| Background:Lung cancer is a common disease of respiratory system,which is difficult to treat.In the past 20 years,the incidence rate and mortality of lung cancer ranked first among all malignant tumors,seriously threatening human health.Among them,non-small cell lung cancer is the most common,accounting for more than 80%of lung cancer patients.Among them,lung adenocarcinoma accounts for nearly 50%of non-small cell lung cancer,which is the focus and difficulty of current research and clinical treatment.Histone is a family of highly conserved basic proteins that bind to DNA in the chromatin of eukaryotic cells.It is the basic nuclear protein that constitutes the nucleosome structure.Studies over the past two decades have shown that histones change gene expression through covalent modifications,including methylation,acetylation,ubiquitin like,phosphorylation,ubiquitination and so on.Histones also play important roles in the occurrence and development of malignant tumors.At the same time,in addition to the core conserved unit H2A,H2B,H3,H4 and H1 in histones,abnormal expression of histone variants and specific epigenetic modifications will also change the stability and spatial conformation of nucleosomes,affect the transcriptional activation or silencing of downstream regulatory genes,as well as DNA damage and repair,which lead to the occurrence and development of tumors.The study on the function and covalent modification of histone family members in tumor cells not only helps to understand the physiological and pathological mechanisms of gene expression and regulation,but also provides a new theoretical basis and new ideas for the early diagnosis and follow-up treatment of tumors.Histone cluster 1 H2A family member B(HIST1H2AB)is an important member of histone H2A family.It has only one exon and encodes a replication dependent histone.It is a histone of H2A family that has not been fully studied.HIST1H2AB is considered to be one of the main differentially expressed genes in Alzheimer’s disease.HIST1H2AB is also one of the top 10 up-regulated proteins in osteosarcoma cells,which may play an important role in the development of osteosarcoma.We screened histone variant gene HIST1H2AB with abnormal high expression by analyzing differentially expressed genes in lung adenocarcinoma patients in TCGA database and GEO database.Our clinical sample analysis also further confirmed that the expression of HIST1H2AB in lung adenocarcinoma was significantly higher than that in adjacent tissues.Therefore,based on the above literature search and research results,my subject focuses on histone variant gene HIST1H2AB,and further explores the function of HIST1H2AB in regulating cell proliferation and apoptosis in lung adenocarcinoma cells through cell experiments in vitro and subcutaneous tumorigenesis in vivo.At the same time,I screen the downstream target genes regulated by HIST1H2AB through Prime View Human Gene Expression Array technology,in order to discover the molecular mechanism of HIST1H2AB promoting tumorigenesis and development.All this will provide theoretical and experimental basis for the treatment of lung cancer.Materials and methods:1.TCGA database and geo database were used to screen differentially expressed genes in cancer tissues and adjacent tissues of lung adenocarcinoma patients,and to screen candidate genes with abnormal high or low expression in cancer tissues.Using the cancer tissue samples of lung adenocarcinoma patients collected by the research group,realtime PCR and immunohistochemical techniques will further verify whether the oncogene or tumor suppressor of the candidate gene.2.Using the HIST1H2AB gene DNA template,designed the RNA interference target sequence,constructed the target gene RNA interference lentivirus vector(sh HIST1H2AB),took sh Ctrl as the blank control vector,and transfected the packaged corresponding virus particles into human lung adenocarcinoma cell lines A549 and H1299,established HIST1H2AB knockdown and control stable cell strains,and verified the knockdown efficiency of HIST1H2AB.3.The effect of HIST1H2AB gene on the proliferation of lung adenocarcinoma cells was detected by celligo imaging analysis technology,MTT assay and cell cloning method in vitro.At the same time,flow cytometry was used to detect the effect of knockdown of HIST1H2AB expression on apoptosis.4.The effect of HIST1H2AB knockdown on the tumorigenicity of A549 cells in vivo was detected by subcutaneous tumorigenesis experiment in nude mice.5.Prime View Human Gene Expression Array technology was used to screen the effects of HIST1H2AB knockdown on gene expression profile and signal pathway in A549 cells,and to explore the mechanism of HIST1H2AB regulating the occurrence and development of lung adenocarcinoma.Results:1.The bioinformatics analysis showed that histone variant HIST1H2AB gene expression in lung adenocarcinoma was significantly higher than that in adjacent tissues.We further verified that HIST1H2AB is a potential cancer promoting gene by using real time PCR and immunohistochemical techniques based on the cancer tissue samples of lung adenocarcinoma patients collected by our research group.The expression of HIST1H2AB in human lung adenocarcinoma cell lines A549,H1299and H1975 was significantly higher than that in normal lung epithelial cell lines BEAS-2B.2.The knockdown stable cell lines A549-sh HIST1H2AB and H1299-sh HIST1H2AB were successfully constructed in human lung adenocarcinoma cell lines A549 and H1299.The silencing efficiency of HIST1H2AB was more than 80%,and the knockdown effect was obvious.3.The cell function test in vitro confirmed that knockdown of HIST1H2AB expression could inhibit cell proliferation and induce apoptosis of A549 and H1299cells.4.The subcutaneous tumorigenesis experiment in nude mice further confirmed that subcutaneous injection of 1x10~7 knockdown HIST1H2AB in nude mice could inhibit the tumorigenicity of A549 cells in vivo.The results showed that the tumor formation rate and size of mice in HIST1H2AB knockdown group were lower than those in control group,which further suggested that HIST1H2AB was a cancer promoting gene.5.The detection of human whole gene expression microarray preliminarily confirmed that knocking down the expression of HIST1H2AB could activate interferon signaling pathway,inhibit the proliferation of cancer cells and induce apoptosis.Real time PCR and western blotting experiments confirmed that knockdown of HIST1H2AB expression in A549 cells could attenuate the transcription and translation levels of HMGB1,FOXM1,F2RL1,SLC4A7.ConclusionHistone variant HIST1H2AB gene is a cancer promoting factor.Bioinformatics analysis and clinical sample experiments have proved that HIST1H2AB is abnormally high expressed in cancer tissues of patients with lung adenocarcinoma.Cell function test in vitro and subcutaneous tumorigenesis test in nude mice confirmed that knockdown of HIST1H2AB expression could inhibit cell proliferation and induce apoptosis of lung cancer cells.Knockdown of HIST1H2AB expression in A549 lung cancer cells could activate interferon signaling pathway and inhibit the expression of HMGB1,FOXM1,F2RL1,SLC4A7and other genes;It can inhibit the proliferation of cancer cells and induce apoptosis.Therefore,HIST1H2AB gene may be an important regulator of interferon signaling pathway,play an important regulatory role in the occurrence and development of lung cancer,and have the potential to become a prognostic marker and a new therapeutic target for lung adenocarcinoma. |
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