The Oncogenic Function And Mechanism Of The Key Transcription Factor RXRG In Papillary Thyroid Carcinoma

Background:Thyroid cancer is one of the most common types of cancers in China.Papillary thyroid carcinoma(PTC)is the most common pathological subtype of thyroid cancer.Epigenetic changes play an important role in development of PTC.Transcription factors(TFs)are a group of proteins which bind to the gene expression regulatory regions including promoters and/or enhancers and play a crucial part in regulation of gene transcription.Abnormal gene expression regulation due to TFs contribute to tumorigenesis and progression of cancers.Therefore,it is important to identify the key TFs and their target genes in malignancies.Super Enhancer(SE)is a large cluster of transcriptionally active enhancers.Multiple master transcription factors,cofactors and histone modification markers are enriched in SEs,which are critically involved in regulating cell fate and cancer development.The master transcription factors enriched in SEs are usually expressed in cell type-specific or lineage-specific ways and can regulate gene transcription and expression of in a cancer-specific way.However,SEs in PTC are still largely unknown.Long non-coding RNAs(lncRNAs)can regulate gene expression at the epigenetic level,the transcriptional level and the post-transcriptional level,and have been found to impact proliferation,invasion and metastasis of PTC.As one of the most common epigenetic modifications in eukaryotes,DNA methylation is crucial for gene expression regulation,transposon silencing and gene imprinting.It has been shown that DNA hypomethylation is significantly associated with active enhancers and increased binding of TFs.In this study,we identified Retinoid X Receptor γ(RXRG)as the key TF of PTC by using Enhancer Linking by Methylation/Expression Relationships(ELMER)algorithm as well as functional studies in vitro and in vivo.We found that RXRG enhances transcription and expression of CD44 and FOXC2-AS1 oncogenes through binding to the SE of CD44 and the promoter of lncRNA FOXC2-AS1,up-regulates the phosphorylation levels of SRC,activates both PI3K/AKT and MAPK/ERK signaling pathways,and,thus,promotes PTC development.Methods and Results:1.The PTC key TF RXRG which was significantly up-regulated in PTC tissues,was predicted via the ELMER algorithm:The ELMER algorithm was used to predict eight key TFs including RXRG through analyzing the DNA methylation and RNA-seq data of PTC tissue in the TCGA database.The expression levels of RXRG in PTC tissues are significantly higher than those in normal tissues.2.RXRG significantly promotes proliferation,invasion,and metastasis of PTC cells:It has been showed that silencing of RXRG significantly inhibited proliferation,clone formation,invasion and metastasis of PTC cells,while overexpression of RXRG significantly promoted malignant phenotypes of PTC cells.The nude mouse xenograft assays confirmed that RXRG can promote the proliferation and distant metastases of PTC cells in vivo.3.RXRG binds to the SE of CD44 gene and promotes transcription and expression of CD44:Through integrated analyses of RNA-seq,H3K27ac ChIP-seq,ATAC-seq and RXRG ChIP-seq data of PTC cells and tissues,we found that transcription factor RXRG can bind to the SE region of CD44 gene and promotes transcription and expression of CD44,and,thus,activates the downstream PI3K/AKT and MAPK/ERK signaling pathways.4.RXRG binds to the promoter region of the lncRNA FOXC2-AS1 and up-regulates expression of FOXC2-AS1:Through integrated analyses of RNA-seq,ATAC-seq and RXRG ChIP-seq data of PTC cells and tissues and validation through RT-qPCR assays and ChIP-qPCR assays,we found that TF RXRG can promote FOXC2-AS1 transcription by binding to the promoter region of FOXC2-AS1.5.LncRNA FOXC2-AS1 can significantly promote malignant phenotypes of PTC cells:It has been showed that silencing of FOXC2-AS1 significantly suppressed proliferation,clone formation,invasion and metastasis of PTC cells,while overexpression of FOXC2-AS1 significantly enhanced malignant phenotypes of PTC cells.6.LncRNA FOXC2-AS1 binds to PYK2 protein,promotes the interactions between PYK2 and SRC,and up-regulates the phosphorylation levels of SRC:The RNA pull-down assays and RIP assays indicated that lncRNA FOXC2-AS1 can bind to PYK2 protein in PTC cells.The CoIP assays showed that FOXC2-AS1 can promote the interactions between PYK2 and SRC and up-regulate the phosphorylation levels of SRC and PYK2,and,thus,activate both PI3K/AKT and MAPK/EKR signaling pathways in PTC cells.Conclusions:In the current study,we for the first time identified RXRG as a key TF in PTC.RXRG is significantly overexpressed in PTC tissues compared to normal tissues and can promote proliferation,invasion,and metastasis of PTC cells.RXRG binds to the SE region of CD44 gene,promotes CD44 transcription and up-regulates expression levels of CD44,which activating both PI3K/AKT and MAPK/ERK signaling pathways in PTC cells.Moreover,RXRG also binds to the promoter region of lncRNA FOXC2-ASl,up-regulates expression levels of FOXC2-AS1 in PTC cells.LncRNA FOXC2-AS1 directly binds to the PYK2 protein,elevates the phosphorylation levels of SRC protein and activates PI3K/AKT and MAPK/ERK signaling pathways.