Role of epithelial-mesenchymal transition in gastric cancer initiation and progression

Gastric cancer is one of the most common malignant tumors worldwide.Due to its intricate initiation and progression mechanisms,early detection and effective treatment of gastric cancer are difficult to achieve.The epithelial-mesenchymal transition(EMT)is characterized as a fundamental process that is critical for embryonic development,wound healing and fibrotic disease.Recent evidence has established that aberrant EMT activation in the human stomach is closely associated with gastric carcinogenesis and tumor progression.EMT activation endows gastric epithelial cells with increased characteristics of mesenchymal cells and reduces their epithelial features.Moreover,mesenchymal cells tend to dedifferentiate and acquire stem cell or tumorigenic phenotypes such as invasion,metastasis and apoptosis resistance as well as drug resistance during EMT progression.There are a number of molecules that indicate the stage of EMT(e.g.,E-cadherin,an epithelial cell biomarker);therefore,certain transcriptional proteins,especially E-cadherin transcriptional repressors,may participate in the regulation of EMT.In addition,EMT regulation may be associated with certain epigenetic mechanisms.The aforementioned molecules can be used as early diagnostic markers for gastric cancer,and EMT regulation can provide potential targets for gastric cancer therapy.Here,we review the role of these aspects of EMT in gastric cancer initiation and development.

Interleukin-34 promotes the proliferation and epithelial-mesenchymal transition of gastric cancer cells

BACKGROUND Interleukin(IL)-34 is a pro-inflammatory cytokine involved in tumor development.The role of IL-34 in the proliferation and epithelial-mesenchymal transition(EMT)of gastric cancer(GC)remains to be investigated.AIM To investigate whether and how IL-34 affects the proliferation of GC cells and EMT.METHODS Using immunohistochemical staining,the expression of IL-34 protein was detected in 60 paired GC and normal paracancerous tissues and the relationship between IL-34 and clinicopathological factors was analyzed.The expression of IL-34 mRNA and protein in normal gastric epithelial cell lines and GC was detected using quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting,respectively.Stable IL-34 knockdown and overexpression in AGS cell lines were established by lentiviral infection and validated by qRT-PCR and western blotting.The cholecystokinin-8 assay,clone formation assay,cell scratch assay,and transwell system were used to detect GC cell proliferation,clone formation,migration,and invasion capacity,respectively.The effects of IL-34 on the growth of GC transplant tumors were assessed using a subcutaneous transplant tumor assay in nude mice.The effects of IL-34 on the expression level of EMT-associated proteins in AGS cells were examined by western blotting.RESULTS Expression of IL-34 protein and mRNA was higher in GC cell lines than in GES-1 cells.Compared to matched normal paraneoplastic tissues,the expression of IL-34 protein was higher in 60 GC tissues,which was correlated with tumor size,T-stage,N-stage,tumor,node and metastasis stage,and degree of differentiation.Knockdown of IL-34 expression inhibited the proliferation,clone formation,migration,and invasion of AGS cells,while overexpression of IL-34 promoted cell proliferation,clone formation,migration,and invasion.Furthermore,the reduction of IL-34 promoted the expression of E-cadherin in AGS cells but inhibited the expression of vimentin and N-cadherin.Overexpression of IL-34 inhibited E-cadherin expression but promoted expression of vimentin and N-cadherin in AGS cells.Overexpression of IL-34 promoted the growth of subcutaneous transplanted tumors in nude mice.CONCLUSION IL-34 expression is increased in GC tissues and cell lines compared to normal gastric tissues or cell lines.In GC cells,IL-34 promoted proliferation,clone formation,migration,and invasion by regulating EMT-related protein expression cells.Interference with IL-34 may represent a novel strategy for diagnosis and targeted therapy of GC.

Effect of h GC-MSCs from human gastric cancer tissue on cell proliferation, invasion and epithelial-mesenchymal transition in tumor tissue of gastric cancer tumor-bearing mice

Objective: To study the effect of h GC-MSCs from human gastric cancer tissue on cell proliferation, invasion and epithelial-mesenchymal transition in tumor tissue of gastric cancer tumor-bearing mice. Methods: BABL/c nude mice were selected as experimental animals and gastric cancer tumor-bearing mice model were established by subcutaneous injection of gastric cancer cells, randomly divided into different intervention groups. h GC-MSCs group was given different amounts of gastric cancer cells for subcutaneous injection, PBS group was given equal volume of PBS for subcutaneous injection. Then tumor tissue volume were determined, tumor-bearing mice were killed and tumor tissues were collected, m RNA expression of proliferation, invasion, EMT related molecules were determined. Results: 4, 8, 12, 16, 20 d after intervention, tumor tissue volume of h GC-MSCs group were significantly higher than those of PBS group and the more the number of h GC-MSCs, the higher the tumor tissue volume; m RNA contents of Ki-67, PCNA, Bcl-2, MMP-2, MMP-7, MMP-9, MMP-14, N-cadherin, vimentin, Snail and Twist in tumor tissue of h GC-MSCs group were higher than those of PBS group, and m RNA contents of Bax, TIMP1, TIMP2 and E-cadherin were lower than those of PBS group. Conclusions: h GC-MSCs from human gastric cancer tissue can promote the tumor growth in gastric cancer tumor-bearing mice, and the molecular mechanism includes promoting cell proliferation, invasion and epithelial-mesenchymal transition.

E3 ubiquitin ligase TRIM55 promotes metastasis of gastric cancer cells by mediating epithelial-mesenchymal transition

BACKGROUND Gastric cancer(GC)is considered a major global health problem.The role of TRIM55,a member of the three-domain protein family,in GC is unknown.AIM To determine the expression of TRIM55 in GC tissues and its relationship with clinicopathological characteristics,and to investigate the effects of TRIM55 on the malignant biological behavior of GC cells.METHODS Differential expression of TRIM55 in GC and para-cancer tissues was detected by immunohistochemistry,and the relationship between TRIM55 level and clinicopathological characteristics and prognosis was analyzed.Gain-of-function,lossof-function,cell counting kit-8 assay,colony formation,transwell assay,wound healing assay,and western blot analysis were used to assess the potential role of TRIM55 in the development of GC.RESULTS TRIM55 expression was significantly increased in GC tissues compared with adjacent normal tissues.High expression of TRIM55 was associated with advanced pathological stage and poor prognosis.Overexpression of TRIM55 promoted invasion and metastasis of GC cells in vitro by regulating epithelial-mesenchymal transition(EMT),whereas knockdown of TRIM55 had the opposite effect.Our data showed that TRIM55 is highly expressed in GC tissues,and is associated with poor prognosis.TRIM55 plays the role of an oncogene in GC,and it promotes metastasis of GC through the regulation of EMT.CONCLUSION TRIM55 may be a possible target for the diagnosis and prognosis of GC patients.

F-box and leucine-rich repeat 6 promotes gastric cancer progression via the promotion of epithelial-mesenchymal transition

BACKGROUND F-box and leucine-rich repeat 6(FBXL6)have reportedly been associated with several cancer types.However,the role and mechanisms of FBXL6 in gastric cancer(GC)require further elucidation.AIM To investigate the effect of FBXL6 in GC tissues and cells and the underlying mechanisms.METHODS TCGA and GEO database analysis was performed to evaluate the expression of FBXL6 in GC tissues and adjacent normal tissues.Reverse transcription-quantitative polymerase chain reaction,immunofluorescence,and western blotting were used to detect the expression of FBXL6 in GC tissue and cell lines.Cell clone formation,5-ethynyl-2’-deoxyuridine(EdU)assays,CCK-8,transwell migration assay,and wound healing assays were performed to evaluate the malignant biological behavior in GC cell lines after transfection with FBXL6-shRNA and the overexpression of FBXL6 plasmids.Furthermore,in vivo tumor assays were performed to prove whether FBXL6 promoted cell proliferation in vivo.RESULTS FBXL6 expression was upregulated more in tumor tissues than in adjacent normal tissues and positively associated with clinicopathological characteristics.The outcomes of CCK-8,clone formation,and Edu assays demonstrated that FBXL6 knockdown inhibited cell proliferation,whereas upregulation of FBXL6 promoted proliferation in GC cells.Additionally,the transwell migration assay revealed that FBXL6 knockdown suppressed migration and invasion,whereas the overex pression of FBXL6 showed the opposite results.Through the subcutaneous tumor implantation assay,it was evident that the knockdown of FBXL6 inhibited GC graft tumor growth in vivo.Western blotting showed that the effects of FBXL6 on the expression of the proteins associated with the epithelial-mesenchymal transition-associated proteins in GC cells.CONCLUSION Silencing of FBXL6 inactivated the EMT pathway to suppress GC malignancy in vitro.FBXL6 can potentially be used for the diagnosis and targeted therapy of patients with GC.

Epithelial-mesenchymal transition contributes to malignant phenotypes of circulating tumor cells derived from gastric cancer

Circulating tumor cells(CTCs)are crucial to tumor metastasis,and they usually undergo epithelial-mesenchymal transition(EMT)in order to disseminate from the primary tumor.However,very little is currently known about the relationship between EMT and malignant phenotypes of CTCs in the context of gastric cancer.Therefore,this study aimed to investigate the contribution of EMT to malignant phenotypes of CTCs derived from gastric cancer cells.We xenografted MKN28 gastric cancer cells pretreated with transforming growth factor-beta 1(TGFβ-1)into nude mice by intravenous injection.Next,we isolated CTCs from the blood of nude mice by gradient centrifugation and found that CTCs derived from MKN28 cells pretreated with TGFβ-1 had a significantly increased viability and invasion ability compared to MKN28 cells without TGFβ-1 treatment.Immunocytochemical staining showed lower expression of E-cadherin and higher expression of N-cadherin,vimentin,and β-catenin in CTCs derived from MKN28 cells pretreated with TGFβ-1.Furthermore,the expression of Wnt3a,β-catenin,cyclin D1,and c-Myc was significantly higher in CTCs derived from MKN28 cells pretreated with TGFβ-1.Taken together,these findings suggest that TGFβ promotes EMT and malignant phenotypes of gastric cancer cells.Furthermore,the malignant phenotypes of gastric cancer cells induced by TGFβ are maintained in CTCs derived from these cells.Targeting EMT in CTCs is a new approach to the treatment of gastric cancer relapse and metastasis.

Effect of PCDH10 on infiltrative growth and epithelial-mesenchymal transition of cancer cells in gastric cancer

Objective:To study the effect of protocadherin 10 (PCDH10) on infiltrative growth and epithelial-mesenchymal transition of cancer cells in gastric cancer.Methods: The patients with gastric cancer who underwent surgical resection in Panzhihua Central Hospital were selected as the research subjects, and the gastric cancer lesions and adjacent lesions were collected after surgical resection to determine the mRNA expression of PCDH10 gene and epithelial-mesenchymal transition genes as well as the protein levels of proteases and their inhibitory molecules.Results: PCDH10, TIMP1, TIMP2, RASAL2 and E-cadherin mRNA expression levels in gastric cancer lesions were significantly lower than those in adjacent lesions whereas Vav2, Vav3, CatB, MMP9, ZEB1, Twist1 and Vimentin contents were significantly higher than those in adjacent lesions;Vav2, Vav3, CatB, MMP9, ZEB1, Twist1 and Vimentin contents in gastric caner lesions with lower PCDH10 expression were significantly higher than those in gastric caner lesions with higher PCDH10 expression whereas TIMP1, TIMP2, RASAL2 and E-cadherin contents were significantly lower than those in gastric caner lesions with higher PCDH10 expression.Conclusion: The low expression of PCDH10 in gastric cancer can promote the infiltrative growth and epithelial-mesenchymal transition of cancer cells.

Silencing Neuropilin 1 gene reverses TGF-β1-induced epithelial mesenchymal transition in HGC-27 gastric cancer cell line

Objective The aim of this study was to determine Neuropilin 1(NRP1)contribution to transforming growth factorβ1(TGF-β1)-induced epithelial mesenchymal transition(EMT)of HGC-27 gastric cancer cells and study its mechanism.Methods In this study,TGF-β1 was used to induce EMT in HGC-27 cells.Further,these cells were stably transfected with siRNA targeting NRP1.Wound healing and transwell assays were used to measure cell migration and invasion,respectively.NRP1 and EMT markers were measured using quantitative real time reverse transcription polymerase chain reaction and western blotting.Results Exposure of TGF-β1 conferred a fibroblastic-like shape to cancer cells and significantly increased the expression of NRP1 in HGC-27 cells.TGF-β1 subsequently promoted migration and invasion of HGC-27 cells.Furthermore,silencing NRP1 inhibited the invasion and migration of TGF-β1-induced cells undergoing EMT.Conclusion Silencing NRP1 can inhibit cell migration,invasion,and metastasis and reverse the TGF-β1-induced EMT process of gastric cancer.

MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1-extracellular signal-regulated kinase-1/2 axis

BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.

Regulation of CTNNB1 signaling in gastric cancer and stem cells

Recent research has shown that the alteration of combinations in gene expression contributes to cellular phenotypic changes. Previously, it has been demonstrated that the combination of cadherin 1 and cadherin 2 expression can identify the diffuse-type and intestinal-type gastric cancers. Although the diffuse-type gastric cancer has been resistant to treatment, the precise mechanism and phenotypic involvement has not been revealed. It may be possible that stem cells transform into gastric cancer cells, possibly through the involvement of a molecule alteration and signaling mechanism. In this review article, we focus on the role of catenin beta 1 (CTNNB1 or β-catenin) and describe the regulation of CTNNB1 signaling in gastric cancer and stem cells.

MAWBP and MAWD inhibit proliferation and invasion in gastric cancer

AIM: To investigate role of putative mitogen-activated protein kinase activator with WD40 repeats (MAWD)/ MAWD binding protein (MAWBP) in gastric cancer (GC). METHODS: MAWBP and MAWD mRNA expression level was examined by real-time reverse transcriptasepolymerase chain reaction and semi-quantitative polymerase chain reaction in six GC cell lines. Western blotting was used to examine the protein expression levels. We developed GC cells that stably overexpressed MAWBP and MAWD, and downregulated expression by RNA interference assay. Proliferation and migration of these GC cells were analyzed by 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), soft agar, tumorigenicity, migration and transwell assays. The effect of expression of MAWBP and MAWD on transforming growth factor (TGF)-β1-induced epithelialmesenchymal transition (EMT) was examined by transfection of MAWBP and MAWD into GC cells. We detected the levels of EMT markers E-cadherin, N-cadherin and Snail in GC cells overexpressing MAWBP and MAWD by Western blotting. The effect of MAWBP and MAWD on TGF-β signal was detected by analysis of phosphorylation level and nuclear translocation of Smad3 using Western blotting and immunofluorescence. RESULTS: Among the GC cell lines, expression of endogenous MAWBP and MAWD was lowest in SGC7901 cells and highest in BGC823 cells. MAWBP and MAWD were stably overexpressed in SGC7901 cells and knocked down in BGC823 cells. MAWBP and MAWD inhibited GC cell proliferation in vitro and in vivo . MTT assay showed that overexpression of MAWBP and MAWD suppressed growth of SGC7901 cells (P < 0.001), while knockdown of these genes promoted growth of BGC823 cells (P < 0.001). Soft agar colony formation experiments showed that overexpression of MAWBP and MAWD alone or together reduced colony formation compared with vector group in SGC7901 (86.25±8.43, 12.75±4.49, 30±6.41 vs 336.75±22.55, P < 0.001), and knocked-down MAWBP and MAWD demonstrated opposite effects (131.25±16.54, 88.75±11.12, 341.75±22.23 vs 30.25±8.07, P < 0.001). Tumorigenicity experiments revealed that overexpressed MAWBP and MAWD inhibited GC cell proliferation in vivo (P < 0.001). MAWBP and MAWD also inhibited GC cell invasion. Transwell assay showed that the number of traverse cells of MAWBP, MAWD and coexpression group were more than that in vector group (84±16.57, 98.33±9.8, 29±16.39 vs 298±11.86, P < 0.001). Coexpression of MAWBP and MAWD significantly decreased the cells traversing the matrix membrane. Conversely, knocked-down MAWBP and MAWD correspondingly promoted invasion of GC cells (100.67±14.57, 72.66±8.51, 330.67±20.55 vs 27±11.53, P < 0.001). More importantly, coexpression of MAWBP and MAWD promoted EMT. Cells that coexpressed MAWBP and MAWD displayed a pebble-like shape and tight cell-cell adhesion, while vector cells showed a classical mesenchymal phenotype. Western blotting showed that expression of E-cadherin was increased, and expression of N-cadherin and Snail was decreased when cells coexpressed MAWBP and MAWD and were treated with TGF-β1. Nuclear translocation of p-Smad3 was reduced by attenuating its phosphorylation. CONCLUSION: Coexpression of MAWBP and MAWD inhibited EMT, and EMT-aided malignant cell progression was suppressed.

Ubiquitin-specific protease 15 contributes to gastric cancer progression by regulating the Wnt/β-catenin signaling pathway

BACKGROUND Ubiquitin-specific protease 15(USP15)is an important member of the ubiquitinspecific protease family,the largest deubiquitinase subfamily,whose expression is dysregulated in many types of cancer.However,the biological function and the underlying mechanisms of USP15 in gastric cancer(GC)progression have not been elucidated.AIM To explore the biological role and underlying mechanisms of USP15 in GC progression.METHODS Bioinformatics databases and western blot analysis were utilized to determine the expression of USP15 in GC.Immunohistochemistry was performed to evaluate the correlation between USP15 expression and clinicopathological characteristics of patients with GC.A loss-and gain-of-function experiment was used to investigate the biological effects of USP15 on GC carcinogenesis.RNA sequencing,immunofluorescence,and western blotting were performed to explore the potential mechanism by which USP15 exerts its oncogenic functions.RESULTS USP15 was up-regulated in GC tissue and cell lines.The expression level of USP15 was positively correlated with clinical characteristics(tumor size,depth of invasion,lymph node involvement,tumor-node-metastasis stage,perineural invasion,and vascular invasion),and was related to poor prognosis.USP15 knockdown significantly inhibited cell proliferation,invasion and epithelialmesenchymal transition(EMT)of GC in vitro,while overexpression of USP15 promoted these processes.Knockdown of USP15 inhibited tumor growth in vivo.Mechanistically,RNA sequencing analysis showed that USP15 regulated the Wnt signaling pathway in GC.Western blotting confirmed that USP15 silencing led to significant down-regulation ofβ-catenin and Wnt/β-catenin downstream genes(c-myc and cyclin D1),while overexpression of USP15 yielded an opposite result and USP15 mutation had no change.Immunofluorescence indicated that USP15 promoted nuclear translocation ofβ-catenin,suggesting activation of the Wnt/β-catenin signaling pathway,which may be the critical mechanism promoting GC progression.Finally,rescue experiments showed that the effect of USP15 on gastric cancer progression was dependent on Wnt/β-catenin pathway.CONCLUSION USP15 promotes cell proliferation,invasion and EMT progression of GC via regulating the Wnt/β-catenin pathway,which suggests that USP15 is a novel potential therapeutic target for GC.

Regulated genes in mesenchymal stem cells and gastric cancer

AIM: To investigate the genes regulated in mesenchymal stem cells(MSCs) and diffuse-type gastric cancer(GC),gene expression was analyzed. METHODS: Gene expression of MSCs and diffuse-type GC cells were analyzed by microarray. Genes related to stem cells, cancer and the epithelial-mesenchymal transition(EMT) were extracted from human gene lists using Gene Ontology and reference information. Gene panels were generated, and messenger RNA gene expression in MSCs and diffuse-type GC cells was analyzed. Cluster analysis was performed using the NCSS software.RESULTS: The gene expression of regulator of G-protein signaling 1(RGS1) was up-regulated in diffuse-type GC cells compared with MSCs. A panel of stem-cell related genes and genes involved in cancer or the EMT were examined. Stem-cell related genes, such as growth arrest-specific 6, musashi RNA-binding protein 2 and hairy and enhancer of split 1(Drosophila), NOTCH family genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated, and genes related to stem cells and NOTCH signaling are altered in diffuse-type GC compared with MSCs.

Ubiquitin-like modifier activating enzyme 2 promotes cell migration and invasion through Wnt/β-catenin signaling in gastric cancer

AIM To investigate the function and mechanism of ubiquitinlike modifier activating enzyme 2(Uba2) in progression of gastric cancer(GC) cells.METHODS Uba2 level in patients with GC was analyzed by Western blotting and immunohistochemistry. MTT and colony formation assays were performed to examine cell proliferation.Flow cytometry was used for cell cycle analysis.Wound healing and Transwell assays were conducted to examine the effects of Uba2 on migration and invasion.Expression levels of cell cycle-related proteins, epithelial-mesenchymal transition(EMT) biomarkers, and involvement of the Wnt/β-catenin pathway was assessed by Western blotting. Activation of the Wnt/β-catenin pathway was confirmed by luciferase assay.RESULTS Uba2 expression was higher in GC than in normal tissues.Increased Uba2 expression was correlated with tissue differentiation, Lauren's classification, vascular invasion,and TNM stage, as determined by the analysis of 100 GC cases(P < 0.05). Knock-down of Uba2 inhibited GC cell proliferation, induced cell cycle arrest, and altered expression of cyclin D1, P21, P27, and Bcl-2, while upregulation of Uba2 showed the opposite effects. The wound healing and Transwell assays showed that Uba2 promoted GC cell migration and invasion. Western blotting revealed alterations in EMT biomarkers, suggesting the role of Uba2 in EMT. Furthermore, the luciferase reporter assay indicated the involvement of the Wnt/β-catenin signaling pathway as a possible modulator of Uba2 oncogenic functions.CONCLUSION Uba2 plays a vital role in GC cell migration and invasion,possibly by regulating the Wnt/β-catenin signaling pathway and EMT.

Circ_0003356 suppresses gastric cancer growth through targeting the miR-668-3p/SOCS3 axis

BACKGROUND Circular RNAs(circRNAs)have attracted extensive attention as therapeutic targets in gastric cancer(GC).Circ_0003356 is known to be downregulated in GC tissues,but its cellular function and mechanisms remain undefined.AIM To investigate the role of circ_0003356 in GC at the molecular and cellular level.METHODS Circ_0003356,miR-668-3p,and SOCS3 expression were assessed via quantitative real time-polymerase chain reaction(qRT-PCR).Wound healing,EdU,CCK-8,flow cytometry and transwell assays were used to analyze the migration,proliferation,viability,apoptosis and invasion of GC cells.The subcellular localization of circ_0003356 was monitored using fluorescence in situ hybridization.The interaction of circ_0003356 with miR-668-3p was confirmed using RIP-qRT-PCR,RNA pull-down,and dual luciferase reporter assays.We observed protein levels of genes via western blot.We injected AGS cells into the upper back of mice and performed immunohistochemistry staining for examining E-cadherin,N-cadherin,Ki67,and SOCS3 expressions.TUNEL staining was performed for the assessment of apoptosis in mouse tumor tissues.RESULTS Circ_0003356 and SOCS3 expression was downregulated in GC cells,whilst miR-668-3p was upregulated.Exogenous circ_0003356 expression and miR-668-3p silencing suppressed the migration,viability,proliferation,epithelial to mesenchy-mal transition(EMT)and invasion of GC cells and enhanced apoptosis.Circ_0003356 overexpression impaired tumor growth in xenograft mice.Targeting of miR-668-3p by circ_0003356 was confirmed through binding assays and SOCS3 was identified as a downstream target of miR-668-3p.The impacts of circ_0003356 on cell proliferation,apoptosis,migration,invasion and EMT were reversed by miR-668-3p up-regulation or SOCS3 downregulation in GC cells.CONCLUSION Circ_0003356 impaired GC development through its interaction with the miR-668-3p/SOCS3 axis.

槲皮素抑制胃癌上皮-间质转化的研究

目的:研究槲皮素对TGFβ-1诱导的人胃癌BGC-803细胞上皮间质转化的影响及机制。方法:将胃癌BGC-803细胞分为3组,分别为正常对照组、TGFβ-1组、TGFβ-1+槲皮素组,分别在第12、24、48和72h后,采用MTT法检测各组细胞增殖能力,划痕修复实验检测各组细胞迁移能力;并采用Western blot实验检测上皮间质转化因子,E-cadherin、N-cadherin和Vimentin的表达情况。同时对PI3K/AKT信号转导通路的活性进行评价。结果:与TGFβ-1组相比,槲皮素降低了人胃癌BGC-803细胞的增值能力、迁移能力;TGFβ-1+槲皮素组E-cadherin表达水平上调,N-cadherin、Vimentin的表达下调。TGFβ-1+槲皮素组的AKT、p-AKT表达水平下调,PI3K/AKT信号转导通路活性降低。结论:槲皮素能够抑制TGFΒ-1诱导的胃癌细胞系的EMT过程,其发生机制可能与调控PI3K/AKT信号转导通路活性有关。

CDX2 Inhibits Invasion and Migration of Gastric Cancer Cells by Phosphatase and Tensin Homologue Deleted from Chromosome 10/Akt Signaling Pathway

Background： Gastric cancer （GC） is one of the most prevalent malignancies in the world today, with a high mortality rate. CDX2 is a Drosophila caudal-related homeobox transcription factor that plays an important role in GC. Phosphatase and tensin homologue deleted from chromosome 10 （PTEN） is an important tumor suppressor which is widely expressed in normal human tissues. The aim of the study was to determine the relationship and mechanism between CDX2 and PTEN in invasion and migration of GC cells.Methods： pcDNA3-CDX2 plasr^lids were transfected into MGC-803 cells to up-regulate CDX2 protein, and small interfering RNA-CDX2 was transfected to down-regulate CDX2. The influence of CDX2 or PTEN on cell migration and invasion was measured by invasion, migration and wound healing assays. Western blotting assay and immunofluorescence were used to detect the expression ofCDX2, PTEN, phosphorylation ofAkt, E-cadherin and N-cadherin. Statistical significance was determined by one-way analysis of variance.Results： The results showed that CDX2 reduced the migration and invasion of GC cells （P 〈 0.05）, and inhibited the activity of Akt through down-regulating PTEN expression （P 〈 0.05）. CDX2 also restrained epithelial-mesenchymal transition of GC cells.Conclusions： CDX2 inhibited invasion andmigration of GC cells by PTEN/Akt signaling pathway, and that may be used for potential therapeutic target.

N6-methyladenosine modification of CENPF mRNA facilitates gastric cancer metastasis via regulating FAK nuclear export

Background:N6-methyladenosine(m^(6)A)modification is the most common modification that occurs in eukaryotes.Although substantial effort has been made in the prevention and treatment of gastric cancer(GC)in recent years,the prognosis of GC patients remains unsatisfactory.The regulatory mechanism between m^(6)A modification and GC development needs to be elucidated.In this study,we examined m^(6)A modification and the downstream mechanism in GC.Methods:Dot blotting assays,The Cancer Genome Atlas analysis,and quantitative real‑time PCR(qRT-PCR)were used to measure the m^(6)A levels in GC tissues.Methylated RNA-immunoprecipitation sequencing and RNA sequencingwere performed to identify the targets ofm^(6)Amodification.Western blotting,Transwell,wound healing,and angiogenesis assays were conducted to examine the role of centromere protein F(CENPF)in GC in vitro.Xenograft,immunohistochemistry,and in vivo metastasis experiments were conducted to examine the role of CENPF in GC in vivo.Methylated RNA-immunoprecipitation-qPCR,RNA immunoprecipitation-qPCR and RNA pulldown assays were used to verify the m^(6)A modification sites of CENPF.Gain/loss-of-function and rescue experiments were conducted to determine the relationship between CENPF and the mitogen-activated protein kinase(MAPK)signaling pathway in GC cells.Coimmunoprecipitation,mass spectrometry,qRT-PCR,and immunofluorescence assays were performed to explore the proteins that interact with CENPF and elucidate the regulatory mechanisms between them.Results:CENPF was upregulated in GC and facilitated the metastasis of GC both in vitro and in vivo.Mechanistically,increasedm^(6)A modification of CENPF was mediated by methyltransferase 3,and this modified molecule could be recognized by heterogeneous nuclear ribonucleoprotein A2/B1(HNRNPA2B1),thereby promoting its mRNA stability.In addition,the metastatic phenotype of CENPF was dependent on the MAPK signaling pathway.Furthermore,CENPF could bind to FAK and promote its localization in the cytoplasm.Moreover,we discovered that high expression of CENPF was related to lymphatic invasion and overall survival in GC patients.Conclusions:Our findings revealed that increased m^(6)A modification of CENPF facilitates the metastasis and angiogenesis of GC through the CENPF/FAK/MAPK and epithelial-mesenchymal transition axis.CENPF expression was correlated with the clinical features of GC patients;therefore,CENPF may serve as a prognostic marker of GC.