| Objective:According to the latest tumor registry data,the incidence and mortality of gastric cancer in China ranked third among malignant tumors.Due to the insidious onset of gastric cancer,90%of gastric cancer patients diagnosed in China are already in the advanced stage,so the 5-year survival rate of gastric cancer in China is only 35.1%.Gastric cancer is a highly heterogeneous tumor that lacks effective biomarkers for efficacy prediction and long-term prognosis assessment.The regulator of G-protein signaling(RGS)family is a family of regulators of G-protein-coupled receptors.Previous studies have found that the abnormal expression of its family members can be directly involved in the development of various tumors and lead to poor prognosis of the patients by affecting the malignant functions of the tumor cells,such as proliferation,migration,and invasion,and also indirectly participate in the occurrence and development of tumors by influencing the chemotaxis and infiltration of the immune cells in the tumor microenvironment.However,whether the RGS family is abnormally expressed in gastric cancer tissue and involved in the clinical characteristics and long-term prognosis,and whether it may play a regulatory role through the immune pathway remains to be elucidated.In this study,first,we used TCGA data to screen and identify RGS family member that was abnormallly expressed in gastric cancer tissue and closely associated with the clinical characteristics and long-term prognosis,and validated it using a gastric cancer cohort from our project group;Second,we explored and validated the relationship between the screened and identified RGS family member and immune cell infiltration in the tumor-immune tumor microenvironment using TCGA data and gastric cancer cohort;Finally,we used cytological experiments to clarify the potential regulatory mechanisms of the screened RGS family member on gastric cancer cells as well as immune cells.Method:1.Based on TCGA data,the expression levels of each RGS family member in gastric cancer tissues and paracancerous control tissues were compared at the m RNA level,and the relationships between each RGS family member and gastric cancer progression and prognosis were explored,and the RGS family member closely related to gastric cancer progression and prognosis(RGSx)were screened.2.Subjects of the gastric cancer cohort were obtained from gastric cancer patients who underwent radical gastric cancer surgery at the first hospital of Jilin University from 2011 to 2016.Tissue samples,demographic data,and clinicopathologic data were collected after informed consent.Immunohistochemistry was used to determine the expression levels of RGSx protein in gastric cancer tissues and paired paracancerous control tissues.Thec~2 test,Fisher’s exact test,and survival analysis were used to verify the relationship between RGSx and the clinical characteristics and long-term prognosis at the protein expression level.3.Based on the TCGA data,the transcriptome data of patients in the lowest RGSx expression group(Q1)and the highest expression group(Q4)were compared to screen out the differential genes,and the GO and KEGG enrichment analysis based on the differential genes were conducted to predict the potential functions and the potential regulatory pathways of RGSx.“Estimate”was used to calculate the immune microenvironment score of gastric cancer tissues,and“EPIC”was used to estimate the degree of immune cell infiltration.Spearman correlation coefficients were calculated to examine the correlation between RGSx expression and estimated score,stromal score,immunity score,degree of immune cell infiltration,and expression levels of immune cell markers.4.Based on the gastric cancer cohort,the correlation between RGSx protein and immune cell infiltration was analyzed.Based on the TCGA data and the gastric cancer cohort,the relationships between RGSx expression combined with the level of immune cell infiltration and the clinical characteristics and long-term prognosis were further analyzed using thec~2 test,Fisher’s exact test,and survival analysis.5.The expression levels of RGSx in gastric cancer cells and immortalized gastric mucosal epithelial cells were compared by qRT-PCR and Western blot assays.Gastric cancer cell lines with RGSx knockdown and overexpression were established,respectively,and the regulatory effects of RGSx on proliferation,cycling and apoptosis,migration,and invasion ability of gastric cancer cells were investigated by CCK8 assay,flow cytometry assay,and transwell assay.6.The qRT-PCR and ELISA assay were used to screen the target genes regulated by RGSx that were enriched by the KEGG analysis.The co-culture system of gastric cancer cells and immune cells was constructed to explore the recruitment effect and polarization effect of gastric cancer cells with different RGSx expression levels on immune cells by transwell assay,qRT-PCR and flow cytometry assay.Next,using the rescue experiments to confirm the effects of RGSx on immune cells were achieved by regulating the target genes.Finally,the effects of immune cells on the function of gastric cancer cells with different RGSx expression levels were explored based on the co-culture system.Result:1.Among the RGS family,RGS1 was highly expressed in gastric cancer tissue and was associated with higher malignant degrees and poor long-term prognosis of patients.The TCGA data included 373 patients with gastric cancer,after excluding patients lost to follow-up at the first visit and perioperative deaths,a total of 333 patients were included in the survival analysis,with a median follow-up of 25.1 months,and deaths occurred in 142(42.0%)patients.In the TCGA data,RGS1,RGS2,RGS3,RGS5,RGS9,RGS11,RGS13,RGS16,and RGS19 were abnormal expressions in gastric cancer tissue;RGS1,RGS2,and RGS18were associated with indicators of clinical characteristics and RGS1,RGS4,and RGS20were independently associated with the long-term prognosis of with gastric cancer patients.The member of the RGS family that was abnormally expressed in gastric cancer tissue and closely related to the malignant degree and poor long-term prognosis was RGS1.So,we took RGS1 as the target gene in this study,and further verified the expression level of RGS1 in gastric cancer tissue and the relationships between RGS1and the malignant clinical characteristics and poor long-term prognosis in our gastric cancer cohort.A total of 375 gastric cancer patients who met the inclusion and exclusion criteria were included in the gastric cancer cohort,and after excluding 2 patients lost to first follow-up and 2 patients who died perioperatively,a total of 371 patients were included in the survival analysis with a median follow-up of 100.6 months,and death occurred in 244(65.8%)patients.In the gastric cancer cohort,immunohistochemical staining showed that RGS1protein was expressed in the cell membrane and cytoplasm.The proportion of high expression of RGS1 protein in gastric cancer tissue was higher than that in paracancerous control tissues(P<0.001).Higher RGS1 protein expression was associated with larger tumor diameter(P=0.005)and positive neural infiltration(P=0.043).After multivariate adjustment for TNM stage and other factors that may affect gastric cancer prognosis,higher RGS1 protein expression was still found to be an independent risk factor for poor long-term prognosis in gastric cancer patients(HR:1.38,95%CI:1.02-1.85).2.RGS1 expression was associated with M2 macrophages infiltration,and the combination of the two is associated with higher malignancy and shorter overall survival in gastric cancer.The TCGA data showed that 689 gene expressions were up-regulated and 36 gene expressions were down-regulated in the RGS1 high-expression group,compared with the RGS1 low-expression group.GO function analysis of the differentially expressed genes focused on immune system processes,and immune response processes,suggesting that RGS1 may function through immune regulation.Correlation analysis of immunity showed that RGS1 expression level was positively correlated with estimate score(r_s=0.71,P<0.001),immune infiltration score(r_s=0.71,P<0.001)and stromal score(r_s=0.59,P<0.001);the strongest correlation was found between RGS1 expression level and level of macrophages infiltration(r_s=0.63,P<0.001),and further analysis showed that at the m RNA expression level,RGS1 expression was correlated with the classic M2 macrophages markers CD163,CD206 and VSIG4(r_s=0.62,0.57,0.58,respectively,all P<0.05).In the gastric cancer cohort,the expression level of RGS1protein also showed a strong correlation with the protein expression levels of CD163,CD206,and VSIG4(r=0.44,0.36,0.39,respectively,all P<0.05).The correlation between RGS1 expression combined with M2 macrophages infiltration and gastric cancer progression and prognosis was further analyzed using TCGA data and a gastric cancer cohort.The TCGA results showed that at the m RNA expression level,compared to RGS1~-&CD163~-,RGS1~-&CD206~-,RGS1~-&VSIG4~-,the correlation was stronger between RGS1~+&CD163~+,RGS1~+&CD206~+,and RGS1~+&VSIG4~+groups,all of which had higher proportions of gastric cancer patients with higher tumor grade(all P<0.05);After adjustment for other possible confounders,the risk of death in patients in the RGS1~+&CD163~+,RGS1~+&CD206~+and RGS1~+&VSIG4~+group show increasing trends.The results of the gastric cancer cohort showed that at the protein expression level,the proportion of patients with tumor size>5 cm was higher in both the RGS1~+&CD163~+,RGS1+&CD206~+,and RGS1~+&VSIG4~+groups(all P<0.05),after adjustment for other influencing factors,the RGS1~+&CD163~+,RGS1~+&CD206~+,and RGS1~+&VSIG4~+groups of gastric cancer patients had a significantly higher risk of death(HR 1.73,95%CI:1.24-2.42;HR:1.67,95%CI:1.19-2.36;HR:1.67,95%CI:1.18-2.38,respectively).3.RGS1 expression promoted the proliferation,cell cycle,migration,and invasion and inhibited apoptosis of gastric cancer cellsThe results of qRT-PCR and Western blot showed that the expression of RGS1m RNA and protein was significantly increased in gastric cancer cells MKN28,BGC823,AGS,and SGC7901 compared with immortalized gastric mucosal epithelial cells GES-1.AGS and SGC7901 gastric cancer cell lines with relatively high RGS1 expression were selected to construct RGS1 knockdown cells,and MKN28 and BGC823 gastric cancer cells with relatively low RGS1 expression were selected to construct RGS1overexpression cells.Compared with the control group,the RGS1 knockdown AGS,and SGC7901 gastric cancer cell lines exhibited reduced cell proliferation ability,cell cycle arrest at the G1/S phase,cell cycle inhibition,an increased proportion of apoptosis,fewer cells passing through transwell chambers and chambers with stromal gel,and reduced cell migration and invasion ability;on the contrary,in the RGS1-overexpressing MKN28 and BGC823 gastric cancer cell lines,ion of cells in the G2phase of the cell cycle was increased,the cell cycle was promoted compared with the control group,the cell proliferation ability was increased,the proportion of apoptosis was decreased,the number of cells passing through transwell chambers and chambers with stromal gel was increased,and the migration and invasion ability of cells was increased.4.RGS1 further promoted macrophages recruitment and polarization by inducing the expression of chemokine CCL4KEGG enrichment analysis of differentially expressed genes suggested that RGS1might be involved in the regulation of the chemokine signaling pathway.qRT-PCR and ELISA results showed that the expression of chemokine CCL4 was decreased in gastric cancer cells with RGS1 knockdown and their culture medium,and the expression of CCL4 was increased in gastric cancer cells with RGS1 overexpression and their culture medium.In the co-culture system,M0 macrophages were cultured in the medium of gastric cancer cells with knockdown and overexpression of RGS1,respectively.Compared with the control group,after cultured in the medium of gastric cancer cells with knockdown of RGS1,the number of M0 macrophages passing through the transwell chambers decreased,the m RNA expression level of CD163 in M0macrophages and the number of CD163 positive M0 macrophages decreased(P<0.05).The recruitment and polarization effects of RGS1 knockdown gastric cancer cells on M0 macrophages were reduced.After adding CCL4 recombinant protein to the RGS1knockdown gastric cancer cell culture medium,compared to the group without CCL4,the number of M0 macrophages passing through the transwell chambers increased,the m RNA expression level of CD163 in M0 macrophages and the number of CD163positive M0 macrophages increased(P<0.05).CCL4 partially restored the recruitment and polarization of RGS1-knockdown gastric cancer cells on M0 macrophages.After culturing M0 macrophages in the medium of RGS1-overexpressing gastric cancer cells,there was an increase in the number of M0 macrophages cells that crossed the transwell chambers,the m RNA expression level of CD163 in M0 macrophages and the number of CD163 positive M0 macrophages increased(P<0.05).Similarly,after the addition of CCL4 to the medium of RGS1-overexpressing gastric cancer cells,it was observed that CCL4 further enhanced the recruitment and polarization effect of RGS1-overexpressing gastric cancer cells on M0 recruitment and polarization.5.RGS1 combined with M2 macrophages to promote the proliferation,cell cycle,migration,and invasion and inhibited apoptosis of gastric cancer cellsAfter culturing RGS1 knockdown or overexpressed gastric cancer cells with M2macrophages-conditioned medium(M2 CM),the cell function results showed that compared with RGS1 knockdown gastric cancer cells without the addition of M2 CM,the addition of M2 CM resulted in increased proliferative capacity of RGS1 knockdown gastric cancer cells,the proportion of cells in the G2 phase of the cell cycle was increased,the cell cycle was promoted,the proportion of apoptosis was decreased,the number of cells passing through transwell chambers and chambers with stromal gel was increased,and the migration and invasion ability of the cells was enhanced.Compared with RGS1-overexpressing gastric cancer cells without M2 CM,the addition of M2 CM further increased the promotion of proliferation,cell cycle,migration,and invasion of gastric cancer cells after RGS1 overexpression,and further inhibited the inhibitory effect of apoptosis of gastric cancer cells after RGS1 overexpression.Conclusion:1.Among the RGS family,RGS1 was highly expressed in gastric cancer tissues,and high RGS1 expression was correlated with higher malignant degree and poor long-term prognosis.RGS1 may serve as a biomarker for the malignant degree and poor long-term prognosis of gastric cancer.2.Higher levels of M2 macrophages infiltration were found in gastric cancer tissues with high RGS1 expression;RGS1 expression combined with M2 macrophages infiltration was associated with higher malignant degree and poor long-term prognosis of gastric cancer.3.RGS1 was highly expressed in gastric cancer cells,and RGS1 expression promoted the proliferation,cycle,migration,and invasion of gastric cancer cells and inhibited apoptosis of gastric cancer cells.4.RGS1 can promote macrophages recruitment and polarization into M2macrophages by promoting the expression of chemokine CCL4.RGS1 expression and M2 macrophages infiltration synergistically promoted the proliferation,cycling,migration,and invasion of gastric cancer cells,and inhibited apoptosis of gastric cancer cells. |
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