Levels Of Snail Research Articles

SNAIL Transctiption factor in prostate cancer cells promotes neurite outgrowth

Neurite outgrowth involves reciprocal signaling interactions between tumor cells and nerves where invading tumor cells have acquired the ability to respond to pro-invasive signals within the nerve environment. Neurite outgrowth could serve as a mechanism leading to invasion of cancer cells into the nerve sheath and subsequent metastasis. Snail transcription factor can promote migration and invasion of prostate cancer cells. We hypothesized that prostate cancer cell interaction with nerve cells will be mediated by Snail expression within prostate cancer cells. For this study we utilized various prostate cancer cell lines: C4-2 non-silencing (NS, control); C4-2 Snail shRNA, (stable Snail knockdown); LNCaP Neo (empty vector control) and LNCaP Snail (stably over-expressing Snail). Cancer cell adhesion and migration towards nerve cells (snF96.2 or NS20Y) was examined by co-culture assays. Conditioned media (CM) collected from C4-2 cells was cultured with nerve cells (PC-12 or NS20Y) for 48 h followed by qualitative or quantitative neurite outgrowth assay. Our results showed that cancer cells expressing high levels of Snail (LNCaP Snail/C4-2 NS) displayed significantly higher migration adherence to nerve cells, compared to cells with lower levels of Snail (LNCaP Neo/C4-2 Snail shRNA). Additionally, LNCaP Snail or C4-2 NS (Snail-high) CM led to a higher neurite outgrowth compared to the LNCaP Neo or C4-2 Snail shRNA (Snail-low). In conclusion, Snail promotes migration and adhesion to nerve cells, as well as neurite outgrowth via secretion of soluble factors. Therefore, targeting cancer cell interaction with nerves may contribute to halting prostate cancer progression/metastasis.

Paternally Expressed Gene 10 (PEG10) Promotes Growth, Invasion, and Survival of Bladder Cancer.

Paternally expressed gene 10 (PEG10) has been associated with neuroendocrine muscle-invasive bladder cancer (MIBC), a subtype of the disease with the poorest survival. In this work, we further characterized the expression pattern of PEG10 in The Cancer Genome Atlas database of 412 patients with MIBC, and found that, compared with other subtypes, PEG10 mRNA level was enhanced in neuroendocrine-like MIBC and highly correlated with other neuroendocrine markers. PEG10 protein level also associated with neuroendocrine markers in a tissue microarray of 82 cases. In bladder cancer cell lines, PEG10 expression was induced in drug-resistant compared with parental cells, and knocking down of PEG10 resensitized cells to chemotherapy. Loss of PEG10 increased protein levels of cell-cycle regulators p21 and p27 and delayed G1-S-phase transition, while overexpression of PEG10 enhanced cancer cell proliferation. PEG10 silencing also lowered levels of SLUG and SNAIL, leading to reduced invasion and migration. In an orthotopic bladder cancer model, systemic treatment with PEG10 antisense oligonucleotide delayed progression of T24 xenografts. In summary, elevated expression of PEG10 in MIBC may contribute to the disease progression by promoting survival, proliferation, and metastasis. Targeting PEG10 is a novel potential therapeutic approach for a subset of bladder cancers.

Deubiquitinase USP29 promotes gastric cancer cell migration by cooperating with phosphatase SCP1 to stabilize Snail protein.

Snail is a master inducer of epithelial-mesenchymal transition (EMT) and metastasis, however, Snail protein is labile and is quickly degraded through the predominate ubiquitination-mediated proteasome pathway. Deubiquitinases (DUBs) can counteract the Snail degradation process to maintain high level of Snail protein in cancer cells. In this study, we screened a cDNA library containing 79 DUBs, and discovered that a panel of DUBs consisting of USP13, USP28, USP29, USP37, OTUD6A, and DUB3 can markedly stabilize Snail protein, with USP29 displaying the strongest activity to prevent Snail degradation. Mechanistically, USP29 enhances the interaction of Snail and SCP1, resulting in simultaneous dephosphorylation and deubiquitination of Snail and thereafter cooperative prevention of Snail degradation. Biologically, ectopic expression of USP29 promotes gastric cancer cell migration, and depletion of Snail abolishes USP29-mediated cell migration; and USP29 can be induced by major EMT and metastatic inducing factors such as TGFβ, TNFα, and hypoxia. More importantly, high expression levels of Snail, USP29, and SCP1 are associated with poor survival and prognosis. Collectively, these data indicate that Snail is a crucial substrate for USP29 to promote cell migration and USP29/SCP1 complex may be new therapeutic targets to treat metastatic cancer.

Overexpression of NCAPG inhibits cardia adenocarcinoma apoptosis and promotes epithelial-mesenchymal transition through the Wnt/β-catenin signaling pathway.

Cardia adenocarcinoma (CA) is a distinct form of gastric cancer, and the optimal means of treating it remains controversial. At present, the role of the condensation complex gene non-SMC condensin I complex subunit G (NCAPG) in CA is uncertain, and as such the present study was designed to elucidate its importance in this oncogenic context. We first used bioinformatics approaches to assess NCAPG expression profiles in CA using public databases. Protein profiling was also used to examine the expression of this protein in CA tumors and adjacent tissues from 20 patients. Then the expression of NCAPG in CA samples was quantified via qRT-PCR and Western blotting. NCAPG knockdown and overexpression in the SGC-7901 and AGS cell lines were subsequently performed, after which the expression of key proteins associated with epithelial-mesenchymal transition (EMT; E-cadherin, vimentin, N-cadherin, Snail, Slug) and the regulation of apoptotic responses (caspase-3, Bax, Bcl-2) was measured. The mechanistic role of NCAPG in CA was additionally studied by analyzing proteins associated with Wnt/β-catenin signaling including Wnt1, phosphorylated GSK3β, β-catenin, and phosphorylated β- catenin. The impact of NCAPG on the migration, survival, and invasion of CA cells was further examined. CA samples exhibited high NCAPG expression. When this gene was overexpressed in cell lines, it reduced caspase-3, Bax, and E-cadherin levels whereas it elevated Bcl-2, vimentin, N-cadherin, Snail, and Slug levels. NCAPG overexpression also resulted in the enhanced expression of Wnt1, phosphorylated GSK3β, and total β-catenin and the reduced expression of phosphorylated β-catenin. The knockdown of NCAPG, in contrast, yielded the opposite phenotype. At a functional level, the overexpression of NCAPG improved the apoptotic resistance of CA cells while driving them to undergo EMT and to become more invasive and migratory. NCAPG overexpression can promote EMT and suppress tumor cell apoptosis via the activation of Wnt/β-catenin signaling.

Activation of autophagy inhibits epithelial to mesenchymal transition process of human lens epithelial cells induced by high glucose conditions

Subcapsular cataracts are common phenotype of diabetic cataracts, and abnormal lens epithelial cells (LECs) under the lens capsules have been considered to involve in the pathogenesis. Our previous studies have shown that the epithelial to mesenchymal transition (EMT), which is responsible for the LECs to lose their original polarity and tight junctions, occurs in a diabetic cataract mouse model. Autophagy is known to function in the EMT process in multiple tissues. However, the relationship between autophagy and EMT process in LECs has not yet been fully demonstrated. We found that high glucose retreatment reducing expression level of E-cadherin, an epithelial marker, but increasing that of α-smooth muscle actin (α-SMA), a mesenchymal marker, by Western blot and immunoflurence staining assays, and increased the cell migration by Transwell assay in human lens epithelial cell line HLE-B3. High glucose retreatment also led to impairment of autophagy, representing by downregulation of Beclin, LC3II/LC3I, and reducing the number of autophagosomes. Activation of autophagy by rapamycin could prevent high glucose-induced EMT. In addition, the levels of p62 and Snail were increased in high glucose-treated HLE-B3 cells, and their interactions were demonstrated by co-immunoprecipitation and immunoflurence staining, but all these changes were attenuated by application of rapamycin. These findings delineated a novel autophagy-mediated mechanism, p62 might mediate Snail underlying high glucose-induced EMT in LECs, suggesting a potential therapeutic approach for diabetic cataract by regulating autophagy.

DPAGT1 Inhibitors of Capuramycin Analogues and Their Antimigratory Activities of Solid Tumors.

Capuramycin displays a narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY). In our program of development of new N-acetylglucosaminephosphotransferase1 (DPAGT1) inhibitors, we have identified that a capuramycin phenoxypiperidinylbenzylamide analogue (CPPB) inhibits DPAGT1 enzyme with an IC50 value of 200 nM. Despite a strong DPAGT1 inhibitory activity, CPPB does not show cytotoxicity against normal cells and a series of cancer cell lines. However, CPPB inhibits migrations of several solid cancers including pancreatic cancers that require high DPAGT1 expression in order for tumor progression. DPAGT1 inhibition by CPPB leads to a reduced expression level of Snail but does not reduce E-cadherin expression level at the IC50 (DPAGT1) concentration. CPPB displays a strong synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic adenocarcinoma, PD002: paclitaxel (IC50: 1.25 μM) inhibits growth of PD002 at 0.0024-0.16 μM in combination with 0.10-2.0 μM CPPB (IC50: 35 μM).

<p>Knockdown of <em>LINC02471</em> Inhibits Papillary Thyroid Carcinoma Cell Invasion and Metastasis by Targeting miR-375</p>

BackgroundLncRNAs play important roles in papillary thyroid carcinoma (PTC). LINC02471 has been reported to be related to PTC prognosis. The current study aimed to investigate the effects of LINC02471 on human PTC cells.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine LINC02471 expression in PTC tissues and cells and miR-375 expression in PTC cells. SiLINC02471, miR-375 mimic and miR-375 inhibitor were used for cell transfection. Cell proliferation, apoptosis, migration, and invasion were detected by performing Cell Counting Kit-8 (CCK-8), clone formation assay, flow cytometry, scratch assay, and transwell assay. Western blot was carried out to detect protein levels of E-cadherin, N-cadherin and Snail. The target gene for LINC02471 was verified by dual-luciferase reporter assay.ResultsLINC02471 was highly expressed in PTC tissues and cells. After silencing LINC02471, cell proliferation, migration and invasion were reduced, but cell apoptosis was increased. SiLINC02471 increased the expressions of E-cadherin and miR-375, and inhibited the expressions of N-Cadherin and Snail. LINC02471 directly targeted miR-375 in PTC cells. Overexpression of miR-375 inhibited the proliferation, migration, invasion of PTC cells and reduced the expressions of N-Cadherin and Snail but promoted the cell apoptosis and increased E-cadherin expression, while miR-375 inhibitor produced opposite effects to overexpressed miR-375. After inhibiting miR-375 expression, siLINC02471 reversed the effect of miR-375 inhibitor.ConclusionLINC02471 could promote the development of PTC. Knocking down LINC02471 could inhibit invasion and metastasis and promote PTC cell apoptosis through directly targeting miR-375.

TP53INP2 Modulates Epithelial-to-Mesenchymal Transition via the GSK-3β/β-Catenin/Snail1 Pathway in Bladder Cancer Cells.

BackgroundThe tumor protein p53-inducible nuclear protein 2 (TP53INP2), an autophagy protein, is essential for autophagosome formation. The deregulation of autophagy is associated with multiple human diseases, including cancer. The present study aims to explore the role of TP53INP2 in bladder cancer.Materials and MethodsQuantitative real-time polymerase chain reaction was used to detect the mRNA level. Relative TP53INP2 protein expression was detected by immunohistochemistry and Western blot. The effect of TP53INP2 silencing on the proliferation, migration, and invasion of bladder cancer cells was investigated by CCK-8 detection kit and transwell assay. In addition, transfection and immunofluorescence were performed.ResultsIn this study, we report that high expression of TP53INP2 is correlated with poor patient survival in bladder cancer. Results demonstrate that the depletion of TP53INP2 inhibits the migration, invasion, and epithelial-to-mesenchymal transition (EMT) of bladder cancer cells. The underlying mechanism was explored. Results show that the TP53INP2 knockdown suppresses EMT by inhibiting the active non-phosphorylated β-catenin and decreasing the Snail1 levels. Furthermore, the glycogen synthase kinase-3 beta (GSK-3β) inhibitor IM-12 abrogates the effect of TP53INP2 silencing. Interestingly, the induction of autophagy partially abrogates the TP53INP2 knockdown-induced decrease in active β-catenin and inhibition of migration and invasion in bladder cancer cells.ConclusionIn summary, our results show that the downregulation of TP53INP2 inhibits EMT via the GSK-3β/β-catenin/Snail1 pathway in bladder cancer. The findings of this study uncover the novel role of TP53INP2 and offer new insights into bladder cancer clinical therapy.

Plantamajoside inhibits hypoxia-induced migration and invasion of human cervical cancer cells through the NF-κB and PI3K/akt pathways

Plantamajoside (PMS) is a major compound of Plantago asiatica and possesses anti-tumor property in several types of cancers. However, the effect of PMS on cervical cancer has not been investigated. This study aimed to investigate the effect of PMS on the migration and invasion of cervical cancer cell lines under hypoxic condition. Our results demonstrated that PMS significantly inhibited hypoxia-caused increases in cell migration and invasion of cervical cancer cells. The hypoxia-induced epithelial-mesenchymal transition (EMT) process was prevented by PMS with increased E-cadherin expression, and decreased expression levels of N-cadherin and vimentin in cervical cancer cells. Besides, the expression levels of transcription factors slug and snail were suppressed by PMS in hypoxia-induced cervical cancer cells. The increased mRNA and protein levels of hypoxia-inducible factor 1alpha (HIF-1α) in hypoxia-induced cervical cancer cells were prevented by PMS. Furthermore, PMS blocked the hypoxia-induced activation of NF-κB and PI3K/Akt pathway in cervical cancer cells. Taken together, these findings suggest that PMS exerted an anti-tumor activity in cervical cancer through preventing the hypoxia-induced EMT. Thus, PMS might serve as a therapeutic agent for the treatment of cervical cancer.

Abstract 1442: Identifying the molecular basis by which βIII-tubulin promotes tumor cell survival and drug resistance in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer-related mortality worldwide with the most common sub-type, non-small cell lung carcinoma (NSCLC), accounting for >80% of cases. Despite advances in treatments, 5-year survival rates for this disease remain dismal. Advanced NSCLC responds poorly to current therapy and the mechanisms underlying its resistance and aggressive nature are not well defined. Microtubules form a component of the cell cytoskeleton and alterations in the expression of microtubule proteins are increasingly correlated with more aggressive and treatment-refractory cancers. βIII-tubulin is a neuronal-specific isotype that is aberrantly expressed in a range of cancers including NSCLC, and has been found to be correlated with reduced patient survival. Our group has recently demonstrated that βIII-tubulin contributes to activation of the PI3K/AKT cell-survival pathway in NSCLC cells[1]. One of the key negative regulators of this pathway is the tumor suppressor PTEN. PTEN can inhibit cell growth, migration and invasion, and a loss of PTEN function is often associated with cancer progression through AKT hyperactivation. Here, using forced-genetics cell models, we show that AKT activation is reduced by βIII-tubulin knockdown, and that re-expression of βIII-tubulin restores these levels in NSCLC cells. Interestingly, we also found that βIII-tubulin knockdown resulted in increased PTEN mRNA along with altered levels of Snail-1/β-catenin, suggesting a possible role for βIII-tubulin in regulating the transcriptional pathway of the PTEN tumor suppressor. Building on these findings, we also identified, through a high-throughput cell-based drug screen, several receptor tyrosine kinase inhibitors that synergize with βIII-tubulin knockdown to reduce NSCLC cell proliferation and tumoroid growth. Together, our data demonstrate an important role for βIII-tubulin in modulating the activation and/or expression levels of signaling proteins which form part of a major pro-survival pathway in NSCLC. In addition, our results suggest that inhibition of βIII-tubulin in combination with drugs that target multiple kinases may offer a novel therapeutic strategy for the treatment of NSCLC to improve patient outcomes. [1] McCarroll et al., TUBB3/betaIII-tubulin acts through the PTEN/AKT signaling axis to promote tumorigenesis and anoikis resistance in non-small cell lung cancer. Cancer Research. 2015;75:415-425. Citation Format: Simon Brayford, Wee Siang Teo, Amelia Parker, Laura McVeigh, Joshua A. McCarroll, Maria Kavallaris. Identifying the molecular basis by which βIII-tubulin promotes tumor cell survival and drug resistance in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1442.

Carboplatin Inhibits the Progression of Retinoblastoma Through IncRNA XIST/miR-200a-3p/NRP1 Axis.

ObjectiveThis study was set out to explore the expression and related mechanism of XIST and miR-200a-3p in retinoblastoma (Rb).Patients and MethodsFifty-four children with Rb who came to our hospital for surgery from January 2018 to September 2019 were collected. In addition, Rb cells and human retinal epithelial cells were purchased. XIST-siRNA (si-XIST), XIST-shRNA (sh-XIST), empty vector plasmid (siRNA-NC), miR-200a-3p-mimics and miR −200a-3p-inhibition were transfected into Y79 cells. The expression of XIST and miR-200a-3p in the samples were determined by qRT-PCR. β-catenin, cyclin B1, cyclin D1, Bax, Caspase-3, N-cadherin, vimentin, Snail, E-Cadherin and ZO-1 protein levels were measured by WB. MTT, Transwell and flow cytometry were utilized to detect cell proliferation, invasion, and apoptosis, respectively.ResultsXIST was highly expressed while miR-200a-3p was lowly expressed in patients’ tissues, and the AUC of both was over 0.8. XIST and miR-200a-3p was related to differentiation degree in Rb patients. Y79 cells were selected for transfection. Compared with the siRNA-NC group, XIST was significantly reduced in the siRNA-XIST group, and it was significantly increased in the shRNA-XIST group (P<0.01). The proliferation capacity of siRNA-XIST group was decreased, while that of shRNA-XIST group was up-regulated. The apoptosis rate of siRNA-XIST group was significantly up-regulated, while that of shRNA-XIST group was decreased (P<0.001). The invasive capacity of siRNA-XIST group was decreased, while that of shRNA-XIST group was up-regulated (P<0.001). Silencing XIST and over-expressed miR-200a-3p could inhibit cell epithelial–mesenchymal transition (EMT), proliferation, invasion, and promote apoptosis. WB detection showed that Carboplatin + LncRNA XIST intervention group could more significantly inhibit β-catenin, cyclin B1, cyclin D1, N-cadherin, vimentin, Snail protein, and promote the up-regulation of Bax, Caspase-3, E-Cadherin and ZO-1 expression.ConclusionInhibition of XIST expression can up-regulate miR-200a-3p-mediated PI3K-Akt/MAPK-ERK signaling pathway and affect cell EMT, proliferation, invasion, and apoptosis, which is expected to be a potential therapeutic target for Rb.

CD147 promotes epithelial‑mesenchymal transition of prostate cancer cells via the Wnt/β‑catenin pathway

The majority of deaths among patients with prostate cancer (PCa) occur following metastasis; therefore, there is a critical need for effective treatment of metastatic PCa. Epithelial-mesenchymal transition (EMT) is vital in the early stage of cancer cell metastasis and CD147 has been reported to be associated with various types of cancer. The goal of this study was to investigate the role of CD147 in the EMT of PCa cells via short hairpin (sh)RNA-mediated knockdown of CD147 in lymph node carcinoma of the prostate (LNCaP) cells. Reverse transcription-quantitative PCR and western blotting were performed to examine gene and protein expression. Cell migration and invasion were detected using a Transwell assay. Cell Counting Kit-8 assay was performed to investigate cell viability. The knockdown of CD147 in LNCaP cells (LNCaP/shCD147 cells) resulted in an increase in the expression of E-cadherin (an epithelial marker), and a decrease in the expression of N-cadherin and vimentin (mesenchymal markers). Importantly, the downregulation of CD147 in LNCaP cells inhibited the expression levels of nuclear β-catenin and Snail, and phosphorylation of glycogen synthase kinase (GSK)-3β on Ser 9, and increased the expression of phosphorylated (p)-β-catenin (Ser33/37/Thr41). Treatment with lithium chloride (LiCl), a Wnt/β-catenin pathway agonist or a GSK-3β inhibitor, attenuated CD147 downregulation-induced p-β-catenin (Ser33/37/Thr41) expression, which resulted in the upregulation of β-catenin in the nucleus. LiCl treatment prompted β-catenin-mediated expression of target proteins such as Snail and vimentin in LNCaP/shCD147 cells, and prevented E-cadherin expression, a molecule downstream to Snail. In conclusion, these findings revealed an important role of CD147 in the regulation of the invasive and metastatic potential of PCa cells. CD147, via modulation of the Wnt/β-catenin pathway, may be implicated in the regulation of EMT of PCa cells and could be a potential therapeutic target for PCa.

Transmembrane protein 92 performs a tumor-promoting function in breast carcinoma by contributing to the cell growth, invasion, migration and epithelial-mesenchymal transition

ObjectiveWe try to examine the role of transmembrane protein 92 (TMEM92) in the progression of breast carcinoma (BC) and assess its prognostic value. Moreover, the effects of TMEM92 on BC cell phenotypes was explored. MethodsThe levels of TMEM92 in BC tissues were evaluated using bioinformatics analysis according to the Oncomine and The Cancer Genome Atlas databases. mRNA levels of TMEM92 in BC cells were measured by qRT-PCR. Kaplan-Meier methods together with log-rank tests were used to conduct survival analysis, and chi-square tests were employed to assess the relationship between TMEM92 levels and clinicopathological parameters. Cox regression analysis was carried out to identify the independent prognosticators. Small interference RNA targeted to TMEM92 and plasmid vectors pcDNA3.1-TMEM92 were respectively used to silence and over-express TMEM92. Protein levels of molecules in this study were tested by western blot. Cell viability, invasiveness and motility of BC cells were determined by cell counting kit 8, clone formation assay and Transwell assay, appropriately. ResultsThe data showed that TMEM92 was upregulated in BC tissues or cells in comparison with control. High expression of TMEM92 was notably correlated with stage and metastasis, and led to a poor overall survival. Moreover, cox multivariate analysis model demonstrated that TMEM92 can be seen as an independent prognostic factor. Functional experiments demonstrated that downregulation of TMEM92 showed a significantly inhibitory effect on MDA-MB-231 cell viability, invasiveness and motility, whereas overexpression of TMEM92 could promote the changes of these phenotypes. Furthermore, western blot analysis revealed that depletion of TMEM92 inactivated the epithelial-mesenchymal transition (EMT) process with raised E-cadherin protein levels, while declined N-cadherin, Vimentin and Snail levels. However, enhancement of TMEM92 showed the opposite outcomes on these EMT-related markers. ConclusionTMEM92 had an independent prognostic value for BC patients, and might act as an oncogene to facilitate tumor cells growth, invasiveness and motility by modulating the EMT relative proteins.

Tight junction protein claudin-1 is downregulated by TGF-β1 via MEK signaling in benign prostatic epithelial cells.

Benign prostatic hyperplasia (BPH) is arguably the most common disease in aging men. Although the etiology is not well understood, chronic prostatic inflammation is thought to play an important role in BPH initiation and progression. Our recent studies suggest that the prostatic epithelial barrier is compromised in glandular BPH tissues. The proinflammatory cytokine transforming growth factor beta 1 (TGF-β1) impacts tight junction formation, enhances epithelial barrier permeability, and suppresses claudin-1 messenger RNA expression in prostatic epithelial cells. However, the role of claudin-1 in the prostatic epithelial barrier and its regulation by TGF-β1 in prostatic epithelial cells are not clear. The expression of claudin-1 was analyzed in 22 clinical BPH specimens by immunohistochemistry. Human benign prostate epithelial cell lines BPH-1 and BHPrE1 were treated with TGF-β1 and transfected with small interfering RNAs specific to claudin-1. Epithelial monolayer permeability changes in the treated cells were measured using trans-epithelial electrical resistance (TEER). The expression of claudin-1, E-cadherin, N-cadherin, snail, slug, and activation of mitogen-activated proteins kinases (MAPKs) and AKT was assessed following TGF-β1 treatment using Western blot analysis. Claudin-1 expression was decreased in glandular BPH tissue compared with adjacent normal prostatic tissue in patient specimens. TGF-β1 treatment or claudin-1 knockdown in prostatic epithelial cell lines increased monolayer permeability. TGF-β1 decreased levels of claudin-1 and increased levels of snail and slug as well as increased phosphorylation of the MAPK extracellular signal-regulated kinase-1/2 (ERK-1/2) in both BPH-1 and BHPrE1 cells. Overexpression of snail or slug had no effect on claudin-1 expression. In contrast, PD98059 and U0126, inhibitors of the upstream activator of ERK-1/2 (ie, MEK-1/2) restored claudin-1 expression level as well as the epithelial barrier. Our findings suggest that downregulation of claudin-1 by TGF-β1 acting through the noncanonical MEK-1/2/ERK-1/2 pathway triggers increased prostatic epithelial monolayer permeability in vitro. These findings also suggest that elevated TGF-β1 may contribute to claudin-1 downregulation and compromised epithelial barrier in clinical BPH specimens.

Silencing of BRF2 inhibits the growth and metastasis of lung cancer cells.

Transcription factor II B (TFIIB)-related factor 2 (BRF2) is involved in the development of cancer, but its role in lung cancer is underreported. The present study aimed to explore the role of BRF2 in the regulation of lung cancer cells. Immunofluorescence staining and immunohistochemistry were performed to detect BRF2 protein expression in human lung cancer cells and tissues. Following cell transfection with small interfering RNA for silencing BRF2, the cell proliferation was examined by Cell Counting Kit-8 and MTT assays. Cell apoptosis, migration and invasion were determined by flow cytometry, wound-healing and Transwell assay. The expression levels of Akt, phosphorylated (p)-Akt, Bax, E-cadherin, Bcl-2, N-cadherin, Snail and epidermal growth factor receptor (EGFR) in human lung cancer A549 cells were detected by western blotting. The results demonstrated that BRF2 expression was increased in human lung cancer cells and tissues, and that silencing of BRF2 promoted cell apoptosis but inhibited cell proliferation and migration. The protein expression levels of Akt, E-cadherin, p-Akt, Bcl-2, N-cadherin, Snail and EGFR in A549 cells were inhibited by silencing of BRF2, while expression levels of Bax and E-cadherin were increased by silencing BRF2. In conclusion, BRF2 demonstrates high expression in lung cancer and silencing of BRF2 inhibits the growth and metastasis of lung cancer cells. The current findings provide a novel approach for the treatment of lung cancer.

LncRNA PVT1 promotes the migration of gastric cancer by functioning as ceRNA of miR-30a and regulating Snail.

Although the incidence and mortality of gastric cancer (GC) are slowly decreasing, the overall prognosis of GC patients with distal metastasis remains dismal. Long non-coding RNA PVT1 has been verified to function as a tumor promoter in several types of cancer. However, the role of PVT1 in GC metastasis remains obscure. Herein, we found that PVT1 was highly expressed in GC tissues and high PVT1 level was associated with tumor stage, lymph node metastasis, and poor prognosis. Overexpression of PVT1 significantly elevated epithelial-to-mesenchymal transition (EMT) marker (N-cadherin, ZEB1, and ZEB2) levels and promoted GC cell EMT process and tumor metastasis in vitro and in vivo. Mechanistically, Snail was identified as a direct target of miR-30a. PVT1 could bind with miR-30a and increase the expression of Snail by acting as a competing endogenous RNA, whereas re-expression of miR-30a in GC cells rescued the EMT markers, decreased Snail level, and inhibited GC cell migration. Taken together, these findings provide a new light on PVT1 in the pathogenesis and development of GC and an important implication for future therapy of the GC.

The Gli1-Snail axis contributes to Salmonella Typhimurium-induced disruption of intercellular junctions of intestinal epithelial cells.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that damages gastrointestinal tissue and causes severe diarrhoea. The mechanisms by which Salmonella disrupts epithelial barrier and increases the paracellular permeability are incompletely understood. Our present study aims to determine the role of Gli1, a transcription factor activated in the sonic hedgehog (Shh) pathway, in decreasing the levels of apical junction proteins in a Salmonella-infected human colonic epithelial cancer cell line, Caco-2, and in the intestinal tissue of Salmonella-infected mice. Here, we report that S. Typhimurium increased the mRNA and protein levels of Gli1 and Snail, a downstream transcription factor that plays an important role in the epithelial-to-mesenchymal transition (EMT). S. Typhimurium also decreased the levels of E-cadherin and three tight junction proteins (ZO-1, claudin-1, and occludin). Gli1 siRNA and GANT61, a Gli1-specific inhibitor, blocked S. Typhimurium-induced Snail expression, restored the levels of E-cadherin and tight junction proteins, and prevented S. Typhimurium-increased paracellular permeability. Further study showed that Gli1 was cross-activated by the MAP and PI-3 kinase pathways. S. Typhimurium devoid of sopB, an effector of the Type 3 secretion system (T3SS) responsible for AKT activation, was unable to induce Snail expression and to decrease the expression of apical junction proteins. Our study uncovered a novel role of Gli1 in mediating the Salmonella-induced disruption of the intestinal epithelial barrier.

The m6A methyltransferase METTL3 aggravates the progression of nasopharyngeal carcinoma through inducing EMT by m6A-modified Snail mRNA

This study aims to elucidate the role of METTL3 in aggravating the progression of NPC through m6A modification on Snail and thus the stimulated epithelial-mesenchymal transition (EMT). Differential expressions of METTL3 in 48 paired NPC tissues and paracancerous tissues were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Its level in NPC patients with different clinical stages and metastatic states was examined. Prognostic potential of METTL3 in NPC patients was assessed by Kaplan-Meier method. After knockdown of METTL3, expression changes of Snail and EMT-related genes, as well as invasive and migratory abilities in SUNE-1 cells were detected. The interaction between Snail with METTL3 and IGF2BP2 was verified by RIP (RNA-Binding Protein Immunoprecipitation) assay. At last, the roles of METTL3/Snail regulatory loop in influencing EMT and metastasis of NPC were clarified. METTL3 was upregulated in NPC tissues than that of paracancerous ones. NPC patients with advanced stage or lymphatic metastasis expressed higher level of METTL3. Kaplan-Meier curves revealed that NPC patients expressing high level of METTL3 suffered worse prognosis. Knockdown of METTL3 downregulated protein levels of Snail and N-cadherin, while E-cadherin was upregulated in SUNE-1 cells. Meanwhile, knockdown of METTL3 inhibited invasive and migratory abilities in NPC cells. RIP assay confirmed the interaction between Snail and METTL3. Besides, knockdown of METTL3 decreased the enrichment abundance of Snail in anti-IGF2BP2. Overexpression of Snail partially reversed the regulatory effects of METTL3 on EMT-related gene expressions and metastatic abilities in NPC. METTL3 is upregulated in NPC, which regulates EMT and metastasis in NPC cells through m6A-modified Snail mRNA.

H1N1 Influenza Virus Cross-Activates Gli1 to Disrupt the Intercellular Junctions of Alveolar Epithelial Cells

Influenza A virus (IAV) primarily infects the airway and alveolar epithelial cells and disrupts the intercellular junctions, leading to increased paracellular permeability. Although this pathological change plays a critical role in lung tissue injury and secondary infection, the molecular mechanism of IAV-induced damage to the alveolar barrier remains obscure. Here, we report that Gli1, a transcription factor in the sonic hedgehog (Shh) signaling pathway, is cross-activated by the MAP and PI3 kinase pathways in H1N1 virus (PR8)-infected A549 cells and in the lungs of H1N1 virus-infected mice. Gli1 activation induces Snail expression, which downregulates the expression of intercellular junction proteins, including E-cadherin, ZO-1, and Occludin, and increases paracellular permeability. Inhibition of the Shh pathway restores the levels of Snail and intercellular junction proteins in H1N1-infected cells. Our study suggests that Gli1 activation plays an important role in disrupting the intercellular junctions and in promoting the pathogenesis of H1N1 virus infections.

Long Noncoding RNA NEAT1 Regulates TGF-β2-Induced Epithelial-Mesenchymal Transition of Lens Epithelial Cells through the miR-34a/Snail1 and miR-204/Zeb1 Pathways.

The aim of this study was to explore whether the long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1)/miR-34a/Snail1 and NEAT1/miR-204/Zeb1 pathways are involved in epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). Primary human LECs (HLECs) were separated and cultured. Our results identified that TGF-β2 induces NEAT1 overexpression in a dose-dependent manner and a time-dependent manner. Additionally, TGF-β2 induced downregulation of E-cadherin and upregulation of fibronectin in primary HLECs through a NEAT1-dependent mechanism. Microarray analysis showed that NEAT1 overexpression inhibited the miR-34a and miR-204 levels in the LECs. The expression of miR-34a and miR-204 was decreased, and the levels of Snail1 and Zeb1 were elevated in human posterior capsule opacification- (PCO-) attached LECs and the LECs obtained from anterior subcapsular cataract (ASC) by quantitative RT-PCR (qRT-PCR). Mechanistic studies revealed that NEAT1 negatively regulates miR-34a or miR-204, and miR-34a or miR-204 directly targets Snail1 or Zeb1 by luciferase assay and RNA-binding protein immunoprecipitation assay, respectively. Overall, the NEAT1/miR-34a/Snail1 and NEAT1/miR-204/Zeb1 pathways are involved in TGF-β2-induced EMT of HLECs. In summary, TGF-β2 induces NEAT1 overexpression, which in turn suggests that NEAT1 acts as a ceRNA targeting Snail1 or Zeb1 by binding miR-34a or miR-204, and promotes the progression of EMT of LECs.