微小RNA-153-3p/α-突触核蛋白轴通过抑制破骨细胞分化调节骨质疏松症的机制

The mechanism of microRNA-153-3p/snca axis promoting osteoporosis by regulating osteoclast differentiation

目的:探讨微小RNA(miR)-153-3p如何与α-突触核蛋白(snca)相互作用影响小鼠的骨质疏松进程。方法:采用切除双侧卵巢构建骨质疏松(OVX)小鼠模型(品系),通过苏木精-伊红(HE)染色明确病理学改变。通过实时荧光定量反转录聚合酶链反应、免疫蛋白印记实验和免疫组织化学染色对目标基因的信使RNA(mRNA)及蛋白表达进行定量。核因子-κB活化因子受体配体(RANKL)处理小鼠单核巨噬细胞白血病细胞(RAW264.7细胞)后,显微镜下观察细胞形态,噻唑蓝(MTT)法评价细胞活力,Transwell实验检测破骨细胞迁移能力;双荧光素酶实验验证miR-153-3p与snca的相互作用。结果:snca在OVX小鼠中低表达(t=11.436,P<0.05),miR-153-3p在OVX小鼠中高表达(t=15.833,P<0.05)。抑制miR-153-3p或过表达snca抑制骨质疏松,snca在RANKL诱导的RAW264.7细胞中低表达(t=11.796,P<0.05),miR-153-3p在RANKL诱导的RAW264.7细胞中高表达(t=10.448,P<0.05)。过表达snca抑制破骨细胞分化[抗酒石酸酸性磷酸酶(TRAP):t=7.493,P<0.05;Cathepsin K:t=9.536,P<0.05;基质金属蛋白酶(MMP)-9:t=20.371,P<0.05;MMP-2:t=31.924,P<0.05]。miR-153-3p通过抑制snca表达促进破骨细胞分化(F=123.390,P<0.05),过表达snca能恢复过表达miR-153-3p的效果(F=136.515,P<0.05)。双荧光素酶及蛋白检测实验结果表明miR-153-3p能负向调控snca的表达(F=92.528,P<0.05)。结论:抑制miR-153-3p通过促进snca表达抑制破骨细胞功能,进而抑制小鼠体内骨质疏松进程。

Objective To investigate how the interactions between microRNA(miR)-153-3p andα-synuclein(SNCA)contribute to osteoporosis in mice.Methods The moust osteoporosis(OVX)model was constructed by bilateral ovariectomy,and the pathological changes were confirmed by hematoxylin-eosin(HE)staining.Real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-qPCR)and Western blotting were used to quantify the messenger RNA(mRNA)and protein expression of target genes.After treating mouse RAW264.7 cells with receptor activator of nuclear factor-κB ligand(RANKL),morphological changes were observed under the microscopy.Methyl thiazolyl tetrazolium(MTT)assay was used to evaluate cell viability,Transwell experiment was used to detect osteoclast migration ability,and dual-luciferase reporter(DLR)assay was used to verify the interaction between miR-153-3p and snca.Results The expression of snca was low in OVX mice(t=11.436,P<0.05),while the expression of miR-153-3p was high(t=15.833,P<0.05).Inhibiting miR-153-3p or overexpressing snca suppressed osteoporosis.The expression of snca was low in RANKL-induced RAW264.7 cells(t=11.796,P<0.05),while the expression of miR-153-3p was high(t=10.448,P<0.05).Overexpression of snca inhibited osteoclast differentiation,evidenced by the expression changes in a series of osteoclast markers[tartrate resistant acid phosphatase(TRAP):t=7.493,P<0.05;Cathepsin K:t=9.536,P<0.05;matrix metalloproteinase(MMP)-9:t=20.371,P<0.05;MMP-2:t=31.924,P<0.05].miR-153-3p promoted osteoclast differentiation by inhibiting snca expression(F=123.390,P<0.05),and overexpression of snca restored the effect of overexpressing miR-153-3p(F=136.515,P<0.05).DLR assay showed that miR-153-3p negatively regulated the expression of snca(F=92.528,P<0.05).Conclusion Inhibition of miR-153-3p suppresses the function of osteoclasts by promoting snca expression,and then inhibits the process of osteoporosis in mice.