Chen Shiyu, Su Xin, Liu Junping, Shi Yutong, Wu Minmin, Xu Mengqi, Zhang Fengmei, Tang Min
College of Laboratory Medicine, Chongqing Medical University//Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education, Chongqing 400016, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2021 Jan 30;41(1):79-86. doi: 10.12122/j.issn.1673-4254.2021.01.11.
To investigate the role of NOV/CCN3 in regulating the proliferation of mesenchymal stem cells (MSCs) and its regulatory mechanism and assess the value of CCN3 as a proliferative factor in bone tissue engineering.
Mouse embryonic fibroblasts (MEFs) were used as the MSC model, in which CCN3 expression was up-regulated and downregulated by transfection with the recombinant adenovirus vectors Ad-CCN3 and Ad-siCCN3, respectively. Flow cytometry was used to analyze the changes in cell cycle and apoptosis of the transfected cells. Western blotting was used to detect the expression levels of the proliferation indicators (PCNA, cyclin E, and cyclin B1) and the apoptosis indicators (Bax and Bcl-2) to assess the effect of modulation of CCN3 expression on MEF proliferation and apoptosis. CCN3 protein secretion by the cells was detected using ELISA. RT-qPCR and Western blotting were employed to analyze the changes in the expressions of Notch1, ligand DLL1, the downstream key proteins or genes (Hey1, P300, H3K9) and MAPK pathway-related proteins ERK1+2 and p-ERK1+2.
Flow cytometry showed that compared with the control cells, MEFs transfected with Ad-CCN3 exhibited significantly increased cell proliferation index ( < 0.01) and lowered cell apoptosis rate ( < 0.05) with obviously enhanced expressions of PCNA, cyclin E and Bcl-2 proteins ( < 0.05). The results of RT-qPCR and Western blotting demonstrated that CCN3 overexpression significantly promoted the expression of Notch1 in the Notch signaling pathway ( < 0.001), inhibited the expressions of DLL1, Hey1, P300, and H3K9 ( < 0.05), and increased the protein expressions of ERK1+2 and P-ERk1+2 in the MAPK pathway ( < 0.01).
CCN3 over-expression promotes the proliferation and inhibits apoptosis of MEFs possibly by inhibiting the classical Notch signaling pathway and activating the MAPK pathway binding to Notch1, suggesting the potential value of CCN3 as a proliferative factor of MSCs in bone tissue engineering.
探讨NOV/CCN3在调节间充质干细胞（MSCs）增殖中的作用及其调控机制，并评估CCN3作为骨组织工程中增殖因子的价值。
以小鼠胚胎成纤维细胞（MEFs）作为MSCs模型，分别用重组腺病毒载体Ad-CCN3和Ad-siCCN3转染上调和下调CCN3表达。采用流式细胞术分析转染细胞的细胞周期和凋亡变化。用蛋白质免疫印迹法检测增殖指标（PCNA、细胞周期蛋白E和细胞周期蛋白B1）和凋亡指标（Bax和Bcl-2）的表达水平，以评估CCN3表达调节对MEF增殖和凋亡的影响。用酶联免疫吸附测定法检测细胞分泌的CCN3蛋白。采用逆转录-定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法分析Notch1、配体DLL1、下游关键蛋白或基因（Hey1、P300、H3K9）以及丝裂原活化蛋白激酶（MAPK）通路相关蛋白细胞外信号调节激酶1+2（ERK1+2）和磷酸化细胞外信号调节激酶1+2（p-ERK1+2）表达的变化。
流式细胞术显示，与对照细胞相比，用Ad-CCN3转染的MEFs细胞增殖指数显著升高（<0.01），细胞凋亡率降低（<0.05），PCNA、细胞周期蛋白E和Bcl-2蛋白的表达明显增强（<0.05）。RT-qPCR和蛋白质免疫印迹法结果表明，CCN3过表达显著促进Notch信号通路中Notch1的表达（<0.001），抑制DLL1、Hey1、P300和H3K9的表达（<0.05），并增加MAPK通路中ERK1+2和P-ERk1+2的蛋白表达（<0.01）。
CCN3过表达可能通过抑制经典Notch信号通路并激活与Notch1结合的MAPK通路来促进MEFs的增殖并抑制其凋亡，提示CCN3作为骨组织工程中MSCs增殖因子的潜在价值。
