HN1 Promotes Dedifferentiation And Stemness Of Anaplastic Thyroid Cancer By Epigenetically Inhibiting CTCF Expression

【purpose】Anaplastic thyroid cancer(ATC)is the most malignant thyroid cancer,and dedifferentiation is an important malignant feature.However,its molecular mechanism is not clear.Therapies targeting to differentiation arrest and stemness had emerged as robust strategies for cancer,but curing ATC remained challenging,mainly on account of superficial mechanistic understanding of the process.Epigenetic modification is critical for tumorigenesis and progression,while its role in facilitating thyroid cancer dedifferentiation has been poorly understood.Therefore,there is an urgent need to discover the key factors that affect ATC differentiation and stemness expression and find out the regulatory mechanisms.【method】The GSE33630 microarray data set was analyzed to assess the correlation between HN1 expression and differentiation related genes.Western Blotting(WB)and q RTPCR were used to detect the protein and m RNA expression levels of stemness and differentiation markers of cells after different treatments.Immunofluorescence assay and flow cytometry were used to detect stemness index(CD133)and differentiation index(NIS)to assist in verifying the differentiation and stemness expression differences of cells after different treatments.Sphere formation assay was used to verify the stemness expression difference between cells with different degrees of HN1 expression.In animal experiments,lentiviral or si RNA was used to knock down or over-express target genes HN1 or CTCF.Thus,HN1-KD,HN1-OE,HN1-WT,CTCF-KD,CTCF-OE,CTCF-WT cell lines were constructed,and their stable cell lines were constructed by purine screening.To investigate the effect of HN1 on tumor growth,ATC cells(8505C)pre-transfected with luciferase were subjected to HN1 knockdown(HN1-KD)and control without treatment(HN1-WT).These cells were then injected subcutaneously into nude mice.Tumor size was measured after tumor growth in nude mice for seven days.D-fluorescein(100 μg/g)was injected at the end of the experiment,and the fluorescence intensity was observed and compared.In the xenograft model of zebrafish tumor transplantation,the differences in fluorescence intensity and area of ATC cells after different treatments were compared three days after injection into zebrafish.Downstream mechanisms were then explored: WB and q RT-PCR were used to detect the difference of CTCF expression between HN1 low expression and HN1 high expression cells or tissue samples,and then WB and q RT-PCR were used to detect the changes of cell stemness and differentiation related indicators after CTCF knockdown or CTCF overexpression.And the changes of dryness and differentiation related indexes after HN1 and CTCF were knocked down in ATC.ATAC-seq was compared between CTCF-OE group and CTCF-WT group.In order to describe the peak value of ATAC-seq,the high-expression genes of CTCF-WT and CTCF-OE groups in the TCGA transcriptome database were compared by enrichment algorithm.And the motif was calculated to obtain the potentially activated transcription factor binding regions of CTCF-WT and CTCF-OE cells,respectively.To explore the pathway mechanism of HN1 regulating CTCF,immunoprecipitation combined with LC-MS/MS was used to detect the potential binding proteins of HN1 in ATC cells,and the protein-protein interaction network was further constructed by using the proteomics results and the STRING database.The histone proteins associated with CTCF were searched through the database,and Ch IP-PCR was used to analyze the H3K27ac-bound DNA fragments.Immunofluorescence co-localization technology was used to verify the colocalization relationship between HN1 and H3K27 ac,and co-IP experiment was used to verify the binding and regulatory relationship between HN1,CTCF and H3K27 ac.【result】In our study,novel roles for hematological and neurological expressed 1(HN1)in promoting dedifferentiation and stemness in ATC were uncovered.It was found that HN1 is associated with tumor stem and differentiation expression,and knockdown of HN1 could effectively impede the sphere formation capacity of ATC cells and increase NIS,TG and other differentiation markers,while overexpression of HN1 was the opposite.Nude mice and zebrafish xenograft models showed that deletion of HN1 effectively inhibit ATC growth in vivo.Moreover,ATAC-seq and CHIP-seq analysis indicated CTCF could promote the synergistic effects of differentiation-associated transcription factors by controlling chromatin accessibility,thus modulating the stemness and dedifferentiation of ATC.And we found that HN1 hindered the acetylation of H3K27 by recruiting HDAC2,thereby inhibiting the transcriptional activation of CTCF.【conclusion】Taken together,our study not only provided new strategies for targeting stemness and differentiation in ATC,but also revealed novel biological functions of the HN1-CTCF signaling axis in blocking differentiation and promoting cancer stem cell-like characteristics.