微小RNA-29a-3p调节卷曲蛋白4表达影响高脂血症大鼠种植体骨整合的实验研究

Micro RNA-29a-3p regulates osteoblast differentiation and peri-implant osseointegration in a rat model of hyperlipidemia by modulating Frizzled 4 expression

目的研究microRNA-29a-3p(miR-29a-3p)对高脂环境下大鼠骨髓间充质干细胞(BMSCs)成骨分化和高脂血症大鼠种植体骨整合的影响及其作用位点。方法 1)体外实验:对BMSCs分别进行普通和高脂成骨诱导,通过逆转录实时定量聚合酶链反应和Western blot检测miR-29a-3p及成骨相关因子碱性磷酸酶(ALP)、Runt相关基因2(Runx2)的基因和蛋白质表达;高脂培养的BMSCs分别转染miR-29a-3p模拟物、抑制物及阴性对照(NC)质粒,RT-qPCR检测miR-29a-3p、ALP及Runx2基因表达情况,Western blot检测ALP、Runx2蛋白表达情况。通过靶基因预测软件(Target Scan、MiRNA.org等)预测miR-29a-3p与成骨相关的靶基因为卷曲蛋白4(Fzd4),双荧光素酶报告检测miR-29a-3p与Fzd4的相互作用关系。2)体内实验:高脂血症大鼠为实验组,普通大鼠为对照组,两组分别植入种植体,检测种植体周围骨组织中miR-29a-3p、ALP、Runx2的表达差异;行种植体-骨组织的硬组织切片,亚甲基蓝-酸性品红染色,进行组织学观察。分别将miR-29a-3p过表达慢病毒载体及空白对照慢病毒载体注射入高脂血症大鼠,种植体植入后3、10 d检测种植体周围骨组织中ALP、Runx2的表达变化以研究miR-29a-3p对高脂血症大鼠成骨的作用。结果高脂组与普通组相比,ALP、Runx2和miR-29a-3p表达下调,BMSCs成骨分化能力下降。miR-29a-3p模拟物组与抑制物组相比,ALP、Runx2表达升高,BMSCs成骨分化能力增强。体内实验也得到了相似结果。双荧光素酶报告基因分析证实miR29a-3p通过直接结合3’-UTR抑制Fzd4表达。结论miR-29a-3p对高脂环境下大鼠BMSCs成骨分化起正向调节作用,可直接与Fzd4结合调节成骨分化;mi R-29a-3p能够促进高脂血症大鼠种植体周围成骨标志基因的表达,有利于骨整合。

Objective This work aimed to study and identify the influence and target gene of microRNA-29a-3p (miR-29a-3p) in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a high-fat environment in vitro and in vivo. Methods 1) In vitro: BMSCs were randomly allocated into two groups and were then induced to undergo osteogenic differentiation in a normal or high-fat environ-ment. Next, a miR-29a-3p mimic/inhibitor was transfected into the two groups of cells. The mRNA expression levels of alkaline phosphatase (ALP), Runt related gene 2 (Runx2), and miR-29a-3p and the protein expression levels of ALP and Runx2 were detected before and after transfection through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Moreover, Frizzled (Fzd) 4 was predicted as the target gene of miR-29a-3p by using an online database (Target Scan, MiRNA.org). The interactive relationship between miR-29a-3p and Fzd4 was confirmed through dual-luciferase assays. 2) In vivo: Rats were randomly divided into two groups and fed with a standard or high-fat diet. Titanium implants were grown in rats. Then, the expression levels of miR-29a-3p, ALP, and Runx2 were detected in bone tissues surrounding implants. Moreover, hard tissue sections were subjected to methylene blue-acid magenta staining and observed under microscopy to study bone formation around implants. In addition, miR-29a-3p-overexpressing lentiviral vectors were transfected into rats, and the expression levels of ALP, Runx2, and miR-29a-3p in bone tissues surrounding implants were detected at 3 and 10 days after transfection. Results The expression levels of ALP, Runx2, and miR-29a-3p and the osteogenic differentiation of BMSCs were suppressed in high-fat groups in vitro and in vivo. Conclusion MiR-29a-3p plays a positive role in the regulation of BMSCs in a high-fat environment. It can increase ALP and Runx2 expression levels in bone tissues surrounding implants in hyperlipidemia models. This result implies that miR-29a-3p can promote implant osseointergration in a rat model of hyperlipidemia.