of Wnt signaling pathway using siRNA methods, the effects of TMPyP4 on proliferation and apoptosis of SW480 cells were significantly reduced. Conclusion: It indicates that the TMPyP4-inhibited proliferation and -induced apoptosis in SW480 cells was accompanied by the suppression of Wnt/β-catenin signaling pathway. Therefore, TMPyP4 may represent a potential therapeutic method for the treatment of colon carcinoma.     

Oncogenic retinoic acid receptor γ knockdown reverses multi-drug resistance of human colorectal cancer via Wnt/β-catenin pathway

Retinoic acid receptor γ (RARγ), a unique member of the nuclear receptor superfamily, plays an important role in the progression of several cancers such as hepatocellular carcinoma, esophageal cancer, and cholangiocarcinoma. However, little is known about the regulatory mechanism of the RARγ expression in colorectal cancer (CRC) progression. In the present study, we found that RARγ was frequently overexpressed in human CRC specimens and CRC cell lines, and it mainly resided in the cytoplasm in CRC specimens. Tissue microarrays showed that RARγ indicated vital clinical significance in CRC. RARγ knockdown neither affected CRC cell proliferation nor blocked the cell cycle of CRC cells. However, RARγ knockdown increased the sensitivity of CRC cells to chemotherapeutics through downregulation of multi-drug resistance 1(MDR1). Further studies suggested that RARγ knockdown resulted in downregulation of MDR1, in parallel with suppression of the Wnt/β-catenin pathway. Moreover, a significantly positive association between RARγ and MDR1 was demonstrated in CRC tissue microarrays. Collectively, these results suggested that overexpression of RARγ contributed to the multidrug chemoresistance of CRC cells, at least in part due to upregulation of MDR1 via activation of the Wnt/β-catenin pathway, indicating that RARγ might serve as a potential therapeutic target for chemoresistant CRC patients.     

Inhibition of Rac1 activity by NSC23766 prevents cartilage endplate degeneration via Wnt/β-catenin pathway

Cartilage endplate (CEP) degeneration has been considered as one of important factors related to intervertebral disc degeneration (IVDD). Previous researches have showed that Rac1 played a pivotal role in chondrocyte differentiation. However, the effect of Rac1 during the process of CEP degeneration remains unclear. Herein, we explored the effect of Rac1 on CEP degeneration and elucidated the underlying molecular mechanism. We found expression of Rac1-GTP increased in human-degenerated CEP tissue and IL-1β-stimulated rat endplate chondrocytes (EPCs). Our study revealed that Rac1 inhibitor NSC23766 treatment promoted the expression of collagen II, aggrecan and Sox-9, and decreased the expression of ADTAMTS5 and MMP13 in IL-1β-stimulated rat EPCs. Moreover, we also found that NSC23766 could suppress the activation of Wnt/β-catenin pathway, suggesting that the beneficial effects of Rac1 inhibition in EPCs are mediated through the Wnt/β-catenin signalling. Besides, puncture-induced rats models showed that NSC23766 played a protective role on CEP and disc degeneration. Collectively, these findings demonstrated that Rac1 inhibition delayed the EPCs degeneration and its potential mechanism may be associated with Wnt/β-catenin pathway regulation, which may help us better understand the association between Rac1 and CEP degeneration and provide a promising strategy for delaying the progression of IVDD.