| Objective: In China,the incidence and mortality of gastric cancer ranks the second among malignant tumors,which seriously threatens people’s life and health.The onset of most gastric cancers is insidiousness,and there are no obvious clinical symptoms in the early stage,or non-specific symptoms(such as nausea,belching,appetite loss and upper abdominal discomfort,etc.)are ignored,so the early diagnosis rate of gastric cancer is still low,and most patients often have missed the best period of treatment when they seek medical treatment.Although the standard tumor treatment based on D2 radical gastrectomy for gastric cancer is widely applied,the 5-year survival rate of gastric cancer patients in China is still only about 50%.Borrmann type 4 gastric cancer is considered as the most special type of gastric cancer,also known as diffuse cancer.It is different from general gastric cancer.Borrmann type 4 gastric cancer tends to grow rapidly and can spread and metastasize to the whole abdominal cavity quickly.Therefore,surgical resection rate of Borrmann 4 is low,and peritoneal recurrence and metastasis are more common after radical surgery,and the prognosis is worse than that of other Borrmann types of gastric cancer.Therefore,in-depth study on the occurrence and development mechanism of Borrmann type 4gastric cancer and the search for key biomarkers and good therapeutic targets are of great significance for the prevention and treatment of Borrmann type 4 gastric cancer.Sphingosine-1-phosphate transporter(Spinster homolog 2,SPNS2),a member of the SPNS/Spinster family,is a membrane transport protein that can transport substrates by means of the electroosmotic potential energy caused by the different concentration of substances in the cell membrane and outside.S1P is a conservative,cell membrane phospholipids decomposition and metabolites by sphingosine by sphingosine kinase(sphingosine kinase,SPHK)phosphorylation is formed,the study found that S1 P is widely involved in the immune regulation,development and regeneration of blood vessels,heart development and nervous system development,a variety of physiological and pathological process,and S1 P has regulating cell proliferation,migration,blood vessels and lymphatic vessel formation and other functions,these functions are closely related to the development of tumor,but SPNS2 ascurrently found only organize the activity of S1 P transporter,Previous studies on SPNS2 and immunomodulation,nervous system,cardiovascular system and other physiological and pathological processes have been abundant.However,studies on SPNS2 and the occurrence and development of tumors are still scarce.Bradley et al.found that the expression of SPNS2 is down-regulated,which is a potential risk factor for ns2 in NSCLC.Weyden et al.found that the degree of lung metastasis in spns2-deficient mice injected with metastatic melanoma cells was significantly reduced.Jin rong et al.found that the high content of SPNS2 in colorectal cancer and the positive expression of SPNS2 protein were related to the occurrence and development of colorectal cancer.And at present,SPNS2 in tables in the case of gastric cancer and its effect on the regulation of gastric cancer has not been reported,based on the above research status Borrmann type 4and the gastric cancer cell lines and low differentiation of gastric cancer cell line AGS OCUM-1 analysis of differentially expressed genes,the purpose of this study is to clear SPNS2 differentially expressed genes in Borrmann type 4 expression in gastric cancer,and explore SPNS2 affect Borrmann type 4 gastric cancer biological behavior and its potential mechanism.Methods: We performed high-throughput sequencing on Borrmann type 4 gastric cancer cell line ocum-1 and low-differentiation gastric cancer cell line AGS to screen differentially expressed genes.Appropriate number of candidate genes were selected from the differentially expressed genes.GEPIA was used to analyze the distribution of candidate genes in tumor and normal tissues of human organs,and the expression levels of candidate genes in gastric cancer and normal tissues were analyzed.Kaplan-meier plotter analyzed the effect of candidate genes on the survival rate of gastric cancer patients.MRNA and protein expression levels of the remaining candidate genes were verified by rt-pcr and Western Blot in Borrmann type 4 gastric cancer cell line ocum-1,low-differentiated gastric cancer cell line AGS,moderately differentiated gastric cancer cell line SGC7901,highly differentiated gastric cancer cell line mgc-803 and human normal gastric mucosal epithelial cell line ges-1.After determining the target gene to be studied,Kaplan Meier-plotter online analysis tool was used to analyze and verify the relationship between the expression of the target gene in gastric cancer tissues and the prognosis of gastric cancer patients in detail again,and stratified analysis was conductedfor different TNM stages,lymph node metastasis and distant metastasis.Immunohistochemistry was used to detect the expression of candidate genes in290 cases of gastric cancer and 158 cases of adjacent normal tissues,and immunohistochemistry was used to detect the expression of candidate genes in Borrmann type 4 gastric cancer and other Borrmann type gastric cancer tissues.To explore the relationship between candidate gene expression and clinicopathological characteristics and prognosis of Borrmann type 4 gastric cancer.Ocum-1 cell lines with stable silencing candidate genes were constructed by lentivirus transfection,and the transfection efficiency was detected by RT PCR and Western Blot.The group with the highest silencing efficiency and the negative control group were selected for subsequent experimental studies.After stable silencing,we detected the difference in invasion and migration ability between the stable silenced cell lines and the negative control cell lines by Transwell chamber experiment,detected the difference in the proliferation ability between the cells by cck-8 experiment,and detected the difference in cell cycle changes and apoptosis by flow cytometry.In order to further explore the specific mechanism of candidate genes on the biological behavior of gastric cancer.We first used rt-pcr and Western Blot to detect the mRNA levels and protein expression levels of emt-related factors e-cadherin,n-cadherin,Snail,Twist,and invasion related factors mmp-2 and mmp-9 in the silence group and the control group.Meanwhile,mRNA content and protein expression levels of PI3K/AKT pathway-related factors in the silenced group and control group were also detected by rt-pcr and Western Blot.T test,rank sum test or chi-square test were used to compare the data between the two groups.Kaplan-meier method was used to analyze the survival of patients,and log-rank test was used to compare the differences between the survival curves.Cox proportional risk regression model was used to screen prognostic independent risk factors.Bilateral test was used for all statistical analysis,and P<0.05 was considered statistically significant.SPSS 22.0 and GraphPad Prism 7.0 software were used for all statistical analysis and image production.Results: After high-throughput sequencing of Borrmann type 4 gastric cancer cell line ocum-1 and low-differentiation gastric cancer cell line AGS,a total of 2,544 up-regulatedand 2,775 down-regulated genes of ocum-1 were detected.We preliminarily screened 4candidate genes from 2544 upregulated genes,including SPNS2,ERN2,HMGCS2 and EEPD1.Two candidate genes,SULT1C4 and C4 BPB,were preliminarily screened out from 2775 up-regulated genes.Firstly,the distribution of SPNS2,ERN2,HMGCS2,EEPD1,SULT1C4 and C4 BPB candidate genes in tumor and normal tissues of human organs were obtained by GEPIA,and the mRNA levels of the six candidate genes in gastric cancer and normal tissues were analyzed.Kaplan-meier plotter was used to analyze the influence of six candidate genes on the survival rate of gastric cancer patients.HMGCS2 and SULT1C4 genes were deleted after preliminary screening by online analysis tool of network database.Subsequently,rt-pcr was used to verify the mRNA contents of the remaining four candidate genes in the Borrmann type 4 gastric cancer cell line ocum-1,low-differentiated gastric cancer cell line AGS,moderately differentiated gastric cancer cell line SGC7901,highly differentiated gastric cancer cell line mgc-803 and human normal gastric mucosal epithelial cell line ges-1.Two candidate genes,EEPD1 and C4 BPB,were deleted after rescreening and comparison.Western Blot was used to detect the protein content of the remaining two candidate genes in the same 5cell lines.After verification and comparison,the ERN2 candidate genes were removed,and finally SPNS2 was retained as the target gene for Borrmann type 4 gastric cancer in subsequent studies.Using Kaplan Meier-plotter online analysis tools are analyzed in detail again SPNS2 expression in gastric cancer tissue relationship with the prognosis of patients with gastric cancer,we found that SPNS2 expression in gastric cancer has relationship with the prognosis of patients with gastric cancer,the prognosis of patients with high expression is poorer,and from different TNM stages,lymph node metastasis or not,with or without distant metastases of hierarchical analysis,the basic present a TNM staging the longer,the heavier the extent of lymph node metastasis,distant metastasis SPNS2 positive expression of poor prognosis,Therefore,we speculated that the expression of SPNS2 in gastric cancer might be related to the metastasis of gastric cancer.Subsequently,we used immunohistochemistry to detect the expression of SPNS2 in 290 gastric cancer tissues and 158 adjacent normal tissues.We found that SPNS2 was almost not expressed in the adjacent normal tissues,but the positive expression rate was higher in gastriccancer tissues,and the positive expression rate of SPNS2 in Borrmann type 4 gastric cancer tissues was significantly higher than that in other Borrmann type gastric cancer tissues.Through the analysis of the correlation between the expression of SPNS2 and the clinicopathological characteristics of Borrmann type 4 gastric cancer cases,it was found that the cases with positive expression of SPNS2 had deeper infiltration depth,heavier lymph node metastasis,and later TNM stage,and the cases with positive expression of SPNS2 had higher proportions of N3 a,N3b and TNM III.By analyzing the relationship between the expression of SPNS2 and the prognosis of Borrmann’s type 4 gastric cancer,we found that in Borrmann’s type 4 gastric cancer,the long-term survival of patients with positive expression of SPNS2 was significantly worse than those with negative expression of SPNS2.The results of univariate analysis and multivariate analysis suggested that positive SPNS2 expression was an independent adverse prognostic factor for Borrmann type 4 gastric cancer,and the risk of death of patients with positive SPNS2 expression was 4.129 times higher than that of patients with negative SPNS2 expression.After the endogenous expression of SPNS2 was knocked down,the migration and invasion ability of ocum-1 cells was significantly reduced,suggesting that the expression of SPNS2 gene could promote the migration and invasion of Borrmann type 4 gastric cancer cells.Cck-8 experiment showed that after knockdown of endogenous expression of SPNS2,its proliferation ability was lower than that of the negative control group,suggesting that the expression of SPNS2 gene can promote the proliferation ability of Borrmann type 4 gastric cancer cells.Further cell cycle detection by flow cytometry showed that after knockdown of endogenous expression of SPNS2,the proportion of g0/1 phase decreased,while the proportion of G2/M phase increased,indicating that the cell cycle may be retarded in the G2/M phase,resulting in the slowing down of cell proliferation.However,there was no significant change in apoptosis after the endogenous expression of SPNS2 knockdown.Through the above experiments,it is suggested that the expression of SPNS2 gene can promote the invasion,migration and proliferation ability of Borrmann type 4 gastric cancer cells,and affect the cell cycle.To further explore the molecular mechanism by which SPNS2 regulates the migration and invasion ability of Borrmann type 4 gastric cancer,we used rt-pcr and Western Blot to detect the mRNA and protein expression levels of emt-related factors e-cadherin,n-cadherin,Snail,Twist,andinvasion-related factors mmp-2 and mmp-9 in the KD group and the NC group.We found that the expression of e-cadherin was up-regulated by knockdown of endogenous SPNS2,while the expression of n-cadherin,Snail,twist and mmp-9 was down-regulated.Meanwhile,mRNA contents and protein expression levels of PI3K/AKT pathway-related factors in KD group and NC group were also detected by rt-pcr and Western Blot.We found that knockdown of endogenous SPNS2 expression could down-regulate the protein expressions of PIK3 CA,pAKT1 and AKT1.Therefore,we speculated that SPNS2 may regulate EMT and invasion-related factors through the PI3K/AKT pathway,thereby regulating the invasion and metastasis ability of Borrmann type 4 gastric cancer.Conclusion: The results of kaplan-meier-plotter analysis suggest that the prognosis of patients with high SPNS2 content in gastric cancer is worse.Immunohistochemical analysis results suggested that compared with normal gastric mucosal tissues adjacent to cancer,SPNS2 expression was significantly increased in gastric cancer tissues,and its expression was higher in Borrmann type 4 gastric cancer tissues than in other Borrmann type gastric cancer tissues.In Borrmann type 4 gastric cancer,high expression of SPNS2 was significantly correlated with deeper tumor invasion,more severe lymph node metastasis and higher TNM stage,and the survival time of patients with high expression was shorter.Positive expression of SPNS2 was an independent adverse prognostic factor for Borrmann type 4 gastric cancer.After silencing,SPNS2 can significantly inhibit the invasion,migration and proliferation of Borrmann type 4 gastric cancer cell line ocum-1,and can affect the change of cell cycle,but not the apoptosis.SPNS2 may regulate the expression of emt-related factors and invasion-related factors of ocum-1 through the PI3K/AKT pathway,so as to further regulate the migration ability and invasion ability of ocum-1. |
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