基于转录组学筛选左归丸促进MC3T3-E1细胞成骨细胞增殖分化的关键基因

作者	单位
霍少川;熊仪;俞建萍;郭海;廖昔威;程文昌;钟嘉漫;温刘莹;黄婷婷;韩霞	1.广州中医药大学深圳医院（福田）研究院（深圳 518000）,2.柳州市中医医院骨伤科（广西 545001）,3.广州中医药大学第六临床医学院（深圳 518000）

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中文摘要:

目的探讨左归丸治疗骨质疏松（OP）多靶点的分子机制。方法采用0、100、200、400、1 000μg/mL浓度的左归丸溶液干预MC3T3-E1细胞，Western Blot检测总β-连环蛋白（β-catenin）、活性β-catenin、成骨细胞特异性转录因子2（Runx2）、成骨细胞特异性转录因子（Osx）蛋白表达，qPCR检测Runx2、Osx、Ⅱ型α 胶原蛋白基因（Col2a1）、骨钙蛋白（Ocn）、β-catenin mRNA表达，CCK-8检测MC3T3-E1细胞增殖活力，碱性磷酸酶（ALP）染色及茜素红染色（ARS）检测MC3T3-E1细胞成骨细胞分化能力，RNA-Seq筛选左归丸促进MC3T3-E1细胞成骨细胞增殖分化的差异基因并进行qPCR验证。结果不同浓度左归丸溶液（100、200、400、1 000μg/mL）干预MC3T3-E1细胞48 h，成骨标志基因（Runx2、Osx、Col2A1、Ocn、β-catenin）表达水平明显升高（P＜0.05），MC3T3-E1细胞增殖率明显升高（P＜0.05），ALP、ARS染色明显增强（P＜0.05），其中以400μg/mL左归丸溶液干预效果最明显。当左归丸溶液干预浓度达到1 000μg/mL，MC3T3-E1细胞成骨分化能力显著下降（P＜0.05）。采用400μg/mL左归丸溶液干预MC3T3-E1细胞48 h，RNA-seq筛选出10个差异最大基因，其中6个上调基因和4个下调基因，qPCR验证发现，EF-Hand 钙结合域9（Efcab9）、叉头框蛋白J1（Foxj1） mRNA表达水平升高（P＜0.05），Ras 蛋白特异性鸟嘌呤核苷酸释放因子 1（RasGRF1）、生长分化因子 -9（GDF9）、Ⅳ型胶原蛋白 α -3 链（Col4a3） mRNA表达水平明显降低（P＜0.05）。结论左归丸溶液（400μg/mL）能够调节Efcab9、FOXj1、RasGRF1、GDF-9、Col4a3 mRNA表达水平，促进MC3T3-E1细胞成骨细胞增殖分化。

英文摘要:

Objective To investigate the molecular mechanism of osteoporosis （OP） in MC3T3-E1 regulated by Zuogui Pill solution. Methods MC3T3-E1 cells were treated with Zuogui Pill solution at concentrations of 0， 100， 200， 400， and 1 000 μg/mL. Western Blot was performed to assess the protein expressions of total β-catenin，active β-catenin，Runt-related transcription factor 2（Runx2），and osxterix（Osx）. qPCR was used to detect the mRNA expressions of Runx2，Osx，typeⅡ alpha 1 collagen gene（Col2a1），osteocalcin（Ocn），and β-catenin. The proliferation activity of MC3T3-E1 cells was measured using CCK-8 assay，while alkaline phosphatase（ALP） staining and alizarin red staining（ARS） were used to evaluate the osteoblast differentiation ability. Differential gene expression related to MC3T3-E1 cell proliferation and differentiation induced by Zuogui Pill solution was confirmed through qPCR analysis of RNA-Seq results. Results Different concentrations of Zuogui Pill solutions （0，100， 200， 400， 1 000 μg/mL） were used to treat MC3T3-E1 cells for 48 hours，the expression levels of osteogenic marker genes （Runx2，Osx，Col2A1，Ocn，β-catenin） were significantly increased （P＜0.05）. Additionally，the proliferation rate of MC3T3-E1 cells was significantly increased （P＜0.05）， and ALP and ARS showed significant enhancement （P＜0.05）. Notably，the intervention effect of the 400 μg/mL Zuogui Pill solution was the most prominent among the tested concentrations. The osteogenic differentiation ability of MC3T3-E1 cells was significantly reduced （P＜0.05） when the intervention concentration of Zuogui Pill solution reached 1 000 μg/mL. After being intervened with 400 μg/mL Zuogui Pill solution for 48 h，RNA-seq analysis identified 10 genes with the most differential expression，including 6 up-regulated genes and 4 down-regulated genes. qPCR verification confirmed a significant increase in the mRNA expression levels of EF-hand calcium-binding domain-containing protein 9（Efcab9），forkhead box J1（Foxj1）（P＜0.05）， while the mRNA expression levels of Ras protein specific guanine nucleotide releasing factor 1（RasGRF1）， growth differentiation factor 9（GDF9），and achain of type Ⅳ collagen（Col4a3） were significantly reduced （P＜0.05）. Conclusion Zuogui Pill solution （400 μg/mL） can regulate the mRNA expression levels of Efcab9，FOXj1，RasGRF1，GDF-9，and Col4a3，and promote osteoblast proliferation and differentiation of MC3T3-E1 cells.

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