Effect of long non-coding RNA NR_033474 on proliferation of C3H10T1/2 mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.05.019

Abstract

Abstract:

BACKGROUND: Recent studies have found that long non-coding RNAs (lncRNAs) can regulate stem cell proliferation and differentiation. But it is unclear that how lncRNA NR_033474 regulate stem cell proliferation and cell cycle.
OBJECTIVE: To investigate the effect of lncRNA NR_033474 on the proliferation and cell cycle regulation in C3H10T1/2 mesenchymal stem cells after the NR_033474 overexpressed by lentivirus, and to study the possible regulation mechanism of NR_033474 on mesenchymal stem cells.
METHODS: LncRNA NR_033474 was cloned into a lentivirus vector. Lentivirus particles were infected into C3H10T1/2 cells to upregulate the expression of NR_033474. The NR_033474 expression level was detected by real-time PCR. Compared with the empty lentivirus vector, the proliferation of C3H10T1/2 cells which overexpressed NR_033474 was detected by cell counting assay and cell cycle was detected using flow cytometry. The expression of cell cycle-associated proteins such as CDK1, Cyclin B1, Cyclin D1 and P53 were detected by western blot assay.
RESULTS AND CONCLUSION: Compared with the control group, lncRNA NR_033474 in C3H10T1/2 cells which overexpressed NR_033474 was increased by about 100 times (P < 0.01), and the proliferation of C3H10T1/2 cells was significantly inhibited after NR_033474 overexpression by lentivirus (P < 0.05). In addition, flow cytometry showed that C3H10T1/2 cells overexpressing NR_033474 were arrested in G2/M phase compared to the control group. Western blot showed that the expression levels of CDK1 and Cyclin B1 were downregulated, while there were no changes in Cyclin D1 and P53 expression. To conclude, these findings suggest that the NR_033474 overexpression significantly inhibits the cell growth of C3H10T1/2 cells, at least in part, through induction of cell cycle arrest at G₂/M phase.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mesenchymal Stem Cells, Cell Proliferation, Cell Cycle, Cell Cycle Proteins, Flow Cytometry, Tissue Engineering

CLC Number:

R394.2

Pan Ya-qiong, Dai Zhong, Zuo Chang-qing, Wang Zong-gui. Effect of long non-coding RNA NR_033474 on proliferation of C3H10T1/2 mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 766-772.

Figures/Tables 2

2.1 长链非编码RNA NR_033474编码蛋白性能鉴定根据CPAT软件分析结果，NR_033474序列是长度为1 334 bp的RNA分子，其编码蛋白的概率得分为0.059 7，远低于蛋白编码阈值0.44(当编码蛋白的概率≥0.44，表明其为编码序列；而当编码蛋白的概率<0.44时，表明其为非编码序列)，表明是非编码序列，属于长链非编码RNA。 2.2 qRT-PCR鉴定NR_033474在C3H10T1/2间质干细胞生长过程中的时序表达模式 qRT-PCR结果显示，细胞生长汇合后长链非编码RNA NR_033474表达逐渐增加，与对数生长期比较差异有显著性意义(P < 0.05)，见图1。 2.3 长链非编码RNA NR_033474过表达对C3H10T1/2间质干细胞生长的影响以2-??Cq法统计lncRNA NR_033474的相对含量，以β-actin为内参基因。与对照组相比，实验组长链非编码RNA NR_033474表达量约上调100倍(P < 0.01)，见图2。将对照组和实验组稳定株种植6孔板，从生长第3 天开始，实验组稳定株生长速度明显受到抑制，第3，5天细胞生长与对照组比较差异有显著性意义(P < 0.05)，见图3，表明过表达长链非编码RNA NR_033474具有抑制C3H10T1/2间质干细胞增殖的作用。 2.4 长链非编码RNA NR_033474过表达对C3H10T1/2间质干细胞周期的影响与对照组比较，实验组能够使G2/M期细胞数由7.48%增加到15.94%，相应处于S期的细胞则从38.77%降到33.35%(P < 0.05)，见图4。 2.5 过表达NR_033474对C3H10T1/2间质干细胞周期相关蛋白表达水平的影响与对照组相比，实验组G2/M期调控蛋白Cyclin B1和CDK1表达水平下调，G0/G1期调控蛋白P53和Cyclin D1表达水平无变化，见图5。实验结果从蛋白水平揭示，长链非编码RNA NR_033474通过减少Cyclin B1和CDK1蛋白的表达诱导C3H10T1/2间质干细胞出现G2/M期阻滞，并未对C3H10T1/2细胞的G0/G1期产生影响。"

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