| Background:Colorectal cancer is now a leading deadly cancer worldwide,and it causes about10%of new diagnosis and cancer-related deaths of all types of cancers annually.In addition to conventional treatments,current therapies include gene targeting therapy and immunotherapy.Due to effective screening,early intervention,and better treatment strategies,worldwide mortality of colorectal cancer has declined in recent decades.However,the prognosis of patients with advanced colorectal cancer is still poor,and in developing countries and regions,epidemiological studies have shown that the incidence of colorectal cancer will increase along with the regional development.In addition,detections have found an increasing number of young colorectal cancer patients in recent years.The incidence of colorectal cancer is related to many factors,among which,some patients with colorectal cancer have a family history.Poor lifestyle habits such as smoking,alcohol abuse and obesity also increase the risk of colorectal cancer.In China,with the continuous development of society and the change of daily living habits of citizens,the prevalence of colorectal cancer has gradually increased in recent years.Colorectal cancer has now become the second largest cancer prevalence in China,causing a serious burden on the life health and quality of life of Chinese residents.Therefore,in order to cope with the rising trend of colorectal cancer prevalence in China and reduce the threat of colorectal cancer to people's health and the burden of life,further exploration of the pathogenesis of colorectal cancer is expected to enhance the understanding of the its formation,and provide theoretical support and new therapeutic targets for the treatment on colorectal cancer.Gene associated with retinoid-interferon induced Mortality 19(GRIM-19)is a novel tumor suppressor whose coding Gene is located on human chromosome 19p13.2and is highly conserved in eukaryotes.GRIM-19 was initially found in an experiment screening for regulators associated with cell apoptosis induced by interferon and retinoic acid.Studies have shown the downregulation and mutations of GRIM-19 in a variety of cancers.A number of preclinical studies have shown that upregulated GRIM-19 inhibited growth of cancer cell.Studies found that GRIM-19 protein is the major component of the mitochondrial respiratory chain complex?,and play an important role in the mitochondrial oxidative phosphorylation.In addition,GRIM-19 has also been found existing in the nucleus and cytoplasm,playing a number of non-mitochondria-related functions,which are related to cancer development.Reports indicated that the expression of GRIM-19 is down regulated in colorectal tumor tissues.Exogenous GRIM-19 protein can inhibit the growth of colorectal cancer cells.Additionally,GRIM-19 has also been reported to be associated with metastasis in colorectal cancer.However,as a tumor suppressor,the specific mechanism of GRIM-19 in the development of colorectal cancer has not been fully explored.As cancer suppressor,p53 has been widely studied in regulating cell cycle,DNA repair,apoptosis,senescence,autophagy and other aspects.Most colorectal cancer patients have p53 gene mutation or wild-type expression down regulation,resulting in p53 dysfunction.The therapeutic strategy targeting p53 is still one of the important research directions of cancer gene therapy at present.However,the selective activation of p53 targets is regulated by a large number of intracellular signals,and clinical restoration of p53 alone can not effectively induce tumor cell death.Therefore,the regulatory mechanism of p53 expression and selective activation of its downstream pathway still needs to be further explored.It has been found that GRIM-19 may be involved in the regulation of p53 signal pathway in some cancers,but its mechanism has not been elucidated.Abnormal p53expression is one of the important pathogenesis of colorectal cancer.Therefore,the study on the effect of GRIM-19 on the occurrence and development of colorectal cancer and its relationship with the regulation of p53 pathway is expected to provide a new theoretical basis for gene therapy of colorectal cancer.Objective:To explore the relationship between GRIM-19 and the occurrence and development of colorectal cancer,and to explore the effect of GRIM-19 on the growth of colorectal cancer cells by overexpressing GRIM-19 gene in vivo and in vitro,and to further explore the anti-tumor mechanism of GRIM-19.Methods and Results:1.Expression and effect of GRIM-19 in Colorectal Cancer(1)UALCAN database,Western blot and immunohistochemistry were used to detect the expression of GRIM-19 in human colorectal tumor tissues and adjacent normal colorectal tissues.The results showed that the expression of GRIM-19 in colorectal tumors was significantly lower than that in normal colorectal tissues.(2)GEPIA database was used to detect the relationship between the expression of GRIM-19 gene and the prognosis of patients with colorectal cancer.The results showed that the overall survival time and disease-free survival time of patients with high expression of GRIM-19 in colorectal cancer were better than those of patients with low expression of GRIM-19.2.Inhibitory effect of GRIM-19 on the growth of Colorectal Cancer in vivo and in vitro(1)si GRIM-19 promotes the proliferation of colorectal cancer cellsGRIM-19 in colorectal cancer HCT-8 and HCT-116 cells was silenced by si RNA transfection;the effect of si GRIM-19 on the growth count of colorectal cancer cells was detected by CCK-8 assay;the effect of si GRIM-19 on the clone formation ability of colorectal cancer cells was detected by clone formation assay.The results showed that the growth count of colorectal cancer cells silenced by si GRIM-19 was significantly increased,and the number of colony formation was significantly increased.(2)Construction of colorectal cancer cell overexpressing GRIM-19Colorectal cancer cells(HCT-8 and HCT-116)which were stably transfected with GRIM-19 plasmid and control plasmid were screened by G418.The expression of GRIM-19 in colorectal cancer cells was detected by q PCR and Western blot assay.The results showed that the expression of GRIM-19 was significantly increased in HCT-8/GRIM-19 and HCT-116/GRIM-19 cells,and the stable transfection cell lines were established successfully.(3)Overexpressed GRIM-19 inhibits the proliferation of colorectal cancer cellsThe effect of overexpressed GRIM-19 on the growth count of colorectal cancer cells was detected by CCK-8 assay,the effect of overexpressed GRIM-19 on the colony formation ability of colorectal cancer cells was detected by clone formation assay,and the effect of overexpressed GRIM-19 on the proliferation of colorectal cancer cells was detected by Ed U staining assay.The results showed that the growth count of colorectal cancer cells overexpressing GRIM-19 decreased significantly,the number of clone formation decreased significantly,and the number of Ed U staining cells decreased significantly.(4)Overexpressed GRIM-19 induces cell cycle arrest in colorectal cancer cellsFlow cytometry was used to detect the effect of overexpressed GRIM-19 on cell cycle of colorectal cancer cells,and Western blot assay was used to detect the effect of overexpressed GRIM-19 on the cycle-related proteins of colorectal cancer cells.The results showed that the number of cells in G1 phase of colorectal cancer cells overexpressing GRIM-19 was significantly increased,the expression of G1 phase arrest related protein p21 was significantly upregulated and the expression of Cyclin D1protein was significantly downregulated.(5)Apoptosis of colorectal cancer cells induced by Overexpressed GRIM-19Flow cytometry was used to detect the effect of overexpressed GRIM-19 on apoptosis of colorectal cancer cells.Hoechst staining was used to detect the effect of overexpressed GRIM-19 on apoptosis of colorectal cancer cells.Western blot assay was used to detect the effect of overexpressed GRIM-19 on apoptosis-related proteins of colorectal cancer cells.The results showed that the proportion of apoptotic cells in colorectal cancer cells overexpressing GRIM-19 was significantly increased;after Hoechst staining,the colorectal cancer cells overexpressing GRIM-19 were bright blue,and the cell size and morphology were uneven;the expression of apoptosis-related proteins BAX and Cleaved Caspase-3 was significantly upregulated,while the expression of Bcl-2 protein was significantly downregulated.(6)Overexpressed GRIM-19 inhibits the growth of colorectal cancer xenografts in nude miceNude mice were divided into two groups and stably transfected colorectal cancer cells HCT-8/EV(Empty Vehicle)and HCT-8/GRIM-19 were implanted subcutaneously.The tumor volume was measured and the tumor growth curve was drawn.The results showed that the tumor volume growth of HCT-8/GRIM-19 group was significantly lower than that of HCT-8/EV group.TUNEL assay was used to detect the effect of overexpressed GRIM-19 on apoptosis of colorectal tumor cells.Western blot assay was used to detect the effect of overexpressed GRIM-19 on the expression of Cleaved Caspase-3 in colorectal cancer tumors.Immunohistochemical staining assay was used to detect the expression of proliferation and apoptosis-related proteins in colorectal cancer tumors.The results showed that the number of apoptotic cells in colorectal cancer tissues with overexpressed GRIM-19 was significantly increased,the expression of Cleaved Caspase-3 was significantly upregulated,the expression of cell proliferation-related protein PCNA was significantly downregulated,the expression of apoptosis-related protein BAX was significantly up-egulated,and the expression of Bcl-2 was significantly downregulated.3.GRIM-19 induces apoptosis of colorectal cancer cells through SIRT7/MDM2/p53 pathway(1)GRIM-19 induces colorectal cancer cell apoptosis through upregulating p53RNA-seq analysis compared the changes of m RNA expression of differential genes in HCT-8/EV and HCT-8/GRIM-19 cells;KEGG enrichment analysis detect the effect of GRIM-19 on the signal pathway of colorectal cancer cells;Western blot assay to detect the effect of GRIM-19 on p53 and related pathways in colorectal cancer cells;CCK-8 to detect the effect of p53 on the growth inhibition of colorectal cancer cells induced by GRIM-19;The effect of p53 on apoptosis of colorectal cancer cells induced by GRIM-19 was detected by flow cytometry.The results showed that compared with EV group,612 genes were upregulated and 900 genes were downregulated in HCT-8cells overexpressing GRIM-19;p53 signal pathway was significantly enriched in HCT-8/GRIM-19 cells,and the expression of p53 transcriptional regulatory targets were generally upregulated;the expression of p53 protein was significantly upregulated in HCT-8 and HCT-116 cells overexpressing GRIM-19;p21 and PUMA proteins were upregulated,and MDM2 protein was downregulated.In HCT-8 cells overexpressing GRIM-19,p53 and its downstream signal pathway were significantly upregulated after cisplatin-induced DNA injury;after adding p53 specific inhibitor,the growth inhibition of HCT-8 cells induced by GRIM-19 could be reversed by PFT;the apoptosis of HCT-8 cells induced by GRIM-19 could be inhibited by PFT;the expression of p53downstream signal proteins induced by GRIM-19 was reversed after adding PFT.(2)ROS participates in p53 upregulation and apoptosis induced by GRIM-19DCFH-DA method was used to detect the effect of GRIM-19 on the level of ROS in colorectal cancer cells;NAC,a ROS scavenger,was used to detect the effect of ROS on the inhibitory effect of GRIM-19 on the growth of colorectal cancer cells;Flow cytometry and Western blot assay were used to detect the effect of NAC on GRIM-19-induced apoptosis of colorectal cancer cells.The results showed that compared with EV group,ROS levels in HCT-8 and HCT-116 cells overexpressing GRIM-19 was significantly upregulated;ROS scavenger NAC could reverse the growth inhibition of colorectal cancer cells induced by GRIM-19;NAC could inhibit the apoptosis of colorectal cancer cells induced by GRIM-19;NAC could reverse the upregulation of p53 expression induced by GRIM-19 and the protein changes of apoptosis-related proteins Bcl-2 and BAX in colorectal cancer cells.(3)GRIM-19 inhibits the ubiquitin degradation of p53 proteinThe effect of GRIM-19 on p53 and its downstream genes in colorectal cancer cells was detected by q PCR;the effect of GRIM-19 on the degradation rate of p53 protein was detected by Western blot assay;the effect of GRIM-19 on the ubiquitination of p53protein was detected by protein co-immunoprecipitation assay.The results showed that the m RNA levels of p53 targets MDM2,PUMA and FAS were upregulated after overexpression of GRIM-19,but the m RNA level of p53 did not change significantly,the degradation rate of p53 in colorectal cancer cells overexpressing GRIM-19decreased after the addition of cycloheximide,and the ubiquitin level of p53 protein decreased in colorectal cancer cells overexpressing GRIM-19.(4)GRIM-19 stabilizes p53 protein by inhibiting MDM2Western blot assayt and immunohistochemistry assay were used to detect the effect of GRIM-19 on MDM2 protein expression;Western blot assay was used to detect the effect of GRIM-19 on MDM2 protein degradation rate;protein co-immunoprecipitation assay was used to detect the effect of GRIM-19 on MDM2 ubiquitination.The results showed that the expression of MDM2 protein in colorectal cancer cells was significantly downregulated after overexpression of GRIM-19.After the addition of cycloheximide,the degradation rate of MDM2 in colorectal cancer cells overexpressing GRIM-19 was increased,and the ubiquitin level of MDM2 protein in colorectal cancer cells overexpressing GRIM-19 was upregulated.(5)GRIM-19 upregulates p53 through SIRT7/PCAF/MDM2 axis.NAD~+/NADH detection kit was used to detect the effect of GRIM-19 on the expression of NAD~+/NADH in colorectal cancer cells;Western blot assay was used to detect the effect of SIRT7 on p53 expression induced by GRIM-19 in colorectal cancer cells;Protein co-immunoprecipitation assay was used to detect the mechanism of GRIM-19 stabilizing p53 protein through SIRT7.The results showed that the proportion of NAD~+/NADH in HCT-8 cells was positively correlated with the expression of GRIM-19,and the upregulation of p53 protein induced by GRIM-19 in HCT-8 cells induced by GRIM-19 was inhibited by si SIRT7 or ROS scavenger,NAC.Deacetylation of PCAF occurs in colorectal cancer HCT-8 cells overexpressing GRIM-19,and the interaction between PCAF and MDM2 protein is enhanced,while si SIRT7 or NAC inhibit the deacetylation of PCAF and its protein interaction with MDM2.4.Combined treatment of GRIM-19 and oxaliplatin on colorectal cancer(1)GRIM-19 enhances the sensitivity of colorectal cancer cells to oxaliplatinThe effect of GRIM-19 and oxaliplatin on the expression of p53 in colorectal cancer cells was detected by Western blot assay;the IC50 of the inhibitory effect of oxaliplatin on colorectal cancer cells was detected by CCK-8 assay.The results showed that after overexpression of GRIM-19,the expression of p53 protein in colorectal cancer cells was significantly upregulated,IC50 of oxaliplatin to colorectal cancer cells decreased and the drug sensitivity increased.(2)GRIM-19 and oxaliplatin inhibit colorectal cancer cell growthCCK-8 assay was used to detect the effect of GRIM-19 combined with oxaliplatin on the proliferation of colorectal cancer cells,and flow cytometry was used to detect the effect of GRIM-19 combined with oxaliplatin on apoptosis of colorectal cancer cells.The results showed that the inhibitory effect of combined treatment group on proliferation of colorectal cancer cells was significantly better than that of GRIM-19group or oxaliplatin group;proportion of apoptosis in combined treatment group was the highest,and there was significant difference compared with GRIM-19 group or oxaliplatin group.(3)Combined GRIM-19 and oxaliplatin against xenograft colorectal tumorColorectal cancer stably transfected cells HCT-8/EV and HCT-8/GRIM-19 were implanted subcutaneously in nude mice.After tumor formation,the tumor was treated by intraperitoneal injection of oxaliplatin according to the groups.After that,the tumor volume was measured and calculated every two days,and the tumor growth curve was drawn.On the 20th day after administration,the nude mice were killed and the tumor was removed.The results showed that GRIM-19 combined with oxaliplatin had the strongest inhibitory effect on colorectal cancer tumor growth,and there was a significant difference compared with the treatment group alone.Conclusions:1.GRIM-19 is downregulated in colorectal tumors,and the patients with relatively low GRIM-19 levels own a poor prognosis.2.Overexpressed GRIM-19 inhibits colorectal cancer cells proliferation,induces apoptosis and cell cycle arrest.3.GRIM-19 induces colorectal cancer cell apoptosis through the SIRT7/MDM2/p53 pathway4.GRIM-19 enhances the therapeutic effect of oxaliplatin on colorectal cancer... |
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