CircOMA1调控多巴胺受体促进泌乳素瘤耐药性

CircOMA1 Promotes Resistance in Prolactinoma by Regulating Dopamine Receptors

【目的】探究环状 RNA hsa_circ_0002316(circOMA1)是否参与泌乳素瘤耐药调控及其作用机制是什 么。【方法】通过 RT-qPCR检测临床泌乳素瘤标本中 circOMA1表达情况(5例敏感泌乳素瘤标本和 12例耐药泌乳 素瘤标本)。通过慢病毒载体构建稳定表达外源性 circOMA1 MMQ细胞系模型进行体外实验,分为 MMQ NC组及 MMQ OMA1组。通过构建裸鼠皮下肿瘤模型进行体内实验,分为 MMQ NC+卡麦角林(CAB)组、MMQ NC+溴隐亭 (BRC)组、MMQ OMA1+CAB组、MMQ OMA1+BRC组。通过 CCK8、Western blot、ELISA等表型实验检测 circOMA1对 耐药机制中的细胞增殖和泌乳素(PRL)分泌两个方面的影响。通过 Western blot、ELISA、免疫组化等实验检测 cir⁃ cOMA1 对多巴胺受体表达的影响。通过 TargetScan、CircInteractome 等软件分析预测、microRNA-145-5p(miR 145-5p)minic补救实验探究 circOMA1调控多巴胺受体机制。【结果】RT-qPCR结果显示多巴胺受体激动剂(DAs) 耐药泌乳素瘤中 circOMA1表达升高(P<0.01)。在细胞实验及裸鼠皮下肿瘤实验中,与 MMQ NC 组相比,MMQ OMA1组 DAs敏感性降低,细胞增殖水平和泌乳素分泌水平升高(P<0.05)。Western blot和免疫组化结果显示, MMQ OMA1组多巴胺 2型受体(DRD2)表达下调,多巴胺 5型受体(DRD5)表达上调,环 3’,5’-单磷酸腺苷(cAMP) 水 平 上 调(P<0.05)。 此 外 ,在 MMQ OMA1 组 中 miR-145-5p 表 达 下 调 ,kelch 重 复 序 列 和 BTB 结 构 域 蛋 白 7 (KBTBD7)mRNA及蛋白表达水平上调(P<0.05),然而 MMQ OMA1组转染 miR-145-5p minic后,KBTBD7表达下 调,DRD2表达上调(P<0.05),表明过表达 miR-145-5p逆转了 circOMA1对 KBTBD7的促进作用。【结论】circOMA1 通过 miR-145-5p/KBTBD7轴下调 DRD2表达,降低泌乳素瘤对 DAs敏感性,并且调控多巴胺受体表达激活 cAMP通 路促进泌乳素合成释放,从而促进泌乳素瘤耐药性。

【Objective】To investigate whether hsa_circ_0002316(circOMA1)is involved in drug resistance in prolac⁃tinoma and its mechanism.【Methods】RT-qPCR was used to dectect the expression of circOMA1 in clinical prolactinoma specimens including 5 dopamine receptor agonists(DAs)-sensitive prolactinomas and 12 DAs-resistant prolactinomas.MMQ cell lines with stable expression of exogenous circOMA1 were constructed by lentivirus vector infection for in vitro ex⁃periment,and were divided into MMQ NC group and MMQ OMA1 group.Xenograft tumor models in nude mice were estab⁃lished for in vivo experiment,and were divided into MMQ NC+cabergoline(CAB)group,MMQ NC+bromocriptine(BRC)group,MMQ OMA1+CAB group and MMQ OMA1+BRC group.CCK8,Western blot and ELISA were performed to detect the effects of circOMA1 on cell proliferation and secretion of prolactin(PRL)in drug resistance mechanism.West⁃ern blot,ELISA and immunohistochemistry were used to examine the effect of circOMA1 on dopamine receptor expression.The mechanism of circOMA1 regulating dopamine receptor was explored by TargetScan,CircInteractome and microRNA 145-5p(miR-145-5p)minic remediation experiment.【Results】RT-qPCR showed that circOMA1 expression was in⁃creased in DAs-resistant prolactinomas(P<0.01).Compared with MMQ NC group,MMQ OMA1 group had lower DAs sensitivity and increased cell proliferation and prolactin secretion(P<0.05)in vivo and in vitro.Western blot and immu⁃nohistochemical analysis showed that in MMQ OMA1 group,the expression of dopamine receptor type 2(DRD2)was down-regulated,and the expression of dopamine receptor type 5(DRD5),the cyclic 3'and 5'-adenosine monophos⁃phine(cAMP)was up-regulated(P<0.05).In MMQ OMA1 group,miR-145-5p expression was down-regulated,kelch repeat sequence and BTB domain-containing protein 7(KBTBD7)mRNA and protein expression was up-regulated(P<0.05).After transfected with miR-145-5p minic,MMQ OMA1 group exhibited down-regulated KBTBD7 expression and up-regulated DRD2 expression(P<0.05),which indicated that overexpression of miR-145-5p reversed the promot⁃ing effect of circOMA1 on KBTBD7.【Conclusion】CircOMA1 down-regulated DRD2 expression through miR-145-5p/KBTBD7 pathway to reduce the sensitivity of prolactinoma to DAs,and regulated the expression of dopamine receptors to activate the cAMP pathway and promote the synthesis and release of prolactin,thus promoting the drug resistance in prolactinoma.