Snail Protein Expression Research Articles

KLF5 silence attenuates proliferation and epithelial-mesenchymal transition induction in Hep-2 cells through NF-κB signaling pathway.

This study aimed at exploring the role and mechanism of Krüppel-like factor 5 (KLF5) in the migration, invasion, epithelial-mesenchymal transition (EMT) induction and proliferation in laryngeal cancer human epithelial type 2 (Hep-2) cells, and to provide a new sight for the treatment of laryngeal carcinoma. Hep-2 cells were randomly divided into three groups: control group (Control), KLF5 siRNA group (siKLF5) and control-siRNA group (NC). The effects of KLF5 inhibition on cell proliferation and apoptosis were assessed by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometer, respectively. Wound healing assay and transwell invasion experiments were used to determine cell migration and invasion. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot were used to compare the levels of KLF5, EMT-related genes E-cadherin, N-cadherin, Vimentin and Zinc finger transcription factors (Snail, Slug) expressions. The levels of nuclear transcription factor-κB (NF-κB-p65) and IκBα were also detected by Western blot. Compared with the Control group, the proliferation rate of Hep-2 cells in the siKLF5 group was significantly decreased while the apoptosis rate was increased (p<0.05). Meanwhile, the migration and invasion ability of Hep-2 cells were markedly decreased (p<0.05). E-cadherin protein expression was up-regulated while Vimentin, N-cadherin, Snail, and Slug protein expression levels were downregulated in siKLF5 group (p<0.05). Silencing KLF5 could inhibit the expression of NF-κB phosphorylation at p65 and the IκBα degradation (p<0.05). These results revealed that silencing KLF5 expression reduced the proliferation, migration and invasion and EMT abilities by inhibiting the NF-κB pathway in Hep-2 cells. Our results suggest that KLF5 may be a potential therapeutic target in laryngeal carcinoma.

Study on the mechanism of interleukine-11 promoting anaplastic thyroid carcinoma cells invasion and metastasis

Objective To explore the mechanism of hypoxia inducing interleukine (IL)-11 secretion in anaplastic thyroid carcinoma (ATC) cells and the mechanism of IL-11 promoting epithelial mesenchymal transition of ATC cells. Methods Knockdown of IL-11, hypoxia-inducible factor (HIF)-1α in ATC cells or treatment of ATC cells with Cobalt Chloride, recombinant human interleukin (rhIL)-11, IL-11 antibody, then the effects of these interventions on the invasion and migration ability of ATC cells and its mechanism were detected by enzyme-linked immunosorbent assay (ELISA), Transwell, Real-time polymerase chain reaction (PCR), Western blotting. Results Under treatment with Cobalt Chloride, the protein and mRNA expression of IL-11 increased in ATC cells. The number of invasive cells in the knockdown HIF-1α group (61.7±4.9, 30.0±4.6) was less than that of the control group 261.7±8.7 (P<0.01), and the number of migrated cells (87.3±4.5, 73.8±5.4) was less than the control group 337.3±7.1 (P<0.01). The expression ofphosphorylated Akt (p-Akt), phosphorylated glycogen synthase kinase 3β (p-GSK3β) and Snail protein was significantly increased after treatment with rhIL-11. The protein expression of p-Akt, p-GSK3β and Snail were significantly decreased after knockdown of IL-11 expression in ATC cells. Conclusion Hypoxia induced epithelial mesenchymal transition of ATC cells is the main mechanism of high invasion and migration of ATC cells. Key words: Anaplastic thyroid carcinoma; Interleukine-11; Epithelialmesenchymal transition; Invasion and metastasis

LKB1 suppresses glioma cell invasion via NF-κB/Snail signaling repression.

BackgroundLiver kinase B1 (LKB1) is involved in various human diseases. Aberrant expression of LKB1 expression is involved in glioma progression and associated with prognosis, however, the specific mechanism involving NF-κB/Snail signaling pathways remain unknown.Materials and methodsIn the present study, quantitative real-time PCR analysis was used to investigate the expression of LKB1 tumor tissue samples and cell lines. In glioma cell lines, CCK-8 assay, transwell invasion and migration assays were used to investigate the effects of LKB1on proliferation and invasion.ResultsWe observed that LKB1 knockdown promoted glioma cell proliferation, migration and invasion. This effect was induced through NF-κB/Snail signaling activation. Also, LKB1 overexpression suppressed proliferation, migration, and invasion, which could be rescued by Snail overexpression.ConclusionTaken together, our results show that LKB1 knockdown promotes remarkably glioma cell proliferation, migration and invasion by regulating Snail protein expression through activating the NF-κB signaling. This may serve as a potential prognostic marker and therapeutic target for glioma.

Expression of lysine-specific demethylase 1 in hepatocellular carcinoma tissues and its effect on cell proliferation and invasion

Objective To investigate the expression of lysine-specific demethylase 1 (LSD1) in hepatocellular carcinoma tissues, and to explore the effects of down-regulation of LSD1 gene expression on proliferation and invasion of human hepatoma HepG2 cells. Methods A total of 91 cases of hepatocellular carcinoma undergoing elective surgery in our hospital and Henan Cancer Hospital were selected. Immunohistochemistry was used to detect the expression of LSD1 protein in hepatocellular carcinoma tissues and adjacent tissues. Human hepatoma HepG2 cells were cultured and divided into LSD1 interference sequence group, negative control group and blank group. Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of LSD1 gene in cells. Methyl thiazol tetrazolium (MTT) assay was used to detect cell proliferation. Transwell chamber was used to detect cell invasion. Western blotting was used to detect the expression of LSD1, Snail, E-cadherin and N-cadherin proteins in cells. Results The positive expression rate of LSD1 protein in hepatocellular carcinoma tissues was 81.32%, which was significantly higher than that in adjacent tissues (35.16%, P<0.01). The expression of LSD1 protein in hepatocellular carcinoma tissues was related to the degree of differentiation, TNM staging and vascular invasion (P<0.05). The relative expression level of LSD1 mRNA in LSD1 interference sequence group was 1.19±0.10, which was significantly lower than that in the negative control group and blank group (2.29±0.14 and 2.34±0.11, P<0.05). The absorbance (A) values at 24, 48, 72 and 96 h in the LSD1 interference sequence group were 0.23±0.05, 0.32±0.08, 0.38±0.08 and 0.52±0.03, respectively, which were significantly lower than those in the negative control group (0.38±0.08, 0.49±0.05, 0.67±0.04 and 0.77±0.10) and blank group (0.33±0.03, 0.52±0.09, 0.70±0.08 and 0.78±0.07, P<0.05). The number of invasive cells in the LSD1 interference sequence group was (86.3±3.9), which was significantly less than that in the negative control group (110.6±9.4) and the blank group (117.7±7.0, P<0.05). The relative expression levels of LSD1, Snail, and N-cadherin proteins in the LSD1 interference sequence group were (0.25±0.03, 0.30±0.02 and 0.32±0.03), which were significantly lower than those in the negative control group (0.77±0.06, 0.76±0.10 and 0.67±0.04) and blank group (0.84±0.14, 0.74±0.07 and 0.69±0.06, P<0.05), and the relative expression level of E-cadherin protein (0.71±0.05) was significantly higher than in the negative control group (0.37±0.02) and blank group (0.34±0.04, P<0.05). Conclusion LSD1 protein was highly expressed in hepatocellular carcinoma tissues, and might play an important role in the proliferation and invasion of human hepatoma HepG2 cells through epithelial-mesenchymal transition. Key words: Carcinoma, hepatocellular; Lysine-specific demethylase 1; Proliferation; Invasion; Epithelial-mesenchymal transition

LncRNA‑AB209371 promotes the epithelial‑mesenchymal transition of hepatocellular carcinoma cells.

The zinc finger protein Snail1 is an important factor in the regulation of the epithelial‑mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) cells. The present study demonstrated that the expression of Snail1 in HCC tissues was significantly higher compared with its expression in tissues adjacent to primary sites, as determined via western blotting. Furthermore, the results of a dual luciferase assay revealed that hsa‑microRNA(miR)199a‑5p negatively regulated the protein expression of Snail1 by binding to its 3' untranslated region. However, in a comparative analysis of primary HCC and its metastatic tissues using reverse transcription‑quantitative polymerase chain reaction and western blotting, it was demonstrated that the expression of hsa‑miR199a‑5p and Snail1 in HCC metastatic tissues were significantly higher compared with primary lesions and an association between them identified that hsa‑miR199a‑5p lost its ability to negatively regulate Snail1. This result is contradictive to the fact that hsa‑miR199a‑5p inhibits the expression of the Snail1 protein. The present study hypothesized that the aberrant expression of long non‑coding RNA was the cause of hsa‑miR199a‑5p inactivation based on loss of function rather than a reduction in content. The data collected in the present study confirmed the hypothesis that AB209371 binds to hsa‑miR199a‑5p and weakened the inhibitory effect of hsa‑miR199a‑5p on Snail1 expression. In addition, an invitro EMT model was established in the present study by inducing HCC cells with TGF‑β1. The results revealed that AB209371 silencing effectively reversed the hsa‑miR199a‑5p mediated inhibition of EMT by negatively regulating Snail1 protein expression. Therefore, AB209371 silencing in combination with hsa‑miR199a‑5p expression may serve as an effective means to inhibit EMT in HCC cells. The present study also revealed that hsa‑miR199a‑5p/Snail1 exhibits a dominant regulatory effect in the EMT of HCC cells via a Snail1 recovery experiment. In conclusion, to the best of our knowledge, the present study confirmed for the first time that the high expression of AB209371 is favorable for the EMT in HCC cells and may be a direct cause of hsa‑miR199a‑5p inactivation (an HCC metastasis suppressor). Additionally, AB209371 silencing combined with hsa‑miR199a‑5p overexpression may be an effective means to inhibit the metastasis of HCC and the EMT of HCC cells.

Long non-coding RNA CRNDE regulates cell proliferation, migration, invasion, epithelial-mesenchymal transition and apoptosis in oral squamous cell carcinoma.

The present study aimed to investigate whether the long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) can promote the migration and invasion of human oral squamous cell carcinoma (OSCC) cells via the regulation of epithelial-mesenchymal transition (EMT). CAL-27 and SCC-15 cells were classified into a control group, a small interfering negative control (si-NC) group (cells transfected with control siRNA) and an si-CRNDE group (cells transfected with CRNDE siRNA). The expression of CRNDE in OSCC tissues and cell lines was detected by in situ hybridization (ISH) and reverse transcription-quantitative polymerase chain reaction. An MTT assay was used to detect cell proliferation, flow cytometry was performed to determine cell apoptosis, wound-healing and Transwell assays were conducted to evaluate cell metastasis, and immunofluorescence staining and western blotting were performed to measure the expression of proteins associated with EMT. Tumor-bearing mouse models were established, and the tumor volumes were recorded. An immunohistochemical assay was performed to determine the expression of EMT-related proteins. CRNDE expression was increased in OSCC tissues and cell lines compared with that in normal tissues and cell lines. Compared with the control group, the si-CRNDE group displayed a reduction in the expression of CRNDE, in the proliferation, migration and invasion of cells, in the protein expression of N-cadherin, vimentin and Snail, and in the expression of proteins in the Wnt/β-catenin pathway. However, an increase was displayed in the apoptosis of cells and the expression of E-cadherin. Compared with the control group of tumor-bearing nude mice, the sh-CRNDE group demonstrated slowed tumor growth, reduced tumor weight and elevated E-cadherin, as well as reduced expression of N-cadherin, vimentin and Snail. In conclusion, silencing CRNDE may inhibit EMT, thus decreasing the migration and invasion of human OSCC cells by repressing the activation of the Wnt/β-catenin signaling pathway, thereby restricting cell growth and promoting cell apoptosis.

14-3-3ζ promotes gliomas cells invasion by regulating Snail through the PI3K/AKT signaling.

14‐3‐3ζ has been reported to function as critical regulators of diverse cellular responses. However, the role of 14‐3‐3ζ in gliomas progression remains largely unknown. The expression level of 14‐3‐3ζ and Snail was detected by Western blot analysis and quantitative polymerase chain reaction in different grades of human gliomas. The effect of 14‐3‐3ζ on gliomas progression was measured using cell migration and invasion assay, the colony formation experiment, and CCK‐8 assay. The effect of 14‐3‐3ζ on PI3K/AKT/Snail signaling protein expression levels was tested by Western blotting. Firstly, 14‐3‐3ζ was often up‐regulated in high‐grade gliomas relative to low‐grade gliomas, and this overexpression was significantly related to tumor size, Karnofsky Performance Scale score and weaker disease‐free survival. Secondly, the overexpression of 14‐3‐3ζ promoted gliomas cells proliferation, migration, and invasion. Conversely, the knockdown of 14‐3‐3ζ suppressed gliomas cells proliferation, migration, and invasion. Furthermore, subsequent mechanistic studies showed that 14‐3‐3ζ could activate PI3K/AKT /Snail signaling pathway to facilitate gliomas cells proliferation, migration, and invasion. This study shows that the overexpression of 14‐3‐3ζ can promote remarkably gliomas cells proliferation, migration, and invasion by regulating the Snail protein expression through activating PI3K/AKT signaling, and it may serve as a potential prognostic marker and therapeutic target for gliomas.

MiR-940 inhibits TGF-β-induced epithelial-mesenchymal transition and cell invasion by targeting Snail in non-small cell lung cancer.

Increased evidence reveals that miR-940 inhibits the migration and invasion of cancer cells. Considering transforming growth factor β (TGF-β) signaling is crucial to cellular epithelial-mesenchymal transition (EMT) process and metastasis of cancer, it is in urgent to explore whether and how miR-940 plays an essential role in regulating TGF-β-induced EMT in lung cancer progression. In the present study, we observed a reciprocal expression with down-regulated miR-940 and up-regulated Snail mRNA in non-small-cell lung cancer (NSCLC) tissues. we further found that the expression of miR-940 was decreased in NSCLC tissues with lymph node metastasis, advanced TNM stages and poor cell differentiation, in which, on the contrary, the expression of Snail was increased. Overexpression of miR-940 significantly inhibited Snail mRNA and protein expression in A549 and H226 cells. Mechanistically, Snail mRNA was identified as target of miR-940. In addition, miR-940 repressed TGF-β-induced EMT and further hampered the cell migration and invasion. Finally, siRNA-mediated knockdown of Snail copied the phenotype of miR-940 overexpression in A549 and H226 cells. Taken together, our study reveals that miR-940 can suppress TGF-β-induced EMT and cell invasion by targeting Snail 3'-UTR mRNA in NSCLC.

GAS5 promotes podocyte injury in sepsis by inhibiting PTEN expression.

To investigate the potential role of long noncoding RNA (lncRNA) growth arrest specific transcript 5 (GAS5) in sepsis-induced podocyte injury and its underlying mechanism. The sepsis model was established by lipopolysaccharide (LPS) induction in podocytes. The expression levels of Nephrin and GAS5 were detected by quantitative Real-time polymerase chain reaction (qRT-PCR) after LPS induction in podocytes for 12 h, 24 h and 36 h, respectively. Western blot was used to detect the expression level of Nephrin in sepsis-induced podocytes. The mRNA expressions of GAS5 and Nephrin in podocytes were detected after transfection of GAS5 siRNA. Albumin influx in podocytes after GAS5 knockdown was detected by Transwell assay. Western blot was used to detect the protein expression of Snail in sepsis after GAS5 knockdown. The target gene of GAS5 was predicted by bioinformatics analysis. QRT-PCR and Western blot were used to detect the protein and mRNA levels of PTEN (phosphatase and tensin homolog deleted on chromosome ten). Nephrin expression and the albumin inflow after PTEN knockdown were then measured. The expression of PI3K/AKT/GSK3β was also detected after GAS5 was downregulated while PTEN was upregulated. LPS stimulation downregulated the mRNA expression of Nephrin in podocytes and achieved the lowest level at 24 h. The protein expression change of Nephrin was consistent with its mRNA expression. In the septic state, the albumin influx of podocytes remarkably increased, but the function of podocyte barrier was weakened. Besides, GAS5 expression decreased in a time-dependent manner in LPS-induced podocytes. After GAS5 knockdown by siRNA, Nephrin expression and the function of podocyte barrier were significantly reduced. Snail expression was also upregulated in septic state, and GAS5 knockdown increased the expressions of phosphorylated Snail and PI3K/AKT/GSK3β. After knockdown of GAS5, the mRNA and protein levels of PTEN significantly decreased, which was contract to the expression of Snail. However, overexpression of PTEN could reverse the promotive effect of GAS5 on PI3K/AKT activation. GAS5 expression decreased in sepsis-induced podocyte injury, and GAS5 was involved in regulating sepsis-induced podocyte injury by reducing PTEN expression.

Mechanisms underlying the increased chemosensitivity of bortezomib-resistant multiple myeloma by silencing nuclear transcription factor Snail1.

The Snail family transcriptional repressor 1 gene (Snail1) was screened in multiple myeloma cells (MMCs) from bortezomib-resistant MM patients and was found to be significantly associated with the development of drug-resistance mechanisms. In the present study, we first confirmed that the protein expression of Snail1 in bortezomib-resistant MMCs was significantly higher than that in MMCs without bortezomib resistance. The mechanistic studies confirmed that the enhancement of Snail1 expression in bortezomib-resistant MMCs directly upregulated transcription of the intracellular MDR1 gene to immediately develop multiple drug resistance mechanisms and inhibited P53 protein expression through the Snail1/hsa-miRNA-22-3p/P53 pathway to inhibit tumor cell apoptosis. By upregulating MDR1 and downregulating P53, Snail1 induced the drug resistance of MMCs to bortezomib, while Snail1 gene silencing effectively improved the drug sensitivity of MMCs to bortezomib chemotherapy. The present study further elucidated the drug resistance mechanisms of MMCs and provides evidence for increased clinical efficacy of bortezomib in MM patients.

FOXC2 promotes epithelial-mesenchymal transition and cisplatin resistance of non-small cell lung cancer cells.

Platinum-based drugs, particularly cisplatin (DDP), are used in the treatment of non-small cell lung cancer (NSCLC). However, development of drug resistance remains the major therapeutic barrier in NSCLC. The potential cisplatin resistance-related genes were identified from the global transcriptomes of cisplatin-resistant A549/DDP cells using microarray analysis. Gain- and loss-of-function assays were performed to analyze the effects of Forkhead Box Protein C2 (FOXC2) on the in vitro and in vivo sensitivity of NSCLC cells to cisplatin and its possible molecular mechanisms. Using global transcriptome analysis, we found that FOXC2 was one of the most upregulated molecules in A549/DDP cells compared with A549 cells. We further confirmed that the expression of FOXC2 was significantly increased in cisplatin-resistant NSCLC tissues. FOXC2 knockdown significantly increased the in vitro and in vivo sensitivity of A549/DDP cells to cisplatin, whereas overexpression of FOXC2 increased cisplatin resistance in cisplatin-sensitive NSCLC cells. Moreover, we found that FOXC2 promoted cisplatin resistance by induction of epithelial-mesenchymal transition (EMT) in NSCLC cells. Furthermore, FOXC2 activated the AKT/GSK3β signaling pathway, and then increased the protein expression of EMT-related transcription factor Snail. Inhibition of AKT or knockdown of Snail reversed FOXC2-induced EMT and cisplatin resistance of NSCLC cells. FOXC2 enhanced cisplatin resistance of NSCLC cells through activating AKT/GSK3β/Snail/EMT signaling pathway, which may be a potential novel therapeutic target for overcoming drug resistance in human NSCLCs.

Expression of Apelin and Snail protein in breast cancer and their prognostic significance

Objective: To investigate the expression of Apelin and Snail proteins in breast cancer and their relationship with the clinicopathological features and prognosis. Methods: The expression of Apelin and Snail proteins was detected by immunohistochemistry in 89 cases of breast cancer and 50 cases of mammary adenosis collected from January to June in 2008 at Fujian Cancer Hospital; the expression was correlated with the clinicopathological features and outcome of the patients. Results: Apelin and Snail were expressed in 42 cases(47.2%)and 36 cases(40.4%)of breast cancers, respectively, and the expression was higher than that of control group (P<0.01). The expression of Apelin was positively correlated with Snail (r=0.230, P<0.05). Apelin expression was associated with lymph node metastasis and TNM staging(P<0.05). Snail expression was associated with lymph node metastasis(P<0.05). Kaplan-Meier survival curve showed that the prognosis of Apelin positive group was worse than that of Apelin negative group (P<0.01). There was no significant difference in prognosis between Snail negative and positive groups (P>0.05). The prognosis of Apelin and Snail in both positive groups was worse than that of Apelin and Snail both negative groups (P<0.01). Multivariate COX regression analysis showed that Apelin and TNM staging could be used as independent prognostic factors for breast cancer (P<0.05). Conclusions: Apelin and Snail are highly expressed in breast cancer, and associated with lymph node metastasis and TNM stage. There is a positive correlation between Apelin and Snail expression, which may suggest a role in breast carcinogenesis. The prognosis of breast cancer with expression of Apelin and co-expression of Apelin and Snail is poor. Therefore, Apelin may be used as an effective indicator to evaluate the prognosis of breast cancer patients.

Continuous exposure of nicotine and cotinine retards human primary pterygium cell proliferation and migration.

Pterygium is a triangular-shaped hyperplastic growth, characterized by conjunctivalization, inflammation, and connective tissue remodeling. Our previous meta-analysis found that cigarette smoking is associated with a reduced risk of pterygium. Yet, the biological effect of cigarette smoke components on pterygium has not been studied. Here we reported the proliferation and migration properties of human primary pterygium cells with continuous exposure to nicotine and cotinine. Human primary pterygium cells predominantly expressed the α5, β1, and γ subunits of the nicotinic acetylcholine receptor. Continuous exposure to the mixture of 0.15 μM nicotine and 2 μM cotinine retarded pterygium cell proliferation by 16.04% (P = 0.009) and hindered their migration by 11.93% ( P = 0.039), without affecting cell apoptosis. SNAIL and α-smooth muscle actin protein expression was significantly downregulated in pterygium cells treated with 0.15 μM nicotine-2 μM cotinine mixture by 1.33- ( P = 0.036) and 1.31-fold ( P = 0.001), respectively. Besides, the 0.15 μM nicotine-2 μM cotinine mixture also reduced matrix metalloproteinase (MMP)-1 and MMP-9 expressions in pterygium cells by 1.56- ( P = 0.043) and 1.27-fold ( P = 0.012), respectively. In summary, this study revealed that continuous exposure of nicotine and cotinine inhibited human primary pterygium cell proliferation and migration in vitro by reducing epithelial-to-mesenchymal transition and MMP protein expression, partially explaining the lower incidence of pterygium in cigarette smokers.

Oridonin prevents epithelial-mesenchymal transition and TGF-β1-induced epithelial-mesenchymal transition by inhibiting TGF-β1/Smad2/3 in osteosarcoma

Osteosarcoma is the most common primary bone tumor with highly invasive characteristic and low long-term survival. Recently, epithelial-mesenchymal transition (EMT) is reported as a key event in cancer invasion and metastasis. Oridonin, a bioactive diterpenoid, has been proved to possess anti-cancer effects. However, the effect of oridonin on EMT and metastasis of osteosarcoma is unclear. In this study, we investigated the underlying mechanism of oridonin on EMT and metastasis of osteosarcoma. We found that oridonin inhibited migration and invasion of MG-63 and 143B cells. Moreover, oridonin increased the protein expression of E-cadherin and decreased that of N-cadherin and Vimentin. Oridonin upregulated the transcription of E-cadherin and downregulated N-cadherin and Vimentin. Oridonin inhibited the protein and mRNA levels of Snail and Slug. Furthermore, oridonin inhibited TGF-β-induced phosphorylation of Smad 2/3, prevented Smad dimer translocation into the nucleus. Finally, we established metastatic models of osteosarcoma 143B cells, and found that oridonin inhibited lung metastasis in vivo. Oridonin increased the protein expression of E-cadherin and reduced N-cadherin and Vimentin. Oridonin inhibited the protein expression of Snail and Slug as well as Smad 2/3 activation. In conclusion, our study demonstrated that oridonin inhibited EMT and TGF-β1-induced EMT by inhibiting TGF-β1/Smad2/3 signaling pathway in osteosarcoma.

HOXB9 inhibits proliferation in gastric carcinoma cells via suppression of phosphorylated-Akt and NF-κB-dependent Snail expression

BackgroundHOXB9 is a homeobox transcription factor which plays an important role in carcinoma development. This protein has been shown to inhibit cancer cell proliferation. However, the mechanisms that underpin HOXB9-mediated inhibition of cellular proliferation remain to be elucidated. MethodsIn this study, two gastric cancer cell lines, SGC7901 and MKN45, were transfected with plasmids pLVX-HOXB9 and shHOXB9. These transfections resulted in the over-expression of the HOXB9 gene in the SGC7901/HOXB9 cells and knockdown of the HOXB9 gene in the MKN45/shHOXB9 cells. ResultsOver-expression of the HOXB9 gene in the SGC7901/HOXB9 cells caused an increase in the apoptotic rate and a concomitant reduction in metastatic ability compared with the knocked-down MKN45/shHOXB9 cells. Moreover, a reduction in the expression of the phosphorylated-Akt protein was observed in the SGC7901/HOXB9 cells, while an increase in expression of the same protein was observed in the MKN45/shHOXB9 cells. We also observed that HOXB9 mediated a reduction in both NF-κB and N-cadherin and Snail protein expression. Conversely, HOXB9 caused an increase in the expression of E-cadherin. ConclusionsIn summary, this study reports that HOXB9 can suppress both phosphorylated-Akt expression and NF-κB activity. The latter phenomenon affects Snail protein expression and the inhibition of gastric carcinoma proliferation.

MiR-125a-3p is responsible for chemosensitivity in PDAC by inhibiting epithelial-mesenchymal transition via Fyn

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and resistance to cytotoxic chemotherapy is the major cause of mortality in PDAC patients. miR-125a-3p was found to be down-regulated in PDAC cells; however, the function of miR-125a-3p in PDAC has been elusive. Here, we explored the role of miR-125a-3p in chemosensitivity in PDAC cells. MethodsWe used qRT-PCR to detect miR-125a-3p expression in two PDAC cell lines. And we measured cell viability and apoptosis by MTT assay and flow cytometry, respectively. Scratch wound healing assay and transwell invasion assay were used to test the effects of miR-125a-3p and Fyn on cell EMT process. In addition, we validated the interaction of miR-125a-3p and Fyn by dual luciferase reporter assay. qRT-PCR and western blot were used to detect the mRNA and protein expressions of E-cadhrein, N-cadhrein, Snail and Fyn. ResultsWe found that miR-125a-3p was down-regulated in a time-dependent manner following treatment with gemcitabine in PDAC cells. Meanwhile, we found that overexpression of miR-125a-3p significantly increased chemosensitivity to gemcitabine and suppressed epithelial-mesenchymal transition (EMT) of PDAC cells. Mechanistically, miR-125a-3p directly targeted Fyn and decreased the expression of Fyn that functions to promote EMT process in PDAC. Furthermore, overexpression of Fyn could partially reverse the effects of miR-125a-3p on chemosensitivity to gemcitabine. ConclusionOur study is the first to show that miR-125a-3p is responsible for chemosensitivity in PDAC and could inhibit epithelial-mesenchymal transition by directly targeting Fyn. This provides a novel potential therapeutic strategy to overcome chemoresistance in PDAC.

3,6-dihydroxyflavone suppresses the epithelial-mesenchymal transition, migration and invasion in endometrial stromal cells by inhibiting the Notch signaling pathway.

Endometriosis is a common disease in women of reproductive age. Characteristics of endometriosis include invasion, metastasis, and recurrence, which are similar to those of malignant tumors. However, the etiology and pathogenesis of endometriosis are still not clear. This study aims to explore the mechanism of 3,6-dihydroxyflavone (3,6-DHF) in the development of endometriosis. Primary cultured ovarian ectopic endometrial stromal cells (OvESCs) were utilized as the in vitro model of endometriosis. OvESCs were treated with different concentrations of 3,6-DHF. The expressions of proteins related to epithelial-mesenchymal transition (EMT) and Notch signal pathway were detected by Western blot. The mRNA expressions of related genes were detected by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The viability of treated cells was detected by transwell assay. The impact of 3,6-DHF on ectopic lesions was explored after the animal model of endometriosis was successfully established. With the increased concentration of 3,6-DHF in OvESCs, the protein and mRNA expressions of E-cadherin were gradually increased, while the protein and mRNA expressions of N-cadherin, Twist, Snail, and Slug were decreased. 3,6-DHF treatment inhibited the migration and invasion ability of OvESCs in a dose-dependent manner. In the endometriosis model of severe combined immunodeficient (SCID) mice, lesions in the 3,6-DHF treated group were significantly smaller than those of the control group. The same changes were found in the endometriosis model of Sprague Dawley (SD) rats. Protein expressions of Notch1, NICD, and Hes-1 in OvESCs were inhibited by 3,6-DHF in a dose-dependent manner. 3,6-DHF can inhibit the binding of NICD-CSL-MAML complex in OvESCs, thereby inhibiting the expressions of proteins related to Notch signaling pathway in vitro. 3,6-DHF can inhibit the development of EMT, migration, and invasion of endometrial stromal cells by inhibiting the Notch signaling pathway.

Downregulation of HOXA13 sensitizes human esophageal squamous cell carcinoma to chemotherapy.

BackgroundChemoresistance often develops in esophageal squamous cell carcinoma (ESCC), leading to poor prognosis. HOX genes play a crucial role in embryonic development and cell differentiation. Studies have recently linked HOX with chemoresistance, thus we explored whether HOXA13 is involved in ESCC chemoresistance.MethodsOne hundred thirty‐one ESCC patients who received neoadjuvant chemotherapy were enrolled. HOXA13 expression was examined by immunohistochemistry. RNA interference was used to knock down the HOXA13 expression in KYSE70 and transfected HOXA13 plasmid to overexpress HOXA13 in KYSE510 cells. We examined half‐maximal inhibitory concentration of cisplatin, apoptosis, and epithelial‐to‐mesenchymal transition (EMT) in ESCC cell lines with different HOXA13 expression levels by cell counting kit‐8, flow cytometry, and transwell analysis.ResultsThe median survival of patients with high HOXA13 expression was significantly shorter than those with low expression (P = 0.027). HOXA13 was associated with worse tumor regression grade (P = 0.009). Low HOXA13 expressed cells decreased the half‐maximal inhibitory concentration of cisplatin (P < 0.05), increased cisplatin‐induced apoptosis (P < 0.05), and decreased EMT (P < 0.05) compared with high HOXA13 expressed cells. In low HOXA13 expressed cells, cleaved caspase‐3 and cleaved PARP expression induced by cisplatin increased, while expression of E‐cadherin and Snail protein, markers of EMT, was upregulated and downregulated, respectively. EMT decreased in low HOXA13 expressed cells.ConclusionHigh HOXA13 expression was associated with inferior tumor regression grade and poor overall survival in ESCC patients treated with neoadjuvant chemotherapy. HOXA13 increased cisplatin‐resistance and promoted EMT in ESCC cells.

Molecular mechanism of Poria cocos combined with oxaliplatin on the inhibition of epithelial-mesenchymal transition in gastric cancer cells

PurposeNatural product Poria cocos possesses antitumor effect. This study will explore the molecular mechanism of Poria cocos combined with chemotherapy in the inhibition of gastric cancer cell EMT process. MethodsThe experiment was divided into blank control group, Poria cocos group, oxaliplatin group and Poria cocos combined with oxaliplatin group. Scratch and Transwell assay were used to detect cell migration and invasion respectively. RT-qPCR and Western Blot analyses were used to detect mRNA and protein expression of the epithelial-mesenchymal transition (EMT) related factors including Snail, Twist, Vimentin, E-cadherin and N-cadherin respectively. Morphologic assessment was performed with HPIAS-1000 automated image analysis system. ResultsThe migration and invasion abilities of gastric cancer cells in the Poria cocos combined with oxaliplatin group were significantly decreased (P < 0.01). The mRNA and protein expression of Snail, Twist, Vimentin and N-cadherin were significantly decreased while the mRNA and protein expression of E-cadherin were significantly increased (P < 0.01) compared with blank control group. Nude mice model of gastric cancer was successfully established. Poria cocos combined with oxaliplatin could significantly inhibit gastric tumor progression. The expression of EMT related factors were consistent with in vitro study. Morphologic assessment showed that the nucleus area, perimeter, mean diameter, volume, long diameter and shape factor in the Poria cocos combined with oxaliplatin group were significantly different compared with the blank control group (P < 0.01) but not significantly different compared with the normal control. ConclusionsPoria cocos combined with oxaliplatin could significantly inhibit the migration and invasion of gastric cancer cells. Through both in vitro and in vivo studies, it is confirmed that Poria cocos combined with oxaliplatin could significantly inhibit the EMT process of gastric cancer. Poria cocos combined with oxaliplatin could significantly affect the morphology changes of gastric cancer cells. These findings may provide a theoretical guidance for the clinical treatment of gastric cancer.

Nur77 suppression facilitates androgen deprivation-induced cell invasion of prostate cancer cells mediated by TGF-β signaling.

Androgen deprivation therapy (ADT) remains a standard treatment for advanced prostate cancers. However, recent studies revealed that while inhibiting the growth of certain types of prostate cancer cells, ADT promotes invasion. In the current study, we explored the effects of Nur77, an orphan nuclear receptor, on prostate cancer cell invasion following ADT. Androgen receptor (AR) and Nur77 protein expression in patient tissues and cell lines were quantified via ELISA and western blot. The effects of AR-signaling on Nur77 expression were examined. The effects of Nur77 over-expression and knockdown on ADT-induced prostate cancer cell invasion were characterized. The results showed that AR and Nur77 are both highly expressed in prostate cancers of patients. Nur77 is positively regulated by AR-signaling at transcriptional level in NCI-H660, a widely used prostate cancer cell line. AR antagonists, Casodex and MDV3100 treatment resulted in significant inhibition of prostate cancer cell growth but enhanced cancer cell invasion. Nur77 over-expression blocked invasion-promoting effect of ADT, which is consistent with the down-regulation of MMP9 and Snail protein expression. Further mechanistic investigations showed that Nur77 inhibited transcription of TGF-β target genes (Snail and MMP9), and thereby inhibits TGF-β-mediated prostate cancer cell invasion following androgen antagonism. In addition, our data suggested the nature of this inhibitory effect of Nur77 on TGF-β-signaling is selective, for Smad3-signaling, the classical effector of TGF-β-signaling, was not interrupted by Nur77 over-expression. Considering the limited success of management of prostate cancer metastasis following ADT, our data strongly suggest that Nur77 regulation could be a promising direction for search of complementary therapeutic strategy on top of classic ADT therapy.