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

LncRNA-PACER通过NF-κB通路上调COX-2和PGE2促进结直肠癌细胞的增殖和侵袭

作　　者：Peng Sun Ji-Chuan Quan Song Wang Meng Zhuang Zheng Liu Xu Guan Gui-Yu Wang Hong-Ying Wang Xi-Shan Wang 

机构地区：[1]Department of Colorectal Surgery,The Second Affiliated Hospital of Harbin Medical University,Harbin,Heilongjiang,P.R.China [2]Department of Gastrointestinal Surgery,Shenzhen Hospital,National Cancer Center/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Shenzhen,Guangdong,P.R.China [3]Department of Colorectal Surgery,National Cancer Center/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing,P.R.China [4]Department of State Key Laboratory of Molecular Oncology,National Cancer Center/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing,P.R.China

出　　处：《Gastroenterology Report》2021年第3期257-268,I0003,共13页胃肠病学报道（英文）

基　　金：supported by National Natural Science Foundation of China [No.81572930];Beijing Science and Technology Plan [No.D171100002617004];the Scientific Research Foundation of Graduate School of Harbin Medical University [YJSCX2017-63HYD].

摘　　要：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背景:p50蛋白相关的环氧合酶-2外源性RNA(PACER)是新近发现的位于环氧合酶-2(COX-2)启动子上游的反义长链非编码RNA(lncRNA)。初步研究显示,PACER参与调节巨噬细胞和骨肉瘤细胞中COX-2的表达,但这种lncRNA在结直肠癌中的作用尚不清楚。我们研究了PACER在结直肠癌中的表达及其对细胞增殖和侵袭的影响。方法:采用实时定量PCR(RT-qPCR)方法分析PACER在结直肠癌组织和细胞系中的表达,采用MTT实验研究敲除PACER对细胞增殖的抑制作用,并通过划痕愈合实验、克隆形成实验、细胞侵袭实验分析该lncRNA对结直肠癌细胞侵袭的促进作用。然后,我们使用荧光原位杂交实验(FISH)确定PACER的亚细胞定位,在PACER敲除后通过western blot对COX-2进行蛋白定量,通过荧光素酶实验分析PACER对COX-2启动子区域的调节。建立裸鼠肿瘤移植模型,分析PACER对体内结直肠癌细胞的影响。最后,采用酶联免疫吸附实验(ELISA)检测PACER敲除后前列腺素E2(PGE2)水平。结果:RT-qPCR结果显示,PACER在结直肠癌组织和细胞中均呈高表达,其高表达与预后不良有关。MTT分析、划痕愈合实验、克隆形成实验和细胞侵袭实验结果显示,PACER促进结直肠癌细胞在体外的增殖、侵袭和转移。FISH实验显示,lncRNA主要定位于细胞核中。RTqPCR检测发现,PACER增加COX-2 mRNA水平。Western blot分析表明,在正常情况下,敲除PACER会降低COX-2蛋白水平;但在缺少p50蛋白的情况下,PACER敲除后COX-2蛋白表达增加并保持高表达。萤光素酶实验显示,PACER调控COX-2启动子区域。裸鼠结直肠癌异种移植模型显示,PACER可促进体内结直肠肿瘤的发生。ELISA检测结果表明,敲除PACER可抑制PGE2的产生。结论:PACER通过NF-jB通路调节结直肠癌中COX-2表达。PACER高表达可以通过增加COX-2和PGE2的合成来促进结直肠肿瘤细胞的增殖、迁移和侵袭。

关 键 词：p50-associated cyclooxygenase-2 extragenic RNA(PACER) colorectal cancer(CRC) lncRNA cyclooxygenase-2(COX-2) 

分 类 号：R735.3[医药卫生—肿瘤]