Mechanism And Clinical Significance Of HnRNPA2/B1 Promotes Lung Cancer Growth By Upregulating COX-2 Expression

Objective: Cyclooxygenase-2(COX-2)is highly expressed in tumor cells and has been regarded as a hallmarker for cancers.Our studies have shown that COX-2 promoter region has many functional binding sites in cancer cells.However,the excise regulation mechanism by which these factors specifically bind to COX-2 promoter,regulates COX-2 expression and tumor growth remains largely unknown.Here,we combined pull down and proteomic mass spectrometry assay,identified a novel COX-2 regulator,heterogeneous nuclear ribonucleoprotein A2/B1(hn RNPA2/B1),which could specifically bind to COX-2 core promoter.To definite the mechanism and biological function of hn RNPA2/B1 in the regulation of COX-2,we investigated how hn RNPA2/B1 could regulate COX-2 promoter activity and expression as well as the production of PGE2,the COX-2 downstream product.To determine whether p300 plays a role in the hn RNPA2/B1-mediated regulation of COX-2,we determined the interaction of p300 with hn RNPA2/B1.To illustrate whether p300 could also acetylate hn RNPA2/B1 and promote its binding activity,we evaluated the effect of overexpression of p300 and its HAT deletion mutant(?HAT)on hn RNPA2/B1 acetylation and its binding on COX-2 promoter.We also further validate whether p300 HAT is important for the hn RNPA2/B1-mediated activation of COX-2.In xenograft mouse model,we also studied the relationship between hn RNPA2/B1 and COX-2 and the effect of hn RNPA2/B1 on tumor weight and volume.Moreover,we evaluated the correlation between high expression of hn RNPA2/B1/COX-2 with tumor development,prognosis,and stage,survival,metastasis,recurrence of lung cancers.The results from our study may provide new insights into understanding the regulatory mechanism of COX-2 and exploring new therapeutic targets for lung cancer treatment.Methods: We used biotin-labeled DNA probe streptavidin-agarose pulldown assay and mass spectrum and proteomic techniques in our study.One protein band with molecular weight about 36 k Da was identified as hn RNPA2/B1.We further verified the binding of hn RNPA2/B1 on COX-2 promoter by Ch IP and pulldown.The expression of hn RNPA2/B1 and COX-2 protein was examined by Western blot and immunohistochemistry in NSCLC cells and tissues.We selected H322,H460 and H1299 cell lines to overexpress or inhibit hn RNPAB,hn RNPA2/B1 and COX-2 expression were analyzed by Western blot,RT-PCR.PGE2 was detected by ELISA.COX-2 promoter activity was analyzed by Luciferase reporter assays.Cell viability was measured by MTT assay.To determine the effect of hn RNPA2/B1 on cell migration and proliferation,we used scratch and MTT assays.Next,we analyzed the subcellular co-localization of p300 and hn RNPA2/B1 in H460 cells by immunofluorescence confocal assay.We used Co-IP to verify hn RNPA2/B1 interacted with p300 directly and was acetylated by p300 histon acetyltransferase(HAT).We overexpressed p300 to analyze COX-2 protein expression,promoter activity,PGE2 production,as well as cell proliferation in H460 cells.In xenograft mouse model,we recorded nude mice weight,volume,dynamic volume,detected hn RNPA2/B1 and COX-2 expression by IHC and Western blot.We next evaluated the expression of hn RNPA2/B1 and COX-2 in tumor tissues and their adjacent samples in 150 tumor samples from 75 NSCLC patients by tissue microarray and immunohistochemical assay.Results: 1.We discovered and identified hn RNPA2/B1 as a novel COX-2 promoter-regulating factor in A549,H1299,H322,H460 NSCLC cells and lung normal cell line HLF.2.In H460 and H322 cell lines,knockdown of hn RNPA2/B1 inhibited COX-2 promoter activity,COX-2 expression,PGE2 production and lung cancer cell growth in NSCLC cells in vitro and in vivo.3.In H1299,H460 and H322 cell lines,over-expression of hn RNPA2/B1 up-regulated COX-2 promoter activity,COX-2 expression and PGE2 production and promoted lung cancer cell growth.4.Transfection of H460 cells with hn RNPA2/B1 sh RNA considerably suppressed cell migration and cell proliferation and viability.By contrast,overexpression of hn RNPA2/B1 in H1299 cells by transfecting its hn RNPA2/B1 expressing vector markedly increased cell proliferation,and the addition of celecoxib(CB)reduced the hn RNPA2/B1-mediated promotion of cell proliferation.5.We also validated the hn RNPA2/B1-mediated regulation of tumor growth via COX-2 signaling in a mouse model.Knockdown of hn RNPA2/B1 by si RNA significantly inhibited tumor volume and tumor weight.However,treatment with LPS,a COX-2 inducer,effectively rescued the inhibitions by hn RNPA2/B1 si RNA.The hn RNPA2/B1 si RNA also considerably suppressed COX-2 protein expression in the mice groups treated with hn RNPA2/B1 si RNA.Moreover,immunohistochemical staining also confirmed that the expression of hn RNPA2/B1 was positively correlated with COX-2 expression.6.A direct interaction between hn RNPA2/B1 and p300 were observed in A549,H1299 and H460 cell lines by immunoprecipitation experiments.To further confirm the interaction between p300 and hn RNPA2/B1,we also analyzed the subcellular co-localization of p300 and hn RNPA2/B1 in H460 cells by immunofluorescence confocal assay,and found that most of hn RNPA2/B1 and p300 proteins colocalized in cell nuclei.Overexpression of p300,but not its HAT deletion mutant(?HAT),rescued the sh RNPA2/B1 sh RNA-mediated inhibitions of COX-2 protein expression,promoter activity,PGE2 production,as well as cell proliferation in H460 cells.7.hn RNPA2/B1 and COX-2 were highly expressed in lung cancer cells and lung adenocarcinoma tissues.The expression level of COX-2 was positively correlated with hn RNPA2/B1 expression.The overall survival in these patients was significantly better than in those with low expression of hn RNPA2/B1.In addition,the worse statues of clinical TNM stage was significantly correlated with the high expression of hn RNPA2/B1(HR=0.306).Our results therefore suggest that the hn RNPA2/B1/COX-2 pathway is a potential new therapeutic target for the treatment of lung cancers.Conclusions: In summary,we have demonstrated an important mechanism of hn RNPA2/B1 in the regulation of COX-2 expression and tumor growth in NSCLC cells.P300 interacted with and acetylated hn RNPA2/B1 to augment the binding of hn RNPA2/B1 to the COX-2 promoter,thereby activating COX-2 expression,COX-2 promoter activity,PGE2 production,and promoting tumor growth.High expression of hn RNPA2/B1 contributed to a poor prognosis in human NSCLCs.Our results therefore suggest that the hn RNPA2/B1/COX-2 pathway is a potential new therapeutic target for the treatment of lung cancers.