| Although the incidence of gastric cancer is decreasing worldwide,it remains the most common type of cancer,and its mortality rate is still high.Even though a large number of routine and targeted therapies have been used in clinic,a large number of gastric cancer patients have been diagnosed with tumor metastasis.Therefore,exploring the important signaling pathways and transmission mechanisms related to tumor invasion and metastasis will be beneficial to early diagnosis and targeted therapy of gastric cancer.The Notch signaling pathway is evolutionarily conserved and plays an important role in cell differentiation,proliferation,apoptosis and self-renewal of stem cells.Studies have shown that Notch signaling pathway is closely related to tumor invasion,metastasis,recurrence,low survival rate and poor prognosis.In recent years,numerous studies have shown that Notch signaling pathway also plays an important role in the EMT process.Notch signaling pathway plays a role between adjacent cells and does not require the participation of second messengers.When the Notch receptor is activated after binding with the ligand Jagged1on the adjacent cell membrane,it releases the intracellular region of the Notch receptor（Notch intracellular domain,NICD）,which then turns to the nucleus and interacts with the transcriptional complex to activate the downstream target gene of the Notch pathway.Epithelial-mesenchymal transition（EMT）is an important way to express malignant behavior of epithelial cancer,and plays an important role in tumor invasion and metastasis.It is characterized by the destruction of epithelial cell connections,loss of cell polarity and epithelial phenotype,gaining a more mesenchymal-like phenotype.In addition,the most important change in the EMT process is that it confers increased invasion and migration ability to tumor cells.The deletion of the epithelial marker gene E-cadherin is a marked change in the EMT event,and the interstitial marker genes include Vimentin,N-cadherin and so on.The expression of these markers can be detected to verify the occurrence of EMT.Transforming growth factor-β1（TGF-β1）is a multi-functional cytokine that plays an important role in cell differentiation,proliferation,apoptosis and motor ability.It is expressed in a variety of pathological conditions,such as chronic inflammation and cancer.Studies have shown that in the tumor microenvironment,TGF-β1 can stimulate angiogenesis,promote the deposition of extracellular matrix and increase the synthesis of proteolytic enzyme.Multiple studies have confirmed that changes in the tumor microenvironments such as hypoxia and the increase of TGF-β1 can induce EMT in many types of epithelial cells.At present,the role of Notch signaling in gastric epithelial stroma has not yet been elucidated,and to explore the mechanism may be beneficial to the diagnosis and treatment of gastric cancer.ObjectiveThe expression of Jagged1,N1ICD,E-cadherin and Vimentin in the blank control group,the induction group,the negative control group and the transfected group were detected by the fluorescence quantitative PCR test（Real time-PCR）,the Western blot test,and the changes of the invasiveness of the cells before and after the treatment were detected by the Transwell invasion test.To explore the effect of Notch signaling in TGF-β1-induced EMT of gastric cancer and its influence on cell invasiveness.Materials and Methods1 MaterialsThe gastric cancer cell line SGC7901 was cultured in a constant temperature incubator at 37°C and 5%CO 2 with complete medium containing 9:1 RPMI 1640and FBS.2 MethodsWhen the cells was fused to 80%,they are randomly divided into four groups,blank control group,induction group,negative control group and transfected group.The blank control group was no treatment,induction group was treated with 10ng/ml TGF-β1 for 24 hours,the transfected group were transiently transfected with Jagged1siRNA,and the negative control cells were transfected with nonsense siRNA.After the tansfected group and negative control group were transfected for 48h,then they were induced by 10ng/ml of TGF-β1 for 24h.The m RNA expression and protein expression of E-cadherin,Vimentin Jagged1and N1ICD in each group were detected by Real-time PCR and Western Blot.Transwell invasion assay was used to detect the change of invasiveness in each group.3 Statistical analysisSPSS21.0 software was applicated for data processing and statistical analysis.Quantitative data were expressed by the（mean±SD）.Comparison of two groups was performed using Student’s t,and comparison of multi-groups were performed using one-way ANOVA.Significance was considered when P<0.05.Results1 Effect of TGF-β1 on the protein expression of E-cadherin and Vimentin in SGC7901 cellsThe results of Western Blot showed that the protein expression of E-cadherin in TGF-β1-inducing group was 0.15±0.12,which was significantly down-regulated than the blank control group（0.54±0.09）.The protein expression of Vimentin in the induced group was 0.81±0.20,which was significantly up-regulated than the blank control group（0.58±0.08）.The difference was statistically significant（P<0.05）.2 Effect of TGF-β1 on the expression of Jagged1 and N1ICD in SGC7901 cells2.1 Effect of TGF-β1 on the mRNA expression of Jagged1 and N1ICD in SGC7901cellsAfter the induction of TGF-beta 1,the results of fluorescence quantitative PCR showed that the relative expression level of Jagged1 mRNA in the induced group was1.59+0.20.Compared with the blank control group,the mRNA expression level of Jagged1 in the induced group was up-regulated,and the difference was statistically significant（P<0.05）.2.2 Effect of TGF-β1 on the protein expression of Jagged1 and N1ICD in SGC7901cellsThe protein expression level of Jagged1 and N1ICD detected by Western Blot showed that the relative expression of Jagged1 protein in the blank control group was0.36+0.22,and the induced group was 0.61+0.20.The expression level of Jagged1protein in the induced group was higher than the blank control group（P<0.05）.The protein expression level of N1ICD in the induced group was 0.74+0.19,which was higher than that in the blank control group（0.30+0.18）,and the difference was statistically significant（P<0.05）.3 The expression of N1ICD,E-cadherin and Vimentin in SGC7901 cells after the transfection and induction3.1 The mRNA expression of Jagged1,E-cadherin and Vimentin after the transfection and inductionAfter transient transfection of Jagged1 siRNA to SGC7901 cells and then induced by TGF-β1,the relative expression level of Jagged1,E-cadherin and Vimentin mRNA was detected by fluorescence quantitative PCR.The results showed that the relative expression level of Jagged1 mRNA in the transfected group was 0.61±0.04,which was down-regulated comparison with the negative control group（1.48±0.03）,the induction group（1.36±0.18）and the blank control group（1.00±0.00）.The relative expression of E-cadherin mRNA in the transfected group was 1.64±0.03,which was up-regulated comparison with the negative control group（0.38±0.12）and the induction group（0.40±1.48）and the blank control group（1.00±0.00）.The relative expression of Vimentin mRNA in the transfected group was 0.43±0.28,which was down-regulated compared with the negative control group（1.67±0.04）,the induction group（1.61±0.03）and the blank control group（1.00±0.00）.And the difference was statistically significant（P<0.05）.3.2 The protein expression of N1ICD,E-cadherin and Vimentin after the transfection and inductionThe protein expression of N1ICD,E-cadherin and Vimentin was detected by Western Blot.The results showed that the protein expression level of N1ICD in the transfected group was 0.22±0.12,which was more lower than the negative control group（0.79±0.30）,the induction group（0.77±0.19）and the blank control group（0.41±0.21）.The protein expression level of E-cadherin in the transfected group was0.95±0.19,which was more higher than the negative control group（0.15±0.27）,the induction group（0.18±0.13）and the blank control group（0.47±0.02）.The protein expression of Vimentin in the transfected group was 0.21±0.32,which was down-regulated compared with the negative control group（0.82±0.19）,the induced group（0.79±0.26）and the blank control group（0.54±0.18）.The difference was statistically significant（P<0.05）.4 The changes in the invasiveness of SGC7901 cells in each treatment groupTranswell invasion assay was used to detect the invasiveness of SGC7901 cells in each group.The number of invasived cells in induction group was 74.67±2.60,and the blank control group was 55.0±2.65.The number of invasived cells in the induction group was significantly higher than the control group（P<0.05）.The number of invasived cells in transfected group was 36.33±4.73,the negative control group was 77.33±3.79.Compared with the negative control,the number of invasived cells in the transfected group significantly decreased,the difference was statistically significant（P<0.05）.ConclusionsTGF-β1 may induce EMT of gastric cancer cells by enhancing the Notch signaling pathway,and enhance the invasiveness of gastric cancer cells,which indicatd that Notch signaling pathway plays an important role in gastric cancer invasion. |
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