Epithelial-mesenchymal Transition Protein Expression Research Articles

Transforming growth factor-beta and Epithelial-mesenchymal transition is considered an attractive pivotal Genetic-Biomarker for Hepatocellular carcinoma with hepatitis C virus-infection patients

Abstract Introduction/Objective Hepatocellular carcinoma (HCC) is the most common cause of cancer worldwide. HCC is induced by hepatitis C-virus (HCV) which represents a major health problem in Egypt. The transforming growth factor- beta (TGF-β) is a potent cytokine that has a multifunctional pro-fibrotic activity, for enhancing liver-inflammation, fibrosis, tumor-progression, and metastasis. TGF-β has a vital role in the regulation of tumorigenesis, by controlling epithelial-mesenchymal-transition (EMT), apoptosis, proliferation, and differentiation process. The dysregulation of TGF-β, Wnt/β-catenin signaling pathways, by HCV-core protein is implicated in the development of HCC. Methods/Case Report The study aims to determine the effect of the HCV-infection in HCC patients with a set of growth-factors, EMT, and anti-apoptotic protein expression levels by using western blotting and immunohistochemistry staining analysis. Results (if a Case Study enter NA) These results observed an insignificant increase in the expression of TGF-β protein for HCC/HCV-infected cells compared to the HCV-uninfected cells, and the expression of the β-catenin protein level is elevated in the nucleus. Also, it showed that Bcl-2 protein is a significant increase in HCV-infected when compared to HCV-free groups. Positive staining for p53 protein was detected in the HCC/HCV-infected, more than in HCC/HCV-uninfected cells. This data indicated that the core-protein of HCV can switch TGF-β from a tumor suppressor to a tumor promoter by suppressing apoptosis of hepatocytes and increasing EMT expression by activation of Wnt/β-catenin signaling pathway. Therefore, HCV-core protein enhances the nuclear accumulation of β-catenin expression protein to activate downstream target genes, which regulate cell-growth and cell-cycle progression. Conclusion It is speculated that HCV may exert an anti-apoptotic effect, and thus enhance tumorigenesis. Also, the accumulation of nuclear Wnt/β-catenin and p53 correlates with TGF-β expression, typically a late event in hepato- carcinogenesis. Taken together, the expression level of TGF-β, Wnt/β-catenin, Bcl-2, and p53 genotype in HCV is considered an indicator of shortened survival in patients, and may be an attractive clinically pivotal genetic biomarker to mediate liver pathogenesis.

P4HB regulates the TGFβ/SMAD3 signaling pathway through PRMT1 to participate in high glucose-induced epithelial-mesenchymal transition and fibrosis of renal tubular epithelial cells

BackgroundDiabetic nephropathy (DN) is a common complication of diabetes mellitus, and Prolyl 4-Hydroxylase Subunit Beta (P4HB) expression is increased in high glucose (HG)-induced renal tubular epithelial cells (TECs). But it’s role in HG-induced TECs remains to be elucidated.MethodsThe HK-2 cells were induced using HG and transfected with SiRNA-P4HB. DCFH-DA staining was utilized for the detection of cellular levels of ROS. WB and immunofluorescence were utilized to detect the expression of P4HB, epithelial-mesenchymal transition (EMT), fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells. Online databases were utilized for predicting the interaction target of P4HB, and immunoprecipitation (IP) experiments were employed to validate the binding of P4HB with the target. SiRNA and overexpression vectors of target gene were used to verify the mechanism of action of P4HB.ResultsHG induced an increase in the expression of P4HB and TGFβ, p-SMAD3, and ROS in HK-2 cells. Furthermore, HG downregulated the expression of E-cadherin and upregulated the expression of N-cadherin, Vimentin, α-SMA, Fibronectin, Collagen IV, SNAIL, and SLUG in HK-2 cells. Interfering with P4HB significantly reversed the expression of these proteins. Database predictions and IP experiments showed that P4HB interacts with PRMT1, and the expression of PRMT1 was increased in HG-induced HK-2 cells. Interfering with PRMT1 inhibited the changes in expression of EMT and fibrosis related proteins induced by HG. However, overexpression of PRMT1 weakened the regulatory effect of P4HB interference on the EMT, fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells.ConclusionP4HB regulated the TGFβ/SMAD3 signaling pathway through PRMT1 and thus participates in HG-induced EMT and fibrosis in HK-2 cells.

Exosomes derived from gastric cancer cells promote phenotypic transformation of hepatic stellate cells and affect the malignant behavior of gastric cancer cells.

This study aimed to evaluate the effect of exosomes derived from gastric cancer cells on the phenotypic transformation of hepatic stellate cells (HSCs) and the effect of HSC activation on the malignant behavior of gastric cancer cells, including its molecular mechanism. Exosomes derived from the human gastric adenocarcinoma cell line AGS were extracted and purified by polymer precipitation and ultrafiltration, respectively. The exosomes' morphologic characteristics were observed using transmission electron microscopy, particle size was determined through nanoparticle-tracking analysis, and marker proteins were detected using western blotting. Exosome uptake by LX-2 HSCs was observed through fluorescence-based tracing. Reverse transcription quantitative PCR (RT-qPCR) was used to detect the messenger RNA (mRNA) expression of alpha-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP). Using functional assays, the effects of LX-2 HSC activation on the biological behavior of malignant gastric cancer cells were evaluated. The effects of LX-2 HSC activation on the protein expression of epithelial-mesenchymal transition (EMT)-related genes and β-catenin were evaluated via western blotting. The extracted particles conformed to the definitions of exosomes and were thus considered gastric cancer cell-derived exosomes. Fluorescence-based tracing successfully demonstrated that exosomes were enriched in LX-2 HSCs. RT-qPCR revealed that the mRNA expression of the cancer-associated fibroblast markers α-SMA and FAP was significantly increased. LX-2 HSC activation considerably enhanced gastric cancer cell proliferation, invasion, and migration. Western blotting showed that the expression of the EMT-related epithelial marker E-cadherin was significantly downregulated, whereas the expression of interstitial markers (N-cadherin and vimentin) and β-catenin was remarkably upregulated in gastric cancer cells. Exosomes derived from gastric cancer cells promoted phenotypic transformation of HSCs and activated HSCs to become tumor-associated fibroblasts. Gastric cancer cell-derived cells significantly enhanced gastric cancer cell proliferation, invasion, and migration after HSC activation, which may promote EMT of gastric cancer cells through the Wnt/β-catenin pathway.

Antiplatelet drug ticagrelor suppresses triple negative breast cancer metastasis by targeting PI3K

Metastatic recurrence is still a major challenge in breast cancer treatment. Patients with triple negative breast cancer (TNBC) develop early recurrence and relapse more frequently. Due to the lack of specific therapeutic targets, new targeted therapies for TNBC are urgently needed. Phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway is one of the active pathways involved in chemoresistance and survival of TNBC, being considered as a potential target for TNBC treatment. Our present study identified ticagrelor, an anti-platelet drug, as a pan-PI3K inhibitor with potent inhibitory activity against four isoforms of class I PI3K. At doses normally used in clinic, ticagrelor showed weak cytotoxicity against a panel of breast cancer cells, but significantly inhibited the migration, invasion and the actin cytoskeleton organization of human TNBC MDA-MB-231 and SUM-159PT cells. Mechanistically, ticagrelor effectively inhibited PI3K downstream mTOR complex 1 (mTORC1) and mTORC2 signaling by targeting PI3K and decreased the protein expression of epithelial-mesenchymal transition (EMT) markers. In vivo, ticagrelor significantly suppressed tumor cells lung metastasis in 4T1 tumor bearing BALB/c mice model and experimental lung metastasis model which was established by tail vein injection of GFP-labeled MDA-MB-231 cells. The above data demonstrated that ticagrelor can inhibit the migration and invasion of TNBC both in vitro and in vivo by targeting PI3K, suggesting that ticagrelor, a pan-PI3K inhibitor, might represent a promising therapeutic agent for the treatment of metastatic TNBC.

GDF-15 Attenuates the Epithelium-Mesenchymal Transition and Alleviates TGFβ2-Induced Lens Opacity.

We sought to evaluate the efficacy of growth differentiation factor (GDF)-15 treatment for suppressing epithelial-mesenchymal transition (EMT) and alleviating transforming growth factor β2 (TGFβ2)-induced lens opacity. To test whether GDF-15 is a molecule that prevents EMT, we pretreated the culture with GDF-15 in neural progenitor cells, retinal pigment epithelial cells, and lens epithelial cells and then treated with factors that promote EMT, GDF-11, and TGFβ2, respectively. To further investigate the efficacy of GDF-15 on alleviating lens opacity, we used mouse lens explant culture to mimic secondary cataracts. We pretreated the lens culture with GDF-15 and then added TGFβ2 to develop lens opacity (n = 3 for each group). Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to measure EMT protein and gene expression, respectively. In cell culture, GDF-15 pretreatment significantly attenuated EMT marker expression in cultured cells induced by treatment with GDF-11 or TGFβ2. In the lens explant culture, GDF-15 pretreatment also reduced mouse lens opacity induced by exposure to TGFβ2. Our results indicate that GDF-15 could alleviate TGFβ2-induced EMT and is a potential therapeutic agent to slow or prevent posterior capsular opacification (PCO) progression after cataract surgery. Cataracts are the leading cause of blindness worldwide, with the only current treatment involving surgical removal of the lens and replacement with an artificial lens. However, PCO, also known as secondary cataract, is a common complication after cataract surgery. The development of an adjuvant that slows the progression of PCO will be beneficial to the field of anterior complications.

Lipopolysaccharide Stimulates A549 Cell Migration through p-Tyr 42 RhoA and Phospholipase D1 Activity.

Cell migration is a crucial contributor to metastasis, a critical process associated with the mortality of cancer patients. The initiation of metastasis is triggered by epithelial-mesenchymal transition (EMT), along with the changes in the expression of EMT marker proteins. Inflammation plays a significant role in carcinogenesis and metastasis. Lipopolysaccharide (LPS), a typical inflammatory agent, promoted the generation of superoxide through the activation of p-Tyr42 RhoA, Rho-dependent kinase 2 (ROCK2), and the phosphorylation of p47phox. In addition, p-Tyr42 RhoA activated phospholipase D1 (PLD1), with PLD1 and phosphatidic acid (PA) being involved in superoxide production. PA also regulated the expression of EMT proteins. Consequently, we have identified MHY9 (Myosin IIA, NMIIA) as a PA-binding protein in response to LPS. MYH9 also contributed to cell migration and the alteration in the expression of EMT marker proteins. Co-immunoprecipitation revealed the formation of a complex involving p-Tyr42 RhoA, PLD1, and MYH9. These proteins were found to be distributed in both the cytosol and nucleus. In addition, we have found that p-Tyr42 RhoA PLD1 and MYH9 associate with the ZEB1 promoter. The suppression of ZEB1 mRNA levels was achieved through the knockdown of RhoA, PLD1, and MYH9 using si-RNAs. Taken together, we propose that p-Tyr42 RhoA and PLD1, responsible for producing PA, and PA-bound MYH9 are involved in the regulation of ZEB1 expression, thereby promoting cell migration.

NQO1 alleviates renal fibrosis by inhibiting the TLR4/NF-κB and TGF-β/Smad signaling pathways in diabetic nephropathy

ObjectiveDiabetic nephropathy (DN) is one of the main complications of diabetes, and inflammation and fibrosis play an important role in its progression. NAD(P)H: quinone oxidoreductase 1 (NQO1) protects cells from oxidative stress and damage caused by toxic quinones. In the present study, we aimed to investigate the protective effects of NQO1 against diabetes-induced renal inflammation and fibrosis and the underlying mechanisms. MethodsIn vivo, the kidneys of type 2 diabetes model db/db mice were infected with adeno-associated virus vectors to induce NQO1 overexpression. In vitro, human renal tubular epithelial (HK-2) cells transfected with NQO1 pcDNA3.1(+) were cultured under high-glucose (HG) conditions. Gene and protein expression was assessed by quantitative real-time PCR, Western blotting, immunofluorescence, and immunohistochemical staining. Mitochondrial reactive oxygen species (ROS) were detected with MitoSOX Red. ResultOur study revealed that the expression of NQO1 was markedly downregulated and that Toll-like receptor (TLR)4 and TGF-β1 expression was upregulated in vivo and in vitro under diabetic conditions. Overexpression of NQO1 suppressed proinflammatory cytokine (IL-6, TNF-α, MCP-1) secretion, extracellular matrix (ECM) (collagen IV, fibronectin) accumulation and epithelial-mesenchymal transition (EMT) (α-SMA, E-cadherin) in the db/db mouse kidneys and HG-cultured HK-2 cells. Furthermore, NQO1 overexpression ameliorated HG-induced TLR4/NF-κB and TGF-β/Smad pathways activation. Mechanistic studies demonstrated that a TLR4 inhibitor (TAK-242) suppressed the TLR4/NF-κB signaling pathway, proinflammatory cytokine secretion, EMT and ECM-related protein expression in HG-exposed HK-2 cells. In addition, we found that the antioxidants N-acetylcysteine (NAC) and tempol increased the expression of NQO1 and decreased the expression of TLR4, TGF-β1, Nox1, and Nox4 and ROS production in HK-2 cells cultured under HG conditions. ConclusionsThese data suggest that NQO1 alleviates diabetes-induced renal inflammation and fibrosis by regulating the TLR4/NF-κB and TGF-β/Smad signaling pathways.

Aspartame consumption during pregnancy impairs placenta growth in mice through sweet taste receptor-reactive oxygen species-dependent pathway

The prevalence of obesity has risen dramatically over recent years, and so has the prevalence of adverse obesity-associated pregnancy outcomes. To combat obesity, the calorie contents of many foods and beverages may be reduced by the use of artificial sweeteners, such as aspartame. However, animal studies suggest that aspartame and its metabolites may exhibit toxicity, and the effects of aspartame on pregnancy are largely unknown. In this study, we treated pregnant mice with aspartame by oral gavage and found that the treatment decreased fasting blood glucose level, whereas systolic blood pressure was elevated. Importantly, the aspartame-treated animals also had low placenta and fetus weights, as well as reduced thickness of the placenta decidua layer. Moreover, aspartame decreased the expression of epithelial-mesenchymal transition proteins and manganese superoxide dismutase (MnSOD) in mouse placentae. In order to clarify the mechanisms though which aspartame affects placenta, we performed experiments on 3A-sub-E trophoblasts. In the cells, aspartame treatments induced cell cycle arrest and reduced the proliferation rate, epithelial-mesenchymal transition, migration activity and invasion activity. We also found that aspartame increased reactive oxygen species (ROS) levels to hyper-activate Akt and downregulate MnSOD expression. Pretreatment with antioxidants or sweet taste receptor inhibitors reversed the effects of aspartame on trophoblast function. We also found that the aspartame metabolite phenylalanine similarly induced ROS production and affected proliferation of trophoblasts. Taken together, our data suggest that aspartame consumption during pregnancy may impact the structure, growth and function of the placenta via sweet taste receptor-mediated stimulation of oxidative stress.

Caulerpa racemosa extract inhibits HeLa cancer cells migration by altering expression of epithelial-mesenchymal transition proteins.

Introduction: Cervical cancer is caused by persistent infections of human papillomavirus types 16 and 18. Also, it is classified as a malignancy since it is able to spread itself to other sites and form a metastasis. Lymph nodes metastasis is an important factor related to cervical cancer survival. The previous study reported that Caulerpa racemosa has an anti-cancer effect by inducing apoptosis by inhibiting p53 protein degradation in HeLa cancer cells. In this study, we conducted a follow-up test to determine the anticancer effect of Caulerpa racemosa as an antimetastatic agent on HeLa cancer cells. Methods: A true experimental study with a post-test-controlled group design was carried out on four groups of HeLa cell cultures by presenting different concentrations of Caulerpa racemosa extract. Moreover, to identify the antimetastatic effect, HeLa cells treated with Caulerpa racemosa extract were subjected to the woud healing scratch test and immunofluorescence staining assays. Data analysis was gained with qualitative and quantitative approaches. Quantitative methods such as One-way analysis of variance, Tukey's multiple comparison test, and Pearson's correlation were conducted. Result: We found that Caulerpa racemosa significantly inhibit HeLa cells wound healing migration. We also demonstrated the effect of Caulerpa racemosa in downregulating Snail and Vimentin protein expression and upregulating E-Cadherin protein expression. Conclusion: Caulerpa racemosa extract inhibits HeLa cancer cells migration by altering important regulator proteins expressions of epithelial-mesenchymal transition pathways.

Matrine induces hepatocellular carcinoma apoptosis and represses EMT and stemness through microRNA-299-3p/PGAM1 axis

This study explored the impacts of matrine on hepatocellular carcinoma (HCC) cell growth, metastasis, epithelial-mesenchymal transition (EMT), and stemness through regulating the microRNA (miR)-299-3p/phosphoglycerate mutase 1 (PGAM1) axis. The association between miR-299-3p expression with the prognosis of HCC patients was studied. miR-299-3p and PGAM1 sequences were transfected into matrine-treated HCC cells, and cell proliferation, invasion, apoptosis, and stemness were detected, as well as protein expression of EMT- and stemness-related makers. The targeting relationship between miR-299-3p and PGAM1 was identified. Matrine elevated miR-299-3p expression, repressed proliferation, invasion, and anti-apoptosis of HCC cells, and constrained EMT and stemness in vitro. PGAM1 was a target of miR-299-3p. Repression of PGAM1 rescued the effects of miR-299-3p downregulation on HCC cells. Matrine stimulates HCC cell apoptosis and represses the process of EMT and stemness through the miR-299-3p/PGAM1 axis.

Calycosin Inhibits the Malignant Behaviors of Lung Adenocarcinoma Cells by Regulating the circ_0001946/miR-21/GPD1L/HIF-1α Signaling Axis.

Objective To clarify the potential function and molecular mechanism of calycosin in lung adenocarcinoma (LUAD) cells. Methods LUAD cells (A549 and H1299) were treated with calycosin at different concentrations (25 nM, 50 nM, and 100 nM) for 24 h. The colony formation, invasion, and migration of the cells were assessed by colony formation, transwell, and scratch assays, respectively. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the mRNA expression level of circ_0001946, miR-21, glycerol-3-phosphate dehydrogenase 1 like (GPD1L), and hypoxia-inducible factor-1α (HIF-1α) in clinical tissue samples and LUAD cells. RNA pull-down assay and dual-luciferase reporter assay were performed to verify the relationship among circ_0001946, miR-21, GPD1L, and HIF-1α. Western blot was performed to detect the protein expression of epithelial-mesenchymal transition (EMT) process-related genes (E-cadherin, N-cadherin, and snail) and GPD1L as well as HIF-1α. Results Calycosin inhibited colony formation, invasion, migration, and EMT progression in A549 and H1299 cells. Besides, calycosin was able to regulate the expression of circ_0001946, miR-21, GPD1L, and HIF-1α in LUAD cells. According to the findings of QRT-PCR, the expression level of circ_0001946 and GPD1L in LUAD tissues was significantly lower than that in adjacent noncancerous normal tissues, and the expression of miR-21 and HIF-1α was also significantly increased in clinical tissue samples. In addition, there was a targeted regulatory relationship among the above four expressions. Knockdown of circ_0001946 expression in A549 cells treated with calycosin enhanced the malignant behavior of A549 cells and inhibited the anticancer effect of calycosin. However, the knockdown of miR-21 promoted the anticancer effect of calycosin and inhibited the malignant behavior of A549. Conclusion Calycosin can inhibit colony formation, invasion, migration, and EMT process of LUAD cells via regulating the circ_0001946/miR-21/GPD1L/HIF-1α signaling axis and could be a promising therapeutic drug for LUAD.

Circ-GGA3 promotes the biological functions of human lens epithelial cells depending on the regulation of miR-497-5p/SMAD4 axis

The cause of posterior capsular opacification (PCO) is the dysfunction of lens epithelial cells (LECs). Circular RNA (circRNA) was found to regulate cell biological functions, including LECs. However, the role of circ-GGA3 in PCO formation is unclear. Quantitative real-time PCR was used to measure the expression of circ-GGA3, miR-497-5p and SMAD4. Cell proliferation, invasion and migration were determined via MTT assay, EdU staining, transwell assay and wound healing assay. The protein expression of epithelial-mesenchymal transition (EMT) markers, fibrosis markers, TGF-β/SMAD pathway markers and SMAD4 were determined by western blot assay. The interaction between miR-497-5p and circ-GGA3 or SMAD4 was confirmed using dual-luciferase reporter assay. Circ-GGA3 was highly expressed in PCO patients, and its silencing inhibited the proliferation, invasion, migration, EMT process and fibrosis of TGF-β2-induced LECs. Circ-GGA3 could sponge miR-497-5p to regulate SMAD4. Further experiments revealed that miR-497-5p inhibitor recovered the negative regulation of circ-GGA3 knockdown on the biological functions of TGF-β2-induced LECs, and SMAD4 overexpression also abolished the suppressive effect of miR-497-5p. In addition, circ-GGA3/miR-497-5p/SMAD4 axis could activate the TGF-β/SMAD pathway. Our results indicated that circ-GGA3 could enhance the biological functions of LECs, suggesting that circ-GGA3 might be a potential target for PCO therapy.

LINC00511 knockdown suppresses glioma cell malignant progression through miR-15a-5p/AEBP1 axis

BackgroundA strong relationship between long intergenic non-protein coding RNA 511 (LINC00511) and glioma has been previously reported but the mechanism of LINC00511 in glioma is yet to be determined. This study examined the mechanism of LINC00511 in glioma. MethodsThe expression of LINC00511 in glioma was determined by bioinformatics analysis and real-time quantitative PCR (RT-qPCR) analysis. The target relationship between genes was predicted by starBase, TargetScan, and was verified by dual-luciferase. Subsequently, siRNA targeting LINC00511 (siLINC00511) and miR-15a-5p mimic were transfected into glioma cells to examine the effect on biological characteristics using cell counting kit-8, clone formation, flow cytometry, wound-healing, and transwell. MiR-15a-5p inhibitor and AEBP1 were used for in vitro rescue experiments, and tumorigenesis assay and immunohistochemical assays were performed for in vivo experiments. Epithelial-mesenchymal transition (EMT) and p65 phosphorylation were examined by Western blot. ResultsLINC00511 was predicted and verified to be up-regulated in glioma. SiLINC00511 suppressed cell viability, proliferation, migration and invasion, accelerated apoptosis of glioma cells. Mechanically, siLINC00511 promoted E-cadherin expression but suppressed N-cadherin and Snail expressions. MiR-15a-5p bound to LINC00511, and miR-15a-5p inhibitor partially reversed the effect and regulation of siLINC00511 on glioma cells. AEBP1, a target gene of miR-15a-5p, could activate p65 phosphorylation to promote EMT protein expression and partially reverse the inhibitory effect of miR-15a-5p mimic on the malignant phenotype of glioma cells. SiLINC00511 inhibited tumor growth, down-regulated miR-15a-5p expression and up-regulated AEBP1 and Ki67 expressions in vivo. ConclusionLINC00511 knockdown inhibits glioma cell progression via miR-15a-5p/AEBP1 axis.

Long non-coding DANCR targets miR-185-5p to upregulate LIM and SH3 protein 1 promoting prostate cancer via the FAK/PI3K/AKT/GSK3β/snail pathway.

Long non-coding RNA differentiation antagonizing non-protein coding RNA (DANCR) acts as an oncogene in different cancers, although its roles in prostate cancer are not fully reported. We aimed to explore its mechanism in facilitating the malignancy of prostate cancer. The expression of DANCR, microRNA (miR)-185-5p and LIM and SH3 protein 1 (LASP1) in 40 pairs of prostate cancer tissues and normal tissues, five prostate cancer cell lines and one epithelial cell line was assessed by a quantitative real-time polymerase chain reaction, western blotting and immunohistochemistry, respectively. In transfected PC3 and C4-2 cells, cell proliferation, migration, invasion, cell cycle distribution and epithelial-mesenchymal transition (EMT) protein expression were tested via cell counting kit-8, wound healing, transwell, flow cytometry and western blot assays, respectively. The interactions between DANCR, miR-185-5p and LASP1 were verified by a dual-luciferase reporter assay. Rescue experiments were conducted to determine the roles of DANCR on the malignant properties of PC3 and C4-2 cells. The involvement of the signaling pathway was examined using a p-FAK inhibitor. DANCR and LASP1 expression was enhanced, whereas miR-185-5p expression was diminished in prostate cancer tissues and cell lines. Knockdown of DANCR suppressed cell proliferation, migration, invasion, G1-S transition and expression of EMT proteins of the transfected PC3 and C4-2 cells. DANCR sponged miR-185-5p to upregulate LASP1 expression. DANCR-miR-185-5p-LASP1 axis activates the FAK/PI3K/AKT/GSK3β/Snail pathway to promote the malignant properties of PC3 and C4-2 cells. These findings suggest that DANCR exerts oncogenic roles in prostate cancer via the miR-185-5p/LASP1 axis activating the FAK/PI3K/AKT/GSK3β/Snail pathway. It can be a potential biomarker in the diagnosis and monitoring of prostate cancer.

DHEA inhibits proliferation, migration and alters mesenchymal-epithelial transition proteins through the PI3K/Akt pathway in MDA-MB-231 cells

Cancer is one of the leading causes of death worldwide, and breast cancer is the most common among women. Dehydroepiandrosterone (DHEA), the most abundant steroid hormone in human serum, inhibits proliferation and migration of breast cancer cells, modulating the expression of proteins involved in mesenchymal-epithelial transition (MET). However, the underlying molecular mechanisms are not fully understood. DHEA effects on the triple-negative breast cancer cell line MDA-MB-231 (mesenchymal stem-like) could be exerted by binding to receptors tyrosine kinase (RTKs) and signaling through MEK/ERK and/or PI3K/Akt pathways. In this study, MDA-MB-231 cells were exposed to DHEA in the presence of pharmacological inhibitors of these pathways and a siRNA against PIK3CA gene, which blocks PI3K pathway. Cell proliferation was measured by crystal violet staining, migration by the wound healing and transwell assays, and MET protein expression by western blot. A xenograft tumor growth in nude mice (nu−/nu−) using a siRNA against PI3K was also performed.Results showed that neither of the inhibitors used reverted the antiproliferative activity of DHEA. However, wortmannin and LY294002, inhibitors of the PI3K/Akt pathway, abolished the up- and down-regulation of E- and N-cadherin expression respectively, and inhibition of migration induced by DHEA in MDA-MB-231 cells. The siRNA that blocks the PI3K pathway, abolished the effects of DHEA on proliferation, migration, MET proteins expression and the growth of tumors in nude mice.In conclusion, these results suggest that PI3K/Akt pathway participates in the effects of DHEA on breast cancer cells.

Inhibition of Wilms' Tumor Proliferation and Invasion by Blocking TGF-β Receptor I in the TGF-β/Smad Signaling Pathway.

Wilms' tumor (WT) is a common embryonal tumor, and nephrogenic rests play a critical role in WT development. The transforming growth factor β (TGF-β) signaling pathway is fundamental to embryo development and cell growth and proliferation. Moreover, TGF-β contributes to WT development, but the mechanisms of disease pathogenicity are unknown. This study investigated whether the TGF-β signaling pathway was involved in WT and whether blocking TβRI receptor inhibited WT growth, proliferation, and invasion. A total of 60 WT patients with clinical data and surgical specimens were evaluated. Immunohistochemistry (IHC) was used to detect the expression of TGF-β1 and P-smad2/3. In vitro, the proliferation, migration, apoptosis, and epithelial-mesenchymal transition (EMT) protein expression were analyzed using the CCK8 assay, wound healing assay, transwell assay, flow cytometry, and western blot, respectively. In vivo, tumor morphology, tumor size, toxicity, and EMT protein expression were analyzed in tumor-bearing mice treated with a TβRI kinase inhibitor or PBS. High protein levels of TGF-β1 and P-samd2/3 were associated with clinical stage and metastasis or invasion. TβRI inhibition effectively suppressed WT proliferation and migration and promoted apoptosis in the human WT cell line G401, consequently decreasing EMT protein expression. In addition, the TβRI kinase inhibitor significantly impaired the subcutaneous growth of WT. It is worth noting that treatment with the TβRI kinase inhibitor did not cause liver and kidney injury. Our results indicate that the TGF-β/Smad signaling pathway plays a crucial role in WT progression. Blocking the TβRI receptor may be a novel strategy to treat and prevent WT.

MiR-21-5p promotes lung adenocarcinoma cell proliferation, migration and invasion via targeting WWC2.

Studies have suggested that miR-21-5p and WWC2 are key players in most cancer types, yet the underlying mechanisms in lung adenocarcinoma (LUAD) remain elusive. This study made in-depth research on the two factors-dependent mechanisms underlying LUAD occurrence and development. Bioinformatics methods were employed to identify the miRNA and its target gene of interest. In all, 20 pairs of LUAD tumor tissue samples and matched adjacent normal samples along with 5 LUAD cell lines were collected for evaluating the aberrant expression of miR-21-5p and WWC2. Dual-luciferase reporter assay was performed to validate the targeted relationship between miR-21-5p and WWC2. A series of in vitro experiments including colony formation assay, EdU, wound healing assay and Transwell were conducted for assessment of the LUAD cell biological behaviors. In addition, Western blot was carried out to determine the protein expression of epithelial-mesenchymal transition (EMT)-related proteins. miR-21-5p was found to be considerably increased in LUAD tissue and cells relative to that in the adjacent tissue and the human bronchial epithelial cells, whereas WWC2 was significantly decreased. Dual-luciferase reporter assay revealed that miR-21-5p targeted WWC2 and down-regulated its expression. Besides, silencing miR-21-5p or overexpressing WWC2 played an inhibitory role in PC-9 cancer cell proliferation, migration and invasion, but such effect was suppressed when miR-21-5p was overexpressed. Furthermore, Western blot uncovered that WWC2 overexpression impeded the EMT process in LUAD cells. miR-21-5p facilitates LUAD cell proliferation, migration and invasion through targeting WWC2, which provides a novel therapeutic target for LUAD treatment.

Ethyl pyruvate inhibits glioblastoma cells migration and invasion through modulation of NF-κB and ERK-mediated EMT.

BackgroundGlioblastoma is a grade IV glioma with the highest degree of malignancy and extremely high incidence. Because of the poor therapeutic effect of surgery and radiochemotherapy, glioblastoma has a high recurrence rate and lethality, and is one of the most challenging tumors in the field of oncology. Ethyl pyruvate (EP), a stable lipophilic pyruvic acid derivative, has anti-inflammatory, antioxidant, immunomodulatory and other cellular protective effects. It has been reported that EP has potent anti-tumor effects on many types of tumors, including pancreatic cancer, prostate cancer, liver cancer, gastric cancer. However, whether EP has anti-tumor effect on glioblastoma or not is still unclear.MethodsGlioblastoma U87 and U251 cells were treated with different concentrations of EP for 24 h or 48 h. CCK8 assay and Colony-Formation assay were performed to test the viability and proliferation. Wound-healing assay and Transwell assay were carried out to measure cell invasion and migration. Western blot was not only used to detect the protein expression of epithelial-mesenchymal transition (EMT)-related molecules, but also to detect the expression and activation levels of NF-κB (p65) and Extracellular Signal Regulated Kinase (ERK).ResultsIn glioblastoma U87 and U251 cells treated with EP, the viability, proliferation, migration, invasion abilities were inhibited in a dose-dependent manner. EP inhibited EMT and the activation of NF-κB (p65) and ERK. With NF-κB (p65) and ERK activated, EMT, migration and invasion of U87 and U251 cells were promoted. However the activation of NF-κB (p65) and ERK were decreased, EMT, migration and invasion abilities were inhibited in U87 and U251 cells treated with EP.ConclusionEP inhibits glioblastoma cells migration and invasion by blocking NF-κB and ERK-mediated EMT.

HOTAIR induces EGFR-TKIs resistance in non-small cell lung cancer through epithelial-mesenchymal transition

ObjectivePrevious research found that HOTAIR, a long non-coding RNA, is aberrantly expressed and associated with tumor invasion, metastasis and chemo-resistance in many cancers. The aim of this study was to investigate the role of HOTAIR in resistance of EGFR-TKIs in NSCLC. MethodsHOTAIR expression level was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in NSCLC cell lines or tumor tissues. A total of 62 samples with EGFR-mutant and EGFR-TKI-sensitive NSCLCs, 42 with acquired resistance and 27 with primary resistance to EGFR-TKIs were analyzed. The effect of HOTAIR on cell proliferation and apoptosis was undergone by CCK-8 and flow cytometry assays. The expression of EMT proteins was assessed by western blot. ResultsHOTAIR was significantly down-regulated in lung cancer cells (PC9/R, H1975, H1299 and A549) and patients with primary and acquired resistance to EGFR-TKIs. In clinical setting, high levels of HOTAIR expression was significantly correlated with longer progression-free survival (PFS) [P < 0.01] compared with low HOTAIR expression subgroup in tumors which respond to EGFR-TKIs. In vitro, over-expression HOTAIR could restore gefitinib sensitivity in gefitinib-resistant cells (PC9/R, H1299 and A549), but this change in sensitivity was not observed in H1975. Up-regulated HOTAIR induced cell apoptosis in PC9/R, H1299 and A549, and activated epithelial-mesenchymal transition (EMT). ConclusionsHOTAIR expression was associated with primary and acquired resistance to EGFR-TKIs and could regulate cell proliferation through activating cell apoptosis and EMT, which suggest that HOTAIR might be able to act as a biomarker to predict the EGFR-TKIs resistance.

USP5 Promotes Metastasis in Non-Small Cell Lung Cancer by Inducing Epithelial-Mesenchymal Transition via Wnt/β-Catenin Pathway.

Ubiquitin-specific protease 5 (USP5) is a deubiquitinating enzyme that functions as an oncoprotein in a variety of human cancers. However, the expression and role of USP5 in the metastasis of non-small cell lung cancer (NSCLC) have not been addressed. In this study, we examined the expression and prognostic significance of USP5 in NSCLC. The results revealed that USP5 was overexpressed and correlated with metastasis and overall survival in NSCLC tissues. A further in vitro study revealed that the levels of USP5 protein in NSCLC cells were associated with epithelial–mesenchymal transition (EMT) markers. Furthermore, USP5 overexpression significantly enhanced, whereas USP5 silencing significantly decreased the expression of EMT proteins and migration and invasion of NSCLC cells. In addition, the results from western blotting demonstrated that USP5 regulated EMT via the Wnt/β-catenin signaling pathway. Further immunohistochemical analysis revealed that USP5 was significantly associated with the expression of β-catenin and EMT markers in NSCLC tissues. Overall, USP5 upregulation is associated with tumor metastasis and poor prognosis in patients with NSCLC. USP5 promotes EMT and the invasion and migration of NSCLC cells. Therefore, USP5 may serve as a novel prognostic biomarker and provide a potential target for the treatment of metastasis in NSCLC.