| Purpose: Gastric cancer is one of the most invasive types of cancer,which is a serious threat to human health.According to data,the number of patients diagnosed with gastric cancer was 1,033,701 in 2018,while the number of deaths was 782,685.Although the continuously improvement of surgery,targeted chemotherapy and radiotherapy has improved the prognosis of the patients with gastric cancer,a great proportion of patients are at risk of tumor recurrence and metastasis and drug resistance.Therefore,further studies about the molecular mechanism of gastric cancer and the searches for biomarkers that can predict disease recurrence and metastasis are essential for improving the disease outcome of gastric cancer.In recent years,the widespread use of high-channel sequencing data,the use of DNA,RNA,protein spectrum data and clinical outcome data integration to continue to more precise subtype classification of tumors,give us a better understanding about the occurrence and driving factors of tumors.The use of public databases for high-throughput data mining has shown important potential clinical value.Methods: 1.Using the GEO gastric cancer dataset,TCGA gastric cancer data analysis and metastasis-related differential genes,and the online database---Kaplan Meier plotter database for clinical outcome verification;2.Using Oncomine database,TCGA database to analyze RBMS1 mRNA in cancer Correlation with cancer tissues and clinical outcomes;3.Observation of cell morphology and lentiviral transfection efficiency by inverted fluorescence microscopy;4.Construction of chemically synthesized siRNA silencing RBMS1 and eukaryotic plasmid expression vector overexpressing RBMS1 gene;5.Construction the lentiviral expression vector targeting silencing and overexpressing RBMS1 gene was used to establish stable transfected gastric cancer cell lines SGC-7901 and HGC-27;6.Cell viability was determined by MTT assay;7.Scratch test,Transwell migration assay and Matrigel invasion assay was used to determine the migration and invasion ability of gastric cancer cells.8.Western blot analysis of RBMS1,STAT3,p-STAT3 and GAPDH proteins 9.Real-time PCR was used to detect the mRNA expression of RBMS1;10.Immunohistochemistry was used to detect the expression of RBMS1 in adjacent tissues,gastric cancer tissues and metastatic lymph nodes.11.GEO data set GSE62254 was downloaded and analyzed about differential expression genes between High RBMS1 expression and low expression.The results were introduced into the IPA platform for functional analysis;12.All data were analyzed using 3 independent experiments with mean ± standard deviation(±s).Statistical analysis was performed using SPSS 18.0 statistical analysis software.The t test was used for comparison between groups,and P < 0.05 was statistically significant.Results: 1.RBMS1 is an potential biomarker for metastasis of gastric cancer patients.The GEO database GSE62254 dataset and the TCGA gastric cancer dataset N0 compared with N1-3 patients were screened for differential genes analysis.The common differential genes in the two data sets were selected,and the disease recurrence survival analysis was performed in the Kaplan-Meier online database.The top10 potential biomarkers associated with metastasis were obtained.Among them,the high expression of RBMS1 was significantly associated with the metastasis of gastric cancer patients and the shortening of disease recurrence time.2.The expression of RBMS1 mRNA in gastric cancer tissues is increased,and its high expression indicates the malignant invasion characteristics and poor prognosis of gastric cancer patients.Oncomine database analysis showed that RBMS1 mRNA expression in gastric cancer tissues was higher than that in normal gastric epithelial tissues;RBMS1 mRNA expression was higher in patients with tumor-related death in 3 and 5 years than in non-dead patients;Online database Kaplan Meier plotter analysis showed the RFS and OS of patients with high RBMS1 expression of were significantly shortened.The results of TCGA database analysis showed that the time to recurrence and the total survival time of patients with high expression of RBMS1 were shortened.The high expression of RBMS1 combined with clinicopathological parameters compared with clinicopathological parameters only for gastric cancer patients.The accuracy of 3,5 years of tumor-related death risk prediction is higher,and RBMS1 combined with clinical pathology parameters can increase the predictive efficacy of total survival in patients with gastric cancer.Line graph analysis found that RBMS1 combined with clinical pathology parameters can increase the prediction of the overall survival of the patient significantly.These results suggest that high expression of RBMS1 is an potential important biomarker for clinicopathological parameters and poor prognosis of poor prognosis in patients with gastric cancer.3.RBMS1 protein positive expression suggests that gastric cancer patients are more inclined to suffer tumor invasion and lymph node metastasis.The RBMS1 protein immunohistochemical staining was performed on the tissue samples of gastric cancer patients.The results showed that the expression of RBMS1 protein in gastric adenocarcinoma tissues was higher than that in adjacent tissues,and it was positively correlated with tumor invasion and lymph node metastasis.Verify the results of the public database from the protein level.4.High expression of RBMS1 enhances the migration and invasion of gastric cancer cells.The lentiviral expression vector was used to silenced and overexpressed the RBMS1 gene establishing gastric cancer cell lines SCG7901 and HGC27.The results of the scratch test showed that the migration ability of SGC7901 and HGC27 cells after silencing RBMS1 was lower than that of the control group(P = 0.0061 and 0.0005,respectively),and the migration ability of SGC7901 and HGC27 cells after overexpression of RBMS1 was higher than that of the control group(P = 0.0146 and 0.003).In addition,Transwell results and Matrigel invasion assay showed that the migration ability of SGC7901 and HGC27 cells after silencing RBMS1 was lower than that of the control group,and the average mobility decreased to 62.4±5.11% and 44.33±9.97% of the control group,respectively.(P=0.0114 and 0.0109),the average invasive rate decreased to 72.4±8.93% and 74.33±10.91% of the control group(P=0.0025 and 0.0007,respectively);the migration ability of SGC7901 and HGC27 cells after overexpression of RBMS1 was higher than that of the control group,the average mobility increased to 261.2 ± 16.1% and 198.21 ± 5.97% of the control group(P = 0.0074 and 0.0261,respectively),and the average invasive rate increased to 178.1 ± 11.91% and 209.67 ± 8.91% of the control group,respectively(P = 0.0271 and 0.0099,respectively).These results suggest that silencing RBMS1 can reduce the invasion and metastasis of gastric cancer cells,and overexpression of RBMS1 can promote the invasion and metastasis of gastric cancer cells.5.RBMS1 regulates IL-6 pathway.The results of the Regulator Effect of IPA suggest that the high expression of RBMS1 has the most obvious regulation of cytokines.Pathway analysis found that RBMS1 is closely related to inflammatory factor-related pathways such as IL-6 Signaling and IL-8 Signaling(Z values are 2.646 and 1.342,respectively);it is closely related to tumor-related signaling pathways such as Colorectal Cancer,Metastasis Signaling,and cAMP-mediated signaling.Related;also related to signaling pathways such as Osteoarthritis Pathway,G Beta Gamma Signaling,HMGB1 Signaling,and Dendritic Cell Maturation.To verify the association between RBMS1 and IL-6,we correlated the expression profiles of 25 gastric cancer cell lines in the CCEL database with the expression profiles of RBMS1 and IL-6 mRNA in 350 gastric cancer patients in the TCGA database.Analysis,the results suggest that gastric cancer cells(P value of 0.0195,R2 = 0.38)and gastric cancer patients(P value <0.0001,R2 = 0.24)were significantly positively correlated with RBMS1 and IL-6.6.RBMS1 promotes the migration and invasion of gastric cancer cells through the IL-6/STAT3 pathway.We performed ELISA on IL-6 levels in the gastric cancer cell lines SGC-7901 and HGC-27,after knocking out the RBMS1 gene.The results showed that IL-6 levels did not change significantly after 24 hours of knockout of RBMS1(P >0.05),which were significantly reduced after 48 hours and 72 hours of knockout of RBMS1.Further,the expression of IL-6 classical downstream signal was detected.Compared with the control group,the activity of p-STAT3 signaling pathway was significantly decreased after knocking out RBMS1 gene.In order to further clarify the regulatory relationship of RBMS1 to IL-6 signaling pathway,we inhibited IL-6 in gastric cancer cell supernatant by administering human IL-6 neutralizing antibody to SGC-7901 and HGC-27 cells overexpressing RBMS1.Transwell migration assay and Matrigel invasion assay showed that the migration ability of SGC-7901 and HGC-27 gastric cancer cells overexpressing RBMS1 decreased after inhibiting IL-6 secretion,indicating that RBMS1 relies on IL-6 pathway to promote migration and invasion of gastric cancer cells.Conclusion: 1.The mRNA and protein expression of RBMS1 are higher in normal gastric epithelial cells and adjacent normal tissues in gastric cancer cells and gastric cancer tissues;2.The recurrence time and overall survival time of patients with high expression of RBMS1 are shorter;3,RBMS1 High expression can enhance the migration and invasion of gastric cancer cell lines;4.RBMS1 is positively correlated with IL-6 expression and can regulate IL-6/STAT signaling pathway;5.RBMS1 enhances gastric cancer cells by activating IL-6/STAT signaling pathway Migration and invasive capabilities. |
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