GRHL3 Promotes Cancer Cells Migration And Invasion Via Downregulation SNX16

Background and Objective Grainyhead-like 3 belongs to a family of three human genes that encode transcription factor orthologs of the Drosophila gene grainy head(grh).This gene family is required for the development and repair of the epidermal barrier layer.A transcription factor expressed in the differentiated suprabasal layers,plays important roles in epidermal differentiation and barrier formation by controlling multiple genes that regulate the differentiation program in skin ? GRHL3 was recently shown to suppress squamous cell carcinoma due to its activation of phosphatase and tensin homolog(PTEN)expression,and previous studies have shown that GRHL3 plays important roles in epithelial migration during early embryogenesis?SNXs is a large family of proteins that are associated with multiple components of the endosomal system and play a role in endocytosis,intracellular protein trafficking,and cellular signal transduction.The SNX16 protein is composed of 344 amino acid residues and contains two domains,the phagocytic oxidase domain(PX domain)and the colied-coil domain.The PX domain binds to phosphatidylinositol-3-phosphate(PI-3P).SNX16 retents in lysosomes through the colied-coil domain.Also,SNX16 is distributed in the focal adhesions depending on the activity of SNX23(a member of the kinesin family),microtubule and PI-3P kinase.In addition to the presence of cytoplasmic perinuclear zone,SNX16 vesicles exist in the plasma membrane.Previous studies proved SNX16 is distributed in a variety of tumor cells,such as,Hela cells,HepG2 cells,Bel7402 cells,NCI-H460 cells,and MCF-7 cells.Overexpression SNX16 in MCF-7 cells inhibited cell migration and invasion,but knockdown of SNX16 in MCF-7 cells promotes cell migration and invasion.Furthermore,MCF-7 cells with overexpressing SNX16 were injected into nude mice,the tumor formation activity was decreased.Our previous microarray analysis proved that knockdown GRHL3 induced SNX16 gene expression in A431 cells.But little is known about the mechanism related to regulation of GRHL3.In this study,we tried to identify SNX16 as a targeted gene of GRHL3 and to investigate it's regulation mechanism.MethodsPCR was performed to clone the human GRHL3 cDNA from human cDNA bank and the promoter region from genomic DNA in HaCat cells,The GRHL3 cDNA was cloned into expression vector to construct recombinant plasmid with flag tagged pCMV-2B.The recombinant vector pCMV-2B-FLAG-GRHL3 was transfected to A431 cells and MCF7 cells to establish the stable expression GRHL3 cell lines(A431-GRHL3 and MCF7-GRHL3).GRHL3 and SNX16 expression were detected by Western blot.ChIP-seq analysis was performed for A431-GRHL3 to identify whether the SNX16 promoter is a direct target of GRHL3.Transwell analysis was performed to investigate cell migrationand cell invasion.PCR was usedto clone the promoter of the human SNX16 gene(-417 to + 105)from genomic DNA.Then the fragment was subcloned into pGL3 basic vector to construct the recombinant vector.Based on this recombinant vector,the potential GRHL3 binding sites were mutated using PCR.The wild type or mutated pCMV-2B-FLAG-GRHL3 recombinant vectors and PRL TK were cotransfected into293 T cellsto detect activities of the promoter of the SNX16..ResultsOverexpression GRHL3 vector(pCMV-2B-FLAG-GRHL3)and the luciferase reporter vectors with the wild type or mutated promoters of SNX16 were successfully constructed.After transfected with pCMV-2B-FLAG-GRHL3 into A431 and MCF7 cells,Western blotting analysis showed the cell lines with stable overexpression GRHL3 in the A431/grhl3 or MCF7/grhl3 were successfully established;ChIP-seq results proved that there are about 2.49% promoter regions combined specifically with the GRHL3.Among of them,GRHL3 may specifically bind to the promoter of SNX16 gene located in Chr8.Luciferase analysis indicated GRHL3 negatively regulating SNX16 gene expression;and the Immunoblotting further proved that overexpression GRHL3 in A431 or MCF7 cells inhibited SNX16 expression.Interestingly,GRHL3 expresses at higher level and SNX16 expresses at lower level in MDA-MB231 cells with the characteristic of higher invasion comparing to A431 and MCF7 with less invasion.Transwell analysis showed that cell migration and invasion increases in A431 and MCF7 cell with overexpression of GRHL3.ConclusionGRHL3 specifically binds to the promoter of SNX16 gene,and negatively regulates SNX16 gene expression;GRHL3 promotes cancer cells migration and invasion may depend on decreasing SNX16 expression.