| Backgroud:Gastric cancer(GC)is the fourth common cancer in the world,with high incidence and mortality as well as poor prognosis.Despite the steady decline in mortality in recent decades,gastric cancer is still one of the most lethal tumors,especially in patients with advanced gastric cancer with long-distance metastasis and recurrence,and its five-year survival rate is only about 30%.About two-thirds of patients had locally advanced or metastatic lesions at diagnosis,and up to half had recurrences after therapeutic surgery.The average survival time of these patients was only 6-9 months.This is mainly due to the following reasons:limited diagnostic strategies for early diagnosis;weak prognostic value of histological indicators;limited surgical or cytotoxic effects for advanced diseases;and lack of molecular markers for targeted therapy.Therefore,it is necessary to study the molecular mechanism of gastric cancer progression in order to find effective therapeutic strategies and new drug targets.MicroRNAs are endogenous RNA with a length of about 21-23 nucleotides.Up to now,it has been found that it plays an important role in many biological processes,such as cell proliferation,differentiation and canceration.There is abundant evidence that the expression of microRNA in various cancer tissues is imbalanced and can be used as a potential biomarker for different types of cancer.Especially,there are several abnormal expressions of microRNAs in tumour tissues and body fluids of patients with gastric cancer,which show good prospects in clinical application.miR-33 8-3p was initially identified as a specific microRNA expressed in human brain,which participates in the formation of basolateral polarity and the regulation of axonal respiration.Recent studies have extended its function to the field of cancer:miR-338-3p can be used as a tumor suppressor to inhibit the proliferation,migration and invasion of hepatocellular,breast and ovarian cancer.In gastric cancer tissues,miR-338-3p is also down-regulated,and the degree of decline is related to the stage of cancer,which suggests that miR-338-3p may be involved in the progress of gastric cancer.The down-regulation of the expression of miR-338-3p is related to the advanced pathological stage and lymph node metastasis of non-small cell lung cancer and gastric cancer,suggesting that miR-338-3p is involved in the progress of primary tumors and metastatic diseases.It has been reported that miR-338-3p inhibits epithelial-mesenchymal transition(EMT)in gastric cancer cells.EMT is a complex pathological process in cancer metastasis,characterized by the dedifferentiation of epithelial cells and the acquisition of mesenchymal phenotype.The newly transformed mesenchymal cells migrate,invade,resist apoptosis and release lysozyme to degrade extracellular matrix and basement membrane.It is well known that EMT is essential for embryonic development and cancer progression.It is characterized by loss of E-Cadherin and up-regulation of Vimentin expression.The formation of E-Cadherin/beta-catenin complex ensures the integrity of epithelium and stable transcription of cell junction.In addition,beta-catenin is considered to be the main protein in Wnt pathway.Wnt signal transduction is known to regulate many biological processes,including cell proliferation and cell polarity during embryonic development and tissue homeostasis.Typical Wnt signaling affects cadherin-mediated cell adhesion by down-regulating E-Cadherin,up-regulating adhesion molecules that are beneficial to cell movement,inducing protease and other EMT promoters.Erythropoietin-producing hepatocellular(Eph)receptors play a role in EMT of various cancers.EphA2,a member of the Eph family,promotes the Wnt/beta-catenin pathway in gastric cancer cells.In this study,we attempt to describe the role of miR-338 in inducing EMT in gastric cancer and its interaction with Wnt/beta-catenin pathway and EphA2.Objectives:In this study,we attempt to further explore the correlation between the expression of miR-338 gene and the occurrence and progression of gastric cancer,and describe the role of miR-338 in inducing EMT in gastric cancer and its interaction with Wnt/beta-catenin pathway and EphA2,so as to provide new theoretical basis for the development of targeted therapeutic drugs for gastric cancer patients.Methods:The subjects were randomly selected from 64 patients with gastric cancer who were treated in our hospital from March 2012 to June 2017.The gastric cancer tissues and the adjacent normal gastric tissues as well as metastatic lymph nodes of these patients were obtained.RT-PCR was used to detect and compare the expression of miR-338 in tissues.In addition,the expression levels of miR-338 in gastric cancer cell lines NCI-N87,AGS,MKN-45,SGC-7901 and human normal gastric epithelial cell line GES-1 were detected to study the potential relationship between its expression and the occurrence of gastric cancer.The correlation between the expression of miR-338 and clinicopathological parameters in 64 patients with gastric cancer was analyzed.According to the median expression level of miR-338 in gastric cancer tissues,the expression levels of miR-338 in gastric cancer tissues were divided into high-expression group and low-expression group.Statistical methods were used to analyze the correlation between the expression of miR-338 and clinicopathological features such as gender,age,smoking,alcohol consumption,differentiation,clinical stage and lymph node metastasis.The AGS gastric cancer cells were transfected with miR-338 mimics to overexpress miR-338,and the efficiency was tested by RT-PCR.MTT assay was used to analyze the effect of miR-338 overexpression on the viability and apoptosis of AGS gastric cancer cells.AGS cells were transfected with miR-338 mimimics before the proliferation of AGS cells was detected by MTT at 24 h,48 h and 72 h after transfection.We then chose 72 h after transfection as the optimal time to further evaluate the effect of miR-338 on apoptosis by Annexin-FITC/PI flow cytometry.Transwell assay was conducted to investigate its potential effect on cell invasiveness.Next,we studied whether microRNA-338 could regulate the Wnt/beta-Catenin signaling pathway.Western blotting was used to evaluate the expression of Wnt/beta-Catenin signaling pathway targets(including GSK-3beta,p-GSK-3betaSER9 and C-Myc)at protein level.In addition,the expression of EphA2 at protein level was detected to determine the effect of miR-338 on EphA2.To further investigate the inhibitory effect of miR-338 on invasion and migration of gastric cancer cells,we examined the effect of overexpression of miR-338 on the expression level of genes related to epithelial-mesenchymal transformation,including epithelial marker E-cadherin and mesenchymal marker vimentin.Based on the important role of E-cadherin/beta-Catenin complex in the development of EMT,we also examined whether the level of miR-338 affected the expression of beta-Catenin at protein level.E-cadherin,Vmentin and beta-catenin antibodies were used for immunoblotting analysis of lysates.Similarly,we targeted the expression of EphA2 by transfecting specific siRNA into cells,and determined the expression of EMT-induced markers E-Cadherin and Vimentin in cells after interfering with EphA2 by Western blotting.We then studied the effect of miR-338 on the growth of transplanted tumors in vivo.Forty BALB/C nude mice(4-6 weeks old)were randomly divided into four groups(including mimics control,miR-338 mimics,EphA2 + miR-338 mimics,EphA2 siRNA).Nude mice were subcutaneously inoculated with the same amount of AGS cells.Tumor size was measured at different time intervals(15 d,18 d,21 d,24 d,27 d,30 d)after inoculation.The expression of p-GSK-3beta,GSK-3beta and c-Myc proteins in mice xenografts was detected by Western blotting to reflect the activity of Wnt pathway.Results:Quantitative PCR showed that the expression of miR-338 was significantly down-regulated in gastric cancer tissues compared with adjacent normal tissues.At the same time,the expression of miR-338 in metastatic lymph nodes was significantly lower than that in primary gastric cancer.In addition,compared with normal gastric mucosal epithelial cells,the expression level of miR-338 in various gastric cancer cell lines was significantly lower.Among them,the expression of miR-338 in AGS cell line is the lowest,which was used for follow-up functional research.The correlation between the expression of miR-338 and clinicopathological characteristics in 64 patients with gastric cancer was analyzed.Statistical analysis showed that there was no significant correlation between the expression of miR-338 and gender,age,smoking and alcohol consumption.On the contrary,it was significantly correlated with the degree of differentiation,clinical stage and lymph node metastasis.The growth of AGS cells was significantly inhibited by enhanced expression of miR-338 in a time-dependent manner,and the cell apoptosis was promoted.In addition,the invasive ability of AGS cells transfected with miR-338 mimics was significantly inhibited.The up-regulation of miR-338 increased the level of E-cadherin protein and decreased the expression of Vimentin.In addition,the expression of beta-catenin protein in miR-338 transfected cells was lower than that in control cells(P<0.05).Thus,miR-338 can down-regulate the expression of beta-catenin and inhibit the process of EMT mediated by beta-catenin.The phosphorylated GSK-3 beta and c-Myc were decreased due to miR-338 overexpression,while the inactive GSK-3 beta expression level was higher(p<0.05).Notably,overexpression of microRNA-338 also led to a decrease in EphA2 level(P<0.05).In addition,E-Cadherin in gastric cancer cells was significantly increased with EphA2 suppression,and the level of Vimentin decreased significantly.In addition,the content of beta-Catenin in EphA2 gene silenced cells was lower than that in control cells(P<0.05).These results suggest that miR-338 inhibits Wnt/beta-Catenin pathway activity and down-regulates EphA2 in gastric cancer cells,thereby weakening the EMT process.After subcutaneous inoculation,the tumor size was measured at different time intervals(15 days,18 days,21 days,24 days,27 days,30 days).It was found that the overexpression of miR-338 significantly inhibited the growth of tumors in vivo.EphA2 overexpression plasmid was transfected into AGS cells overexpressing miR-338 at the same time,and then subcutaneously inoculated into mice.It was found that the overexpression of EphA2 could restore the growth of tumors.EphA2 interference group was also set up to inhibit the growth of tumors.Therefore,we speculate that mir-338 inhibits the growth of tumors by inhibiting the expression of EphA2 in vivo.The expression of phosphorylated GSK-3beta in allograft tumors over-expressed by miR-338 was significantly lower than that in control group,while the expression of unphosphorylated GSK-3beta was increased,and the expression of c-Myc was also decreased,which indicated that the activation of Wnt pathway could be inhibited by miR 338 in vivo.When EphA2 is overexpressed at the same time,this result will be reversed.Conclusion:In conclusion,our experiments confirm that the expression of miR-338 is down-regulated in gastric cancer tissues and cell lines,and its low expression is associated with differentiation,clinical stage and lymph node metastasis.In addition,miR-338 can inhibit the proliferation,invasion and EMT process of gastric cancer cells in vitro,and promote cell apoptosis,as well as inhibit the growth of tumors in vivo.miR-338 may inhibit cancer by targeting the expression of EphA2,thereby reducing the activity of Wnt/beta-Catenin pathway. |
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