MicroRNA-200a suppresses the Wnt/β-catenin signaling pathway by interacting with β-catenin

The Wnt/β-catenin signaling pathway is crucial for human organ development and is involved in tumor progression of many cancers. Accumulating evidence suggests that the expression of β-catenin is, in part, regulated by specific microRNAs (miRNAs). The purpose of this study was to determine the expression of a recently identified epithelial to mesenchymal transition (EMT)-associated tumor suppressor microRNA (miR)-200a, in cancer cells. We also aimed to identify specific miR-200a target genes and to investigate the antitumor effects of miR-200a on the Wnt/β-catenin signaling pathway. We employed TOP/FOP flash luciferase assays to identify the effect of miR-200a on the Wnt/β-catenin pathway and we confirmed our observations using fluorescence microscopy. To determine target genes of miR-200a, a 3' untranslated region (3' UTR) luciferase assay was performed. Cell viability, invasion and wound healing assays were carried out for functional analysis after miRNA transfection. We further investigated the role of miR-200a in EMT by Western blot analysis. We found fluctuation in the expression of miR-200a that was accompanied by changes in the expression of members of the Wnt/β-catenin signaling pathway. We also determined that miR-200a can directly interact with the 3' UTR of CTNNB1 (the gene that encodes β-catenin) to suppress Wnt/β-catenin signaling. MiR-200a could also influence the biological activities of SGC790 and U251 cells. Our results demonstrate that miR-200a is a new tumor suppressor that can regulate the activity of the Wnt/β-catenin signaling pathway via two mechanisms. MiR-200a is a candidate target for tumor treatment via its regulation of the Wnt/β-catenin signaling pathway.     

miR-122/Wnt/β-catenin regulatory circuitry sustains glioma progression

Malignant glioma is the most common type of human intracranial cancer and has poor prognosis due to its high recurrence and invasiveness. However, the molecular mechanisms underlying its malignant phenotypes have still not been completely explored yet. miR-122 has been well documented to act as a tumor suppressor for hepatocellular carcinoma and breast cancer, but the implication of miR-122 in the progression of glioma is not clear yet. In this study, we found that miR-122 was underexpressed in glioma specimens and glioma cell lines, compared with normal brain tissues and cell lines. The expression of miR-122 levels is inversely correlated with the survival of patients after surgery. Overexpression of miR-122 by an adenoviral vector suppressed the proliferation and colony formation of glioma cells. The in vivo tumorigenicity of U-87 MG cells was also greatly compromised by restoring miR-122. miR-122 suppressed the activation of Wnt/β-catenin pathway in glioma cells. Interestingly, Wnt/β-catenin signaling conversely reduced the expression of miR-122 in glioma cells, maybe in a hepatocyte nuclear factor (HNF)-dependent mechanism. Taken together, we revealed that there is a miR-122/Wnt/β-catenin regulatory circuitry existing in glioma cells that contributes to glioma progression.     

Down-regulation of EZH2 by RNA Interference Inhibits Proliferation and Invasion of ACHN Cells via the Wnt/β- catenin Pathway

Although enhancer of zeste homolog 2 (EZH2) has been reported as an independent prognostic factor inrenal cell carcinoma (RCC), little is known about the exact mechanism of EZH2 in promoting the genesis ofRCC. However, several studies have shown that dysregulation of the Wnt/β-catenin signaling pathway plays acrucial role. Therefore, we determined whether EZH2 could affect ACHN human RCC cell proliferation andinvasion via the Wnt/β-catenin pathway. In the present study, we investigated the effects of short interferingRNA (siRNA)-mediated EZH2 gene silencing on Wnt/β-catenin signaling in ACHN cells. EZH2-siRNA markedlyinhibited the proliferation and invasion capabilities of ACHN, while also reducing the expression of EZH2,Wnt3a and β-catenin. In contrast, cellular expression of GSK-3β (glycogen synthase kinase-3β), an inhibitorof the Wnt/β-catenin pathway, was conspicuously higher after transfection of EZH2 siRNA. These preliminaryfindings suggest EZH2 may promote proliferation and invasion of ACHN cells via action on the Wnt/β-cateninsignaling pathway.     

Dishevelled 2 signaling promotes self-renewal and tumorigenicity in human gliomas

Glioblastoma multiforme is the most common glioma variant in adults and is highly malignant. Tumors are thought to harbor a subpopulation of stem-like cancer cells, with the bulk resembling neural progenitor-like cells that are unable to fully differentiate. Although multiple pathways are known to be involved in glioma tumorigenesis, the role of Wnt signaling has been poorly described. Here, we show that Dishevelled 2 (Dvl2), a key component of the Wnt signaling pathway, is overexpressed in human gliomas. RNA interference-mediated depletion of Dvl2 blocked proliferation and promoted the differentiation of cultured human glioma cell lines and primary, patient-derived glioma cells. In addition, Dvl2 depletion inhibited tumor formation after intracranial injection of glioblastoma cells in immunodeficient mice. Inhibition of canonical Wnt/β-catenin signaling also blocked proliferation, but unlike Dvl2 depletion, did not induce differentiation. Finally, Wnt5a, a noncanonical Wnt ligand, was also required for glioma cell proliferation. The data therefore suggest that both canonical and noncanonical Wnt signaling pathways downstream of Dvl2 cooperate to maintain the proliferative capacity of human glioblastomas.     

miR-1471对胶质瘤细胞增殖、侵袭和迁移能力的影响及其机制探讨

目的:探讨miR-1471对胶质瘤细胞增殖、侵袭和迁移能力的影响及其机制。方法:体外培养人胶质瘤细胞U251,将miR-1471模拟物或阴性对照分别转染至细胞中,记为miR-1471组和阴性对照（NC组）,同时设置对照组。qRT-PCR检测转染效果。CCK8检测各组细胞增殖能力。Transwell实验检测各组细胞侵袭能力。划痕实验检测各组细胞迁移能力。qRT-PCR和Western blot检测转移黏附基因（metadherin, MTDH）及Wnt/β-catenin信号通路相关基因和蛋白的表达。结果:与对照组和NC组比较,miR-1471组细胞增殖活性均显著降低（P＜0.05）,侵袭细胞数均显著减少（P＜0.05）,划痕间距明显较大,细胞中MTDH、Wnt1和β-catenin mRNA和蛋白表达水平均显著降低（P＜0.05）。结论:miR-1471能抑制人胶质瘤细胞U251的增殖、侵袭和迁移能力,其作用机制可能与下调MTDH表达,抑制Wnt/β-catenin信号通路的激活有关。     

Wnt/β-catenin信号转导通路影响神经胶质瘤侵袭的机制

神经胶质瘤的侵袭是一个多步骤、多因素参与的复杂过程,主要包括抑制肿瘤细胞黏附,降解细胞外基质,促进肿瘤细胞移动和肿瘤新生血管的生成四个部分.Wnt/β-catenin信号转导通路的异常激活与神经胶质瘤侵袭密切相关.研究Wnt/β-catenin通路影响神经胶质瘤的侵袭机制,将为神经胶质瘤的治疗提供新的思路和靶点.     

miR-613通过下调Wnt/β-catenin信号通路活性抑制前列腺癌细胞的增殖与侵袭

目的:研究miR-613在人前列腺癌组织中的表达情况,探讨miR-613是否通过下调Wnt信号通路活性抑制前列腺癌细胞系细胞的增殖和侵袭能力。方法:收集临床前列腺癌组织及配对癌旁组织20例,通过实时荧光定量PCR(RT-qPCR)检测各组组织中miR-613的表达情况。进一步在细胞实验中,通过转染miR-613 mimic和miR-NC至离体培养的PC-3、DU-145细胞中,随后,采用MTT法、平板克隆实验检测细胞增殖和Matrigel侵袭实验测定前列腺癌细胞的侵袭情况,采用荧光素酶分析方法评估Wnt信号通路活性变化,采用实时荧光定量PCR(RT-qPCR)检测Wnt/β-catenin信号通路下游靶基因的转录(包括Cyclin D1和c-Myc),WB法检测细胞中β-catenin、c-Myc和Cyclin D1的表达量。结果:相比配对癌旁组织,miR-613在前列腺癌组织中的表达降低(P<0.01);在体外细胞实验中,相比于miR-NC组,转染miR-613 mimic后,PC-3、DU-145细胞增殖能力下降(P<0.05),PC-3、DU-145细胞的迁移侵袭能力下降(P<0.01);miR-613的过表达显著降低Wnt信号通路活性、β-catenin蛋白表达及Wnt信号下游靶基因Cyclin D1和c-Myc的转录及蛋白表达。结论:miR-613通过抑制Wnt/β-catenin信号通路来影响前列腺癌细胞的增殖与侵袭,为前列腺癌的潜在治疗靶点之一。     

Malignant gliomas induce and exploit astrocytic mesenchymal-like transition by activating canonical Wnt/β-catenin signaling

The complex microenvironment of malignant gliomas plays a dynamic and usually cancer-promoting role in glioma progression. Astrocytes, the major stromal cells in the brain, can be activated by glioma microenvironment, resulting in a layer of reactive astrocytes surrounding the gliomas. Reactive astrocytes are universally characterized with the upregulation of glial fibrillary protein and glycoprotein podoplanin. In this work, we investigated the role of reactive astrocytes on malignant glioma microenvironment and the potential mechanism by which glioma cells activated the tumor-associated astrocytes (TAAs). The reactive astrocytes were observed around gliomas in the intracranial syngeneic implantation of rat C6 and mouse GL261 glioma cells in vivo, as well as primary astrocytes cultured with glioma cells condition medium in vitro. Besides, reactive astrocytes exhibited distinct epithelial-to-mesenchymal (-like) transition and enhanced migration and invasion activity, with the decrease of E-cadherin and concomitant increase of vimentin and matrix metalloproteinases. Furthermore, canonical Wnt/β-catenin signaling was activated in TAAs. The Wnt/β-catenin pathway inhibitor XAV939 and β-catenin plasmid were used to verify the regulation of Wnt/β-catenin signaling on TAAs and their invasion ability. Taken together, our findings established that glioma cells remarkably activated astrocytes via upregulating Wnt/β-catenin signaling, with obviously mesenchymal-like transition and increased migration and invasion ability, indicating that glioma cells may stimulate adjacent astrocytes to degrade extracellular matrix and thereby promoting tumor invasiveness.     

miR-139-5p通过调节Wnt/β-catenin信号通路在卵巢癌中发挥抑癌作用

目的:探讨miR-139-5p表达的改变对卵巢癌的影响,并进一步探索其中的信号通路。方法:通过RT-PCR（real time-polymerase chain reaction）测量卵巢癌组织样品中miR-139-5p的表达水平。通过细胞实验,包括CCK-8（cell counting kit-8）、集落形成和Transwell测定,探索miR-139-5p对卵巢癌细胞功能的影响。通过荧光素酶基因测定确认miR-139-5p的靶基因,并通过质粒转染检验miR-139-5p对Wnt/β-catenin信号通路的作用。结果:miR-139-5p在卵巢癌组织及卵巢癌细胞中表达明显降低。miR-139-5p过表达可明显抑制卵巢癌细胞的活性、增殖和迁移能力,并且在裸鼠体内可明显抑制卵巢癌的体积和质量。miR-139-5p过表达可抑制β-catenin蛋白的表达,从而抑制卵巢癌的增长。结论:miR-139-5p的过表达可通过调节Wnt/β-catenin信号通路抑制卵巢癌的发展,其可能是一种抑制卵巢癌进展的肿瘤抑制因子。     

miR-205-5p/E2F1信号抑制经典Wnt/β蛋白通路调控脑胶质瘤细胞放射敏感性

目的 探索miR-205-5p/E2F1信号轴在脑胶质瘤U251、U87细胞放射耐受中的调控机制。方法 利用X射线逐步递增递间歇诱导方法照射U251、U87细胞,建立放射耐受的U251/TR、U87/TR细胞。对两种细胞进行形态学、细胞运动、侵袭及增殖能力分析。通过荧光素酶基因检测系统及点突变技术分析E2F1基因对U251/TR、U87/TR细胞的调控机制。结果 放射耐受的U251/TR、U87/TR细胞分别比251、U87细胞的增殖活力增强,运动、侵袭能力增强,X射线照射下细胞凋亡下降。miR-205-5p mimics转染能够下调U251/TR细胞E2F1因子表达,抑制细胞增殖、侵袭及运动,增加放射敏感性。miR-205-5p mimics转染协同E2F1下调是通过抑制细胞Wnt/β-catenin信号通路活性发挥抑制肿瘤作用,并降低细胞耐受。结论 逐步递增递间歇诱导方法能较好地建立U251/TR、U87/TR细胞。miR-205-5p/E2F1信号轴通过经典Wnt/β-catenin信号通路发挥抑癌作用,可以作为提高胶质瘤放射敏感性的治疗靶点。     

Wnt-β-catenin信号通路相关长链非编码RNA在胶质瘤中的研究进展

胶质瘤是中枢神经系统中最常见且侵袭性高的恶性肿瘤之一,Wnt-β-catenin信号通路为经典Wnt通路,参与胶质瘤等多种肿瘤的发生发展。长链非编码RNA(LncRNA)为无蛋白编码功能的功能性RNA分子,在多种肿瘤的发生、发展中发挥着调控作用。研究表明,LncRNA与Wnt-β-catenin信号通路共同参与胶质瘤的生长、侵袭、迁移等过程,但其中的复杂机制目前并未得到深入的阐述。文章就Wnt-β-catenin信号通路及其相关的LncRNA对胶质瘤的影响进行综述。     

β-Catenin/LEF1 transactivates the microRNA-371-373 cluster that modulates the Wnt/β-catenin-signaling pathway

The microRNA-371-373 (miR-371-373) cluster is specifically expressed in human embryonic stem cells (ESCs) and is thought to be involved in stem cell maintenance. Recently, microRNAs (miRNAs) of this cluster were shown to be frequently upregulated in several human tumors. However, the regulatory mechanism for the involvement of the miR-371-373 cluster in human ESCs or cancer cells remains unclear. In this study, we explored the relationship between this miRNA cluster and the Wnt/β-catenin-signaling pathway, which has been shown to be involved in both stem cell maintenance and tumorigenesis. We show that miR-371-373 expression is induced by lithium chloride and is positively correlated with Wnt/β-catenin-signaling activity in several human cancer cell lines. Mechanistically, three TCF/LEF1-binding elements (TBEs) were identified in the promoter region and shown to be required for Wnt-dependent activation of miR-371-373. Interestingly, we also found that miR-372&373, in turn, activate Wnt/β-catenin signaling. In addition, four protein genes related to the Wnt/β-catenin-signaling pathway were identified as direct targets of miR-372&373, including Dickkopf-1 (DKK1), a well-known inhibitor of Wnt/β-catenin signaling. Using a lentiviral system, we showed that overexpression of miR-372 or miR-373 promotes cell growth and the invasive activity of tumor cells as knockdown of DKK1. Taken together, our study demonstrates a novel β-catenin/LEF1-miR-372&373-DKK1 regulatory feedback loop, which may have a critical role in regulating the activity of Wnt/β-catenin signaling in human cancer cells.     

Activation of canonical WNT/β-catenin signaling enhances in vitro motility of glioblastoma cells by activation of ZEB1 and other activators of epithelial-to-mesenchymal transition

Here we show that activation of the canonical WNT/β-catenin pathway increases the expression of stem cell genes and promotes the migratory and invasive capacity of glioblastoma. Modulation of WNT signaling alters the expression of epithelial-to-mesenchymal transition activators, suggesting a role of this process in the regulation of glioma motility. Using immunohistochemistry in patient-derived glioblastoma samples we showed higher numbers of cells with intranuclear signal for β-catenin in the infiltrating edge of tumor compared to central tumor parenchyma. These findings suggest that canonical WNT/β-catenin pathway is a critical regulator of GBM invasion and may represent a potential therapeutic target.     

lncRNA BCAR4通过调节Wnt/β-catenin信号通路对胃癌发生发展的影响

目的:探讨lncRNA BCAR4通过调节Wnt/β-catenin信号通路调控胃癌细胞的增殖和凋亡。方法:通过实时定量PCR法检测lncRNA BCAR4在胃癌组织和胃癌细胞中的表达;免疫组化方法分析胃组织中c-myc和cyclin D1蛋白的表达;通过MTT、克隆形成和流式细胞术分别检测lncRNA BCAR4对胃癌细胞SGC7901增殖和凋亡的影响,并通过Western blot法检测lncRNA BCAR4敲除前后对SGC7901细胞中c-myc、cyclin D1蛋白以及Wnt/β-catenin信号通路的影响。结果:相比于癌旁组织以及胃黏膜上皮细胞,lncRNA BCAR4在人胃癌组织和胃癌细胞中表达显著升高（P＜0.01）;同时,c-myc和cyclin D1蛋白的表达在胃癌组织和胃癌细胞中也有所增加,尤其在SGC7901胃癌细胞中上调最为显著（P＜0.01）; lncRNA BCAR4敲除能够显著降低细胞活力,促进细胞凋亡（P＜0.01）。lncRNA BCAR4敲除能够显著抑制Wnt/β-catenin信号通路的活化（P＜0.01）,进而降低其下游增殖相关蛋白c-myc和cyclin D1的表达（P＜0.01）。结论:降低lncRNA BCAR4能够通过抑制Wnt/β-catenin信号通路的活化,从而降低下游c-myc和cyclin D1蛋白表达,导致胃癌细胞的增殖抑制,促进细胞凋亡。     

miR-942 promotes cancer stem cell-like traits in esophageal squamous cell carcinoma through activation of Wnt/β-catenin signalling pathway

The Wnt/β-catenin signalling pathway is known to play a vital role in the maintenance of cancer stem cells (CSCs), which are reported to be the origine of malignant cancers, and result in poor prognosis of multiple kinds of cancer. Therefore, it is of great importance to illuminate the mechanism by which the Wnt/β-catenin pathway regulates the cancer stem cell-like traits in cancers. Here, we report that miR-942 is significantly upregulated in esophageal squamous cell carcinoma (ESCC), and miR-942 levels are associated with poor prognosis in ESCC patients. Overexpression of miR-942 promotes, whereas inhibition of miR-942 decreases, the tumor sphere formation, the CD90⁺ subpopulation cells and the expression of pluripotency associated markers. Moreover, in vivo assay shows that miR-942 overexpressing cells form larger tumors and display higher tumourigenesis. Furthermore, we demonstrate that miR-942 upregulates the Wnt/β-catenin signaling activity via directly targeting sFRP4, GSK3β and TLE1, which are multiple level negative regulators of the Wnt/β-catenin signaling cascade. In addition, our results indicate that c-myc directly binds to the miR-942 promoter and promotes its expression. Taken together, our findings establish an oncogenic role of miR-942 in ESCC and indicate that miR-942 might be an effective therapeutic target for ESCC.     

MiR-1207 overexpression promotes cancer stem cell–like traits in ovarian cancer by activating the Wnt/β-catenin signaling pathway

Wnt/β-catenin signaling pathway is strictly controlled by multiple negative regulators. However, how tumor cells override the negative regulatory effects to maintain constitutive activation of Wnt/β-catenin signaling, which is commonly observed in various cancers, remains puzzling. In current study, we reported that overexpression of miR-1207 in ovarian cancer activated Wnt/β-catenin signaling by directly targeting and suppressing secreted Frizzled-related protein 1 (SFRP1), AXIN2 and inhibitor of β-catenin and TCF-4 (ICAT), which are vital negative regulators of the Wnt/β-catenin pathway. We found that the expression of miR-1207 was ubiquitously upregulated in both ovarian cancer tissues and cells, which inversely correlated with patient overall survival. Furthermore, overexpression of miR-1207 enhanced, while silencing miR-1207 reduced, stem cell-like traits of ovarian cancer cells in vitro and in vivo, including tumor sphere formation capability and proportion of SP+ and CD133+ cells. Importantly, upregulating miR-1207 promoted, while silencing miR-1207 inhibited, the tumorigenicity of ovarian cancer cells. Hence, our results suggest that miR-1207 plays a vital role in promoting the cancer stem cell-like phenotype in ovarian cancer and might represent a potential target for anti-ovarian cancer therapy.     

Wnt/β-catenin pathway in bone cancers

The Wnt signaling pathway regulates some of the crucial aspects of cellular processes. The beta-catenin dependent Wnt signaling (Wnt/β-catenin) pathway controls the expression of key developmental genes, and acts as an intracellular signal transducer. The association of Wnt/β-catenin pathway is often reported with different cancers. In this study, we have reviewed the association of Wnt/β-catenin pathway with bone cancers, focusing on carcinogenesis and therapeutic aspects. Wnt/β-catenin pathway is a highly complex and unique signaling pathway, which has ability to regulate gene expression, cell invasion, migration, proliferation, and differentiation for the initiation and progression of bone cancers, especially osteosarcoma. Association of Wnt/β-catenin pathway with chondrosarcoma, Ewing’s sarcoma and chondroma is also documented. Recently, targeting Wnt/β-catenin pathway has gained significant interests as a potential therapeutic application for the treatment of bone cancers. Small RNA technology to knockdown aberrant Wnt/β-catenin or inhibition of β-catenin expression by natural component has shown promising effects against bone cancers. Advances in understanding the mechanisms of Wnt signaling and new technologies have facilitated the discovery of agents that can target and regulate Wnt/β-catenin signaling pathway, and these may provide a basement for the innovative therapeutic approaches in the treatment of bone cancers.