LINC00472 通过海绵吸附 miR-300 促进成骨细胞分化并缓解骨质疏松症，从而上调 FGFR2 的表达。

LINC00472 promotes osteogenic differentiation and alleviates osteoporosis by sponging miR-300 to upregulate the expression of FGFR2.

机构信息

Shi's Center of Orthopedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Institute of Traumatology and Orthopedics, Shanghai Academy of Traditional Chinese Medicine, Shanghai, China.

出版信息

Eur Rev Med Pharmacol Sci. 2020 May;24(9):4652-4664. doi: 10.26355/eurrev_202005_21151.

DOI:10.26355/eurrev_202005_21151

PMID:32432728

Abstract

OBJECTIVE

To investigate the expression of LINC00472 in osteoporotic issues of patients, ovariectomized (OVX) mice and mice bone marrow mesenchymal stem cells (BMSCs), its effect on osteogenic differentiation of BMSCs and its mechanism.

PATIENTS AND METHODS

The expression of LINC00472 and miR-300 in osteoporosis patients (n=55), ovariectomized (OVX) mice (n=10) and mice BMSCs (n=3) was detected by RT-qPCR and the correlation between the expression of miR-300 and LINC00472 was analyzed. After transferring sh-LINC00472 and overexpression LINC00472 plasmids into mice BMSCs, the expression of ALP, Bglap, OPN, Runx2 was detected by RT-qPCR and Western blot, which were related with osteogenic differentiation. In addition, Luciferase activity was used to detect whether miR-300 combined with LINC00472 and FGFR2 in mice BMSCs directly. Finally, Western blot (WB) was used to detect the change of FGFR2 protein expression by miR-300 inhibitor and sh-LINC00472.

RESULTS

We found there was a negative correlation between the expression of miR-300 and LINC00472 in osteoporosis patients, bone tissues of OVX mice and mice BMSCs. The expression of LINC00472 in mice BMSCs was gradually increased with osteogenic differentiation. Transferring overexpression plasmid of LINC00472 into BMSCs, the expression of ALP, Bglap, OPN, Runx2 was increased both in mRNA and protein levels. Transferring sh-LINC00472 to BMSCs, the results were the opposite. Luciferase results showed that miR-300 could directly bind to LINC00472 and FGFR2 in mice BMSCs. What's more, RT-qPCR and WB results showed that transferring sh-LINC00472 could decrease the expression of FGFR2 mRNA and protein, while miR-300 inhibitor could recover this tendency.

CONCLUSIONS

According to these results, this study revealed the previously neglected LINC00472/miR-300/FGFR2 regulatory axis for the regulation of osteogenic differentiation in osteoporosis, which may be a potential target for the treatment of osteoporosis.

摘要

目的

探讨 LINC00472 在骨质疏松症患者、去卵巢（OVX）小鼠和小鼠骨髓间充质干细胞（BMSCs）中的表达，及其对 BMSCs 成骨分化的影响及其机制。

方法

采用 RT-qPCR 检测骨质疏松症患者（n=55）、去卵巢（OVX）小鼠（n=10）和小鼠 BMSCs（n=3）中 LINC00472 和 miR-300 的表达，并分析 miR-300 与 LINC00472 表达的相关性。将 sh-LINC00472 和过表达 LINC00472 质粒转染入小鼠 BMSCs 后，采用 RT-qPCR 和 Western blot 检测碱性磷酸酶（ALP）、骨钙素（Bglap）、骨桥蛋白（OPN）、 runt 相关转录因子 2（Runx2）等与成骨分化相关的基因的表达。此外，采用荧光素酶活性检测 miR-300 是否能直接与小鼠 BMSCs 中的 LINC00472 和成纤维细胞生长因子受体 2（FGFR2）结合。最后，采用 Western blot（WB）检测 miR-300 抑制剂和 sh-LINC00472 对 FGFR2 蛋白表达的影响。

结果

我们发现，在骨质疏松症患者、OVX 小鼠骨组织和小鼠 BMSCs 中，miR-300 与 LINC00472 的表达呈负相关。随着成骨分化，小鼠 BMSCs 中 LINC00472 的表达逐渐增加。将过表达质粒转染入 BMSCs 后，ALP、Bglap、OPN、Runx2 的 mRNA 和蛋白水平均升高。将 sh-LINC00472 转染入 BMSCs 后，结果则相反。荧光素酶结果表明，miR-300 可直接与小鼠 BMSCs 中的 LINC00472 和 FGFR2 结合。此外，RT-qPCR 和 WB 结果表明，转染 sh-LINC00472 可降低 FGFR2 mRNA 和蛋白的表达，而 miR-300 抑制剂可恢复这种趋势。