The Mechanism Of YWHAZ Regulating Migration And Invasion Of Gastric Cancer Cells

Research background:Gastric cancer(GC)is a malignant tumor originating from gastric epithelial cells.It is also one of the most common malignant tumors in the world.Because of its insidious onset,early symptoms are not obvious,so patients have developed symptoms when they seek medical diagnosis.In the middle and late stages of gastric cancer,it manifests as migration,invasion and distant metastasis of surrounding tissues of the tumor,affecting the comprehensive treatment effect of surgery and postoperative chemotherapy,resulting in poor prognosis and low survival rate[1,3,33]。.Metastasis of malignant tumors is a very complex process,including local invasion of the primary tumor.Tumor cells invade the blood vessels and survive in the blood pool,invading the distant organs with the blood circulation and colonizing the tumor.Migration and invasion are particularly important as the initial link of tumor metastasis[19].The results of the research to date show that the YWHAZ gene plays an important role in the migration and invasion of various malignant tumor[20].In 2010,Japanese scholars first proposed that the down-regulation of Micro RNA-375 in gastric cancer tissues regulates cell survival by targeting YWHAZ gene.Subsequent studies by other scholars have shown that YWHAZ protein overexpression is associated with tumor size,venous and lymphatic invasion,depth of invasion,pathological stage and recurrence rate,but not related to histological classification;and in vitro cell experiments confirmed the migration of YWHAZ gene in gastric cancer.And has an important role in invasion[31,32].In recent years,research in this field has gradually attracted the attention of scholars at home and abroad.However,no research report on the molecular mechanism of YWHAZ gene regulation of gastric cancer migration and invasion has been reported so far.Objetive:In this study,we attempted to clarify the effects of YWHAZ on the biological behavior of gastric cancer cells(apoptosis,autophagy,invasion,migration,EMT,chemosensitivity and tumorigenicity)in vivo and in vitro.This study elucidates the molecular mechanism of the mir-375/YWHAZ axis in the migration and invasion of gastric cancer by targeting knockdown the YWHAZ gene to inhibit the migration and invasion of gastric cancer,and provides reliable experimental data for translational medical research.Materials and methods:1、Materials:Fresh GC tissue samples and normal adjacent tissues were collected from 28 patients,Healthy female nude BALB/c mice(4-week-old).Cell lines,miR-375 mimics,mimics control,miR-375 inhibitor,and inhibitor control.2、Methods:(1)YWHAZ m RNA expression in Fresh GC tissue and normal adjacent tissues was measured by q PCR.The protein levels in Fresh GC tissue samples and normal adjacent tissues were assessed by western blot assay.(2)Knockdown of YWHAZ inhibited the migration,invasion and EMT of GC cells: The stable YWHAZ-silenced cells were established by transfection of YWHAZ sh RNA.The migration capability of BGC-823 cells was assessed by scratch assay.The invasion capability of BGC-823 cells was determined by transwell assay.The levels of EMT-related proteins E-cadherin,N-cadherin and Vimentin in GC cells were assessed by western blot assay.(3)In vivo tumor xenograft study:Selection and establishment of stable YWHAZ silencing cell line by G-418.Different groups of gastric cancer cells were inoculated subcutaneously into the axilla of mice to observe the state of nude mice,the time and size of tumorigenesis.(4)Knockdown of YWHAZ increased the chemosensitivity of BGC-823 cells to cisplatin: The cell viability of BGC-823 cells after treatment with various concentrations of cisplatin for 24 h was evaluated by MTT assay.(5)The study on the Targeted Regulation of YWHAZ Gene by Micro RNA-375: These oligonucleotides were transiently transfected into GC cell lines by Lipofectamine 2000(Invitrogen,USA).GC cells were collected for further tests after the transfection for 48 h.The wide-type(WT)or mutant type(MUT)3’-untranslated region(3’-UTR)of YWHAZ was inserted into control plasmid.Then 293 T cells were transiently cotransfected with miR-375 mimics or mimics control,and firefly luciferase plasmidcontaining WT or MUT 3’-UTR of YWHAZ by Lipofectamine 2000.After transfection of 48 h,the cells were lysed and luciferase activities were detected using the Dual-Luciferase Assay kit.(6)Effects of micro RNA-375/YWHAZ/β-catenin on migration,invasion and EMT of GC:Western blot was used to detect the expression of β-catenin in YWHAZ silenced cell lines,and immunofluorescence staining was used to detect the expression of β-catenin in GC cells.The YWHAZ silenced cell lines were transfected with micro RNA-375 Minics or micro RNA-375 inhibitor.Scratch assay was used to evaluate cell migration ability,Transwell assay was used to evaluate the invasive ability of BGC-823 cells,and Weston blot was used to detect EMT-related proteins.Results1 、 the protein level of YWHAZ was upregulated in most tumor tissues compared with the adjacent normal tissues(P<0.001).Moreover,the m RNA expression of YWHAZ in tumor tissues was also significantly increased(P<0.05).2、Knockdown of YWHAZ significantly inhibited the cell migration(P<0.001)and invasion(P<0.01)in BGC-823 cells.Moreover,the levels of EMT-related proteins E-cadherin,N-cadherin and Vimentin in BGC-823 cells were assessed by western blot assay.Silence of YWHAZ up-regulated the protein level of E-cadherin(P<0.001),and down-regulated the protein levels of N-cadherin(P<0.005)and Vimentin(P<0.01)in BGC-823 cells.3、Cisplatin markedly inhibited the viability of BGC-823 cells in a dose-dependent manner and suppression of YWHAZ enhanced the inhibitory effect of cisplatin in BGC-823 cells compared with NC group.Knockdown of YWHAZ further promoted cisplatin-induced apoptosis in BGC-823 cells compared with NC group.4、Knockdown of YWHAZ restrained the activation of wnt/β-catenin pathway: Suppression of YWHAZ significantly decreased the nuclear level of β-catenin in BGC-823 and MGC-803 cells.As detected by immunofluorescent assay,the expression and nuclear accumulation ofβ-catenin were obviously suppressed by YWHAZ silencing in GC cells 5 、 miR-375 targeted YWHAZ in GC cells: the expression of miR-375 was raised by transfection with miR-375 mimics,and reduced by miR-375 inhibitor.Transfection with miR-375 mimics significantly reduced the m RNA expression of YWHAZ,while miR-375 inhibitor effectively increased the m RNA expression of YWHAZ in GC cells.Consistent with this result,the protein level of YWHAZ was restrained by miR-375 mimics,while enhanced by miR-375 inhibitor in BGC-823 and MGC-803 cells.the relative luciferase activity was evidently decreased in WT+miR-375 group in 293 T cells,which could be reversed in MUT+miR-375 group.6、miR-375 inhibited migration,invasion and EMT of GC cells via targeting YWHAZ : transfection with miR-375 inhibitor significantly promoted the migration and invasion abilities in GC cells,which could be inhibited by knockdown of YWHAZ.In addition,the EMT markers E-cadherin was downregulated,while N-cadherin and Vimentin were up-regulated by miR-375 inhibitor in GC cells.However,silencing of YWHAZ remarkably reversed the above changes.Transfection of miR-375 inhibitor resulted in increase in the nuclear level of β-catenin,whereas YWHAZ silencing could inhibit the nuclear accumulation of β-catenin in GC cells.Conclusion:1 、 Silencing of YWHAZ can promote apoptosis,autophagy and chemosensitivity of GC cells in vitro,and inhibit migration,invasion and EMT transformation of GC cells;can inhibit the tumorigenicity of GC cells in vivo.2、miR-375 / YWHAZ/β-catenin YWHAZ can regulating migration and invasion of gastric cancer cells.