MicroRNA-221 promotes cell proliferation, migration, and differentiation by regulation of ZFPM2 in osteoblasts

Bone fracture is a common medical condition, which may occur due to traumatic injury or disease-related conditions. Evidence suggests that microRNAs (miRNAs) can regulate osteoblast differentiation and function. In this study, we explored the effects and mechanism of miR-221 on the growth and migration of osteoblasts using MC3T3-E1 cells. The expression levels of miR-221 in the different groups were measured by qRT-PCR. Then, miR-221 mimic and inhibitor were transfected into MC3T3-E1 cells, and cell viability and migration were measured using the CCK-8 assay and the Transwell migration assay. Additionally, the expression levels of differentiation-related factors (Runx2 and Ocn) and ZFPM2 were measured by qRT-PCR. Western blot was used to measure the expression of cell cycle-related proteins, epithelial-mesenchymal transition (EMT)-related proteins, ZFPM2, and Wnt/Notch, and Smad signaling pathway proteins. miR-221 was significantly up-regulated in the patients with lumbar compression fracture (LCM) and trochanteric fracture (TF). miR-221 promoted ALP, Runx2, and OPN expressions in MC3T3-E1 cells. miR-221 overexpression significantly increased cell proliferation, migration, differentiation, and matrix mineralization, whereas suppression of miR-221 reversed these effects. Additionally, the results displayed that ZFPM2 was a direct target gene of miR-221, and overexpression of ZFPM2 reversed the promoting effects of miR-221 overexpression on osteoblasts. Mechanistic study revealed that overexpression of miR-221 inactivated the Wnt/Notch and Smad signaling pathways by regulating ZFPM2 expression. We drew the conclusions that miR-221 overexpression promoted osteoblast proliferation, migration, and differentiation by regulation of ZFPM2 expression and deactivating the Wnt/Notch and Smad signaling pathways.

骨折是一类常见的临床病症，可由创伤性损伤或相关性疾病引发。已有研究证实，微小RNA（microRNAs）可调控成骨细胞的分化与功能。本研究以MC3T3-E1细胞为模型，探究miR-221对成骨细胞增殖与迁移的影响及其作用机制。

首先采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测各组样本中miR-221的表达水平。随后将miR-221模拟物（mimic）与抑制剂（inhibitor）转染至MC3T3-E1细胞，分别采用CCK-8法与Transwell迁移实验检测细胞活力与迁移能力。此外，通过qRT-PCR检测成骨分化相关因子Runx2、骨钙素（osteocalcin, Ocn）以及ZFPM2的表达水平；采用蛋白质印迹法（Western blot）检测细胞周期相关蛋白、上皮间质转化（epithelial-mesenchymal transition, EMT）相关蛋白、ZFPM2以及Wnt/Notch、Smad信号通路相关蛋白的表达情况。

实验结果显示，腰椎压缩性骨折（lumbar compression fracture, LCM）与转子间骨折（trochanteric fracture, TF）患者的样本中，miR-221的表达显著上调。miR-221可促进MC3T3-E1细胞中碱性磷酸酶（alkaline phosphatase, ALP）、Runx2以及骨桥蛋白（osteopontin, OPN）的表达。过表达miR-221可显著提升细胞增殖、迁移、分化及基质矿化能力，而抑制miR-221则可逆转上述效应。此外，本研究证实ZFPM2是miR-221的直接靶基因，过表达ZFPM2可抵消miR-221过表达对成骨细胞的促增殖与分化作用。机制研究表明，miR-221过表达可通过调控ZFPM2的表达，抑制Wnt/Notch与Smad信号通路的激活。

综上，本研究得出结论：miR-221过表达可通过调控ZFPM2的表达、失活Wnt/Notch与Smad信号通路，从而促进成骨细胞的增殖、迁移与分化。