PCNP promotes ovarian cancer progression by accelerating β‐catenin nuclear accumulation and triggering EMT transition

Ever reports showed that PCNP is associated with human cancers including neuroblastoma and lung cancer. However, the role and underlying molecular mechanism of PCNP in ovarian cancer have not been plenty elucidated. Herein, we first investigated the expression of PCNP in ovarian cancer tissues and cells, the effects of PCNP in ovarian cancer proliferation, apoptosis, migration and invasion, and determined the molecular mechanism of PCNP in ovarian cancer progression. The results indicated that PCNP was significantly overexpressed in human ovarian cancer tissues and cells, and related to poor prognosis in ovarian cancer patients. In addition, we also detected that PCNP promoted ovarian cancer cells growth, migration and invasion, as well as inhibited ovarian cancer cells apoptosis. Mechanistically, PCNP binding to β‐catenin promoted β‐catenin nuclear translocation and further activated Wnt/β‐catenin signalling pathway. Moreover, PCNP regulated the expression of genes involved in EMT and further triggered EMT occurrence. Conclusionally, PCNP may promote ovarian cancer progression through activating Wnt/β‐catenin signalling pathway and EMT, acting as a novel and promising target for treating ovarian cancer.     

HIF‐1α regulates EMT via the Snail and β‐catenin pathways in paraquat poisoning‐induced early pulmonary fibrosis

Paraquat (PQ) poisoning‐induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia‐inducible factor‐1α (HIF‐1α) and epithelial‐mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and β‐catenin are two other factors involved in promoting EMT. However, the relationship among HIF‐1α, Snail and β‐catenin in PQ poisoning‐induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF‐1α in EMT occurs via the Snail and β‐catenin pathways in PQ poisoning‐induced pulmonary fibrosis. Sixty‐six Sprague–Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE‐6TN cell lines were transfected with HIF‐1α siRNA for 48 hrs before being exposed to PQ. Western blotting, real‐time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF‐1α and α‐SMA were increased at 2 hrs and the level of ZO‐1 (Zonula Occluden‐1) was reduced at 12 hrs. In vitro, the transient transfection of HIF‐1α siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and β‐catenin were significantly reduced when HIF‐α was silenced. These data demonstrate that EMT may be involved in PQ poisoning‐induced pulmonary fibrosis and regulated by HIF‐1α via the Snail and β‐catenin pathways. Hypoxia‐inducible factor‐1α may be a therapeutic target for the treatment of PQ poisoning‐induced pulmonary fibrosis.     

IL‐8 promotes cell migration through regulating EMT by activating the Wnt/β‐catenin pathway in ovarian cancer

Interleukin‐8 (IL‐8), as an inflammatory chemokine, has been previously shown to contribute to tumorigenesis in several malignancies including the ovarian cancer. However, little is known about how IL‐8 promotes the metastasis and invasion of ovarian cancers cells. In this study, we found that IL‐8 and its receptors CXCR1 and CXCR2 were up‐regulated in advanced ovarian serous cancer tissues. Furthermore, the level of IL‐8 and its receptors CXCR1 and CXCR2 expression were associated with ovarian cancer stage, grade and lymph node metastasis. In vitro, IL‐8 promoted ovarian cancer cell migration, initiated the epithelial‐mesenchymal transition (EMT) program and activated Wnt/β‐catenin signalling. However, when treated with Reparixin (inhibitor of both IL‐8 receptors CXCR1 and CXCR2), effect of both endogenous and exogenous IL‐8 was reversed. Together, our results indicated that IL‐8 triggered ovarian cancer cells migration partly through Wnt/β‐catenin pathway mediated EMT, and IL‐8 may be an important molecule in the invasion and metastasis of ovarian cancer.     

MicroRNA‐300 promotes apoptosis and inhibits proliferation,migration, invasion and epithelial‐mesenchymal transition via the Wnt/β‐catenin signaling pathway by targeting CUL4B in pancreatic cancer cells

The study aims to verify the hypothesis that up‐regulation of microRNA‐300 (miR‐300) targeting CUL4B promotes apoptosis and suppresses proliferation, migration, invasion, and epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells by regulating the Wnt/β‐catenin signaling pathway. Pancreatic cancer tissues and adjacent tissues were collected from 110 pancreatic cancer patients. Expression of miR‐300, CUL4B, Wnt, β‐catenin, E‐cadherin, N‐cadherin, Snail, GSK‐3β, and CyclinD1 were detected using qRT‐PCR and Western blot. CFPAC‐1, Capan‐1, and PANC‐1 were classified into blank, negative control (NC), miR‐300 mimics, miR‐300 inhibitors, siRNA‐CUL4B, and miR‐300 inhibitors + siRNA‐CUL4B groups. The proliferation, migration, invasion abilities, the cell cycle distribution, and apoptosis rates were measured in CCK‐8 and Transwell assays. Pancreatic cancer tissues showed increased CUL4B expression but decreased miR‐300 expression. When miR‐300 was lowly expressed, CUL4B was upregulated which in‐turn activated the Wnt/β‐catenin pathway to protect the β‐catenin expression and thus induce EMT. When miR‐300 was highly expressed, CUL4B was downregulated which in‐turn inhibited the Wnt/β‐catenin pathway to prevent EMT. Weakened cell migration and invasion abilities and enhanced apoptosis were observed in the CUL4B group. The miR‐300 inhibitors group exhibited an evident increase in growth rate accompanied the largest tumor volume. Smaller tumor volume and slower growth rate were observed in the miR‐300 mimics and siRNA‐CUL4B group. Our study concludes that lowly expressed miR‐300 may contribute to highly expressed CUL4B activating the Wnt/β‐catenin signaling pathway and further stimulating EMT, thus promoting proliferation and migration but suppressing apoptosis of pancreatic cancer cells.     

Oestrogen induces epithelial‐mesenchymal transition in endometriosis via circ_0004712/miR‐148a‐3p sponge function

Endometriosis is a common, chronic gynaecologic disease affecting up to 10% of women in their reproductive age and leading to pain and infertility. Oestrogen (E₂)‐induced epithelial‐mesenchymal transition (EMT) process has been considered as a key factor of endometriosis development. Recently, the dysregulated circular RNAs (circRNAs) have been discovered in endometriosis tissues. However, the molecular mechanism of circRNAs on the E₂‐induced EMT process in endometriosis is still unknown. Here, we demonstrated that circ_0004712 up‐regulated by E₂ treatment in endometrial epithelial cells. Knock‐down the expression of circ_0004712 significantly suppressed E₂‐induced cell migration activity. Meanwhile, we identified miR‐148a‐3p as a potential target miRNA of circ_0004712. Inhibited the expression of miR‐148a‐3p could recovered the effect of circ_0004712 knock‐down in E₂‐treated endometrial epithelial. Furthermore, Western blot assay showed that E₂ treatment could increase the expression and activity of β‐catenin, snail and N‐cadherin and reduce the expression of E‐cadherin. The expression and activity of β‐catenin pathway were recovered by circ_0004712 knock‐down or miR‐148a‐3p overexpression. Altogether, the results demonstrate that circ_0004712/miR‐148a‐3p plays an important role in E₂‐induced EMT process in the development of endometriosis, and the molecular mechanism may be associated with the β‐catenin pathway. This work highlighted the importance of circRNAs in the development of endometriosis and provide a new biomarker for diagnosis and therapies.     

DKK3 blocked translocation of β‐catenin/EMT induced by hypoxia and improved gemcitabine therapeutic effect in pancreatic cancer Bxpc‐3 cell

The Wnt/β‐catenin signalling pathway is activated in pancreatic cancer initiation and progression. Dickkopf‐related protein 3 (DKK3) is a member of the human Dickkopf family and an antagonist of Wnt ligand activity. However, the function of DKK3 in this pathway in pancreatic cancer is rarely known. We examined the expression of DKK3 in six human pancreatic cancer cell lines, 75 pancreatic cancer and 75 adjacent non‐cancerous tissues. Dickkopf‐related protein 3 was frequently silenced and methylation in pancreatic cancer cell lines (3/6). The expression of DKK3 was significantly lower in pancreatic cancer tissues than in adjacent normal pancreas tissues. Further, ectopic expression of DKK3 inhibits nuclear translocation of β‐catenin induced by hypoxia in pancreatic cancer Bxpc‐3 cell. The forced expression of DKK3 markedly suppressed migration and the stem cell‐like phenotype of pancreatic cancer Bxpc‐3 cell in hypoxic conditions through reversing epithelial–mesenchymal transition (EMT). The stable expression of DKK3 sensitizes pancreatic cancer Bxpc‐3 cell to gemcitabine, delays tumour growth and augments gemcitabine therapeutic effect in pancreatic cancer xenotransplantation model. Thus, we conclude from our finding that DKK3 is a tumour suppressor and improved gemcitabine therapeutic effect through inducing apoptosis and regulating β‐catenin/EMT signalling in pancreatic cancer Bxpc‐3 cell.     

miR‐193b represses influenza A virus infection by inhibiting Wnt/β‐catenin signalling

Due to an increasing emergence of new and drug‐resistant strains of the influenza A virus (IAV), developing novel measures to combat influenza is necessary. We have previously shown that inhibiting Wnt/β‐catenin pathway reduces IAV infection. In this study, we aimed to identify antiviral human microRNAs (miRNAs) that target the Wnt/β‐catenin signalling pathway. Using a miRNA expression library, we identified 85 miRNAs that up‐regulated and 20 miRNAs that down‐regulated the Wnt/β‐catenin signalling pathway. Fifteen miRNAs were validated to up‐regulate and five miRNAs to down‐regulate the pathway. Overexpression of four selected miRNAs (miR‐193b, miR‐548f‐1, miR‐1‐1, and miR‐509‐1) that down‐regulated the Wnt/β‐catenin signalling pathway reduced viral mRNA, protein levels in A/PR/8/34‐infected HEK293 cells, and progeny virus production. Overexpression of miR‐193b in lung epithelial A549 cells also resulted in decreases of A/PR/8/34 infection. Furthermore, miR‐193b inhibited the replication of various strains, including H1N1 (A/PR/8/34, A/WSN/33, A/Oklahoma/3052/09) and H3N2 (A/Oklahoma/309/2006), as determined by a viral reporter luciferase assay. Further studies revealed that β‐catenin was a target of miR‐193b, and β‐catenin rescued miR‐193b‐mediated suppression of IAV infection. miR‐193b induced G0/G1 cell cycle arrest and delayed vRNP nuclear import. Finally, adenovirus‐mediated gene transfer of miR‐193b to the lung reduced viral load in mice challenged by a sublethal dose of A/PR/8/34. Collectively, our findings suggest that miR‐193b represses IAV infection by inhibiting Wnt/β‐catenin signalling.     

PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

Protein arginine methyltransferase 5 (PRMT5) has been implicated in the development and progression of human cancers. However, few studies reveal its role in epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells. In this study, we find that PRMT5 is up‐regulated in pancreatic cancer, and promotes proliferation, migration and invasion in pancreatic cancer cells, and promotes tumorigenesis. Silencing PRMT5 induces epithelial marker E‐cadherin expression and down‐regulates expression of mesenchymal markers including Vimentin, collagen I and β‐catenin in PaTu8988 and SW1990 cells, whereas ectopic PRMT5 re‐expression partially reverses these changes, indicating that PRMT5 promotes EMT in pancreatic cancer. More importantly, we find that PRMT5 knockdown decreases the phosphorylation level of EGFR at Y1068 and Y1172 and its downstream p‐AKT and p‐GSK3β, and then results in down‐regulation of β‐catenin. Expectedly, ectopic PRMT5 re‐expression also reverses the above changes. It is suggested that PRMT5 promotes EMT probably via EGFR/AKT/β‐catenin pathway. Taken together, our study demonstrates that PRMT5 plays oncogenic roles in the growth of pancreatic cancer cell and provides a potential candidate for pancreatic cancer treatment.     

MicroRNA‐1275 induces radiosensitization in oesophageal cancer by regulating epithelial‐to‐mesenchymal transition via Wnt/β‐catenin pathway

Acquired radioresistance is one of the main obstacles for the anti‐tumour efficacy of radiotherapy in oesophageal cancer (EC). Recent studies have proposed microRNAs (miRNAs) as important participators in the development of radioresistance in various cancers. Here, we investigated the role of miR‐1275 in acquired radioresistance and epithelial‐mesenchymal transition (EMT) in EC. Firstly, a radioresistant cell line KYSE‐150R was established, with an interesting discovery was observed that miR‐1275 was down‐regulated in KYSE‐150R cells compared to the parental cells. Functionally, miR‐1275 inhibition elevated radioresistance in KYSE‐150 cells via promoting EMT, whereas enforced expression of miR‐1275 increased radiosensitivity in KYSE‐150R cells by inhibiting EMT. Mechanically, we demonstrated that miR‐1275 directly targeted WNT1 and therefore inactivated Wnt/β‐catenin signalling pathway in EC cells. Furthermore, WNT1 depletion countervailed the promoting effect of miR‐1275 suppression on KYSE‐150 cell radioresistance through hampering EMT, whereas WNT1 overexpression rescued miR‐1275 up‐regulation‐impaired EMT to reduce the sensitivity of KYSE‐150R cells to radiation. Collectively, our findings suggested that miR‐1275 suppressed EMT to encourage radiosensitivity in EC cells via targeting WNT1‐activated Wnt/β‐catenin signalling, providing a new therapeutic outlet for overcoming radioresistance of patients with EC.     

Wnt/β‐catenin signalling pathway mediated aberrant hippocampal neurogenesis in kainic acid‐induced epilepsy

Temporal lobe epilepsy is a chronic disorder of nerve system, mainly characterized by hippocampal sclerosis with massive neuronal loss and severe gliosis. Aberrant neurogenesis has been shown in the epileptogenesis process of temporal lobe epilepsy. However, the molecular mechanisms underlying aberrant neurogenesis remain unclear. The roles of Wnt signalling cascade have been well established in neurogenesis during multiple aspects. Here, we used kainic acid‐induced rat epilepsy model to investigate whether Wnt/β‐catenin signalling pathway is involved in the aberrant neurogenesis in temporal lobe epilepsy. Immunostaining and western blotting results showed that the expression levels of β‐catenin, Wnt3a, and cyclin D1, the key regulators in Wnt signalling pathway, were up‐regulated during acute epilepsy induced by the injection of kainic acids, indicating that Wnt signalling pathway was activated in kainic acid‐induced temporal lobe epilepsy. Moreover, BrdU labelling results showed that blockade of the Wnt signalling by knocking down β‐catenin attenuated aberrant neurogenesis induced by kainic acids injection. Altogether, Wnt/β‐catenin signalling pathway mediated hippocampal neurogenesis during epilepsy, which might provide new strategies for clinical treatment of temporal lobe epilepsy. Temporal lobe epilepsy is a chronic disorder of nerve system, mainly characterized by hippocampal sclerosis. Aberrant neurogenesis has been shown to involve in the epileptogenesis process of temporal lobe epilepsy. In the present study, we discovered that Wnt3a/β‐catenin signalling pathway serves as a link between aberrant neurogenesis and underlying remodelling in the hippocampus, leading to temporal lobe epilepsy, which might provide new strategies for clinical treatment of temporal lobe epilepsy.     

Inhibition of GSK‐3β enhances neural differentiation in unrestricted somatic stem cells

GSK‐3β is a key molecule in several signalling pathways, including the Wnt/β‐catenin signalling pathway. There is increasing evidence suggesting Wnt/β‐catenin signalling is involved in the neural differentiation of embryonic, somatic and neural stem cells. However, a large body of evidence indicates that this pathway maintains stem cells in a proliferative state. To address this controversy, we have investigated whether the Wnt/β‐catenin pathway is present and involved in the neural differentiation of newly introduced USSCs (unrestricted somatic stem cells). Our results indicate that the components of Wnt/β‐catenin signalling are present in undifferentiated USSCs. We also show that the treatment of neurally induced USSCs with BIO (6‐bromoindirubin‐3′‐oxime), a specific GSK‐3β inhibitor and Wnt activator, for 5 and 10 days results in increased expression of a general neuronal marker (β‐tubulin III). Moreover, the expression of pGSK‐3β and stabilized β‐catenin increased by BIO in neurally induced USSCs, indicates that the Wnt pathway is activated and functional in these cells. Thus, inhibition of GSK‐3β in USSCs enhances their neural differentiation, which suggests a positive role of the Wnt/β‐catenin signalling pathway towards neural fate.     

β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.     

Resveratrol promotes trophoblast invasion in pre‐eclampsia by inducing epithelial‐mesenchymal transition

Impairment spiral arteries remodelling was considered to be the underlying cause of pathogenesis of pre‐eclampsia (PE). Resveratrol (RE) was reported that it could modulate cellar phenotype to ameliorate diverse human diseases. However, the biological function of RE in PE remains poorly understood. In this report, we investigated the effect of RE on trophoblast phenotype both in vivo and in vitro. We conducted MTT and transwell assays to explore cell proliferation and invasion events in HTR‐8/SVneo. In mice model, the clinical characteristics of PE were established through the injection of NG‐nitro‐l ‐arginine methyl ester (L‐NAME). Furthermore, related experiments were performed to detect cellar phenotype‐associated signalling pathway, including epithelial‐mesenchymal transition (EMT) and Wnt/β‐catenin. Cell assays indicated that RE could increase trophoblasts migration and invasion. In addition, hypertension and proteinuria were markedly ameliorated by RE compared with the controls in PE mice model. Moreover, treatment by RE in trophoblasts or in PE model, we found that RE activated EMT progress through the regulation of E‐cadherin, β‐catenin, N‐cadherin, vimentin expression, and further altered the WNT‐related gene expression, including WNT1, WNT3 and WNT5B. Our findings demonstrated that RE might stimulate the invasive capability of human trophoblasts by promoting EMT and mediating the Wnt/β‐catenin pathway in PE.     

Activation of Wnt/β‐catenin signalling is required for TGF‐β/Smad2/3 signalling during myofibroblast proliferation

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α‐SMA and collagen I) and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis.     

Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β‐catenin signalling and apoptosis regulation in breast cancer cells

Dickkopf‐related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour‐specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial‐mesenchymal transition (EMT) and down‐regulating stem cell markers. DKK3 inhibited canonical Wnt/β‐catenin signalling through mediating β‐catenin translocation from nucleus to cytoplasm and membrane, along with reduced active‐β‐catenin, further activating non‐canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.