H2AZ Acetylation By LincZNF337-AS1 Via KAT5 In The Transcriptional Misregulation In Cancer Signaling Pathway In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the most common cancer among adults and accounts for approximately 740,000 deaths worldwide.It is highly prevalent in China,where new cases of HCC and related deaths are more than half of its global number.Although the combination of multiple therapies has greatly improved the prognosis of HCC,the 5-year survival rate for HCC is still not ideal.The formation of HCC is caused by the disorder of multiple genes and signaling pathways,and these related genes and signaling pathway disorders also confer the characteristics of tumor recurrence and metastasis.Therefore,it is particularly important to understand the formation mechanism,recurrence and metastasis mechanism of HCC in the process of prevention and treatment of HCC.Histone variants are variants of traditional histones that regulate chromatin structure and associated biological processes by replacing traditional histones in chromatin at specific locations.Histone variants are important in eukaryotic chromatin stabilization.With the development of proteomics,histone variation has also been found to play an important role in gene transcription and different tumors.In a study of breast cancer,H4G was reported to promote the proliferation of breast cancer cells.In malignant melanoma,mH2A was proven as a tumor suppressor gene in melanoma cells,with CDK8 possibly being the downstream target gene.So is there a particular histone or variant in HCC that plays a critical role in the development of HCC?We analyzed the transcriptome data of 23 pairs of histones and variants in hepatocellular carcinoma and adjacent tissues by RNA-seq technique.Combined with literature analysis,we found that H2A.Z may be associated with tumors.Subsequent functional and mechanism studies further confirmed that H2A.Z acetylated by LincZNF337-AS1 via KAT5 plays a critical role in the occurrence and development of HCC through the tumor transcription factor dysregulation pathway.Methods and results:1,RNA-seq and data analysis.A previous search in the GEO(Gene expression omnibus)database yielded 23 pairs of raw data(not transcripts)(GSE138485).To verify the reliability of the data,we explored three pairs of genes from HCC cells and paracancerous tissues(BioProject:PRJNA642330).A cumulative distribution curve,based on the analysis of the above two data,indicated the difference to be highly consistent.Then,we analyzed the gene expression levels of all histones and variants in 23 pairs of sequencing data,and found that the expression of H2A.Z was significantly increased in HCC.Based on the literature analysis,we believe that H2A.Z is probably one of the oncogenes leading the occurrence and development of hepatocellular carcinoma.2,The expression of H2A.Z RNA and protein was significantly increased in HCC.We downloaded the transcripts of hepatocellular carcinoma and adjacent tissues as well as relevant clinical data from the TCGA database,and the results showed that H2A.Z was highly expressed in hepatocellular carcinoma tissues.RT-qPCR and WB method were used to test the RNA and protein expression levels of H2A.Z in HCC.We confirmed that H2A.Z is highly expressed in HCC.3,The correlation between H2A.Z and clinicopathological features and its prognostic significance in hepatocellular carcinoma.We selected 96 HCC tissues from the biological sample bank of Taizhou People’s Hospital,and analyzed the expression of H2A.Z RNA qRT-PCR,according to the median,the 96 HCC patients were divided into two groups:the high H2A.Z group and the low H2A.Z group.Combined with the clinicopathological features and follow-up data of the patients,we concluded that the expression of H2A.Z was closely related to the TNM stage and the presence or absence of vascular invasion.In addition,H2A.Z is closely related to survival time and recurrence time of patients,and the high expression of H2A.Z indicates that the tumor is more likely to relapse and has a shorter survival time.4,H2A.Z enhanced the tumor biology of hepatocyte cancer cell lines in vitro.We established stable sh_H2A.Z and clone_H2A.Z cell lines in mhcc-97h and HepG2 cell lines.Compared with the blank control group(NC),interfering with the expression of H2A.Z can reduce the proliferation,migration and invasion of HCC,increase the ability of apoptosis,and keep cells in G1 phase.Overexpression of H2A.Z is opposite to interference of H2A.Z.5,H2A.Z can enhance the tumor biology of hepatocyte cancer cell lines in vivo.We used the established HCC cell line stably transfected with H2A.Z to induce tumor formation in nude mice,and detected the tumorigenesis ability of tumor cells in each group of nude mice.Stable cell lines were injected into the tail vein of nude mice to detect the level of metastatic tumor in the lungs of nude mice.The results showed that H2A.Z could enhance the tumorigenesis and lung metastasis of tumor cell lines.At the same time,the axillary tumors of nude mice were collected for immunohistochemical analysis of related protein expression.6,RNA-seq analysis of H2A.Z knockdown HCC cell lines and control HCC cell lines showed that H2A.Z was associated with tumor transcription factor dysregulation pathway.We performed high-throughput sequencing analysis on H2A.Z knockdown HCC cell lines and control HCC cell lines.Combined with Chip-Seq data of H2A.Z,KEGG pathway analysis showed that H2A.Z was most likely associated with tumor transcription factor maladjustment pathway.The related transcription factors in this pathway were TCF3,CDK14,JUP,Spintl,CDKN1A and IGF1.According to GSEA database analysis,H2A.Z is related to tumor cell characteristics such as cell proliferation,apoptosis,cycle and metastasis.7,BCL6 is the interaction protein of H2A.Z,and the BTB and PHA03247 parts of BCL6 protein are the main binding domains of H2A.Z.The motif analysis of ChIP-Seq data of H2A.Z indicated that BCL6 was the most likely interacting protein of H2A.Z,and the interaction between the two proteins was confirmed by Co-IP method.At the same time,Chip-seq data of BCL6 also showed that BCL6 binds to the promoter region of TCF3,CDK14,JUP,Spint1,CDKN1A and IGF1.In addition,we analyzed the domain of BCL6 protein,and confirmed that BTB and PHA03247 are the main binding domains of BCL6 protein with H2A.Z.At the same time,the migration and invasion ability of hepatocellular carcinoma cell lines were decreased after knockdown of BCL6.8,The acetylation level of H2A.Z was significantly elevated in hepatocellular carcinoma.The analysis of ChIP-seq and RNA-seq data of H2A.Z indicated that H2A.Z mainly played a promoting role in downstream genes.In combination with literature analysis,we believed that the acetylation level of H2A.Z might be increased.Then,we verified the acetylation level of H2A.Z in hepatocellular carcinoma cell lines and tissues,and the results showed that the acetylation level of H2A.Z was increased in hepatocellular carcinoma tissues and cell lines.9,The acetylation of H2A.Z is associated with lincZNF337-AS1.We performed RIP experiment with H2A.Z acetylated antibody,and sequenced the obtained RNA for non-coding protein.At the same time,sequencing data of non-coding protein RNA of hepatocellular carcinoma and its adjacent tissues were downloaded from GEO data,and combined analysis revealed that LincZNF337-AS1 may bind to H2A.Z.The results were verified by RIP-qPCR.In addition,the acetylation level of H2A.Z decreased after lincZNF337-AS1 was knocked down.After knockdown of LincZNF337-AS1 in H2A.Z overexpressed hepatocellular carcinoma cell lines,the cloning ability and proliferation ability of tumor cells were decreased.10,LincZNF337-AS1 is highly expressed in HCC and is related to the clinical prognosis of HCC patients.We downloaded the sequencing data of non-coding protein RNA associated with HCC from the TCGA database.Analysis of bioinformatics results showed that lincZNF337-AS1 was abnormally high in HCC tissues.We also selected 30 cases of paired HCC and paracancerous tissues from the hospital biobank,and qPCR showed that lincZNF337-AS1 was highly expressed in hepatocellular carcinoma.In follow-up data from 96 patients with hepatocellular carcinoma,high expression of lincZNF337-AS1 was associated with poor prognosis.11,In the exon sequence of LincZNF337-AS1,exons 1 and exons 5 bind to H2A.Z.We truncated the full length of the exon sequence of lincZNF337-AS1,and studied the binding of the truncated fragment to H2A.Z.After synthesis,the trunked sequence was inserted into the plasmid vector and co-transfected with the H2A.Z overexpressing vector in HEK393T cells.The results were verified by RIP-qPCR and RNA-pull down.The results show that exons 1 and exons5 bind to H2A.Z.12,LincZNF337-ASl binds to KAT5 and promotes acetylation of H2A.Z.Literature analysis showed that KAT5 and KAT3 were most likely associated with the acetylation of H2A.Z.Co-IP method demonstrated that KAT5 interacts with H2A.Z in hepatocellular carcinoma.RIP-qPCR and RNA-pull down confirmed the binding of lincZNF337-AS1 to KAT5,respectively.The acetylation level of H2A.Z decreased after knockdown of lincZNF337-AS1 and KAT5.Truncation experiments showed that exons3 of lincZNF337-ASl were bound to KAT5.ConclusionIn conclusion,the histone variant H2A.Z is a proto-oncogene in HCC,which promotes the development of tumors and is related to the prognosis of patients.Bcl6 is one of the interacting proteins of H2A.Z in HCC.Acetylation of H2A.Z is an important epigenetic modification in HCC;lincZNF337-AS1 binds to H2A.Z and KAT5 at different sites in order to promote this acetylation.