lncRNA-PACER upregulates COX-2 and PGE2 through the NF-κB pathway to promote the proliferation and invasion of colorectal-cancer cells

Background p50-associated cyclooxygenase-2 extragenic RNA(PACER)is a recently identified antisense long non-coding RNA(lncRNA)located on the upstream of the promoter region of cyclooxygenase-2(COX-2).Preliminary studies have suggested that PACER is involved in the regulation of COX-2 expression in macrophagocyte and osteosarcoma cells.However,the role of this lncRNA in colorectal cancer(CRC)remains elusive.Here,we investigated the expression of PACER and its effect on cell proliferation and invasion to explore the role of PACER in CRC.Methods Real-time quantitative PCR(RT-qPCR)analysis was used to evaluate the expression of PACER in CRC tissues and cells.Methyl thiazolyl tetrazolium(MTT)analysis was then used to investigate the inhibition effect of PACER knock-down in cell proliferation.The promoting role of this lncRNA on invasion by CRC cells was analysed by wound-healing assays,colony-formation assay,and transwell assays.We then used fluorescence in situ hybridization(FISH)to establish the subcellular localization of PACER.COX-2 protein levels were quantified by Western blot analysis and grayscale scanning analysis following the knock-down of PACER.Luciferase assay was carried out to monitor the modulation of the COX-2 promoter region by PACER.Tumor xenografts models were used to investigate the impact of PACER on the tumorigenesis of CRC cells in vivo.Enzyme-linked immunosorbent assay(ELISA)was then used to quantify prostaglandin E2(PGE2)production upon knock-down of PACER.Results RT-qPCR analysis revealed that PACER was highly expressed in CRC tissues and cells,and a high PACER-expression level was associated with poor prognosis.MTT assay,wound-healing assay,colony-formation assay,and transwell assay revealed that PACER enhanced CRC-cell proliferation,invasion,and metastasis in vitro.Analysis of lncRNA localization by FISH showed that it mainly resided in the nucleus.RT-qPCR showed that PACER increased mRNA levels of COX-2.Western blot analysis demonstrated,under normal circumstances,that knock-down of PACER decreased the COX-2 protein level.In the case of p50 absence,COX-2 protein increased rapidly and remained highly expressed after knocking down PACER.Luciferase assay revealed that PACER modulated the COX-2 promoter region.Mouse xenograft models of CRC revealed that PACER promoted colorectal tumorigenesis in vivo.ELISA revealed that PACER knock-down inhibited PGE2 production.Conclusions PACER modulates COX-2 expression through the nuclear factor kappa B(NF-jB)pathway in CRC.An increased level of PACER enhances proliferation,migration,and invasion of tumor cells by increasing COX-2 and PGE2 synthesis.