The Mechanism Of RGS16 Promoting The Progression And Chemoresistance Of Colorectal Cancer And Analysis Of Clinical Relevance

Objective: Colorectal cancer is one of the most common malignant tumors.Heterogeneity of prognosis in CRC patient is significant.The 5-year survival rate of patients with early-stage CRC is as high as 90%,while that of patients with m CRC is only 10%.The outcomes of advanced-stage patients vary greatly.Half will develop to recurrence or metastasis.Therefore,biological markers for predicting the prognosis of patients with colorectal cancer and the sensitivity of chemoradiotherapy have become a genuine clinical need.G protein-coupled receptors are the most prominent family of cell membrane receptors in the human body.They activate intracellular signaling pathways by activating heterotrimeric G proteins and participate in many malignant tumor occurrence and development processes.The Regulator of G protein Signaling that accelerates G protein inactivation is a crucial factor in the negative regulation of the GPCRs-G protein signaling pathways.RGS which expresses differentially in tumors has prognostic value.This study aims to provide a supplementary reference for the pathogenesis and therapeutic targets of colorectal cancer by searching for the target genes with prognostic value and chemosensitivity in colorectal cancer from the RGS family and further exploring their biological functions and mechanisms.Methods: In the first part of the study,we used the TIMER database and the TCGA database to screen genes of the RGS family which differentially expressed between colorectal cancer and adjacent tissues.Kaplan-Meier survival analysis were used to 695 transcriptome data and 627 clinical data to screen target genes with predictive value of prognosis.A retrospective study including 899 colorectal cancer patients operated on in Changhai hospital was performed.Postoperative specimens of patients mentioned above,38 paracancerous tissues,16 liver metastatic tumor specimens,as well as 37 normal colorectal mucosa tissues were made into TMAs for immunohistochemistry.The degree of gene antigen staining was scored.Clinical characteristics such as gender,age,tumor location,TNM stage,degree of differentiation,and adjuvant chemotherapy were recorded.So were recurrence,metastasis,and survival of patients.All patients were followed up for five years from surgery.Kaplan-Meier survival analysis and COX regression analysis were performed to determine the predictive value of the target gene.All patients were divided into different subgroups for survival analysis according to clinical characteristics.In the second part of the study,we verified the expression of the target gene in the colorectal cancer cell line and then selected the Caco-2 and SW480 cell lines for follow-up experiments through RNA interference.By knocking down and overexpressing the target gene,the cell lines were divided into four groups: NC-p RGS16,p RGS16,NC-sh RGS16,and sh RGS16.The effect of the difference in the expression of the target gene on the proliferation,clone formation,migration,and invasion of colorectal cancer cells were verified in the four groups of cells.The genes related to the differential expression of the target gene were analysed through data mining and sequencing.The functional annotation and enrichment analysis of these genes was carried out to predict the mechanism of action of the target gene preliminarily.Western Blot was performed to detected apoptosis-related proteins(Cleaved-Caspase-3,Cleaved-PARP)and core proteins of related pathways to determine the effect and the mechanism of regulating tumor cell apoptosis.Four groups of cells were injected subcutaneously into nude mice and observed for four weeks.The nude mice’s subcutaneous tumor volume and body weight change curves were measured.The expressions of Ki67 and TUNEL in metastatic tumor tissue were detected by immunohistochemistry and immunofluorescence,respectively.In the third part of the study,we filtered sensitive chemotherapeutic drugs related to the differential expression of the target gene in TCGA database.PTX was selected for the follow-up experiment.Caco-2 cells were cultured into four groups: NC-RGS16,si RGS16,PTX + NC-RGS16,and PTX +si RGS16.The proliferation,clone formation,and apoptosis of the four groups were verified in vitro;in vivo,the size of tumorigenesis in nude mice between the four groups was compared.All studies used R 4.1.3 and Graph Pad Prism 8.0 for data analysis,statistical analysis,and visualization.Findings: Among the 20 genes of classical RGS family,we found that three genes(RGS5,RGS14,and RGS16)were upregulated in tumor tissue,and seven genes(RGS1,RGS2,RGS7,RGS9,RGS10,RGS13,and RGS18)were downregulated.Kaplan-Meier survival analysis showed that RGS2,RGS13,and RGS16 had predictive significance for OS,and high expression of RGS2 and RGS16 predicted shorter PFS.We selected RGS16 as the target gene for this study based on the consistency of expression differences and prognostic differences.Further analysis of clinical data found that its high expression was significantly correlated with the depth of primary tumor invasion and pathological stage.In a retrospective study,immunohistochemistry of surgical specimens from 899 colorectal cancer patients operated in Changhai hospital found that the expression of RGS16 in primary tumors and metastatic tissues was significantly upregulated compared with the normal tissues and adjacent tissues.The expression of RGS16 was associated with tumor differentiation(P<0.05),TNM stage(P< 0.01),recurrence(P< 0.001),and death(P< 0.001).Compared with patients with low expression of RGS16,patients with high expression of RGS16 had shorter OS(HR = 3.428,95% CI,2.060-5.704,P < 0.001),DFS(HR = 2.418,95% CI,1.754-3.334,P< 0.001)and DSS(HR = 2.745,95% CI,1.973-3.819,P < 0.001).RGS16 expression was an independent risk factors in CRC patients.Stratified survival analysis found no difference in DFS(HR = 1.374,95% CI,0.750-2.515,P=0.278)in patients with high RGS16 expression in the non-chemotherapy subgroup compared with the lower expression group;higher RGS16 expression in the adjuvant chemotherapy subgroup was associated with shorter OS,DFS and DSS(P < 0.001).This part of the study screened RGS16 as a target gene for followup research and verified the value of RGS16 as a prognostic biomarker for colorectal cancer in TMAs;the study also suggested that high expression of RGS16 may be related to tumor proliferation and invasion.The role of predicting chemotherapy sensitivity is to be verified in the subsequent study.In the second part of the study,we found that m RNA and protein expression of RGS16 were upregulated in colorectal cancer cell lines.It was found that knocking down RGS16 could inhibit the proliferation of Caco-2 and SW480 cells while overexpressing of RGS16 got the opposite result(P < 0.01).Knocking down RGS16 significantly inhibited the clone formation ability(P < 0.01),invasion and migration ability of Caco-2 and SW480 cells(P <0.01).In vivo,the tumor volume of the p RGS16 group was the largest,and the sh RGS16 group had the smallest volume(P < 0.05).FCM found that knocking down RGS16 could increase the proportion of annexin V(+)cells,indicating that the deletion of RGS16 led to the apoptosis of CRC cells.GO and KEGG enrichment analysis in data mining revealed that the genes related to differential expression of RGS16 were enriched in the MAPK signaling pathway.NGS detection was performed on four groups of cells.GO enrichment analysis found that cell death and apoptosis were the main functions of RGS16-related gene,while KEGG enrichment analysis suggested that the TGF-β pathway was the main pathway.The apoptosis-related protein and TAK1,core protein of TGFβ pathway,were detected by Western blot.The results showed that knocking down RGS16 significantly increased expression of Cleaved-Caspase-3,Cleaved-PARP,Phos-TAK1,Phos-P38,and Phos-JNK;in the rescue experiment,after cells of sh RGS16 were treated with 5Z-7-Oxozeaenol,the inhibitor of TAK1,Phos-TAK1,Phos-P38 and Phos-JNK were down-regulated.This part of the study confirmed that down-regulation of RGS16 expression could inhibit tumor cell proliferation,migration,and invasion and promote tumor apoptosis in vivo and in vitro;NGS suggested that the effect of RGS16 on apoptosis of tumor cell may be related to regulating TGF-β/TAK1/JNK/P38 pathway,and this mechanism was verified by rescue experiments.In the third part of the study,43 drugs or small molecule inhibitors were filtered through the database to be more sensitive to CRC patients with low expression of RGS16,and PTX was selected for follow-up research.CRC cells were treated with PTX in vivo and in vitro.In vitro,knocking down RGS16 combined with PTX could significantly inhibit the ability of the proliferation and clone formation of tumor cells,and promote apoptosis;in vivo,tumor volume of the PTX+si RGS16 group was the smallest.Immunohistochemical detection of subcutaneous metastases showed that the expression of Ki67 was the lowest in the PTX+si RGS16 group,while the expression of Tunel was the highest,which suggesting an increase in apoptosis.This part of the study filtered sensitive drugs in colorectal cancer patients with low expression of RGS16 and confirmed that low expression of RGS16 can sensitize the pro-apoptotic and anti-proliferative effects of PTX in vivo and in vitro.Conclusion: RGS16 could become a biomarker to predict the prognosis and chemosensitivity of CRC patients.Downregulation of RGS16 expression promotes cell apoptosis by promoting TGF-β/TAK1/JNK/p38 MAPK pathways,and inhibits proliferation,migration,and invasion of tumor cell.Our findings suggest that RGS16 is expected to be a promising independent prognostic biomarker and potential therapeutic target.