中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2017, Vol. 12 ›› Issue (12): 2084-2091.doi: 10.4103/1673-5374.221168

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

促进损伤周围神经许旺细胞增殖的NONMMUG014387 长链非编码RNA

  

  • 出版日期:2017-12-15 发布日期:2017-12-15
  • 基金资助:

    中国学生创新和创业培训项目，国家自然科学基金(重点项目)，（ 201610062009。81330042）；中国科技部主办的中俄联合研究项目（2014DFR31210）；天津市科学技术委员会项目（13RCGFSY19000，14ZCZDSY00044）

Long non-coding RNA NONMMUG014387 promotes Schwann cell proliferation after peripheral nerve injury,

Bin Pan, Zhong-ju Shi, Jia-yin Yan, Jia-he Li, Shi-qing Feng   

  1. Department of Orthopedics, Tianjin Medical University General Hospital, Tianjin, China
  • Online:2017-12-15 Published:2017-12-15
  • Contact: Shi-qing Feng, M.D.,sqfeng@tmu.edu.cn.
  • Supported by:

    This work was supported by a grant from Student’s Platform for Innovation and Entrepreneurship Training Program in China,No. 201610062009; the National Natural Science Foundation of China (Key Program), No. 81330042; a grant from the Special Program for Sino-Russian Joint Research Sponsored by the Ministry of Science and Technology, China, No. 2014DFR31210; and a grant from the Key Program Sponsored by the Tianjin Science and Technology Committee of China, No. 13RCGFSY19000, 14ZCZDSY00044.

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摘要：

许旺细胞的逆分化和大量增殖在周围神经损伤后的再生过程中发挥关键作用。在前期研究中，我们通过基因芯片检测坐骨神经损伤后远端神经长链非编码RNA的表达变化，应用生物信息预测出长链非编码RNA NONMMUG014387参与调控周围神经损伤后许旺细胞的增殖过程，并且长链非编码RNA NONMMUG014387的靶基因为Cthrc1，而Cthrc1的上调可以激活下游Wnt/PCP通路进而影响多种生物学进程，但生物信息学预测结果最终仍需要生物学手段进行验证。为此，实验在许旺细胞中应用腺病毒载体过表达NONMMUG014387长链非编码RNA，分别采用pHBAd-MCMV-GFP- NONMMUG014387和pHBAd-MCMV-GFP腺病毒载体转染许旺细胞，设为NONMMUG014387过表达组和GFP过表达组，设不做任何处理的许旺细胞作为对照组。采用CCK-8法检测过表达前后许旺细胞的增殖能力，用PCR和Western blot检测靶基因和下游信号通路相关基因和蛋白的不表达。可见NONMMUG014387过表达组许旺细胞较其他两组增殖能力明显增强；与对照组相比，NONMMUG014387过表达组许旺细胞中Cthrc1，Wnt5a，ROR2，RhoA，Rac1，JNK和ROCK的mRNA和蛋白表达水平明显上调。结果证实，NONMMUG014387 长链非编码RNA通过靶向Cthrc1和激活Wnt/PCP途径可以促进损伤周围神经许旺细胞的增殖。

orcid:0000-0001-9437-7674(Shi-qing Feng)

 

关键词: 神经再生, 周围神经损伤, 许旺细胞, 长链非编码RNA, 神经再生, 增殖；Wnt/PCP 信号通路, CCK8实验, 腺病毒过表达, 坐骨神经, Cthrc1

Abstract:

Schwann cells play a critical role in peripheral nerve regeneration through dedifferentiation and proliferation. In a previous study, we performed microarray analysis of the sciatic nerve after injury. Accordingly, we predicted that long non-coding RNA NONMMUG014387 may promote Schwann cell proliferation after peripheral nerve injury, as bioinformatic analysis revealed that the target gene of NONMMUG014387 was collagen triple helix repeat containing 1 (Cthrc1). Cthrc1 may promote cell proliferation in a variety of cells by activating Wnt/PCP signaling. Nonetheless, bioinformatic analysis still needs to be verified by biological experiment. In this study, the candidate long non-coding RNA, NONMMUG014387, was overexpressed in mouse Schwann cells by recombinant adenovirus transfection. Plasmid pHBAd-MCMV-GFP-NONMMUG014387 and pHBAd-MCMV-GFP were transfected into Schwann cells. Schwann cells were divided into three groups: control (Schwann cells without intervention), Ad-GFP (Schwann cells with GFP overexpression), and Ad-NONMMUGO148387 (Schwann cells with GFP and NONMMUGO148387 overexpression). Cell Counting Kit-8 assay was used to evaluate proliferative capability of mouse Schwann cells after NONMMUG014387 overexpression. Polymerase chain reaction and western blot assay were performed to investigate target genes and downstream pathways of NONMMUG014387. Cell proliferation was significantly increased in Schwann cells overexpressing lncRNA NONMMUG014387 compared with the other two groups. Further, compared with the control group, mRNA and protein levels of Cthrc1, Wnt5a, ROR2, RhoA, Rac1, JNK, and ROCK were visibly up-regulated in the Ad-NONMMUGO148387 group. Our findings confirm that long non-coding RNA NONMMUG014387 can promote proliferation of Schwann cells surrounding the injury site through targeting Cthrc1 and activating the Wnt/PCP pathway.

Key words: nerve regeneration, peripheral nerve injury, Schwann cells, long non-coding RNAs, proliferation, Wnt/PCP pathway, Cell Counting Kit-8 assay, adenovirus overexpression, sciatic nerve, Cthrc1, neural regeneration