ATF3 Inhibits The Growth And Migration Of Tongue Squamous Cell Carcinoma By Downregulating New Targets IFI6 And IFI27

Background and objectiveSquamous cell carcinoma(SCC)is a main histological type of head and neck malignancies,accounting for 90%-95%of malignant tumors in these sites.Head and neck squamous cell carcinoma(HNSCC)is the sixth most common malignant tumor in the world.Oral squamous cell carcinoma(OSCC)accounts for about 25%of HNSCCs,and nearly half of OSCC are tongue squamous cell carcinoma(TSCC).TSCC is a highly aggressive malignant tumor that is prone to local recurrence and distant metastasis.In addition,TSCC progresses to invasive cancer without any specific symptoms,making early diagnosis difficult and ultimately leading to poor prognosis.The 5-year survival rate is still not ideal even after surgery,radiotherapy and chemotherapy.A number of factors,such as environmental factors,genetic predisposition to poor lifestyles and viral infections are associated with the risk of TSCC.However,the detailed molecular mechanism involved in TSCC development remains unclear.Therefore,it is urgent to further explore the molecular mechanism of TSCC occurrence and development,and to provide new strategies for early clinical diagnosis and treatment of TSCC.Activating transcription factor 3(ATF3),a member of ATF/CREB family,is a key transcription factor regulating cellular stress responses,the expression and function if which are different in different tissues.In recent years,the role of ATF3 in tumor cells has been drawn wide concern.It is of interest that ATF3 can function as both oncogene and tumor suppressor gene in different tumor types and cellular environments.Recent studies have shown that ATF3 is a downstream target of miR-488 and miR-431,and plays an important role in the progression of TSCC.However,the downstream effects of ATF3 in TSCC cells have not been studied so far.This study aims to explore the function and downstream signaling pathway of ATF3 in TSCC tumor cells,and to provide new ideas for the development of new therapies for TSCC.Materials and methods1.The expression of ATF3 in TSCC cells.The expression of ATF3 in TSCC tissues and normal tongue tissues was detected by H&E staining and immunohistochemistry.The expression of ATF3 in TSCC cell lines,CAL27 and SCC-9 and normal oral keratinocytes(HOK)were stained with ATF3 fluorescence by cellular immunofluorescence.The TCGA database were used to analyze the expression of ATF3 in HNSCC and TSCC.2.Effects of ATF3 on TSCC cells.To study the specific effects of ATF3 on TSCC cell function,this study used CRISPR/Cas9 gene editing technology to knock out ATF3 genes in TSCC cell lines CAL27 and SCC-25,and infected the ATF3 genes in TSCC cell lines SCC-9 and SCC-4 with lentivirus.The effects of ATF3 knockout or overexpression on proliferation of TSCC cell lines were detected by CCK-8 technique.Transwell assay and wound-healing assay were used to detect the effects of ATF3 on the migration ability of TSCC cells.Furthermore,western blot was used to detect the activation of AKT and ERK signaling pathways in ATF3 knockout or overexpression.3.Molecular mechanisms of ATF3 regulation of TSCC cell growth and migration.In order to explore the molecular mechanism of ATF3 regulating the growth and migration of TSCC cells,this study analyzed the mRNA gene expression profile of CAL27 cells with ATF3 knockout by RNA-seq,and obtained related signaling pathways and differentially expressed genes in CAL27 cells with ATF3 knockout by GO analysis and gene sequencing.Two targets IFI6 and IFI27 downstream of ATF3 in TSCC cells were identified by qRT-PCR and ChIP analysis,and the high expression of IFI6 and IFI27 in TSCC tissues was further confirmed by IHC and TCGA databases.To analyze whether ATF3 regulates the function of TSCC cells through IFI6 or IFI27,we used gene editing techniques to knock down or overexpress the IFI6 and IFI27 genes in TSCC cells.CCK-8,transwell and wound-healing assay were used to detect the proliferation,migration and adhesion of TSCC cells with knockdown or overexpression of IFI6 and IFI27 genes.Western blot was used to detect the activation of AKT and ERK signaling pathways.To further prove that ATF3 regulates the function of TSCC cells through IFI6 and IFI27,we used nu/nu mice to conduct in vivo experiments and transplanted CAL27 cells with and without CRISPR/Cas9-mediated ATF3 knockout into nu/nu mice dorsal skin.The gross growth of tumors was compared by morphology.And the proliferation differentiation of tumors was further analyzed by IHC and Ki-67 staining.In addition,SCC-9 cells overexpressing ATF3 were overexpressed with IFI6 and IFI27,and then transplanted into dorsal skin of nu/nu mice.Morphology was used to compare the tumor gross growth,and IHC and KI-67 staining was used to further analyze the differentiation of the tumor.Results1.ATF3 expression was low in tongue squamous cells.Through H&E staining and IHC experiments showed that the expression of ATF3 was decreased in TSCC tissues,and the expression of ATF3 was most significantly decreased in poorly differentiated TSCC tissues.Through cell immunofluorescence technology,ATF3 fluorescence staining of TSCC cell lines CAL27 and SCC-9 and normal HOK cells was found the nuclear staining of ATF3 in cells CAL27 and SCC-9 was significantly lower than that in HOK,which was statistically significant.Moreover,the ATF3 level in the nucleus of SCC-9 was statistically lower than that in the nucleus of CAL27,and the same conclusion was obtained through centralized analysis in TCGA database.2.ATF3 negatively regulates the proliferation and migration of tongue squamous cell carcinoma.ATF3 gene knockout and overexpression were performed in TSCC cell lines.CCK8,transwell and wound-healing assay showed that the proliferation ability,invasion ability and migration ability of TSCC cell lines were significantly improved after ATF3 gene knockout.After overexpression of ATF3 gene,the functions of TSCC cell lines were significantly reduced.In addition,western blot showed that AKT and ERK signaling pathways were activated during ATF3 gene knockout and inhibited during ATF3 gene overexpression.In conclusion,our results suggest that ATF3 may negatively regulate TSCC cell growth and migration by down-regulating the activation of AKT and ERK pathways.3.ATF3 negatively regulates the growth and migration of tongue squamous cell carcinoma cells through expression of IFI6 and IFI27.By mRNA gene expression profile RNA-seq analysis of ATF3 knockout CAL27 cells,GO analysis and gene sequencing,we found that the two genes related to signal pathways and differentially expressed in the ATF3 knockout CAL27 cells were IFI6 and IFI27.The two downstream targets of ATF3 in TSCC cells were further identified as IFI6 and IFI27 by qRT-PCR and ChIP analysis.The high expression of IFI6 and IFI27 in TSCC tissues was reconfirmed by IHC and TCGA databases.In vitro experiments,we used gene editing techniques to knock down or overexpress the IFI6 and IFI27 genes in TSCC cells with ATF3 knockout,and found that knockdown of IFI6 or IFI27 significantly blocked the proliferation,migration and adhesion enhancement of CAL27 cells caused by ATF3 deletion.In TSCC cells overexpressed by ATF3 gene,the overexpression of IFI6 or IFI27 significantly reversed the decrease in proliferation,migration and adhesion of SCC-9 cells caused by ATF3 overexpression.Importantly,western blot analysis showed that the corresponding changes in AKT or ERK activity caused by ATF3 deletion or overexpression were offset by IFI6 or IFI27 expression.Taken together,these data suggest that ATF3 regulates TSCC cell growth and migration by down-regulating IFI6 and IFI27.In vivo experiments,according to tumor morphology,tumors formed by ATF3 knockout TSCC cells were significantly larger than those formed by unknockout TSCC cells.Ki-67 cell proliferation assay and IHC showed that the proliferation and differentiation ability of ATF3 knockout TSCC cells was significantly enhanced.On the contrary,TSCC cells overexpressing ATF3 produced smaller tumors than TSCC cells transfected with empty vectors.However,tumors formed by TSCC cells overexpressing IFI6 and IFI27 genes were significantly larger.Similarly,Ki-67 cell proliferation assay showed that overexpression of ATF3 significantly inhibited the proliferation of tumor cells,while overexpression of IFI6 or IFI27 blocked this inhibition.IHC showed that TSCC cell differentiation ability induced by ATF3 overexpression was reduced,while TSCC cell differentiation ability was significantly increased when ATF3 was overexpressed with IFI6 or IFI27.In summary,these results suggest that ATF3 negatively regulates TSCC tumor growth and differentiation in vivo through the expression level of IFI6 or IFI27.ConclusionsIn this study,we confirmed that ATF3 was low expressed in TSCC and low nuclear location of ATF3 was associated with low differentiation;besides,we found that ATF3 participated in TSCC tumor progression through regulating ERK and AKT signaling pathway by targeting IFI6 and IFI27.Our study may provide new ideas for TSCC prognosis and therapy based on ATF3.