前列腺癌细胞系中结肠癌相关转录因子2与雄激素受体基因的转录调控关系

Transcriptional Regulation between CCAT 2 and AR in Prostate Cancer Cell Lines

雄激素受体(androgen receptor,AR)是经典的前列腺癌主效基因。结肠癌相关转录因子2(colon cancer associated transcript 2,CCAT2)是基于GWAS大数据结果发现的前列腺癌相关长链非编码基因,二者关系尚未见报道。依据AR表达模式的不同,前列腺癌(prostate cancer,PCa)可以划分为普通前列腺癌以及恶性程度更高的去势抵抗性前列腺癌(castration-resistance prostate cancer,CRPC)两种截然不同的发展阶段,其机制尚不明确。AR与CCAT2是否存在调控关系以及是否在其中发挥作用是本研究的切入点。本文采用雄激素依赖型与非依赖型的前列腺癌细胞研究模型LNCaP与DU145细胞系,研究发现了CCAT2与AR存在转录调控关系。首先,采用过表达与敲低的分子生物学研究策略,发现二者的RNA水平存在相互调控。在DU145细胞中,G型CCAT2激活AR mRNA水平到2.6倍,T型CCAT2抑制AR mRNA水平至0.2(P<0.05);在LNCap细胞中,G型CCAT2激活AR mRNA水平1.5倍,T型CCAT2对AR mRNA水平变化无显著意义(P<0.05)。在DU145细胞中过表达AR,CCAT 2的表达量上升2.9倍(P<0.05);在LNCaP细胞中沉默AR的表达,CCAT 2的表达水平下降至0.48(P<0.05)。报告基因分析结果显示,CCAT2对AR启动子相对活性影响与RNA水平改变相一致。由于CCAT2研究的缺乏,本文进一步研究了其与细胞活性以及对AR蛋白质水平的影响,数据显示与转录水平的结果基本一致。最后,本文初步分析了AR与CCAT2可能相关的靶基因,包括CTNNB1,MYC和TCF7L 2,初步探讨可能的机制并验证了转录水平的发现。结果表明,在LNCaP细胞中G型CCAT2分子激活AR的转录,U型CCAT2无作用。在DU145细胞中,G型CCAT2激活AR转录,U型CCAT2抑制AR的转录。在2个细胞系中,AR都激活CCAT 2整体的转录活性,提示二者存在反馈调节机制。研究发现,CCAT2与AR的转录调控关系,为真核基因表达与调控的机制研究以及前列腺癌的发病机制与治疗提供了理论与实验数据。

AR(androgen receptor)and CCAT 2 are two prostate cancer(PCa)-related genes whereas their relationship is not yet reported.AR is the classical major functional gene in PCa progression.CCAT 2,a non-coding gene,was identified based on big-data GWAS(Genome-Wide Association Studies)in the year of 2013.Androgen deprivation therapy(ADT)is usually used to treat PCa in the early stage.After persistent androgen deprivation,PCa would generally lead to castration resistant prostate cancer(CRPC),whereas the mechanism is yet unclear.Here we explore the function of AR and CCAT 2 in PCa progression,especially their relation in androgen sensitive and insensitive cell model LNCap and DU145.We found a loop between AR and CCAT2 transcription by over-expression and knock-down strategies.In DU145 cells,G-CCAT2 activated AR mRNA level 2.6 times,while T-CCAT2 inhibited it to 0.2 times(P<0.05).In LNCaP cells,G-CCAT2 could activate AR mRNA levels 1.5 times,and T-CCAT2 had no significant effect(P<0.05).Under overexpression of AR in DU145 cells,the expression of CCAT2 increased 2.9 times(P<0.05).The abundance of CCAT 2 decreased to 0.48(P<0.05)in LNCaP cells by AR knock-down.Reporter gene analysis showed that CCAT2 could function on the AR promoter.We then performed CCK8 assays and AR protein level detection as supplement for the new gene CCAT 2 studies.Finally we primarily studied some target genes that are related to AR and CCAT 2.The results showed that the G-CCAT2 transcript could activate AR expression in LNCap cells while U-CCAT2 had no significant effect.In DU145 cells,G-CCAT2 exhibited a more relative stronger activation effect on AR,and U-CCAT2 could inhibit AR transcription.AR activates the transcriptional activity of CCAT 2 in both cell lines,suggesting a feedback regulation between them.Our data showed that there would be a feedback loop between CCAT 2 and AR,which may indicate a new method for PCa treatment.