Searching For Candidate Target Genes Of GRHL3 Involved In Cell Migration And Invasion Of Skin Squamous Cell Carcinoma

Background and Objective Cutaneous squamous cell carcinoma is characterized by local invasion and distant metastasis.Although surgical resection of local tumor tissues was the main treatment method,the residual tumor and metastatic lesions were difficult to be cleared.However,the molecular mechanism of migration,invasion and metastasis of skin squamous cell carcinoma is not clear at present.In particular,studies on the involvement of target genes regulated by important transcription factors in the migration and invasion of skin squamous cell carcinoma are rare.The mammalian Grainyhead-like transcription factor family contains GRHL1GRHL2,and GRHL3,which is expressed in a specific tissue-and development-manner.Usually,GRHL3 mainly expresses in the epidermis,olfactory epithelium,oral cavity,cochlear lining cells,esophagus,stomach,lung,kidney and digestive tract and genitourinary ducts.Its functions include: regulating the proliferation and migration of epidermal cells during embryonic development;Participate in the regulation of adult skin injury repair,wound healing,etc.It has been reported to be in close relations with tumors such as breast cancer,esophageal tumor,colorectal cancer,lymphoma,clear cell renal cell tumor,skin non-melanocyte cancer,and head and neck cell squamous carcinoma.GRHL3 can promote the formation of tumors and inhibit the occurrence of tumors as well,playing different and contradictory roles in tumors of different tissues and organs,even in the same tumor,such as breast cancer,suppressing its growth in the early stage while promoting late.However,GRHL3 is involved in the regulation of epithelial cell migration and biological behavior in the physiological processes of skin protective barrier formation,epidermal wound healing,neural tube closure and palpebral fissure closure,suggesting that GRHL3 may play an important role in the migration and invasion of skin squamous cell carcinoma cells.Our previous study found that overexpression of GRHL3 promotes migration and invasion of tumor cells such as swollen skin squamous cell carcinoma and breast cancer by inhibiting the expression of cadherin.However,the species and number of target genes involved in migration and invasion of tumor cells by GRHL3 are unknown.In this study,skin squamous cell carcinoma was selected as the study object to search for the migration and invasion related target genes regulated by GRHL3.Methods Detect GRHL3 expression in skin cancer tissues by immunohistochemistry;and study the effect of GRHL3 on the migration and invasion of skin squamous carcinoma cells.Then transfect the overexpression vector p CMV-2B-FLAG-Grhl3 and the control vector p CMV-2B-FLAG-vector into A431 cells with Lipofectamine3000 to establish the Grhl3-overexpressing A431 cell line.The expression of GRHL3 was detected by Western blot to identify the overexpression of A431 cell lines.Transwell analysis was performed to detect migration and invasion of A431 cells in which overexpressed GRHL3.Total RNA was extracted fromthe A431/Vector cells and the A431/Grhl3 cells to generate c DNA by RT-PCR and perform Microarray analysis for RNA expression profiling.Differentially expressed genes were screened by Nimble Scan RMA(Version 2.5),and then compared with the control group,regarding those whose gene expressions differ by more than 2 fold(Fold change ? 2.0)as candidate genes,followed by functional clustering analysis of differential genes on the online analysis website http://pantherdb.org/ to obtain migration of GRHL3-overexpressing A431 cells.Capture DNA by chromatin immunoprecipitation with Anti-Flag antibody,and analyze the GRHL3-regulated target gene and the binding site of GRHL3 by Ch IP-Seq technique to conduct the analysis of GRHL3 binding peaks by Model-based Analysis of Ch IP-seq(MACS version 1.4),and cluster analysis of GRHL3 regulatory candidate gene function on the online analysis website http://pantherdb.org/.Finally compare the GRHL3-overexpressing cell lines detected by RNA chip expression profiling and Ch IP-Seq with GRHL3-regulated differential genes in the control cell line A431 to screen the target genes in keeping with GRHL3-regulated cell invasion and migrationResults The expression level of GRHL3 in skin cancer tissues was significantly higher than that in normal skin tissues by immunohistochemistry.Results of Western Blot confirmed that GRHL3 was overexpressed in A431 cells at the protein level,and thus established the A431 cell line stably overexpressing GRHL3.Transwell invasion and migration assay showed that overexpression of GRHL3 caused an increase in the migration and invasion ability of A431 cells.Overexpression of GRHL3 resulted in 136 differentially expressed genes in A431 cells associated with cell migration and invasion in the expression profile microarray.According to Ch IP-Seq results,the GRHL3-binding genomic DNA was primarily located in the intergenic region(55.65%),while only 2.49% distributed in the promoter region of the structural genes.The expression profile chip showed the 128 differently expressed genes showed in the expression profile chip and the GRHL3-regulated 149 candidate genes in Ch IP-Seq were all involved in cell migration and invasion,of which 26 genes were in consistency with each other,suggesting to promote the migration and invasion of squamous cell carcinoma cells by GRHL3 regulating 26 candidate genes.Conclusion GRHL3 may promote cell migration and invasion by regulating 26-gene expression involved in migration and invasion in skin squamous cell carcinoma cells;GRHL3-binding DNA is mainly located in the intergenic region(55.65%),only 2.49% in the promoter region of structural genes.