Li Pengfei, Sun Nan, Zeng Jianchun, Zeng Yirong, Fan Yueguang, Feng Wenjun, Li Jie
Guangzhou University of Chinese Medicine, Guangzhou, China.
Deparment of orthopedics, The First Affiliated Hospital of Guangzhou university of Chinese Medicine, Guangzhou, China.
Gene. 2016 Oct 10;591(1):69-73. doi: 10.1016/j.gene.2016.06.045. Epub 2016 Jul 1.
Apoptosis of osteoblasts and osteocytes is one cause of steroid-induced osteonecrosis of the femoral head; however, the molecular mechanism of steroid affecting osteoblasts at the genetic level is unclear. The aim of the present work is to examine differential expression of osteoblasts in rats after steroid intervention and to verify expression by real-time polymerase chain reaction (RT-PCR).
Primary culture, passaging and identification of osteoblasts of SD neonatal rats were conducted; osteoblasts were divided into two groups, the control group, and the steroid group. Total RNA was extracted separately, and quality control was performed; by means of RNA labeling and microarray hybridization, data were collected and then standardized to ascertain differences in miRNA expression between the two groups. The gene expression spectrum was analyzed. Obvious differential expression of miR-672-5p and miR-146a-5p was verified by RT-PCR. Miranda, microcosm and mirdb bioinformatics software were used to predict target genes.
Compared with the control group, morphologically, the osteoblasts in the steroid group were more irregular and showed various shapes. The number of miRNAs (fold change >2) in the steroid group was six. Four miRNAs were upregulated and two miRNAs were downregulated. In particular, upregulated miR-672-5p expression and downregulated miR-146a-5p expression were significant. RT-PCR results showed that the 2(-△△) CT value of miR-672-5p in the steroid group was 3.743-fold of that in the control group, and the 2(-△△) CT value of miR-146a-5p in the steroid group was 0.322-fold of that in the control group. Angptl4, Ccdc51, Ssbp3 and RGD1306991 were predicted as the target gene of miR-672-5p, while Hrp12 was that of miR-146a-5p.
Expression profiles of miR-672-5p and miR-146a-5p had the most significant changes in the osteoblasts of rats with steroid intervention, which may provide a new viewpoint to pathogenesis of osteonecrosis of the femoral head.
成骨细胞和骨细胞凋亡是类固醇诱导的股骨头坏死的原因之一；然而，类固醇在基因水平影响成骨细胞的分子机制尚不清楚。本研究的目的是检测类固醇干预后大鼠成骨细胞的差异表达，并通过实时聚合酶链反应（RT-PCR）验证表达情况。
进行SD新生大鼠成骨细胞的原代培养、传代及鉴定；将成骨细胞分为两组，即对照组和类固醇组。分别提取总RNA并进行质量控制；通过RNA标记和微阵列杂交收集数据，然后进行标准化以确定两组之间miRNA表达的差异。分析基因表达谱。通过RT-PCR验证miR-672-5p和miR-146a-5p的明显差异表达。使用Miranda、microcosm和mirdb生物信息学软件预测靶基因。
与对照组相比，形态上，类固醇组的成骨细胞更不规则，呈现出各种形状。类固醇组中miRNA（倍数变化>2）的数量为6个。4个miRNA上调，2个miRNA下调。特别是，miR-672-5p表达上调和miR-146a-5p表达下调显著。RT-PCR结果显示，类固醇组中miR-672-5p的2（-△△）CT值是对照组的3.743倍，类固醇组中miR-146a-5p的2（-△△）CT值是对照组的0.322倍。Angptl4、Ccdc51、Ssbp3和RGD1306991被预测为miR-672-5p的靶基因，而Hrp12是miR-146a-5p的靶基因。
在类固醇干预的大鼠成骨细胞中，miR-672-5p和miR-146a-5p的表达谱变化最为显著，这可能为股骨头坏死的发病机制提供新的视角。
