BAP18 Up-regulates β-catenin-mediated Gene Transcription And Its Effect On Non-small Cell Lung Cancer Growth

Objective: Lung cancer is a malignant tumor with high morbidity and mortality in the world.The research on lung cancer has always been the focus and hotspot in the field of oncology.Lung cancer can be divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC)according to the pathological type,the latter can account for80% of the total lung cancer.In recent years,due to the development of inspection methods,great progress has been made in the early detection and treatment of lung cancer,but most patients are still diagnosed as advanced patients at the first visit,losing the opportunity for surgery.The 5-year overall survival rate for metastatic lung cancer patients remains below 5%.Although targeted and immunotherapy have partially benefited patients,eventually resistance will inevitably develop,leading to treatment failure.Therefore,finding new therapeutic targets is the main direction for future research on lung cancer.The expression of cytogenetic information begins with transcription.This fundamental,conserved and extremely complex biological process is tightly regulated to ensure that genetic programs adapt to cellular needs.Transcriptional dysregulation can lead to serious diseases,including cancer.Mediators of RNA polymerase II transcription were identified more than 20 years ago as co-regulators of transcription.Since then,it has been established that transcriptional regulators are often required for transcription and can act as bridge factors.Transcriptional regulation needs the participation of many co-regulatory factors,which requires further research in lung cancer.Based on the literature and bioinformatics database,we screened some co-regulators reported in epigenetic regulation,and found that BAP18 is highly expressed in tumors and significantly correlated with proliferation.Our laboratory has rich research experience on BAP18 in other tumors before.Therefore,this research focuses on the biological function and mechanism of BAP18 in lung cancer.BAP18(BPTF associated protein of 18 k Da,BAP18),is a component of the chromatin complex MLL1-WDR5 and NURF/BPTF.BAP18 carries the Swi3,Ada2,N-Co R,(TF)IIIB(SANT)domains,which normally occur in chromatin remodeling complexes.Previous studies have shown that in prostate cancer,BAP18 can act as a co-regulator of AR,upregulate AR-mediated gene transcription,and play a role in promoting the development of prostate cancer;in ER-positive breast cancer,BAP18 binds to the COMPASS complex,up-regulation of ER-mediated gene transcription,can promote the growth and proliferation of ERα-positive breast cancer,and is associated with resistance to the endocrine therapy drug tamoxifen.However,BAP18 is an unidentified protein,and its function and molecular mechanism in lung cancer still needs to be further elucidated.The Wnt signaling pathway is an evolutionarily conserved pathway that plays a crucial role in embryonic development.The Wnt signaling pathway includes the canonical signaling pathway mediated by β-catenin and the non-canonical signaling pathway that does not depend on β-catenin.The canonical Wnt signaling pathway mainly controls cell proliferation and is also considered to be an important oncogenic pathway that mediates the occurrence and development of tumors.Aberrant Wnt signaling pathway has been found in a variety of tumors.In this study,our purpose was to: 1.Clarify the expression of BAP18 in NSCLC tissues;2.Explore the biological function of BAP18 in NSCLC;3.Clarify whether BAP18 can participate in the regulation mediated by β-catenin and promote the occurrence and development of lung cancer.Methods: 1.First,we use bioinformatics methods to analyze the expression of BAP18 in NSCLC tumors and normal tissues,its possible biological functions and its relationship with lung cancer driver genes;2.To clarify the expression of BAP18 in clinical NSCLC tissues: The clinical tissue samples of 30 patients with NSCLC were selected,and the expression levels of BAP18 in the cancer and adjacent normal tissues were analyzed by Western blot.The relationship between BAP18 expression and clinicopathological characteristics was analyzed,and the effect of BAP18 expression on patient prognosis was analyzed in online datasets and in immunohistochemical microarrays was analyzed by Kaplan-Meier plotter method;3.The effect of BAP18 on the growth and proliferation of NSCLC was detected by MTS and cell colony formation assays;the effect of BAP18 on the cell cycle was detected by flow cytometry;4.The effect of BAP18 on the migration ability of NSCLC cell was explored by scratch and Transwell experiments;5.In A549 cells,using si RNA to knock down BAP18,while the control cells were transfected with si Ctrl for treatment,and the differences between samples were detected from a genome-wide perspective by RNA-sequence analysis.The enrichment analysis of differential genes that are regulated and involved in cell growth and tumor development process found that these genes can promote tumor development through the β-catenin signaling pathway;6.Exogenous and endogenous protein Co-Immunoprecipitation(Co-IP)to determine whether BAP18 and β-catenin bind to each other;7.Luciferase assay experiments explored the effect of BAP18 on β-catenin-mediated gene transcription in the case of overexpression of BAP18 and knockdown of BAP18;8.Real-time PCR and Western blot were used to detect the expression level changes ofβ-catenin-mediated Wnt pathway downstream target genes after knockdown and overexpression of BAP18;9.Find BAP18 co-binding protein actin-like 6A(Actin Like6 A,ACTL6A)and RNA polymerase II-related factor 1 homolog(PAF1 Homolog,Paf1/RNA Poly-merase II Complex Component,PAF1)through Shotgun LC-MS/MS mass spectrometry;10.Use Co-IP experiments to verify the interaction between BAP18,ACTL6 A and PAF1;11.Chromatin Immunoprecipitation(Ch IP)to verify the changes of BAP18 knockdown on the recruitment of β-catenin,ACTL6 A and PAF1 in transcriptionally active regions of Wnt target genes.Results: 1.In NSCLC,whether it is Lung Adenocarcinoma(LUAD)or Lung Squamous-cell Carcinoma(LUSC),the expression of BAP18 in tumor tissue is higher than that in normal tissue through analysis of UALCAN online dataset.In the GSE131907 dataset,32,368 epithelial cells were selected for analysis,of which 1,516cells(4.68%)were BAP18-positive.The ssGSEA enrichment analysis of BAP18-negative and positive-expressing cells showed that BAP18-positive cells were up-regulated in proliferation and cell cycle checkpoint-related pathways.The results of the TCGA-LUAD dataset showed that BAP18 expression was not associated with EGFR,ALK,ROS-1,RET,BRAF and KRAS mutation status(p>0.05);2.Western blot results showed that the expression of BAP18 in tumor tissues was significantly higher than that in adjacent noncancerous tissues.IHC detected 92 NSCLC tissues and 78 adjacent noncancerous tissues,which were consistent with Western blot results.Statistical analysis of BAP18 expression between clinical characteristics showed that BAP18 was correlated with lymph node metastasis and clinical stage: in the absence of lymph node metastasis,the expression of BAP18 was low,and with the increase of clinical stage,the expression of BAP18 increased.There were no significant differences in age,sex,pathological grade,tumor size and EGFR mutation.Kaplan-Meier plotter analysis of immunohistochemical arrays and online datasets found that BAP18 expression had no effect on survival;3.Silencing BAP18 inhibited the growth of NSCLC cells in vitro:MTS and cell colony formation experiments showed that silencing BAP18 inhibited the proliferation of NSCLC cells.The flow cytometry results of cell cycle analysis showed that silencing BAP18 significantly increased the proportion of NSCLC cells in G1 phase and decreased the proportion of S phase.Scratch and Transwell experiments showed that silencing BAP18 inhibited the migration ability of NSCLC cells;4.Silencing BAP18 inhibited the growth of NSCLC cells in vivo: BALB/c tumor-bearing mice experiments showed that the subcutaneous tumors formed by A549 cells silenced by BAP18 were smaller than those in the control group.5.In A549 cells,BAP18 was knocked down with si RNA,while the control cells were transfected with si Ctrl for treatment,3 samples in each group,6 samples in total.Transcriptome sequencing analysis was performed to detect differences between the 6 samples from a genome-wide perspective.A total of1143 genes were significantly changed after BAP18 knockdown(p<0.05,|log Fc|>1).There were 563 differentially expressed genes(DEGs)positively regulated by BAP18,which were significantly downregulated after BAP18 knockdown.580 genes were DEGs negatively regulated by BAP18 and were significantly up-regulated after BAP18 knockdown.GO enrichment analysis of the differential genes revealed that the identified genes were involved in a wide range of functions and multiple pathways,including single-organism processes,single-cell processes,protein binding,cellular processes,cell cycle,and biological regulation.KEGG enrichment analysis showed that stem cell pluripotency,Rap1 signaling pathway,tumor,non-small cell lung cancer,metabolism,Hippo signaling pathway,DNA replication and cell cycle were involved.We further selected a subset of genes that were clearly regulated by BAP18 and involved in cell growth and tumor development,which were found to be enriched in cell cycle progression,TGF-β signaling,and the signaling pathway of β-catenin into the nucleus;6.BAP18 mainly affects the development of NSCLC cells through the Wnt signaling pathway:(1)The interaction between BAP18 and β-catenin in NSCLC cells: exogenous and endogenous Co-IP confirmed that BAP18 interacts with β-catenin;(2)Luciferase assay confirmed that overexpression of BAP18 can up-regulate β-catenin-mediated gene transcriptional activity under the stimulation of Li Cl or Wnt3 a recombinant protein,and knockdown BAP18 can down-regulate β-catenin-mediated gene transcriptional activity;(3)Real-time PCR results showed that under the condition of Li Cl stimulation,compared with the control group,the m RNA levels of β-catenin-mediated Wnt signaling pathway target genes CCND1,AXIN2,CD44,c-JUN,c-myc and MMP7 were all significantly increased,and the m RNA levels of CCND1,AXIN2,CD44,c-JUN,c-myc and MMP7 were all decreased after BAP18 knockdown,while the m RNA level of ID2 had no significant effect;(4)Western blot also found that the protein levels of Wnt downstream target genes decreased after knockdown of BAP18 and increased while overexpressing BAP18;7.After mass spectrometry analysis,we found ACTL6 A and PAF1 could interact with BAP18,BAP18 knockdown or overexpression did not affect the protein levels of ACTL6 A and PAF1;Co-IP experiments confirmed the co-binding of BAP18,β-catenin,ACTL6 A and PAF1.8.Ch IP experiments showed that BAP18,β-catenin,ACTL6 A and PAF1 were recruited in the promoter region of the Wnt target gene CCND1,and the recruitment of β-catenin,ACTL6 A and PAF1 decreased after knockdown of BAP18,accompanied by decreased levels of histone modification H3K4me3 and H4 ac.Conclusion: 1.BAP18 is highly expressed in NSCLC and is related to lymph node metastasis and clinical stage;2.BAP18 promotes the proliferation and metastasis of NSCLC cells;3.BAP18 interacts with β-catenin and upregulates β-catenin-mediated gene transcription;4.BAP18 can be recruited to the Wnt responsive element(WRE)of Wnt target genes,promote the recruitment of β-catenin,ACTL6 A and PAF1 to the WRE,and increase the levels of histone modification H3K4me3 and H4 ac in the WRE region.