长链非编码 RNA母系表达基因 8靶向微小 RNA-497-5p调控血管瘤血管内皮细胞的增殖和凋亡

目的 探讨长链非编码RNA母系表达基因8(MEG8)靶向微小RNA(miR)-497-5p对血管瘤血管内皮细胞的增殖和凋亡的影响.方法 实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测河南中医药大学第一附属医院2017年1月至2018年6月收集的52例血管瘤组织和与其对应的瘤旁正常皮肤组织中MEG8和miR-497-5p的表达水平.血管瘤血管内皮细胞HemEC分为si-NC组、si-MEG8组、miR-NC组、miR-497-5p组、si-MEG8+anti-miR-NC组、si-MEG8+anti-miR-497-5p组.四甲基偶氮唑盐微量酶反应比色法(MTT法)检测细胞活力,流式细胞术检测细胞凋亡率,蛋白质印迹法(Western blotting)检测细胞周期蛋白D1(cyclin D1)和P21、B细胞淋巴瘤-2(Bcl-2)和Bcl相关X(Bax)蛋白的表达水平.双荧光素酶报告基因实验验证MEG8和miR-497-5p的靶向关系.结果 与瘤旁正常皮肤组织相比,血管瘤组织中MEG8表达水平(0.82±0.08)比(0.33±0.03)]升高(P<0.05),miR-497-5p表达水平(0.34±0.03)比(0.84±0.08)]降低(P<0.05).与si-NC组比较,si-MEG8组HemEC细胞存活率(39.61±4.06)％比(103.50±11.04)％]、cyclin D1和Bcl-2蛋白表达降低(P<0.05),凋亡率(26.57±2.73)％比(8.33±0.85)％]、P21和Bax蛋白表达升高(P<0.05).与miR-NC组比较,miR-497-5p组HemEC细胞存活率(48.87±5.09)％比(104.11±10.64)％]、cy?clin D1和Bcl-2蛋白表达降低(P<0.05),凋亡率(21.26±2.45)％比(8.33±0.85)％]、P21和Bax蛋白表达升高(P<0.05).MEG8和miR-497-5p直接特异性结合.与si-MEG8+anti-miR-NC组比较,si-MEG8+anti-miR-497-5p组HemEC细胞存活率(86.10±8.81)％比(40.23±4.35)％]、cyclin D1和Bcl-2蛋白表达升高(P<0.05),凋亡率(15.32±1.67)％比(26.81±2.94)％]、P21和Bax蛋白表达降低(P<0.05).结论 抑制MEG8通过靶向miR-497-5p能够抑制血管瘤血管内皮细胞增殖,促进细胞凋亡,进而遏制血管瘤的发生发展.

Long non-coding RNA maternal expression gene 8 regulates the proliferation and apoptosis of hemangioma vascular endothelial cells by targeting microRNA-497-5p

Objective To explore the effect of long non-coding RNA maternal expression gene 8 (MEG8) on proliferation and apoptosis of hemangioma vascular endothelial cells through targeting microRNA (miR)-497-5p.Methods Quantitative real-time PCR polymerase chain reaction (qRT-PCR) was used to detect the expression levels of MEG8 and miR-497-5p in 52 cases of hemangioma tissues and their corresponding adjacent normal skin tissues collected from The First Affiliated Hospital of Henan University of ChineseMedicine from January 2017 to June 2018. Hemangioma vascular endothelial cells HemEC were assigned into si-NC group, si-MEG8 group, miR-NC group, miR-497-5p group, si-MEG8+anti-miR-NC group, and si-MEG8+anti-miR-497-5p group. Methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, and flow cytometry was used to detect apoptosis rate, and the expression levels ofcyclin D1, P21, B-cell lymphoma-2 (Bcl-2) and Bcl-associated X protein (Bax) were detected by Western blotting. Dual luciferase reporter gene assay was used to verify the targeted relationship between MEG8 and miR-497-5p.Results Compared with normal skin tissue adjacent to the tumor, the expression level of MEG8 (0.82±0.08) vs. (0.33±0.03)] in hemangioma tissue was increased (P<0.05), and the expression level of miR-497-5p (0.34±0.03) vs. (0.84±0.08)] was decreased (P<0.05). Compared with the si-NC group, the survival rate of HemEC cells in the si-MEG8 group (39.61±4.06)% vs. (103.50±11.04)%], the expression of cyclin D1 and Bcl-2 protein were decreased, and the apoptosis rate (26.57±2.73) % vs. (8.33±0.85)%], P21 and Bax protein expression were increased (P<0.05). Compared with the miR-NC group, the survival rate of HemEC cells in the miR-497-5p group (48.87±5.09)% vs. (104.11±10.64)%], cyclin D1 and Bcl-2 protein expression were decreased (P<0.05), and the apoptosis rate (21.26± 2.45)% vs. (8.33±0.85)%], P21 and Bax protein expression were increased (P<0.05). MEG8 was directly and specifically bound to miR-497-5p. Compared with the si-MEG8+anti-miR-NC group, the survival rate of HemEC cells in the si-MEG8+anti-miR-497-5p group (86.10±8.81)% vs. (40.23± 4.35)%], cyclin D1 and Bcl-2 protein expression were increased (P<0.05), and the apoptotic rate (15.32±1.67)% vs. (26.81±2.94)%], the expression of P21 and Bax protein were decreased (P<0.05).Conclusion Inhibition of MEG8 could inhibit the proliferation of hemangioma vascular endothelial cells and promote apoptosis by targeting miR-497-5p, thereby inhibiting the occurrence and development of hemangioma.