摘要
目的 分析长非编码RNA(LncRNA)心肌梗死相关转录物(MIAT)在颅内动脉瘤(IA)患者血清中的表达,探究其对血管平滑肌细胞(VSMCs)增殖和凋亡的影响及作用机制.方法 数字减影血管造影诊断的IA患者15例为IA组,同期无IA家族史的健康参与者15例为对照组(C组),qRT-PCR检测两组受试者血清Ln-cRNA MIAT和miR-331-3p水平;体外培养的VSMCs分为空白对照组(BC组)、过表达LncRNA MIAT阴性对照组(oe-NC组)、过表达LncRNA组(oe-MIAT组)、过表达LncRNA+过表达miR-331-3p阴性对照组(oe-MIAT+miR-331-3p-NC组)及过表达LncRNA+过表达miR-331-3p组(oe-MIAT+miR-331-3p mimic组);采用qRT-PCR检测VSMCs中LncRNA MIAT、miR-331-3p表达,MST测定VSMCs 24、48、72 h时的细胞活力(OD值),EdU染色测定VSMCs增殖,AnnexinⅤ/7-AAD双染法测定VSMCs凋亡,Western blot分析VSMCs中Bax、Bcl-2、cleaved-Caspase-3表达;双荧光素酶报告、RNA免疫共沉淀验证LncRNA MIAT、miR-331-3p靶向关系.结果 与C组相比,IA组患者血清LncRNA MIAT相对表达量显著升高,血清miR-331-3p相对表达量显著降低(P<0.05);与BC组及oe-NC组相比,oe-MIAT组、oe-MIAT+miR-331-3p-NC组LncRNA MIAT相对表达量、凋亡率、cleaved-Caspase-3、Bax表达增高,miR-331-3p的相对表达量、24 h、48 h、72 h的OD值、EdU阳性细胞比例、Bcl-2蛋白表达均下降(P<0.05);oe-MIAT+miR-331-3p-mimic组与oe-MIAT+miR-331-3p-NC组、oe-MIAT组相比,凋亡率、cleaved-Caspase-3、Bax表达下降,miR-331-3p的相对表达量、24 h、48 h、72 h的OD值、EdU阳性细胞比例、Bcl-2蛋白水平均显著增高(P<0.05);双荧光素酶报告、RNA免疫共沉淀表明LncRNA MIAT和miR-331-3p之间存在负向调控.结论 LncRNA MIAT在IA患者血清中高表达,高表达的LncRNA MIAT可通过下调miR-331-3p表达诱导VSMCs过度凋亡、抑制VSMCs增殖,参与IA发生或进展.
Abstract
Objective To analyze the expression of long non-coding RNA (LncRNA) myocardial infarction-associated transcript ( MIAT) in the serum of patients with intracranial aneurysm ( IA),and to explore its impact and mechanism on the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). Methods A total of15 patients with IA diagnosed by digital subtraction angiography were classified as IA group,and 15 healthy participants without family history of IA were enrolled in the control group ( C group ) . The expression levels of serum LncRNA MIAT and miR-331-3 p were detected by qRT-PCR in both groups. VSMCs cultured in vitro were divided into blank control group ( BC group ),overexpressed LncRNA MIAT negative control group ( oe-NC group ),overexpressed LncRNA group ( oe-MIAT group ),overexpressed LncRNA+overexpressed miR-331-3 p negative control group (oe-MIAT+miR-331-3p-NC group),and overexpressed LncRNA+overexpressed miR-331-3p group (oe-MIAT+miR-331-3p mimic group). The expression of LncRNA MIAT and miR-331-3p in VSMCs was detected by qRT-PCR;the cell viability of VSMCs at 24 h,48 h and 72 h was measured by MST;the proliferation of VSMCs was detected by EdU staining;Annexin V/7-AAD double staining was applied to detect apoptosis of VSMCs;the expression of Bax,Bcl-2 and cleaved-Caspase-3 in VSMCs were analyzed by Western blot;dual-luciferase reporter and RNA co-immunoprecipitation were used to verify the targeting relationship between LncRNA MIAT and miR-331-3p. Results Compared with the C group,the relative expression of LncRNA MIAT in serum of IA group was markedly increased,and the relative expression of miR-331-3p in serum was obviously decreased (P<0.05). Compared with BC group and oe-NC group,the relative expression of LncRNA MIAT,apoptosis rate,and the expression of cleaved-Caspase-3 and Bax in oe-MIAT group and oe-MIAT+miR-331-3p-NC group were increased,while the relative expression of miR-331-3p,the OD value at 24 h,48 h,and 72 h,the proportion of EdU positive cells,and the protein level of Bcl-2 were decreased ( P<0.05 ) . Compared with the oe-MIAT+miR-331-3 p-NC group and the oe-MIAT group,the apoptosis rate,the expression of cleaved-Caspase-3 and Bax in the oe-MIAT+miR-331-3p-mimic group were decreased,while the relative expression of miR-331-3p,the OD value at 24 h,48 h,and 72 h,the proportion of EdU positive cells,and the protein level of Bcl-2 were obviously increased (P<0.05). Dual-luciferase reporter,RNA co-immunoprecipitation indicated that there was a negative regulation between LncRNA MIAT and miR-331-3p. Conclusion LncRNA MIAT is highly expressed in the serum of IA patients,and the highly expressed LncRNA MIAT can induce excessive apoptosis of VSMCs by down-regulating the expression of miR-331-3p,inhibit the proliferation of VSMCs,and participate in the occurrence or progression of IA.
基金项目
河北省2024年度医学科学研究课题(20240351)
2022年唐山市市级科技计划项目(22130202H)
维普
万方数据