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Title: [Effect of OCT4 Gene Expression Upregulation on the Expression of iPSC-related Transcription Factors in Human Bone Marrow Derived Mesenchymal Stem Cells].
Author: Guo XP, Tang YM.
Journal: Zhongguo Shi Yan Xue Ye Xue Za Zhi; 2020 Aug; 28(4):1086-1095. PubMed ID: 32798382.
Abstract:
OBJECTIVE: To explore the effect of OCT4 over-expression on the expression of induced pluripotent stem cell (iPSC)-related transcription factors (cMYC，KLF4，LIN28，NANOG and SOX2) in human bone marrow derived mesenchymal stem cells (hBMMSCs), so as to provide fundamental basis for exploring the pathogenesis of hematological diseases (leukemia, aplastic anemia, etc.) from the perspective of hemopoietic microenvironment in the future. METHODS: Recombinant plasmid pcDNA3.1-OCT4 was constructed and transferred into the optimal generation P3-4 hBMMSCs by liposome transfection. The cells with stable and high expression of OCT4（hBMMSCs-OCT4）were screened by G418 resistance screening (limited dilution) and subcloning, the expression of OCT4 mRNA and OCT4 protein was verified by RT-PCR and FCM, respectively. The expression of iPSC-related transcription factors (cMYC, KLF4, LIN28, NANOG and SOX2) were also determined by FCM and RT-PCR, so as to evaluate the effect of ectopic high expression of OCT4 on the expression of iPSC related transcription factors in hBMMSCs. RESULTS: Recombinant plasmid pcDNA3.1-OCT4 was successfully constructed and cells with stable and high expression of OCT4 were successfully screened from hBMMSCs by limited dilution and subcloning. The result of flow cytometry showed that the mean expression level of OCT4 protein increased from (3.03±1.49)% to (95.46±1.40)% compared with the untransfected parental MSCs, which was also confirmed by RT-PCR analysis. At the same time, the expression levels of OCT4 protein and mRNA were compared between transient transfection (day 4) and stable expression cells (day 96), respectively, it was showed that the OCT4 protein level increased from (36.36±0.28)% at day 4 to (96.25±1.38)% at day 96, and the OCT4 mRNA level increased from 2.75-folds to 6.23-folds, respectively. Compared with the untransfected parental MSCs, the average expression levels of stemness transcription factors increased from (1.12±0.47)% (cMYC), (0.84±0.30)% (KLF4), (2.14±0.79)% (LIN28), (0.63±0.37)% (NANOG) and (14.34±2.44)% (SOX2) to (80.65±4.75)%, (73.03±4.70)%, (68.08±3.05)%, (39.39±1.85)%and (91.45±4.56)% in hBMMSCs-OCT4, respectively, which were consistent with results of RT-PCR analysis. Moreover, the expression levels of NANOG and SOX2 positively correlated with the mean expression of OCT4 (OCT4 vs NANOG: r=0.7802，OCT4 vs SOX2: r=0.4981；NANOG vs SOX2: r=0.7426). CONCLUSION: Cells with stable and high expression of OCT4 have been successfully established from hBMMSCs. Ectopic high expression of transcription factor OCT4 in hBMMSCs can up-regulate the expression of other iPSC-related transcription factors such as cMYC, KLF4, LIN28, NANOG and SOX2. 题目: 上调OCT4基因表达对人骨髓间充质干细胞的iPSC相关转录因子表达的影响. 目的: 探讨OCT4基因过表达对人骨髓间充质干细胞（human bone marrow derived mesenchymal stem cells, hBMMSC）iPSC相关转录因子（cMYC、KLF4、LIN28、NANOG、SOX2）表达的影响，为从骨髓造血微环境的角度探索血液系统疾病如白血病、再生障碍性贫血等的发生机制提供实验依据. 方法: 首先构建重组质粒pcDNA3.1-OCT4表达载体，通过脂质体转染的方式将上述表达质粒导入第3-4代hBMMSC，分别通过PCR及流式细胞术对OCT4 mRNA和OCT4蛋白的过表达进行验证，然后经G418抗性加压下有限稀释和连续亚克隆的方法筛选出稳定表达OCT4的细胞（hBMMSC-OCT4），分别通过RT-PCR和流式细胞术测定iPSC相关转录因子（cMYC、KLF4、LIN28、NANOG、SOX2）mRNA和蛋白的表达，观察OCT4过表达对iPSC相关转录因子表达的影响. 结果: 成功构建了携带OCT4的重组表达质粒pcDNA3.1-OCT4；通过有限稀释和连续亚克隆的方法筛选出稳定表达OCT4的细胞（hBMMSC-OCT4）；流式细胞术检测结果显示，OCT4的平均表达阳性率从未转染时的（3.03±1.49）%增加至稳定表达时的（95.46±1.40）%；RT-PCR证实了OCT4基因的高表达；Real-time PCR结果显示，与未转染的对照组MSC相比，OCT4 mRNA的相对表达量从瞬时转染时的2.75倍增加至稳定转染时的6.23倍，OCT4蛋白的平均表达阳性率从瞬时转染时的（36.36±0.28）%（d 4）升高至稳定转染时的（96.25±1.38）% （d 96）。hBMMSC-OCT4细胞高水平表达干性转录因子（cMYC、KLF4、LIN28、NANOG、SOX2），其平均表达量分别从未转染时的（1.12±0.47）%、（0.84±0.30）%、（2.14±0.79）%、（0.63±0.37）%和（14.34±2.44）% 增加至（80.65±4.75）%、（73.03±4.70）%、（68.08±3.05）%、（39.39±1.85）%和（91.45±4.56）%，与RT-PCR 结果一致，且NANOG和SOX2的表达水平与OCT4的平均表达量呈正相关（OCT4 vs NANOG: r=0.7802，OCT4 vs SOX2: r=0.4981；NANOG vs SOX2: r=0.7426). 结论: 获得了稳定表达OCT4的细胞（hBMMSC-OCT4），过表达OCT4可上调hBMMSC干性转录因子（cMYC、KLF4、LIN28、NANOG、SOX2）的表达.

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