SOX9/miR-130a/CTR1 axis modulates DDP-resistance of cervical cancer cell

Cisplatin (DDP) -based chemotherapy is a standard strategy for cervical cancer, while chemoresistance remains a huge challenge. Copper transporter protein 1 (CTR1), a copper influx transporter required for high affinity copper (probably reduced Cu I) transport into the cell, reportedly promotes a significant fraction of DDP internalization in tumor cells. In the present study, we evaluated the function of CTR1 in the cell proliferation of cervical cancer upon DDP treatment. MicroRNAs (miRNAs) have been regarded as essential regulators of cell proliferation, apoptosis, migration, as well as chemoresistance. By using online tools, we screened for candidate miRNAs potentially regulate CTR1, among which miR-130a has been proved to promote cervical cancer cell proliferation through targeting PTEN in our previous study. In the present study, we investigated the role of miR-130a in cervical cancer chemoresistance to DDP, and confirmed the binding of miR-130a to CTR1. SOX9 also reportedly act on cancer chemoresistance. In the present study, we revealed that SOX9 inversely regulated miR-130a through direct targeting the promoter of miR-130a. Consistent with previous studies, SOX9 could affect cervical cancer chemoresistance to DDP. Taken together, we demonstrated a SOX9/miR-130a/CTR1 axis which modulated the chemoresistance of cervical cancer cell to DDP, and provided promising targets for dealing with the chemoresistance of cervical cancer.     

miR-937-5p targets SOX17 to modulate breast cancer cell cycle and cell proliferation through the Wnt signaling pathway

Breast cancer is one of the most frequent cancers in women and the globally leading cause of cancer-related deaths. Bioinformatics and experimental analyses found that miR-937-5p may play a proto-oncogenic role in breast cancer; however, the specific effects and the molecular mechanism need further investigation. GSEA-KEGG and GSEA-GO suggested that miR-937-5p might be related to cell cycle and DNA replication. The experimental data indicated that miR-937-5p inhibition significantly repressed the proliferation of breast carcinoma cells and elicited S-phase cell cycle arrest. Meanwhile, the protein levels of proliferating marker ki-67 and cell cycle regulators Cyclin A2, Cyclin B1, CDK1, and Cyclin D1 were also decreased by miR-937-5p inhibition. miR-937-5p could directly bind to and negatively regulate SOX17. SOX17 overexpression also significantly repressed the proliferation of breast carcinoma cells and elicited S-phase cell cycle arrest and decreased ki-67, β-catenin, c-Myc, Cyclin A2, Cyclin B1, Cyclin D1, and CDK1 protein contents. More importantly, the effects of miR-937-5p were reversed by SOX17.     

miR-155-3p对HANK1细胞EAF1 mRNA降解速率及恶性增殖的影响*

目的:探究miR-155-3p对人NK/T细胞淋巴瘤细胞HANK1恶性行为的影响及潜在机制。方法:Targetscan数据库预测miR-155-3p的靶基因,培养对数生长期HANK1细胞,将细胞分为空白组、过表达组、对照组及干扰组,利用细胞转染技术依次转入pENTER-puro空白载体、pENTER-miR-155-3p过表达载体、GV248对照载体、GV248-miR-155-3p siRNA干扰载体。同时放线菌素D(ActD)处理各组细胞,实时荧光定量PCR技术检测各组细胞miR-155-3p、EAF1、β-catenin及c-Myc的表达水平,并分析各组细胞ActD处理后EAF1 mRNA降解速率(n=5),Western blot检测细胞EAF1、β-catenin及c-Myc蛋白表达情况(n=3),CCK-8检测细胞恶性增殖能力变化(n=5)。结果:与空白组相比,过表达组细胞miR-155-3p、β-catenin及c-Myc表达水平显著增高,EAF1表达水平降低且EAF1 mRNA半衰期缩短,细胞恶性增殖能力增强(P均＜0.05);与对照组相比,干扰组细胞miR-155-3p、β-catenin及c-Myc表达水平显著降低,EAF1表达水平升高且EAF1 mRNA半衰期延长,细胞恶性增殖能力降低(P均＜ 0.05)。结论:miR-155-3p可促进EAF1 mRNA降解及HANK1细胞恶性增殖能力。     

miR-140-5p is negatively correlated with proliferation,invasion, and tumorigenesis in malignant melanoma by targeting SOX4 via the Wnt/β-catenin and NF-κB cascades

MicroRNAs (miRNAs) have been validated as critical regulators in the development of melanoma. miR-140 was abnormally downregulated in uveal melanoma samples. However, the expression level and roles of miR-140-5p remain unclear in melanoma for now. We speculate that miR-140-5p is abnormally expressed and may play an important role in melanoma. The expressions of miR-140-5p and SOX4 messenger RNA were determined by quantitative real-time polymerase chain reaction assays. Western blot assays were employed to detect the expression levels of SOX4, Ki67, MMP-2, MMP-7, p-β-catenin, c-Myc, cyclin D1, p65, and IκBα. Luciferase reporter assays were employed to elucidate the interaction between SOX4 and miR-140-5p. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) and transwell invasion assays were applied to evaluate capabilities of cell proliferation and invasion, respectively. Xenograft models of melanoma were established to verify the role and molecular basis of miR-140-5p. Immunohistochemical (IHC) assays were employed to measure the Ki67 and SOX4 at the protein level in xenografted melanoma tissues. Herein, these observations showed that, miR-140-5p was abnormally downregulated in melanoma tissues and cells, while SOX4 was upregulated. miR-140-5p directly targeted SOX4 and inhibited its expression in melanoma cells. miR-140-5p overexpression repressed melanoma cell proliferation and invasion and its effects were partially restored SOX4 overexpression. Moreover, miR-140-5p hindered melanoma growth in vivo by downregulating SOX4. Mechanistically, miR-140-5p suppressed activation of the Wnt/β-catenin and NF-κB pathways by targeting SOX4. Our study concluded that miR-140-5p hindered cell proliferation, invasion, and tumorigenesis by targeting SOX4 via inactivation of the Wnt/β-catenin and NF-κB signaling pathways in malignant melanoma, which provides an underlying molecular mechanism for the treatment for melanoma with miRNAs.     

MiR-34c在奶山羊雄性生殖干细胞增殖分化过程中的作用

microRNA (miRNA)在奶山羊雄性生殖细胞和精子发生过程有重要的调控功能。为研究miR-34c对雄性生殖干细胞增殖与分化中的作用,本文利用视黄酸效应基因8（Stra8）在雄性生殖细胞中随年龄增长,以其表达量上调的表达特征为指针,使用实时定量PCR技术筛选分析miRNAs。结果发现,miR-34c与Stra8的表达规律基本一致。在无精症奶山羊的睾丸组织中,发现miR-34c在无精症奶山羊睾丸组织中表达缺失。利用miR-34c模拟物及抑制剂转染奶山羊雄性生殖干细胞,体外转染miR-34c模拟物及其抑制剂,发现miR-34c能够下调Rarg、Stra8与c-Myc基因的表达,减缓奶山羊雄性生殖干细胞的增殖。结果提示,miR-34c可能具有调控奶山羊雄性生殖干细胞的减数分裂的作用,同时抑制其增殖。     

Suppression of nasopharyngeal carcinoma cell by targeting β-catenin signaling pathway

Aim: To investigate the role of β-catenin in pathogenesis of nasopharyngeal carcinoma (NPC). Methods: Cellular proliferation, apoptosis, matrix penetration assay, and western blotting were employed to determine cell biological changes in NPC cell lines transfected with β-catenin siRNA. Immunohistochemistry staining was used to detect β-catenin and Ki-67 expression in NPC tissue. Results: β-Catenin was upregulated in NPC cell lines and tissues compared with chronic nasopharyngitis tissue. β-Catenin knockdown dramatically inhibited cellular growth, migration and invasion, but induced apoptosis of NPC cells. Further study showed that downstream genes of β-catenin signaling pathway including cyclin D1, c-Myc, MMP2 and MMP9 expression were suppressed in NPC cell lines transfected with β-catenin siRNA. Conclusion: Targeting β-catenin signaling pathway may be a noval strategy for NPC therapy.     

miR-27 promotes osteoblast differentiation by modulating Wnt signaling

Canonical Wnt signaling is particularly important for differentiation of human mesenchymal stem cells into osteoblast. MicroRNAs (miRNAs) also play an essential role in regulating cell differentiation. However, the role of miRNAs in osteoblast differentiation remains poorly understood. Here we found that the expression of miR-27 was increased during hFOB1.19 cells differentiation. Moreover, ectopic expression of miR-27 promoted hFOB1.19 cells differentiation, whereas its repression was sufficient to inhibit cell differentiation. Western blot analysis showed that the expression level of miR-27 was positively correlated with that of β-catenin, a key protein in Wnt signaling. Further, we verified that miR-27 directly targeted and inhibited adenomatous polyposis coli (APC) gene expression, and activated Wnt signaling through accumulation of β-catenin. This study suggests miR-27 is an important mediator of osteoblast differentiation, thus offering a new target for the development of preventive or therapeutic agents against osteogenic disorders.     

Salinomycin inhibits proliferation and induces apoptosis of human nasopharyngeal carcinoma cell in vitro and suppresses tumor growth in vivo

Salinomycin (Sal) is a polyether ionophore antibiotic that has recently been shown to induce cell death in various human cancer cells. However, whether salinomycin plays a functional role in nasopharyngeal carcinoma (NPC) has not been determined to date. The present study investigated the chemotherapeutic efficacy of salinomycin and its molecular mechanisms of action in NPC cells. Salinomycin efficiently inhibited proliferation and invasion of 3 NPC cell lines (CNE-1, CNE-2, and CNE-2/DDP) and activated a extensive apoptotic process that is accompanied by activation of caspase-3 and caspase-9, and decreased mitochondrial membrane potential. Meanwhile, the protein expression level of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and β-catenin was down-regulated, which showed that the Wnt/β-catenin signaling was involved in salinomycin-induced apoptosis of NPC cells. In a nude mouse NPC xenograft model, the anti-tumor effect of salinomycin was associated with the downregulation of β-catenin expression. The present study demonstrated that salinomycin can effectively inhibit proliferation and invasion, and induce apoptosis of NPC cells in vitro and inhibit tumor growth in vivo, probably via the inhibition of Wnt/β-catenin signaling, suggesting salinomycin as a potential candidate for the chemotherapy of NPC.     

ß-Catenin is markedly induced in a murine model of an arteriovenous fistula: the effect of metalloproteinase inhibition

Neointimal hyperplasia contributes to failure of hemodialysis arteriovenous fistulas (AVFs). Increased expression of matrix metalloproteinase (MMP)-9 occurs in AVFs, and MMP-9 is implicated in neointimal hyperplasia and vascular injury. Recent studies demonstrate that MMP-9, by degrading N-cadherin, leads to increased expression of β-catenin and β-catenin-dependent proliferation of smooth muscle cells. The present study examined this pathway in the venous limb of a murine AVF model. Western analyses demonstrate that, in this model, there is diminished expression of N-cadherin accompanied by increased expression of β-catenin, c-Myc, and proliferating cell nuclear antigen (PCNA). By immunohistochemistry, β-catenin and c-Myc localized to proliferating smooth muscle cells in the venous limb of the AVF. Increased expression of β-catenin was accompanied by augmented expression of phosphorylated (p)-glycogen synthase kinase (GSK)-3β, GSK-3β, and integrin-linked kinase. The administration of doxycycline suppressed MMP-9 expression but did not reduce venous histological injury in the AVF, or increase AVF patency assessed 6 wk after its creation. Doxycycline did not influence expression of β-catenin, c-Myc, GSK-3β, or integrin-linked kinase. Thus, in this vascular injury model, the upregulation of β-catenin cannot be readily attributed to MMP-9 upregulation; increased β-catenin expression may reflect either the upregulation of p-GSK-3β, GSK-3β, or integrin-linked kinase. This study provides the first exploration of β-catenin in an AVF, demonstrating substantial upregulation of this mitogenic signaling molecule and uncovering possible mechanisms that may account for such upregulation.     

Retracted: Downregulation of microRNA-367 promotes osteoblasts growth and proliferation of mice during fracture by activating the PANX3-mediated Wnt/β-catenin pathway

A majority of people suffering from bone fractures fail to heal and develop a nonunion, which is a challenging orthopedic complication requiring complex and expensive treatment. Previous data showed the inhibition of some microRNAs (miRNAs or miRs) can enhance fracture healing. The objective of the present study is to explore effects of miR-367 on the osteoblasts growth and proliferation of mouse during fracture via the Wnt/β-catenin pathway by targeting PANX3. Primarily, the femur fracture model was successfully established in 66 (C57BL/6) 6-week–old male mice. To verify whether miR-367 target PANX3, we used the target prediction program and performed luciferase activity determination. Subsequently, to figure out the underlying regulatory roles of miR-367 in fracture, osteoblasts were elucidated by treatment with miR-367 mimic, miR-367 inhibitor, or siRNA against PANX3 to determine the expression of miR-367, siPANX3, β-catenin, and Wnt5b as well as cell proliferation and apoptosis. The results demonstrated that PANX3 was verified as a target gene of miR-367. MiR-367 was found to highly expressed but PANX3, β-catenin, and Wnt5b were observed poorly expressed in fracture mice. downregulated miR-367 increased the mRNA and protein expression of PANX3, β-catenin, and Wnt5b, increased cell growth, proliferation, and migration, while decreased cell apoptosis in osteoblasts. Altogether, our study demonstrates that the downregulation of miR-367 may promote osteoblasts growth and proliferation in fracture through the activation of the PANX3-dependent Wnt/β-catenin pathway.     

Nonsteroidal Anti-inflammatory Drugs Diclofenac and Celecoxib Attenuates Wnt/β-Catenin/Tcf Signaling Pathway in Human Glioblastoma Cells

Glioblastoma, the most common and aggressive primary brain tumors, carry a bleak prognosis and often recur even after standard treatment modalities. Emerging evidence suggests that deregulation of the Wnt/β-catenin/Tcf signaling pathway contributes to glioblastoma progression. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit tumor cell proliferation by suppressing Wnt/β-catenin/Tcf signaling in various human malignancies. In this study, we sought to inhibit Wnt/β-catenin/Tcf signaling in glioblastoma cells by the NSAIDs diclofenac and celecoxib. Both diclofenac and celecoxib significantly reduced the proliferation, colony formation and migration of human glioblastoma cells. Diclofenac and celecoxib downregulated β-catenin/Tcf reporter activity. Western and qRT-PCR analysis showed that diclofenac and celecoxib reduced the expression of β-catenin target genes Axin2, cyclin D1 and c-Myc. In addition, the cytoplasmic accumulation and nuclear translocation of β-catenin was significantly reduced following diclofenac and celecoxib treatment. Furthermore, diclofenac and celecoxib significantly increased phosphorylation of β-catenin and reduced the phosphorylation of GSK3β. These results clearly indicated that diclofenac and celecoxib are potential therapeutic agents against glioblastoma cells that act by suppressing the activation of Wnt/β-catenin/Tcf signaling.     

MicroRNA-27 promotes the differentiation of odontoblastic cell by targeting APC and activating Wnt/β-catenin signaling

MicroRNAs (miRNAs) play an essential role in regulating cell differentiation either by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontoblastic cell differentaion is largely unknown. In the present study, we demonstrate that the expression of miR-27 was significantly increased during MDPC-23 odontoblastic cell differentiation. Furthermore, the up-regulation of miR-27 promotes the differentiation of MDPC-23 odontoblastic cells and accelerates mineralization without cell proliferation. In addition, our results of target gene prediction revealed that the mRNA of adenomatous polyposis coli (APC) associated with Wnt/β-catenin signaling pathway has miR-27 binding site in the its 3′ UTR and is suppressed by miR-27. Subsequentially, the down-regulated APC by miR-27 triggered the activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus. Our data suggest that miR-27 promotes MDPC-23 odontoblastic cell differentiation by targeting APC and activating Wnt/β-catenin signaling. Therefore, miR-27 might be considered a critical candidate as an odontoblastic differentiation molecular target for the development of miRNA based therapeutic agents in the dental medicine.     

miR-186 modulates hepatocellular carcinoma cell proliferation and mobility via targeting MCRS1-mediated Wnt/β-catenin signaling

Previous studies have revealed that miR-186 is involved in the pathogenesis of many malignancies. However, the role of miR-186 in hepatocellular carcinoma (HCC) carcinogenesis and its detailed mechanism are poorly understood. This study was to investigate the function of miR-186 in modulating HCC cell proliferation, cell cycle, migration, and invasion. We found that miR-186 was decreased in HCC tissues and cell lines. Loss-of-function experiments showed that reduction of miR-186 dramatically enhanced tumor cell proliferation and metastasis. Besides, miR-186 also participated in the modulation of the cell cycle. In addition, luciferase reporter assays and Western blot analysis showed that MCRS1 was a novel target of miR-186 in HCC cells. Notably, upregulation of miR-186 suppressed the nuclear β-catenin accumulation and blocked the activation of Wnt/β-catenin signaling in HCC cells. Forced MCRS1 expression abrogated the inhibitory effect of miR-186 on cell growth, metastasis and Wnt/β-catenin signaling in HCC cells. Our findings may provide new insight into the pathogenesis of HCC and miR-186/ MCRS1 might function as new therapeutic targets for HCC.     

KHC-4 inhibits β-catenin expression in prostate cancer cells

ABSTRACT

KHC-4 is a 2-phenyl-4-quinolone analogue that exhibits anticancer activity. Aberrant activation of β-catenin signaling contributes to prostate cancer development and progression. Therefore, targeting β-catenin expression could be a useful approach to treating prostate cancer. We found that KHC-4 can inhibit β-catenin expression and its signaling pathway in DU145 prostate cancer cells. Treatment with KHC-4 decreased total β-catenin expression and concomitantly decreased β-catenin levels in both the cytoplasm and nucleus of cells. KHC-4 treatment also inhibited β-catenin expression and that of its target proteins, PI3K, AKT, GSK3β and TBX3. We monitored the stability of β-catenin with the proteasomal inhibitor, MG132, in DU145 cells and found that MG132 reversed KHC-4-induced proteasomal β-catenin degradation. We verified CDK1/β-catenin expression in KHC-4 treated DU145 cells. We found that roscovitine treatment reversed cell proliferation by arresting the cell cycle at the G2/M phase and β-catenin expression caused by KHC-4 treatment. We suggest that KHC-4 inhibits β-catenin signaling in DU145 prostate cancer cells.     

Long noncoding RNA TALNEC2 regulates myocardial ischemic injury in H9c2 cells by regulating miR-21/PDCD4-medited activation of Wnt/β-catenin pathway

The goal of this study was to explore the role of tumor associated long noncoding RNA expressed on chromosome 2 (TALNEC2) in protecting against myocardial ischemic injury, as well as its underlying molecular mechanism. We established a cell model of myocardial injury through treating H9c2 cells with hypoxia, and the expression level of TALNEC2 was analyzed. Further, in vitro studies investigated the functional role of TALNEC2 dysregulation in hypoxia injury by assessing cell proliferation, migration, invasion, and apoptosis. Moreover, the expression of miR-21 was determined after dysregulation of TALNEC2, and whether TALNEC2-regulated hypoxia injury in H9c2 cells via regulating miR-21 expression were explored. Furthermore, the regulatory relationship between TALNEC2 and Wnt/β-catenin pathway was also investigated. TALNEC2 was highly expressed in the serum from patients with myocardial ischemic compared with that in healthy persons. Hypoxia-induced injury in H9c2 cells. Overexpression of TALNEC2 aggravated hypoxia injury in H9c2 cells. TALNEC2 could negative regulate the miR-21 expression, and overexpression of TALNEC2 aggravated hypoxia injury by downregulation of miR-21. Moreover, miR-21 negatively regulated the PDCD4 expression, and PDCD4 was a target of miR-21. Further studies disclosed that the overexpression of TALNEC2 further activated the Wnt/β-catenin pathway in hypoxia-treated H9c2 cells, implying that the Wnt/β-catenin pathway was a downstream mechanism mediating the role of TALNEC2 in regulating hypoxia injury in H9c2 cells. These findings confirmed the key functions of TALNEC2 in regulating myocardial ischemic injury. Upregulation of TALNEC2 may aggravate hypoxia injury in H9c2 cells via regulating miR-21/PDCD4-medited activation of the Wnt/β-catenin pathway. TALNEC2 may serve as a promising therapeutic target in myocardial ischemia.     

Participation of miR-200a in TGF-β1-mediated hepatic stellate cell activation

Hepatic stellate cell (HSC) activation is a pivotal event in the initiation and progression of hepatic fibrosis since it mediates transforming growth factor beta 1 (TGF-β1)-driven extracellular matrix (ECM) deposition. MicroRNAs (miRNAs), small non-coding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key factors to regulate cell proliferation, differentiation, and apoptosis. Although the function of miR-200a has been discussed in many cancers and fibrotic diseases, its role in hepatic fibrosis is still poorly understood. The aim of this study is to investigate whether miR-200a could attenuate hepatic fibrosis partly through Wnt/β-catenin and TGF-β-dependant mechanisms. Our study found that the expression of endogenous miR-200a was decreased in vitro in TGF-β1-induced HSC activation as well as in vivo in CCl₄-induced rat liver fibrosis. Overexpression of miR-200a significantly inhibited α-SMA activity and further affected the proliferation of TGF-β1-dependent activation of HSC. In addition, we identified β-catenin and TGF-β2 as two functional downstream targets for miR-200a. Interestingly, miR-200a specifically suppressed β-catenin in the protein level, whereas miR-200a-mediated suppression of TGF-β2 was shown on both mRNA and protein levels. Our results revealed the critical regulatory role of miR-200a in HSC activation and implied miR-200a as a potential candidate for therapy by deregulation of Wnt/β-catenin and TGFβ signaling pathways, at least in part, via decreasing the expression of β-catenin and TGF-β2.     

MicroRNA-329 upregulation impairs the HMGB2/β-catenin pathway and regulates cell biological behaviors in melanoma

Melanoma is responsible for the majority of deaths caused by skin cancer. Antitumor activity of microRNA-329 (miR-329) has been seen in several human cancers. In this study, we identify whether miR-329 serves as a candidate regulator in melanoma. Melanoma-related differentially expressed genes were screened with its potential molecular mechanism predicted. Melanoma tissues and pigmented nevus tissues were collected, where the levels of miR-329 and high-mobility group box 2 (HMGB2) were determined. To characterize the regulatory role of miR-329 on HMGB2 and the β-catenin pathway in melanoma cell activities, miR-329 mimics, miR-329 inhibitors, and siRNA-HMGB2 were transfected into melanoma cells. Cell viability, migration, invasion, cell cycle, and apoptosis were assessed. miR-329 was predicted to influence melanoma by targeting HMGB2 via the β-catenin pathway. High level of HMGB2 and low miR-329 expression were observed in melanoma tissues. HMGB2 was targeted and negatively regulated by miR-329. In melanoma cells transfected with miR-329 mimics or siRNA-HMGB2, cell proliferation, migration, and invasion were impeded, yet cell cycle arrest and apoptosis were promoted, corresponding to decreased levels of β-catenin, cyclin D1, and vimentin and increased levels of GSK3β and E-cadherin. Collectively, our results show that miR-329 can suppress the melanoma progression by downregulating HMGB2 via the β-catenin pathway.