Effects Of GRIM-19 On Growth And Invasion Of Human Head And Neck Squamous Cell Carcinoma And Its Mechanisms Research

Background:Head and neck squamous cell carcinoma(HNSCC] is one of the most common cancer,accounting for over 650,000 new cases and 350,000 cancer deaths every year worldwide and HNSCC incidence increases with age. Tobacco smoking, alcohol consumption,environmental exposures, and HPV infection have been identified as important risk factors for HNSCC development. Although there have been improvements in surgery, radiotherapy,and chemotherapy, the 5-year survival for advanced HNSCC remains low. Many efforts have been attempted to identify molecular events that occur during HNSCC development,including the inactivation of TP53, Notch mutations, and altered metabolites. Further elucidation of the molecular mechanisms in head and neck carcinogenesis are expected to accelerate the development of efficacious anticancer agents and the identification of diagnostic or therapeutic biomarkers.Gene associated with retinoid- interferon(IFN]-induced mortality 19(GRIM- 19], a novel IFN- β/retinoic acid- inducible gene product, has been identified as a potential tumor suppressor, which is associated with the inhibition of tumor growth. GRIM- 19 has been demonstrated to be downregulated in the tumour tissue of patients with head and neck squamous cell carcinoma, however, its role in head and neck squamous cell carcinoma remains to be fully elucidated. In the present study, a recombinant eukaryotic expression plasmid carrying GRIM- 19 was constructed and then transfected into the HN5 human head and neck squamous cell carcinoma cell line to examine its effects on squamous cell carcinoma cell growth, migration and invasion using several in vitro approaches. The results demonstrated that upregulation GRIM- 19 in the HN5 cells significantly inhibited cell proliferation, colony formation, migration and invasion, and induced cell apoptosis.Additionally, upregulation of GRIM- 19 also suppressed the secretion of urokinase- typeplasminogen activator(u- PA), matrix metalloproteinase(MMP-2, MMP-9)and vascular endothelial growth factor(VEGF). It was also demonstrated that the activation of signal transducer and activator of transcription 3(STAT3) was downregulated by the expression of GRIM- 19. These results revealed that overexpression of the GRIM- 19 gene may be an effective approach to control the growth and invasion of human head and neck squamous cell carcinoma cells.Objective:The present study aimed to investigate the effects of GRIM- 19 on the proliferation,apoptosis, migration and invasion in head and neck squamous cell carcinoma cells, by transfecting cells with a plasmid constructed to overexpress GRIM- 19. The present study developed a dual expression plasmid that co- expressed GRIM- 19 and P16, and evaluated the combined effects of the two genes against squamous cell carcinoma in vitro and in vivo.The findings of this investigation may contribute to the development of gene therapy for head and neck squamous cell carcinoma, by using GRIM- 19 as a target.Materials and methods Plasmid construction and transfection. A full-length open reading frame of the human GRIM- 19 gene was cloned from the HN5 cells using polymerase chain reaction(PCR).Reverse transcription-quantitative PCR(RT- q PCR). The m RNA expression levels of signal transducer and activator of transcription 3(STAT3)and GRIM- 19 were assessed using RT-q PCR. Western blot analysis. The culture supernatants were collected by centrifugation at 1,000 x g for 5 min at room temperature and concentrated~20- fold using a spinconcentrator(Millipore, Bedford, MA, USA) 72 h after transfection. Cell proliferation assay.Cell proliferation was assessed using an MTT cell proliferation kit(Roche Applied Science,Indianapolis, IN, USA), according to the manufacturer’s instructions. Colony formation assay. The cells were seeded at a density of 1,000 cells/well in 24-well tissue culture plates.Cell apoptosis. Wound- healing assay. A wound- healing assay was performed to assess the effect of GRIM- 19 on cell migration. Invasion assay. Cell invasion was measured using a Matrigel- coated film insert(8 μm pore size)in 24-well invasion chambers(BD Biosciences].Measurement of VEGF production. VEGF production was determined using a competitive ELISA. Recombinant plasmids were transfected into cells with lipofection. Cell proliferation was detected by MTT at different time; cell cycle and apoptosis were detected through flow cytometry(FCM), Annexin V-FITC kit. The target gene and protein(P16,GRIM-19) and associated genes and protins(Stat3, c-Myc, Cyclin D1, Bc L-x L, caspase3 and VEGF) expressions by RT-PCR and Western blot.In order to observe the anti-SCC effect of the recombinant plasmids,we constructed nude SCC xenografts hypodermically and injected different plasmids into peritoneal cavity to the tumor.We measured tumor volumes,compared tumor growth rates and tumor weights between groups at the same time. RT-PCR,Western blot were used to detect the target genes and proteins and associated genes and proteins(Stat3, c-Myc, cyclin D1, Bc L-x L, caspase3 and VEGF); Detect the changes in morphology and apoptosis. Through the assays we evaluated the anti-SCC effects and revealed the related mechanisms of the co-expression plasmid.Result:Transfection with p GRIM-19 increases the expression of GRIM-19 in HN5 cells.Upregulation of the expression of GRIM-19 inhibits the expression of STAT3 in HN5 cells.Upregulation of the expression of GRIM-19 inhibits cell proliferation and cell colony formation in HN5 cells. Upregulation of the expression of GRIM-19 induces apoptosis in HN5 cells. Upregulation of GRIM-19 inhibits the migration and invasion of HN5 cells.Upregulation of the expression of GRIM-19 inhibits the expression levels of MMP-9, VEGF,u-PA and MMP-2 in HN5 cells.Conclusion:In conclusion, the present study provided evidence that the upregulation of GRIM-19 suppressed cell proliferation, colony formation, migration and invasion, and induced cell apoptosis in human squamous cell carcinoma cells. Furthermore, overexpression of GRIM-19 in the HN5 cells suppressed invasion and migration by decreasing the expression levels of MMP-2, MMP-9, VEGF and u-PA, mediated by the STAT3 pathway. Collectively,these data suggested that upregulation of the GRIM-19 gene may be a potential approach tocontrol the invasion and metastasis of Human head and neck squamous cell carcinoma. In conclusion the findings of the present study suggested that transfection with eukaryotic plasmid for the simultaneous expression of GRIM-19 and P16 more effectively suppressed the growth of squamous cell carcinoma in vitro and in vivo, and may therefore have therapeutic potential for the treatment of human squamous cell carcinoma.