微小RNA-643靶向下调细胞色素P450家族成员11B1促进小儿骨肉瘤增殖的临床分析及细胞与小鼠机制研究

Clinical analysis and cell and mouse mechanism of microRNA-643 promoting pediatric osteosarcoma proliferation by down-regulating cytochrome P450 11B1

目的:探讨微小RNA(miR)-643调控细胞色素P450家族成员11B1(CYP11B1)对小儿骨肉瘤增殖的临床与机制研究。方法:收集2017年6月至2018年3月天津市天津医院收治的18例小儿骨肉瘤患者,年龄(12.04±2.68)岁,男12例,女6例,同期收集18例性别和年龄配对的健康儿童。应用Lipofectamine 2000介导转染细胞株,共分为五组:空载组,正常生长组,miR-643模拟物组,miR-643抑制剂组,CYP11B1抑制剂组,每组重复3次。实时定量聚合酶链式反应(RT-qPCR)检测miR-643相对表达量,蛋白质印迹法(Western blot)检测CYP11B1蛋白表达水平,细胞增殖与毒性(CCK-8)法检测细胞增殖能力,流式细胞仪检测细胞凋亡能力,小鼠体内实验探究miR-643对肿瘤体积的影响。多组间比较采用单因素方差分析,两两比较采用t检验。结果:与健康者(0.53±0.06)比较,骨肉瘤患者(3.24±0.81)血清miR-643表达水平显著增高(P<0.05)。在24、48、72 h时,与空载组[吸光度(A)值0.36±0.04、0.44±0.03、0.56±0.05]和正常生长组(0.35±0.06、0.45±0.04、0.58±0.07)比较,miR-643模拟物组(0.51±0.05、0.75±0.07、0.93±0.06)MG-63细胞的增殖能力显著增强(P<0.05),miR-643抑制剂组(0.24±0.05、0.28±0.06、0.33±0.06)增殖能力明显受到抑制(P<0.05)。与空载组[(7.65±0.47)%]和正常生长组[(7.80±0.51)%]比较,miR-643模拟物组[(3.12±0.22)%]MG-63细胞的凋亡能力显著下降(P<0.05),miR-643抑制剂组[(24.32±2.25)%]凋亡能力显著增高(P<0.05)。与空载组(1.04±0.06)和正常生长组(0.98±0.05)MG-63细胞CYP11B1蛋白水平比较,miR-643模拟物组(0.41±0.04)和CYP11B1抑制剂组(0.45±0.05)均显著下降(P<0.05),miR-643抑制剂组(1.64±0.13)显著增高(P<0.05)。皮下注射稳定转染sh-miR-643的MG-63细胞小鼠的肿瘤平均体积为[(621.74±38.63)mm 3],显著低于对照组[(1104.36±57.01)mm 3,t=4.017,P<0.01]。结论:miR-643可促进骨肉瘤MG-63细胞增殖并抑制其凋亡,其机制与调控靶基因CYP11B1密切相关。

Objective To investigate the clinical and mechanism of microRNA(miR)-643 by regulate the cytochrome P450 family member 11B1(CYP11B1)in the proliferation of pediatric osteosarcoma.Methods Eighteen cases of pediatric osteosarcoma admitted to Tianjin Hospital from June 2017 to March 2018 were collected.The average age was(12.04±2.68)years old,and 12 males and 6 females,respectively.Meanwhile,eighteen healthy and age-matched healthy children were collected.The transfected cell lines were mediated by Lipofectamine 2000 and divided into five groups:empty group,normal growth group,miR-643 mimic group,miR-643 inhibitor group,and CYP11B1 inhibitor group,and repeat 3 times per group.The real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the relative expression of miR-643,the expression of CYP11B1 protein was detected by Western blotting,the cell proliferation ability was detected by cell proliferation and toxicity(CCK-8)method,and the apoptosis ability was detected by flow cytometry.The effect of miR-643 on tumor volume was explored in vivo in mice.Results Compared with healthy subjects(0.53±0.06),the expression level of serum miR-643 in patients with osteosarcoma(3.24±0.81)was significantly increased(P<0.05).At 24,48 and 72 h,compared with the empty group[optical density(A)values of 0.36±0.04,0.44±0.03,0.56±0.05]and normal growth groups(0.35±0.06,0.45±0.04,0.58±0.07),the miR-643 mimic group(0.51±0.05,0.75±0.07,0.93±0.06)significantly enhanced the proliferation of MG-63 cells(P<0.05),and the proliferation of MG-63 cells in miR-643 inhibitor group(0.24±0.05,0.28±0.06,0.33±0.06)was significantly decreased(P<0.05).Compared with the empty group[(7.65±0.47)%]and the normal growth group[(7.80±0.51)%],the apoptosis ability of the miR-643 mimic group[(3.12±0.22)%]was significantly decreased(P<0.05),the apoptotic ability of the miR-643 inhibitor group[(24.32±2.25)%]was significantly increased(P<0.05).Western blotting results showed that compared with the empty group(1.04±0.06)and the normal growth group(0.98±0.05),the level of CYP11B1 in the miR-643 mimic group(0.41±0.04)and the CYP11B1 inhibitor group(0.45±0.05)was significantly decreased(P<0.05),while the level was significantly increased in the miR-643 inhibitor group(1.64±0.13,P<0.05).The average tumor volume of MG-63 cells stably transfected with sh-miR-643 was[(621.74±38.63)mm3],which was significantly lower than that of the control group[(1104.36±57.01)mm3,t=4.017,P<0.01].Conclusion MiR-643 can promote the proliferation of osteosarcoma MG-63 cells and inhibit its apoptosis,and its mechanism is closely related to the regulation of the target gene CYP11B1.