| The gene associated with retinoid-interferon-induced mortality-19(GRIM-19)is a new type of tumor suppressor genes,previous studies have reported that tumor cells showed tolerance of apoptosis caused by IFN-?/RA after GRIM-19 gene knockout.Therefore,study on the mechanism of GRIM-19 induced apoptosis in IFN-?/RA,it has great significance for the research and development of tumor therapy based on the molecular mechanism.Initially,GRIM-19 is thought to be a kind of nuclear protein,which has been proved to be localized in mitochondria subsequently,which is an important component of mitochondrial complex I.GRIM-19 may be involved in the process of energy metabolism in mitochondria,with participating in the regulation of apoptosis by activating the signal transduction pathway in nucleus.Therefore,it is necessary to clarify the subcellular localization of GRIM-19.In view of the limitations of imaging technology in the past,it is difficult to determine the intracellular localization of GRIM-19.In this study,the subcellular localization of GRIM-19 before and after IFN-?/RA treating was observed by 3D-SIM imaging system.The results showed that in normal conditions,GRIM-19 is mainly distributed in cytoplasm,with co-localization with mitochondria,and in the process of cell apoptosis,the distribution of GRIM-19 moves to the nucleus and GRIM-19 in cytoplasm decreased obviously.After determine the moving forward nucleus of GRIM-19 in the process of apoptosis,the effect of GRIM-19 on apoptosis related signal transduction pathway was discussed.In order to ensure the subsequent experiments smoothly,this study successfully constructed eukaryotic expression plasmid--p GRIM-19.Detection by RTPCR and Western blotting proved that p GRIM-19 could significantly increase the GRIM-19 expression in tumor cells.In this study,we detected the expression of GRIM-19 on the activation of related signal transduction pathway.We found that the upregulation of GRIM-19 can inhibit the expression of STAT3,Bcl-2,cyclin D1 signaling pathway.In conclusion,in the process of apoptosis,the subcellular localization of GRIM-19 changes from mitochondria to nucleus,and it can inhibit the STAT3 related signal transduction pathway.The above results provide the possibility of GRIM-19 as a target for tumor therapy,and it has been reported that GRIM-19 can inhibit the growth of many tumors,but there is no study on its role in oral squamous cell carcinoma.Therefore,this study attempts to reveal the effect of GRIM-19 on the proliferation,apoptosis,metastasis and invasion of oral squamous cell carcinoma.MTT assay and colony formation assay revealed that upregulation of GRIM-19 could inhibit the proliferation of oral squamous cell carcinoma,acridine orange staining and Caspase activity assay results showed that the expression of GRIM-19 could promote the proliferation of oral squamous cell carcinoma;cell migration experiments show that the upregulation of GRIM-19 could inhibit the migration of oral squamous cell carcinoma;Transwell invasion assay showed the expression of GRIM-19 could inhibit oral squamous cell carcinoma cell invasion,invasion mechanism further proof that the upregulation of GRIM-19 could decrease the expression of MMP-2 and MMP-9 in oral squamous cell carcinoma cells,and inhibit cell invasion.On the basis of the above experiments,we investigated the effect of GRIM-19 on the growth of oral squamous cell carcinoma in mice.The oral squamous cell carcinoma cells were inoculated subcutaneously in BALB nude mice,and the weight and volume of tumor cells were significantly decreased by GRIM-19.To further prove the inhibition of GRIM-19 on oral squamous cell carcinoma,tumor tissues were removed and detected of the cell apoptosis,TUNEL assay showed that the apoptosis of tumor cells in nude mice model significantly enhanced after GRIM-19 upregulated.Therefore,GRIM-19 has the potential to be a target for oral squamous cell carcinoma.It has been proved that GRIM-19 has the potential to be a target for oral squamous cell carcinoma,but it is very difficult to develop GRIM-19 activator in a short time.Therefore,first of all,it is possible to find a feasible strategy for the clinical application in combination with GRIM-19.In the treatment of oral squamous cell carcinoma,cisplatin has a better therapeutic effect,but its toxicity and drug resistance greatly limit its further clinical application.It has been reported that the combination therapy of cisplatin and tumor suppressor gene could overcome the drug resistance and enhance the therapeutic effect.Therefore,this study attempts to investigate the effect of GRIM-19 expression on cisplatin in the treatment of oral squamous cell carcinoma.First,the appropriate concentration of cisplatin was determined to be 10?M,while upregulation of GRIM-19 could enhance the inhibition of cisplatin on the proliferation and migration of oral squamous cell carcinoma cells,and enhanced the apoptosis of cisplatin.Besides,results of experiment in vivo also showed that compared to the single injection of cisplatin,combined treatment of cisplatin and GRIM-19 increased oral squamous cell carcinoma tumor weight and volume were significantly reduced,and could further inhibit the proliferation of murine spleen cells.Therefore,the upregulation of GRIM-19 could enhance the therapeutic effect of cisplatin on oral squamous cell carcinoma.In conclusion,the subcellular localization of GRIM-19 changed in the process of apoptosis,some of GRIM-19 moved from mitochondria to nucleus,and could inhibit the activation of STAT3 signal transduction pathway in the nucleus.At the same time,GRIM-19 has been proved to be the target of oral squamous cell carcinoma,which could enhance the therapeutic effect of cisplatin on oral squamous cell carcinoma.This study reveals the subcellular localization of GRIM-19 and its effects on the signal transduction pathway of GRIM-19.The study on the therapeutic effect of GRIM-19 in oral squamous cell carcinoma has expanded the potential clinical therapeutic range of GRIM-19,and provided preclinical data for GRIM-19 related drug development. |
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