| Background:According to the World Health Organization(WHO),cancer is the second leading cause of death over the world.In 2018,9.6 million people died of cancer[1].According to the International Agency for Research on Cancer(IARC),gastric cancer ranks the fifth in the new cases and the third in the fatal cases of cancer worldwide According to the statistics of the China's National Cancer Center,gastric cancer ranks the second of incidence and the third of mortality in China In clinical diagnosis and treatment,molecular markers such as carcinoembryonic antigen(CEA),carbohydrate antigen 19-9(CA19-9)are applied to assist the clinical diagnosis of gastric cancer,but the specificity is poor Early gastric cancer is usually resected by endoscopy,and advanced gastric cancer is usually resected by laparotomy or laparoscopic surgery combined with postoperative adjuvant chemotherapy.In addition,radiotherapy,radiofrequency ablation.intraperitoneal perfusion and arterial embolization perfusion are more frequently used.However,the prognosis of gastric cancer patients remains unoptimistic.In terms of molecular targeted drug therapy or immunotherapy,molecular targeted drugs such as anti-HER2 Patuzumab,anti-angiogenesis Ramolumab have not been reported as an effective treatment of gastric cancer.The efficiency of immunotherapeutics represented by the Programmed death-1(PD-1)monoclonal antibody Pembrolizumab also remains controversial[5].Therefore,exploring new markers related to the progression of gastric cancer and clearinging the molecular mechanisms of cancer progression regulation may provide important clinical significance for early diagnosis,targeted therapies and prognosis improvement for gastric cancer patients.T-cell lymphoma invasion and metastasis inducing factor 1(Tiam1)was separated from mouse T-lymphoma in 1994 by Habets.The human Tiaml gene was located on chromosome 21 q22.1[6].Normally,Tiaml is relatively overexpressed in brain and testis,and lower expressed in bronchial mucosal epithelium,hepatocyte,myocardium and other tissues.Many studies shown that Tiaml has a dual role in tumor progression(cancer promotion and cancer inhibition):Many studies shown that Tiaml is highly expressed in lung adenocarcinoma[7],esophageal squamous cell carcinoma[8],pancreatic calcer[9],thyroid cancer[10],promoting tumor progressioniind closely related to poor prognosis of patients.On the other side,it has also been found that Tiaml is highly expressed in colon cancer and inhibits tumor progression[11].These studies suggest that Tiaml may invole in the progression of tumors,but its specific mechanism in tumor evolution needs further study.Our previous studies found that Tiam1 was overexpressed in gastric cancer tissues and may predict the poor prognosis of gastric cancer patients,but the molecular mechanisms of its involvement in regulating the progression of gastric cancer is still unclear.The relationship between Tiaml and clinicopathological parameters of gastric cancer needs to be further explored and verified.Objectives:(1)To investigate the expression of Tiam1 in gastric cancer,analyze the correlation between the Tiaml expression,clinicopathological parameters and the prognosis of gastric cancer patients;(2)To clarify the effects of Tiaml on the proliferation,epithelial-mesenchymal transition(EMT)and angiogenesis of gastric cancer cells;(3)To reveal the molecular mechanisms of Tiaml in the progression regulation of gastric cancer.To provide an effective molecular target for early screening and prognosis improvement of gastric cancer.Materials and methods:Materials:The gastric mucosa cell line GES-1,gastric cancer cell line AGS,BGC-823,SGC-7901,MKN-45,MKN-28,human umbilical vein endothelial cell line(HUVEC),human lymphatic endothelial cell line(HLEC)and other cell lines were used;209 paraffin samples and chips were collected,including 153 gastric cancer tissues and 56 adjacent tissues;70 fresh tissue samples were collected from gastric cancer patients,including 10 pairs of early gastric cancer and adjacent fresh tissues of 20 samples,10 pairs of mid-term gastric cancer and adjacent fresh tissues of 20 samples,10 groups of advanced gastric cancer,adjacent cancer and lymph node metastasis fresh tissues of 30 samples.Methods:(1)The expression of Tiaml protein in gastric cancer tissues was detected by retrieving database,and was further verified by immunohistochemistry,HE staining and Western blot assay:The Human Protein Atlas database,cBioPortal database.Oncomine database and GEO database were retrieved to analyze the expression of Tiam1 in gastric cancer tissues;Western blot,HE staining and immunohistochemistry were used to detect the expression of 70 fresh tissues.Immunohistochemical staining was used to detect the expression of Tiam1 in 209 paraffin and tissue chip samples,and analyze the correlation between Tiam1 expression and clinicopathological parameters;Kaplan Meier plotter database was uesd to evaluate the relationship between the expression of Tiaml and the survival of gastric cancer patients.(2)The effects of Tiaml on proliferation,metastasis,invasion and angiogenesis of gastric cancer cells were examined by lentivirus transfection,cloning,scratching,migration and invasion:Western blot was used to detect the expression of Tiaml in various gastric cancer cell lines,and lentivirus transfection was used to establish AGS/sh-Tiam1 cell lines with down-regulation of Tiam1 expression and MKN-45/ex-Tiam1 cell lines with up-regulation of Tiam1;Methyl thiazolyl tetrazohum(MTT)assay and cloning assay were used to detect the effect of Tiaml differential expression on the proliferation of gastric cancer cells;scratch assay,migration assay and invasion assay were used to detect the effect of Tiaml on the migration and infiltration of gastric cancer cells;Western blot and Human umbilical vein endothelial cells(HUVEC)and human lymphocyte endothelial cells(HLEC)microtubule formation assay were used to detect the effect of Tiaml on angiogenesis.(3)Western blot,immunofluorescence and microtubule formation assays were used to explore the mechanisms of Tiam1's involvement in the EMT process and PI3K/AKT/mTOR signaling pathway in gastric cancer cells through the treatment of signaling pathway inhibitors:Western blot and immunofluorescence were used to detect the expression changes of EMT related markers;Western blot was used to detect the changes of PI3K/AKT/mTOR signaling pathway related markers.Reverse verification,PI3K/AKT/mTOR signaling pathway specific inhibitors LY294002 and Rapamycin were used to treat Tiaml high expressed MKN-45-exTiaml cells,and then detect the changes of PI3K/AKT/mTOR signaling pathway related markers in gastric cancer cells by Western blot;Scratch assay,migration assay,invasion assay,HUVEC and HLEC microtubule formation assay were used to detect the changes of cell migration,invasion and angiogenesis.Immunofluorescence and Western blot were used to detect the changes of EMT related markers in gastric cancer cells treated by LY294002 and Rapamycin.Results:(1)Multiple database analysis showed that Tiaml was highly expressed in gastric cancer tissues:The Human Protein Atlas database showed that the mRNA expression level of Tiam1 in gastric cancer tissues was significantly up-regulated,and it was overexpressed in more than 80%of gastric cancer patients.The cBioPortal database showed that Tiam1 was overexpressed in gastric cancer tissues,which was consistent with the analysis results of Oncomine database(P<0.01).GEO database showed that Tiaml mRNA expression level in gastric cancer tissues was significantly higher than that in adjacent tissues(P<0.05).(2)Tiaml was highly expressed in gastric cancer tissues,and it was closely related to clinicoPathological parameters such as lymph node metastasis and serosal infiltration,which may predict the poor prognosis of gastric cancer patients.? Western blot results of 70 fresh tissue samples showed that the expression level of Tiaml in gastric cancer tissues was significantly up-regulated.The results of HE staining and the immunohistochemistry are the same.The expression level of Tiaml in 10 pairs of early,10 pairs of mid-term and 10 pairs of advanced gastric cancer tissues was significantly higher than that in the adjacent normal tissues(P<0.01).In the 10 groups of patients with lymph node metastasis(three parts of tissues including cancer,paracancerous and lymph node metastases were taken from each patient),the expression of Tiam1 in gastric cancer tissues and lymph node metastasis was significantly higher than that in the adjacent normal tissues(P<0.01).Further analysis showed that the expression of Tiaml in 30 gastric cancer tissues was significantly higher than that in adjacent normal tissues,in advanced gastric cancer tissues was higher than that in early gastric cancer tissues,and in lymph node metastasis gastric cancer tissues was higher than that in non-lymph node metastasis gastric cancer tissues(P<0.01).?Immunohistochemical staining results of 209 paraffin specimens and tissue microarray showed that the positive rate of Tiaml in gastric cancer tissues was 86.3%(132/153),significantly higher than that in the adjacent normal tissues(35.7%,20/56)(P<0.01),and the strong positive rate was 66.7%(102/153),significantly higher than that in the adjacent normal tissues(10.7%,6/56)(P<0.01).Further analysis of the relationship between Tiaml and clinicopathological parameters revealed that the strong positive rate of Tiaml was 79.8%(87/109)in patients with moderately and poorly differentiated gastric cancer,significantly higher than that in patients with well differentiated gastric cancer(34.1%,15/44)(P<0.01).The strong positive rate of Tiaml was 75.8%(72/95)in gastric cancer patients with clinical stage?b-?c,which was significantly higher than that of gastric cancer patients with clinical stage ?-?a(51.7%,30/58)(P<0.01).In gastric cancer patients with lymph node metastasis,the strong positive rate was 74.7%(68/91),significantly higher than that of gastric cancer patients without lymph node metastasis(54.8%,34/62)(P<0.01)In gastric cancer patients with serous infiltration,the strong positive rate was 77.6%(66/85),higher than that of gastric cancer patients without serous infiltration(52.9%,36/68)(P<0.05).At the same time,it was also found that Tiaml was expressed in the cytoplasm and nucleus of gastric cancer cells,but the nuclear staining intensity was significantly enhanced in the gastric cancer tissues of low differentiation,?b-?c stage and gastric cancer tissues with lymph node metastasis and serous infiltration.?The Kaplan-Meier plotter database analysis showed that the total survival time of gastric cancer patients with high Tiaml expression was significantly shorter than that of gastric cancer patients with low Tiaml expression(P<0.05).In gastric cancer patients with lymph node metastasis and gastric cancer patients with distal metastasis,the overall survival rate of patients with high expression of Tiaml was significantly shorter than that of patients with low expression(P<0.05).In patients with clinical stage ?,? and ?,the survival rate of patients with high expression of Tiaml was significantly shorter than that of patients with low expression(P<0.05).(3)The overexpression of Tiaml promoted the proliferation,migration and invasion of gastric cancer cells:MTT assayand plate clone formation assay found that the down-regulated expression of Tiaml significantly inhibited the proliferation of gastric cancer cells(P all<0.05),while the high expression of Tiam1 significantly increased the proliferation of gastric cancer cells.The scratch,migration and invasion showed that the migration and infiltration of gastric cancer cells decreased when Tiaml expression was down-regulated,while the migration and infiltration of gastric cancer cells increased when Tiaml was overexpressed(P all<0.01).(4)Tiaml affected the progression of gastric cancer through EMT:Western blot results showed that when Tiam1 expression was down-regulated,the expression of epithelial markers E-cadherin and ZO-1 were up-regulated,while the expressions of stromal markers Snail,ZEB-1 and ZEB-2 were down-regulated.When Tiaml was overexpressed,the result was the opposite(P<0.01).Immunofluorescence experiments showed that when Tiaml expression was down-regulated,the fluorescence intensity of E-cadherin was significantly enhanced,while that of Vimentin was significantly decreased.When Tiaml was overexpressed,the result was the opposite.(5)Overexpression of Tiaml promoted vascular formation in gastric cancer:Metascape database shown that Tiam1 is closely related to VEGF signaling pathway.When Tiaml expression was down-regulated,the forming ability of HUVEC tube structure was significantly reduced.When Tiaml was highly expressed,HUVEC tube structure was significantly enhanced(P<0.01).The experimental results of HLEC microtubule formation were consistent(P<0.01).Western blot results showed that when the expression of Tiaml was down-regulated,the expressions of VEGF,MMP-2 and MMP-9,the molecular markers related to vascular formation,were significantly down-regulated.When Tiam1 was highly expressed,VEGF,MMP-2 and MMP-9 were significantly up-regulated(P<0.01).(6)Tiaml regulated the progression of gastric cancer through the PI3K/AKT/mTOR signaling pathway:the analysis of String database found that Tiaml has a certain correlation with PIK3CA in the PI3K signaling pathway.Western blot results showed that when Tiaml expression was down-regulated,the expressions of related molecular markers p-AKT and p-S6 in PI3K/AKT/mTOR signaling pathway were down-regulated(P<0.01)9 while the expression of AKT and S6 were not significantly changed.When Tiaml was overexpressed,p-AKT and p-S6 were up-regulated(P<0.01),while AKT and S6 were not significantly changed.Reverse validation showed that cells with treatment of MKN-45/ex-Tiaml by PI3K/AKT/mTOR pathway specific inhibitors LY294002 and Rapamycin,the expressions of p-mTOR,p-AKT and p-S6 in the two treatment groups were decreased compared with MKN-45/ex-Tiaml cells(/P<0.01),while the expressions of mTOR,AKT and S6 were not significantly changed.The results of scratch assay,migration assay,invasion assay,HUVEC microtubule formation assay and HLEC microtubule formation assay showed that the proliferation,migration,invasion and angiogenesis of cells in the two inhibitor treatment groups were decreased compared with MKN-45/ex-Tiaml cells(P<0.01).Cellular immunofluorescence staining results showed that,compared with MKN-45/ex-Tiaml cells,the E-cadherin fluorescence signal of LY294002 and Rapamycin treated cells was significantly enhanced,while the Vimentin fluorescence signal was significantly weakened.Western blot analysis showed that the expression of E-cadherin was up-regulated in LY294002 and Rapamycin-treated group compared with MKN-45/ex-Tiam1 cells,and the expression of Snail and Slug were down-regulated(P<0.01).Conclusions:(1)Tiaml was overexpressed in gastric cancer tissues,and closely related to the clinicopathological parameters such as lymph node metastasis and serosal infiltration,which may predict the poor prognosis of gastric cancer patients;(2)Tiaml promoted the proliferation,migration,invasion and angiogenesis of gastric cancer cells;(3)Tiaml promoted the EMT progression and angiogenesis of gastric cancer cells by activating PI3K/AKT/mTOR signaling pathway. |
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