ARID1A Loss Promote Tumorigenesis And Progression Through Derepressing Of Human Endogenous Retrovirus-H

Through evolutionary exaptation,transposable elements(TEs)have become an integral part of the human genome,offering ample regulatory sequences to the organism.Although people have recognized the functional effects of TEs-derived sequences on early embryogenesis,the silencing mechanism of transposons and the possible downstream mechanism of transposons,their upstream regulatory mechanisms,functions and mechanisms in malignant tumors are only starting to emerge.Objective:The incidence and mortality rate of colorectal cancer(CRC)are high,which seriously threaten public health.Therefore,this study takes CRC as an example to explore the role of transposons on tumorigenesis and progression,and to elucidate their specific mechanisms.Methods:(1)We collected RNA-seq data of colorectal cancer patients from the Cancer Genome Atlas and an independent cohort(GSE50760).In total,there are 69 normal tissues and 649 cancer tissue samples.Differential expression analysis was performed to identify abnormally activated transposable elements in CRC.(2)Many endogenous retroviruses(ERVs)play important role during human early embryonic development.Based on the ERVs that activate during early embryonic development,we identified ERVs associated with the tumorigenesis and progression of cancer.(3)Fluorescence in situ hybridization experiments were performed using tissue microarray to verify the abnormal high expression of candidate ERVs in colorectal cancer.(4)Cell viability assay,colony formation assay,tumor sphere formation assay,organoid culture assay derived from colorectal cancer patient tissue,and xenograft tumor assay were used to investigate the functions of candidate ERVs.(5)Whole-exome sequencing and transcriptome data of cancer patients were used to analyze the association between frequently mutated genes and candidate ERVs.Cancer Cell Line Encyclopedia CRC cell lines were used to verify the correlation between genes and candidate ERVs.(6)RNA-seq analysis of the gene knockout cell lines,immunoblotting,and RT-qPCR were performed to explore the causal association between candidate genes and candidate ERVs.(7)In order to explore the possible mechanism by which candidate genes influence candidate ERVs,we analyzed Ch IP-seq and ATAC-seq,and carried out Ch IP-qPCR experiments for verification.(8)In order to explore the mechanism of the candidate ERV effect on tumorigenesis and progression of CRC,we identified the downstream target genes and pathways of the candidate ERV based on virus infection,RNA interference and RNA sequencing experiments.We constructed a candidate ERV-activated cell line and used drug sensitivity experiments to verify the association between candidate ERVs and downstream target genes.Results:(1)Many TEs,especially the pluripotency-related human endogenous retrovirus H(HERVH),are abnormally activated in CRC samples.(2)Suppression of HERVH in CRC cells and patient-derived organoids impairs tumor growth.(3)Transcriptional upregulation of HERVH is positively correlated with mutations of multiple tumor suppressors,including ARID1 A.(4)Knockout of ARID1 A in CRC cells leads to increased transcription of a group of HERVH loci in the genome,which is related to the compensatory contribution of ARID1 B which is ARID1A’s homologous gene.(5)Knockout of ARID1 A in CRC cells leads to increased H3K27 ac and H3K4 me at derepressed HERVH loci.(6)ARID1A and HERVH co-regulate the expression of a group of target genes.Abnormally high expression of HERVH exerts a regulatory effect by affecting the binding of BRD4 to its target genes.Conclusions:(1)HERVH is abnormally activated in colorectal cancer tissues.(2)HERVH is essential for colorectal cell lines survival.(3)There is critical role for ARID1 A in restraining HERVH,which is related to ARID1B-mediated regulatory function.(4)Abnormal activation of HERVH can stimulate BRD4-related transcription.