Zhang Hengshuo, Chen Lu, Wang Ziyu, Sun Zhenqian, Shan Yu, Li Qinghui, Qi Linzeng, Wang Hongliang, Chen Yunzhen
Department of Orthopedics, Qilu Hospital of Shandong University, Jinan, Shandong 250012, PR China.
Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, PR China.
Aging (Albany NY). 2022 May 19;14(10):4486-4499. doi: 10.18632/aging.204088.
Noncoding RNAs play an important role in regulating osteoclast differentiation. We investigated whether and how potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1), a long noncoding RNA, regulates osteoclast differentiation. We found that the expression of KCNQ1OT1 was downregulated in osteoporotic bone tissue. Then transfection of KCNQ1OT1 overexpression vectors or small interfering RNAs showed that the proliferation, migration, and osteoclast differentiation of RAW 264.7 cells were inhibited by KCNQ1OT1 upregulation, while they were promoted by KCNQ1OT1 knockdown. Interestingly, we found and confirmed that miR-128-3p was a target of KCNQ1OT1 using online databases, dual luciferase reporter assays and quantitative real-time polymerase chain reaction, and that it inhibited the expression of miR-128-3p. Moreover, we confirmed that miR-128-3p directly targeted nuclear factor of activated T cell 5 (NFAT5), a protein that combines with osteoprotegerin and thus regulates osteoclastogenesis with the presence of the receptor activator of nuclear factor κB ligand. Furthermore, we demonstrated that both the knockdown of KCNQ1OT1 and the overexpression of miR-128-3p attenuate the expression of NFAT5, while upregulating the osteoclastogenesis markers c-Fos, NFATc1, and Ctsk. The results from overexpression of KCNQ1OT1 and the inhibition of miR-128-3p were contrary to the above. Finally, we found that the inhibition of osteoclast differentiation by KCNQ1OT1 overexpression could be rescued using a miR-128-3p mimic, while the enhancement of migration and osteoclast differentiation by si-NFAT5 could be reversed with a miR-128-3p inhibitor. These results suggested that KCNQ1OT1 regulates the osteoclast differentiation via the miR-128-3p/NFAT5 axis.
非编码RNA在调节破骨细胞分化中发挥着重要作用。我们研究了长链非编码RNA钾离子电压门控通道Q亚家族成员1重叠转录本1（KCNQ1OT1）是否以及如何调节破骨细胞分化。我们发现KCNQ1OT1在骨质疏松性骨组织中的表达下调。然后，KCNQ1OT1过表达载体或小干扰RNA的转染表明，KCNQ1OT1上调抑制了RAW 264.7细胞的增殖、迁移和破骨细胞分化，而KCNQ1OT1敲低则促进了这些过程。有趣的是，我们使用在线数据库、双荧光素酶报告基因检测和定量实时聚合酶链反应发现并证实miR-128-3p是KCNQ1OT1的一个靶点，并且它抑制了miR-128-3p的表达。此外，我们证实miR-128-3p直接靶向活化T细胞核因子5（NFAT5），该蛋白与骨保护素结合，从而在核因子κB配体受体激活剂存在的情况下调节破骨细胞生成。此外，我们证明KCNQ1OT1的敲低和miR-128-3p的过表达均减弱了NFAT5的表达，同时上调了破骨细胞生成标志物c-Fos、NFATc1和Ctsk。KCNQ1OT1过表达和miR-128-3p抑制的结果与上述情况相反。最后，我们发现使用miR-128-3p模拟物可以挽救KCNQ1OT1过表达对破骨细胞分化的抑制作用，而使用miR-128-3p抑制剂可以逆转si-NFAT5对迁移和破骨细胞分化的增强作用。这些结果表明，KCNQ1OT1通过miR-128-3p/NFAT5轴调节破骨细胞分化。
