The Mechanism Of HP1α-HDAC1 Complex Influencing The Proliferation Of Intrahepatic Cholangiocarcinoma Cells By Transcriptionally Regulating STAT1

Objective: To investigate the regulatory effect of HP1α protein on the proliferation of intrahepatic cholangiocarcinoma cells and the related molecular mechanism,and to transform the mechanism into a feasible therapeutic regimen.Methods: Intrahepatic cholangiocarcinoma cell line HUCCT1 and HCCC-9810 were cultured.Immunohistochemical assay was used to measure the expression level of HP1α in tumor and adjacent tissue samples.The effect of HP1α expression change on the proliferation of intrahepatic cholangiocarcinoma cells was evaluated by colony formation assay,CCK-8 assay and flow cytometry.Real-time quantitative PCR and Western Blot were used to evaluate the regulatory effect of HP1α on Type I Interferon signaling pathway.The corresponding regulatory target was then screened out.Protein immunoprecipitation assay,chromatin immunoprecipitation assay,RNA sequencing,electrospray ionization mass spectrometry and CUT&Tag assay were used to investigate the function of histone acetylation markers,the interaction between HP1α and histone deacetylase and the interaction between the protein dimer and STAT1 promoter region.The inhibitory effects of histone deacetylase inhibitor TSA and interferon preparation on the proliferation of intrahepatic cholangiocarcinoma cells were evaluated by colony formation assay,CCK-8 assay and flow cytometry.The conclusion was further verified in murine intrahepatic cholangiocarcinoma model and nude mice model.Results: HP1α was found to be highly expressed in intrahepatic cholangiocarcinoma tissue.HP1α knockdown can significantly inhibit the proliferation of intrahepatic cholangiocarcinoma cells.After HP1α was knocked down,the m RNA level and protein level of STAT1 and interferon-stimulated genes were significantly up-regulated and the Type I Interferon signaling pathway was activated.STAT1 can be regulated by histone acetylation markers,especially H3K27 ac.HP1α can form protein dimer with HDAC1 and be located in the promoter region of STAT1 gene.HP1α-HDAC1 complex can down-regulate H3K27 ac markers,and up-regulate H3K9me3 markers in local environment.TSA treatment down-regulated the expression of HP1α and reduced the distribution of HP1α-HDAC1 complex in the specific region of STAT1 promoter.Interferon preparation treatment and TSA treatment both exhibited significant inhibition on the proliferation of intrahepatic cholangiocarcinoma cells.Compared with interferon preparation monotherapy,the combination of interferon preparation and TSA had stronger inhibiting effects on the proliferation of intrahepatic cholangiocarcinoma cells.Conclusions: HP1α-HDAC1 complex affects the distribution of histone markers,e.g.,H3K27 ac and H3K9me3 by directly binding to the promoter region of STAT1 gene,transcriptionally down-regulating the expression level of STAT1 and inhibiting the Type I Interferon pathway.Combined application of interferon preparation and TSA can inhibit the proliferation of intrahepatic cholangiocarcinoma cells by targeting HP1α-HDAC1 complex,which can provide a certain reference for the clinical practice and drug development of intrahepatic cholangiocarcinoma treatment.