| Objectives:Gastric cancer is one of the most common malignant tumors worldwide.In 2020,approximate 478 thousand new cases of gastric cancer have occurred in China,which ranked second in all types of cancer,accounting for approximately 43.9%of total new cases of gastric cancer in 185 countries of the world.The estimated number of gastric cancer deaths in China has reached 373 thousand,and China accounted for 48.6%of deaths of gastric cancer worldwide.However,GC screening methods are not adequately applied to the Chinese population currently so the first diagnosis of gastric cancer patients is usually advanced gastric cancer,predicting a poor prognosis of patients.And then the 5-year survival rate of gastric cancer patients is only 35.1%in China though surgery and chemotherapy are available to them.Therefore,gastric cancer,which is of heavy burden in China,remains a fatal disease.The pathogenesis of gastric cancer is complex and the overall survival of patients varies significantly.However,there is no molecular marker to predict the long-term overall survival of gastric cancer patients effectively.Previous studies have found that TXNDC9（thioredoxin domain-containing protein 9）is associated with the development,chemotherapy resistance,and prognosis of colorectal cancer,prostate cancer,and liver cancer,but which drew different conclusions on the effect of TXNDC9.For other types of cancer and susceptible races,there may be differences in the expression and prognostic influence of TXNDC9.However,in terms of gastric cancer,there aren’t large-scale cohort studies or experimental studies to explore the association between the expression of TXNDC9 and gastric carcinogenesis and the prognosis of these patients.This study aimed to investigate the association of expression of TXNDC9 m RNA and its protein with gastric cancer carcinogenesis and prognosis influence on gastric cancer patients and to explore the potential molecular mechanism by carrying out a bioinformation study and a prospective cohort study.Methods:（1）The transcriptome data of TXNDC9 gene expression in tumor tissues and their normal tissues across different cancer types were obtained from The Cancer Genome Atlas（"TCGA"）database,including tumor tissues of 375 gastric cancer patients and paired adjacent tissues of 32 subjects.Mann-Whitney U test was performed to investigate the differential expression of TXNDC9 between the tumor tissues and their normal tissues.To explore the association of the expression of TXNDC9 m RNA with the prognosis of gastric cancer patients,transcriptomic data and survival information of 375 patients in the TCGA database combined with data of 740 patients in the GEO（Gene expression omnibus）database were used and Kaplan-Meier method with log-rank test was performed.（2）q PCR method was performed to examine the relative levels of TXNDC9m RNA in the gastric cancer cell lines of KATO-Ⅲ、AGS、MKN-45、MKN-74、HGC-27 and human gastric mucosal epithelial cell line GES-1,and t test was used for examining the differential expression between the two main types of cell lines.（3）A total of 233 patients,who were diagnosed with gastric cancer by histological examination at the First Hospital of Jilin University from February 2011 to February2017,were recruited into our prospective cohort study.Necessary data were collected,including demographic characteristics,clinicopathological information of patients,and history of diagnosis and chemotherapy.And follow-up information such as postoperative chemotherapy and survival status were collected by telephone follow-up at the 3rd,6th,and 12th months,and every year after then.The postoperative gastric cancer tissues specimens and matched adjacent paracancer tissues specimens both under deep hypothermia and paraffin-embedded were collected for PCR and immunohistochemical staining.to detect the level of TXNDC9 m RNA and its protein respectively,and the scores of TXNDC9 protein,assessed by the latter method,were used for dividing subjects into groups of TXNDC9 high-expressed and low-expressed.The correlation between TXNDC9 protein expression and the clinicopathological characteristics of the patients was analyzed with Pearsonχ~2 test or Fisher’s exact probability test.The survival curves between patients with different TXNDC9 protein levels and clinical characteristics were plotted using the Kaplan-Meier method and compared with the log-rank test.Cox regression analysis was performed to analyze independent predictive factors of the prognosis of gastric cancer and calculate hazard ratios（HR）and corresponding 95%confidence intervals（CI）.Subgroup analyses were used to evaluate the consistency of the relationship between the expression level of TXNDC9 and the prognosis of gastric cancer between the clinicopathological feature groups.（4）Based on the gastric cancer transcriptomic data of the GEO dataset（n=740）,the Pearson linear correlation coefficient was used to measure the degree of correlation between TXNDC9 and each gene for screening co-expression genes of TXNDC9 with the criterion of r≤-0.27 or r≥0.27 and meanwhile the P-value<0.001.According to TXNDC9 and its co-expressed gene sets,enrichment analyses and protein-protein interaction networks（PPI）were performed to analyze the biological processes,signaling pathways,and protein-protein interactions which may associate with TXNDC9.Results:（1）The results of pan-cancer analysis of the TCGA database showed that the levels of TXNDC9 m RNA in different types of tumors were varies.The expression of TXNDC9 in pancreatic cancer,liver cancer,prostate cancer,and lung cancer tissues was higher than that in the corresponding adjacent paracancer tissues,however,lower in renal chromophobe carcinoma,renal clear cell carcinoma,and melanoma tissues.For gastric cancer,the level of TXNDC9 in tumor tissue was higher than that in adjacent tissue（P<0.001）,indicating that TXNDC9 might associate with the genesis of gastric neoplasm.And the survival analysis of data from diverse sources showed that there are inconsistent conclusions on the association between the level of TXNDC9 in gastric cancer tumor tissue and the prognosis of gastric cancer patients.For the 350 gastric cancer patients from the TCGA database,the result suggested that patients with a higher level of TXNDC9 in gastric cancer tissues had an increased risk of cancer death（HR=1.58,95%CI:1.14-2.20）with a shorter median survival time than patients with lower level（20.6 months vs.43.1 months,log-rank P=0.006）.However,the prognostic analysis of 740 gastric cancer patients in the GEO database found that patients with high TXNDC9 levels had a longer survival time（70.2 months vs.26.6 months,log-rank P<0.001）with a better prognosis（HR=0.62,95%CI:0.50-0.76）,which implied an association of TXNDC9 level with the prognosis of gastric cancer patients and a demand for a further investigation due to the inconsistent conclusions.（2）At the cellular level,the expression of TXNDC9 m RNA in gastric cancer cell lines tended to be lower than that in gastric normal mucosa epithelial cell line,but the difference did not show statistical significance（all P>0.05）.（3）A total of 233 subjects were included in our prospective cohort.The expression of TXNDC9 m RNA in gastric cancer tissues of 39 subjects was also lower in gastric cancer tissues than that in adjacent tissues（P=0.047）.The result of immunohistochemical staining showed that the levels of TXNDC9 protein in gastric cancer tissues of 233 subjects were lower than that in adjacent paracancer tissues,without statistical significance（P=0.433）.Among all 233 gastric cancer patients,no correlations between TXNDC9 protein expression and clinicopathological features were found.The results of the prognostic analysis showed that gastric cancer patients with high TXNDC9 expression in tumors had a better prognosis（HR=0.56,95%CI:0.38-0.80,log-rank P=0.001）,with a longer median survival time（89.4 months vs.30.7 months,log-rank P=0.001）.Furthermore,multivariate Cox regression showed that a higher level of TXNDC9 protein,earlier TNM staging,without perineuronal infiltration,shorter tumor diameter（<5 cm）,and postoperative chemotherapy were associated with longer overall survival in patients with gastric cancer.And the high level of TXNDC9 protein was an independent predictor of better prognosis in gastric cancer patients（HR=0.58,95%CI:0.40-0.85）after adjusting for potential prognostic factors.Subgroup analyses showed patients with higher expression of TXNDC9 protein tended to survive longer in each subgroup.（4）174 genes co-expressed with TXNDC9 were screened from the GEO database.The enrichment analysis of TXNDC9 and its co-expression genes showed that TXNDC9may be related to biological functions and pathways such as cell cycle,ATPase activities,mismatch repair,and spliceosome.PPI was constructed and it showed that TXNDC9 may be correlated with cell cycle-related proteins AKT1,PDCD10,CDK1,p53,and so on.Conclusions:（1）For Chinese gastric cancer patients,the levels of TXNDC9 m RNA and its protein in tumor tissues were lower than those in adjacent paracancer tissues,and TXNDC9 might associate with the genesis of gastric tumor.（2）High expression of TXNDC9 protein is associated with longer overall survival in gastric cancer patients and better prognosis.（3）Besides of higher level of TXNDC9 protein,earlier TNM staging,no perineural invasion,shorter longest tumor diameter,and postoperative chemotherapy were independent prognosis factors predicting better prognosis in gastric cancer patients.（4）TXNDC9 protein may play its role by inducing cell cycle arrest and inhibiting e EF2 and PI3K/AKT signaling pathways and so on. |
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