| Gastric cancer(GC)is the fifth most common malignancy,and the third leading cause of cancer-related deaths(723,100 in 2012)globally.Currently,a series of risk factors,such as dietary factors,Epstein-Barr virus,and Helicobacter pylori infection have been identified for GC.Moreover,an increasing amount of evidence show that genetic factors and their downstream effects on cellular processes play important roles in GC progression,but,the pathogenesis of GC still remains complex.Additionally,despite the advances in cancer diagnosis and treatment,the prognosis of GC patients still remains unsatisfactory with the 5-year survival rate less than 30% due to its aggressive behavior.Hence,it is urgent to identify novel biomarkers and effective therapeutic targets for patients with GC.With the development of bioinformatics and genomic sequencing,there is hope for further exploration of tumor pathogenesis,diagnosis and treatment,and prevention.TNF-like ligand 1A(TL1A;also known as TNFSF15 or VEGI),produced by endothelial cells,T cells,macrophages,monocytes and dendritic cells,which inhibits proliferation and angiogenesis of endothelial cells.TL1 A has been reported to be involved in modulating vascular homeostasis and inflammation.Previous studies have reported that TL1 A plays an important role in a variety of autoimmune inflammatory diseases,such as rheumatic arthritis(RA),asthma and inflammatory bowel disease.Additionally,TL1 A is involved in the development of multiple tumor types,and is recognized as the potential therapeutic target for cancer therapy.Recently,reduced TL1 A expression was found to be associated with poor prognosis in breast cancer patients.However,the expression of TL1 A in gastric cancer and its role in tumorigenesis and development are still unclear and rarely reported.Objective: In this study,we aim to express TL1 A in gastric cancer tissue by using oncological databases and bioinformatics methods,as well as immunohistochemistry,Western Blot,qPCR and other molecular biology experimental techniques.The relationship with clinical pathological factors and prognosis were analyzed to clarify therole of TL1 A in the development of gastric cancer.In addition,The effect of TL1 A on the biological behavior of gastric cancer cells was studied in vitro.The purpose was to clarify the possible mechanism of TL1A’s role in gastric cancer and provide new targets for the diagnosis and treatment of gastric cancer.Methods: 1.Retrieving the expression data of RNA-seq gene in tissue samples of gastric cancer and normal tissue samples adjacent to carcinoma by using the Tumor Genome Atlas(TCGA),GEPIA,and UALCAN online database,and comparative TL1 A in gastric cancer tissue and normal tissue adjacent to carcinoma mRNA expression level differences.The relationship between TL1 A and clinical pathological factors was analyzed and evaluated on the Kaplan Meier website(Http://kmplot.com)to evaluate the value of TL1 A gene in gastric cancer.2.Collected 104 cases of gastric adenocarcinoma patients surgical treatment in the past three years and 34 cases of normal tissue adjacent to carcinoma(5 cm from the cancer tissue)paraffin buried specimens.The expression of TL1 A was detected by immunohistochemical staining,and the relationship between TL1 A and clinical pathological factors was analyzed.In addition,20 specimens of fresh gastric cancer tissue were collected for surgical resection in 2017,and the expression of mRNA and protein levels was measured using Western Blot and real time fluorescence quantitative PCR(qPCR).3.We observed the expression and localization of TL1 A in gastric cancer cell lines AGS,MGC803,BGC823,SGC7901,HGC27 and normal gastric epithelial cell lines GES-1 by immunofluorescence staining and laser scanning confocal fluorescence microscopy.4.After understanding the expression of TL1 A in the tissue of gastric cancer and its relevance to clinicopathology,in order to further confirm the expression of TL1 A,it is also the next step in the study of biological behavior and molecular biology functions in vitro.Western Blot and qPCR were used to detect expression of AGS,MGC803,BGC823,SGC7901,HGC27 and GES-1 TL1 A in normal gastric epithelial cells.The above are the clear expression of TL1 A in gastric cancer and its relationship with the development of gastric cancer.5.In order to further clarify the biological function of TL1 A on gastric cancer cells,we used two strains of AGS and MGC803 that were significantly higher in the Western Blot test results to transfect TL1 A shRNA(3 items)and over expression TL1 A plasmid respectively.The transfection efficiency was observed and verified by fluorescence microscopy,qPCR and Western Blot.Select sh1-TL1 A,sh3-TL1 A two shRNAs and over expression TL1 A plasmid for cellular function detection.6.In order to further confirm,we selected the TL1A-shRNA with the best knockdown efficiency to package into lentivirus,established AGS cell line with stable knockdown of TL1 A,and conducted further observation of cell function.7.In order to further study the mechanism of TL1 A in gastric cancer,we used two shRNA knockdown plasmids and TL1 A overexpression plasmids to transfect AGS and MGC803 cells bidirectional,and detected proteins that play an important role in tumor biological functions by Western Blot.8.The change trend of protein is closely related to the PI3K/Akt signal pathway,we use the Western Blot detection of PI3K/Akt pathway related proteins,and used the P13K/Akt signal pathway protein kinase inhibitor LY294002 to intervene.The effects of CCK8,plate cloning,and transcell experiments on cell proliferation and migration in gastric cancer were detected.Western Blot detected changes in related proteins to clarify the mechanism of TL1 A in gastric cancer.Results : 1.Based on the TCGA database,we collected the RNA-seq gene expression data and clinical data from 375 patients with gastric cancer tumor and 50 normal gastric tissue samples.The multidimensional scaling(MDS)analysis of the samples revealed that the tumor samples were concentrated in the middle of the image in compared to the normal controls.Moreover,TL1 A expression in GC samples was higher than that in normal controls(Log FC =1.07),and the difference was significant(P<0.05,P=8.90E-07).Based on the GEPIA online database,408 gastric cancer tissue samples and211 normal tissue samples were retrieved,and TL1 AmRNA expression in gastric cancer tissue samples was higher than the adjacent normal tissues.The difference wasstatistically significant(P < 0.01).In addition,The expression of TL1 A was not statistically significant to the clinical staging of gastric cancer.Using the UALCAN online database to retrieve gastric cancer tissue samples and adjacent normal tissue samples,to analyze the relationship between TL1 A expression and clinicopathological factors of gastric cancer.The results showed that the expression of TL1 A was related to the degree of gastric cancer differentiation and the state of Helicobacter pylori infection.The difference is statistically significant(P<0.01,P<0.01).To evaluate the prognostic value of the screened genes,survival analysis was performed,and to improve the reliability of the results with larger samples,we performed a KM plot analysis(http://kmplot.com)using a JetSet best probe set(Only including HGU133 PLUS 2.0microarray data),based on the datasets,GSE14210,GSE15459,GSE22377,GSE29272,GSE51105,and GSE62254.876 patients were divided into the high and low groups based on the gene expression median.The results showed that the patients with high TL1 A levels had shorter overall survival(OS)than those with low expression(P <0.01,P=2.6e-07).The results of these bioinformatics analysis fully show that TL1A’s expression in gastric cancer is of great significance.2.Immunohistochemical staining showed that TL1 A was expressed in cell membrane,cytoplasm and nucleus in 104 gastric cancer tissues collected,among which76 cases had high expression of TL1 A and 28 cases had low expression.The expression of TL1 A in 34 patients with paracancer normal tissues was mainly expressed by cell membrane and cytoplasm,among which the expression of TL1 A was low in 32 patients and high in 2 patients.The expression of TL1 A in gastric cancer was significantly higher than that in adjacent normal tissues(P<0.05).In addition,it was noted in the staining results that the lower the differentiation of gastric cancer,the higher the expression of TL1 A.Western Blot and qPCR results also showed that the expression of TL1 A in gastric cancer tissues was significantly higher than that in paired adjacent normal tissues(P<0.01,P<0.01),suggesting that the increase of TL1 A might be related to the occurrence of gastric cancer.Further,we analyzed the correlation between TL1 A expression and various clinicopathological factors of gastric cancer,and the analysisresults confirmed the correlation between the expression of TL1 A and the differentiation degree and tumor size of gastric cancer,with no other statistical significance.Consistent with the results of bioinformatics analysis,TL1 A was suggested to be related to the development of gastric cancer.3.The application of immunofluorescence staining and laser scanning confocal fluorescence microscopy showed that the TL1 A protein is mainly located in the cell membrane and cytoplasm in GES-1.In other gastric cancer cells,AGS,MGC803,BGC823,SGC7901,and HGC27,cytoplasms were predominant,and cell membranes and nucleus were also expressed.In addition,TL1 A has high expression in gastric cancer cell lines.The results were consistent with the results of immunohistochemical staining.4.Western Blot and qPCR were used to further detect the expression of TL1 A in gastric cancer cell lines AGS,MGC803,BGC823,SGC7901,HGC27 and GES-1(normal gastric epithelial cell lines).The gene and protein of TL1 A in AGS and MGC803 cells were significantly higher than that of GES-1.We also examined the expression of TL1 A in cytoplasmic protein and nuclear protein isolated and extracted from various gastric cancer cell lines,and the results showed consistent with the trend of total protein,further confirming the expression of TL1 A in cytoplasm and nucleus of gastric cancer.5.When TL1 A in AGS cells was down-regulated,the knockdown efficiency of 3TL1A-shRNA(sh1-TL1 A,sh2-TL1 A,sh3-TL1A)groups was 0.1626 ± 0.04,2.729 ±0.41,and 0.3037±0.04,respectively,compared with the negative control shRNA(sh-NC)group(P < 0.01).The protein level was consistent with the results.When TL1 A was down-regulated in MGC803 cells,the knockdown efficiency of 3 TL1A-shRNA(sh1-TL1 A,sh2-TL1 A,sh3-TL1A)groups was 0.0767±0.06,3.156±0.58,0.3136±0.013,respectively,compared with that of the negative control shRNA(sh-NC)group(P< 0.01).The protein level was consistent with the results.Therefore,we selected sh1-TL1 A and sh3-TL1 A for functional detection in the following experiments.When TL1 A was up-regulated in AGS and MGC803 cells,the transfection efficiency of TL1 A overexpressed plasmid pHBLV-TL1A(OE-TL1A)group was 623.7±23.74,29.82±10.3,respectively,compared with the pHBLV empty vector(OE-NC)group(P < 0.01,P <0.01).The protein level was consistent with the results.Through CCK8 and plate cloning and Transwell assay,it was concluded that TL1 A could promote the proliferation and migration of gastric cancer cells.6.In order to further confirm,we selected the TL1A-shRNA with the best knockdown efficiency to package into lentivirus,established AGS cell line with stable knockdown of TL1 A.The mRNA knockdown efficiency was 0.3756±0.077.The protein level verification results are consistent.Use AGS cell line with stable knockdown of TL1 A and co-transfected with TL1 A overexpression plasmid,which were divided into four groups: LV-NC-NC,LV-NC-TL1 A,LV-TL1A-RNAi-NC,LV-TL1A-RNAi-TL1 A for TL1 A expression reply,further verifying the biological function of TL1 A in gastric cancer cell lines,and consistent results were obtained.Therefore,we determined that TL1 A is overexpressed in gastric cancer and promotes the proliferation and migration of gastric cancer.Therefore,it is of great significance to further study TL1 A.7.In order to further study the mechanism of TL1 A in gastric cancer,we transfected AGS and MGC803 cells with two shRNA knockdown plasmids and TL1 A overexpression plasmid,and then detected a series of proteins that play an important role in various biological functions of the tumor by Western Blot.The expressions of PCNA,CDK2,ROCK1 and RhoA in AGS and MGC803 gastric cancer cells were decreased,while the expressions of p21 and p27 were increased.Compared with OE-NC group,PCNA,CDK2,ROCK1 and RhoA in the over-expressing TL1 A group were increased,while p21 and p27 were decreased.These differential proteins were found to be closely related to the PI3K/Akt signaling pathway.8.We used Western Blot to detect PI3K110β and PI3K85α and p-Akt in the PI3K/Akt signal pathway.The results showed that compared with the negative control group shRNA(sh-NC),the gastric cancer cells AGS and MGC803 of TL1A-shRNA(sh1-TL1 A,sh3-TL1A)were transferred,and the expression of PI3K110β,PI3K85α,p-Akt after TL1 A was knocked down was also reduced.Compared with the OE-NC group,the expression of PI3K110β,PI3K85α and p-Akt in the over-expressed TL1 A group was also increased.The results indicated that TL1 A promoted theproliferation and migration of gastric cancer and was related to the PI3K/Akt signaling pathway.To further confirm,we applied the protein kinase inhibitor LY294002 of PI3K/Akt cell signaling pathway.The results confirmed that in the DMSO control group,TL1 A overexpression promoted the proliferation of AGS and MGC803 in gastric cancer cells.After the use of the PI3K/Akt signaling protein kinase inhibitor LY294002,the growth and migration of the cells were inhibited.Western Blotting results showed that:In the DMSO control group,after TL1 A overexpression,the expressions of PI3K110 beta,PI3K85 a,p-Akt,PCNA,CDK2,ROCK1 and RhoA were also increased,and the expressions of p21 and p27 were decreased.These expression changes were inhibited after the use of PI3K/Akt signaling protein kinase inhibitor LY294002.The above experimental results confirmed that TL1 A could promote the proliferation and migration of gastric cancer cells through the PI3K/Akt signaling pathway.Conclusion : 1.TL1 A is mainly located in cytoplasm and nucleus and highly expressed in gastric cancer.2.The high expression of TL1 A is positively correlated with the differentiation degree and tumor size of patients with gastric cancer,and the high expression of TL1 A was negatively correlated with the survival rate of gastric cancer patients.3.TL1 A promotes proliferation and migration of gastric cancer.4.TL1 A promotes the proliferation and migration of gastric cancer by promoting the PI3K/Akt signaling pathway. |
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