Expression Of Trps1 In Gastric Cancer And Its Effect On Proliferation, Migration And Invasion Of Gastric Cancer Cells

BackgroundGastric cancer is one of the most common malignant tumors in digestive tract disease,as well as an important major cancer type and the common cause of cancer-related death in the world.Because of lacking of specific clinical symptoms in early stage,most of patients with gastric cancer diagnosed were late,losing treatment opportunities,or causing poor prognosis.To explore the molec?lar mechanisms of the occurrence and development of gastric cancer will provide theory proof for the diagnosis and treatment of gastric cancer.Transcriptional repressor GATA binding gene 1(TRPS1)is a newly discovered transcription factor in the GATA family,which can inhibit m?ltiple members of the GATA family by specifically binding GATA sequences,then further reg?lates the expression of downstream genes.Up to now,TRPS1 has been reported in breast cancer,prostate cancer,colon cancer,osteosarcoma,epithelial ovarian cancer,and non-small cell lung cancer.However,no research has been reported the value of TRPS1 in gastric cancer.The objective of this study was to determine the differential expression of TRPS1 in gastric cancer tissues and non-caner gastric tissues,then analyze the relationship between TRPS1expression and other clinicopathological features in gastric cancer.Finally,experiments in vitro were used to explore the effect of TRPS1 on the proliferation,migration and invasion of gastric cancer cells.The current study will provide theoretical and experimental basis for further investigating the molec?lar mechanism of TRPS1 in gastric cancer in the future.Materials and methods1 Detecting the expression of TRPS1 in gastric cancer tissues,and analyzing the relationship between TRPS1 and other clinicopathological features of gastric cancer1)The expression profile of TRPS1 in normal gastric tissues and gastric cancer tissues were screened and extracted from TCGA and GTEx public databases,then differential expression of TRPS1 in gastric cancer tissues and its relationship with clinicopathological features were statistically analyzed.Additionally,the clinical information of gastric cancer were also collected from TCGA and GTEx public databases.Univariate Cox analysis and m?ltivariate Cox analysis were then used to investigate the prognostic value of TRPS1 in gastric cancer patients.2)The mRNA level of TRPS1 in gastric cancer were screened and extracted from GEO and Oncomine databases.Then the expression profiles of TRPS1 from TCGA,GTEx,GEO and Oncomine four databases were combined.A meta-analysis was conducted to confirm the expression level of TRPS1 in gastric cancer.2 Sixty pairs of gastric cancer and para-cancerous paraffin tissues,together with the corresponding clinicopathological parameters of gastric cancer patients were collected from the Department of Pathology,the First Affiliated Hospital of Guangxi Medical University.Immunohistochemistry was used to detect the protein expression level of TRPS1 in gastric cancer tissues.The relationship between TRPS1 protein expression and other clinicopathological features in gastric cancer patients were then analyzed.Finally,correlations analysis were conducted between TRPS1 and clinical parameters.3 The effect of TRPS1 on the proliferation,migration and invasion of gastric cancer cell lines were investigated by experiments in vitro.3.1 Detecting and analyzing the mRNA and protein expression levels of TRPS1 in gastric cancer cell lines and normal gastric mucosal cell line.1)Real-time Quantitative polymerase chain reaction(RT-qPCR)was used to analyze the mRNA level of TRPS1 in gastric cancer cell lines(MGC-803,HGC-27)and normal gastric mucosal cell line(GES-1).2)Immunocytochemistry was used to qualitatively detect the protein expression and localization of TRPS1 in gastric cancer cells(MGC-803,HGC-27)and normal gastric mucosal cell line(GES-1).3.2 TRPS1 knockdown plasmid and empty plasmid were transiently transfected into gastric cancer cell lines separately.1)After transfected for 48h,transfection efficiency were observed the in TRPS1 knockdown group and empty group MGC-803 cells.2)RT-qPCR was used to verify the down-reg?lation of TRPS1 in TRPS1knockdown gastric cancer cells.3.3 CCK8 assay was performed to investigate the proliferation differences between TRPS1 knockdown group,empty plasmid group and non-interference group at 36 h.3.4 Cross-scratch test was used to compare the capacity of migration of gastric cancer cells in the TRPS1 knockdown group,the empty plasmid group and the non-interference group.3.5 Transwell migration experiments was further performed to verify the migration capacity of gastric cancer cells in the TRPS1 knockdown group,the empty plasmid group and the non-interference group.3.6 Invasion experiment was performed to compare the invasive ability of the gastric cancer cells between the TRPS1 knockdown group,the empty plasmid group and the non-interference group.Results1 TRPS1 expression in gastric cancer and its relationship with clinicopathological parameters1)Analyzing the expression level and clinicopathological features of TRPS1 in gastric cancer tissues with TCGA and GTEx data revealed that the expression of TRPS1 in 413 cases of gastric cancer was significantly higher than that in 211 cases of non-cancerous gastric tissues(9.477±0.068 vs 8.376±0.116,P<0.001);TRPS1 was up-reg?lated in patients with age?60 years,non-intestinal gastric cancer type,high-grade tumor and T3-4 gastric cancer(P<0.001;P<0.001;P=0.001;P=0.035).The Kaplan-Meier survival analysis showed that the total survival time and recurrence-free survival time of patients with high expression of TRPS1 were significantly lower than those with low expression of TRPS1(P=0.030;P=0.012).Univariate Cox analysis revealed that the hazard ratio of TRPS1 in gastric cancer was 1.428,95%CI:1.030-1.979,P=0.033,while m?ltivariate Cox analysis showed that the hazard ratio of TRPS1 in gastric cancer was 1.242,95%CI:0.867-1.778,P=0.238.2)The expression level of TRPS1 in gastric cancer was evaluated by meta-analysis.A total of 22 datasets(twenty datasets were selected from GEO database,one dataset was extracted from the TCGA and GTEx databases,and another one was extracted from the Oncomine database)were included,which contained 1398 gastric cancer tissue samples and 850 non-cancerous gastric tissue samples.Because significant heterogeneity existed among individual datasets(I~2=99.1%,P<0.001),a random-effects model was chosen.The res?lts showed that TRPS1 expression was significantly increased in gastric cancer(SMD=3.881,95%CI:2.063~5.699,Z=4.180,P<0.001)when compared to normal controls.The Begg funnel plot test revealed no significant publication bias in meta-analysis(P=0.367).2 The res?lt of immunohistochemistry showed that TRPS1 protein was localized in the nucleus.The high expression rate of TRPS1 protein was 45.0%,while the low expression rate was 55.0%in 60 gastric cancer tissues.The high expression rate of TRPS1 protein was 8.3%,while the low expression rate was91.7%in normal gastric mucosa.The protein expression of TRPS1 in gastric cancer tissues was significantly higher than that in adjacent normal gastric mucosa tissues(P<0.001).Moreover,TRPS1 was highly expressed in gastric cancer patients with stage III-IV TNM and lymph node metastasis(P=0.027;P=0.036).Spearman correlation analysis showed that the expression of TRPS1in gastric cancer was positively correlated with TNM stage and lymph node metastasis(r=0.289,P=0.027;r=0.272,P=0.037).3 The effect of TRPS1 on the proliferation,migration and invasion of gastric cancer cell lines.3.1 The mRNA and protein expression levels of TRPS1 were detected and analyzed in gastric cancer cell lines and normal gastric mucosal cell lines1)RT-qPCR res?lts showed that the mRNA expression level of TRPS1 in gastric cancer cell lines(HGC-27 and MGC-803)was significantly higher than that in human normal gastric mucosal cell line GES-1(P<0.001;P=0.005).Significant different expression of TRPS1 mRNA between HGC-27 cell line and human normal gastric mucosal cell line GES-1 co?ld be found.2)The res?lts of immunocytochemistry showed that TRPS1 protein was localized in the nucleus of gastric cancer cells HGC-27 and MGC-803,with brown-yellow staining.TRPS1 protein expression in HGC-27 gastric cancer cells was relatively strong,but relatively weak in MGC-803 gastric cancer cell.TRPS1 protein was negative expressed in human gastric normal mucosal cells GES-1.3.2 TRPS1 knockdown gastric cancer cell lines and empty plasmid gastric cancer cell lines were successf?lly constructed1)Transfection efficiency was detected at 48h.It was found that the ratio of green fluorescent cells in the TRPS1 knockdown group and the empty group was over 70%,the transfection efficiency was satisfactory.2)RT-qPCR res?lts revealed that the expression level of TRPS1 in TRPS1knockdown group was 0.22 times compared with empty plasmid group(P=0.006),indicating that TRPS1 knockdown gastric cancer cells were successf?lly constructed.3.3 The res?lts of CCK8 proliferation assay showed that the average proliferation rates of gastric cancer cells for 36h were 74.8%,267.8%,and 157.3%in the TRPS1 knockdown group,empty-plasmid group and non-interfering groups,respectively(P<0.001).3.4 The res?lts of cell scratch test showed that after 24 hours of scratching,the migration area of TRPS1 knockdown cells was significantly smaller than that of the empty-plasmid group(P<0.001).3.5 Transwell migration assay was further used to verify the effect of TRPS1 on the migration of gastric cancer cells.The res?lts suggested that after24 hours,the average transmembrane cell number of gastric cancer cells was321,291,and 139 in the non-interference group,the empty plasmid group and the TRPS1 knockdown group,respectively.The average number of transmembrane cells in TRPS1 knockdown group was significantly lower than in the empty plasmid group(P<0.001).3.6 The res?lts of invasion experiment showed that after 24h,the average number of perforating cells of gastric cancer cells were 253,161,and 86 in non-interference group,empty plasmid group and TRPS1 knockdown group,respectively.The average number of transmembrane cells in the TRPS1knockdown group was significantly lower than that in the empty cell group(P<0.001).Conclusions1.TRPS1 is up-reg?lated in gastric cancer tissues and cells;2.Down-reg?lation of TRPS1 can inhibit the proliferation,migration and invasion of gastric cancer cells.In summary,TRPS1 may be a tumor promoter in the development of gastric cancer,and TRPS1 may promote the development of gastric cancer by affecting the malignant biological behavior of gastric cancer.