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Journal of Peking University (Health Sciences) ›› 2025, Vol. 57 ›› Issue (2): 227-236. doi: 10.19723/j.issn.1671-167X.2025.02.002

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Gene silencing of Nemo-like kinase promotes neuralized tissue engineered bone regeneration

Mengdi LI1, Lei LEI2, Zhongning LIU1,*(), Jian LI1, Ting JIANG1,*()   

  1. 1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing 100081, China
    2. Department of Stomatology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China
  • Received:2021-10-10 Online:2025-04-18 Published:2025-04-12
  • Contact: Zhongning LIU, Ting JIANG E-mail:lzn45@163.com;jt_ketizu@163.com
  • Supported by:
    the National Natural Science Foundation of China(81771045);the National Natural Science Foundation of China(82170928);the Beijing Nova Program of Science and Technology(Z191100001119096)

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Abstract:

Objective: To identify the role of gene silencing or overexpression of Nemo-like kinase (NLK) during the process of neural differentiation of human mesenchymal stem cells (hBMSCs), and to explore the effect of NLK downregulation by transfection of small interfering RNA (siRNA) on promoting neuralized tissue engineered bone regeneration. Methods: NLK-knockdown hBMSCs were established by transfection of siRNA (the experimental group was transfected with siRNA silencing the NLK gene, the control group was transfected with control siRNA and labeled as negative control group), and NLK-overexpression hBMSCs were established using lentivirus vector transfection technique (the experimental group was infected with lentivirus overexpressing the NLK gene, the control group was infected with an empty vector lentivirus and labeled as the empty vector group). After neurogenic induction, quantitative real-time polymerase chain reaction (qPCR) was used to detect the expression of neural-related gene, and Western blot as well as immunofluorescence staining about several specific neural markers were used to evaluate the neural differentiation ability of hBMSCs.6-week-old male nude mice were divided into 4 groups: ① β-tricalcium phosphate (β-TCP) group, ② β-TCP+ osteogenic induced hBMSCs group, ③ β-TCP+ siRNA-negative control (siRNA-NC) transfection hBMSCs group, ④ β-TCP+ siRNA-NLK transfection hBMSCs group. Four weeks after the subcutaneous ectopic osteogenesis models were established, the osteogenesis and neurogenesis were detected by hematoxylin-eosin (HE) staining, Masson staining and tissue immunofluorescence assay. Statistical analysis was conducted by independent sample t test. Results: After gene silencing of NLK by siRNA in hBMSCs, neural-related genes, including the class Ⅲ β-tubulin (TUBB3), microtubule association protein-2 (MAP2), soluble protein-100 (S100), nestin (NES), NG2 proteoglycan (NG2) and calcitonin gene-related peptide (CGRP), were increased significantly in NLK-knockdown hBMSCs compared with the negative control group(P < 0.05), and the expression levels of TUBB3 and MAP2 of the NLK silencing group were also increased. Oppositely, after NLK was overexpressed using lentivirus vector transfection technique, TUBB3, MAP2, S100 and NG2 were significantly decreased in NLK-overexpression hBMSCs compared with the empty vector group (P < 0.05), and the expression level of TUBB3 was also decreased. 4 weeks after the subcutaneous ectopic osteogenesis model was established, more mineralized tissues were formed in the β-TCP+ siRNA-NLK transfection hBMSCs group compared with the other three groups, and the expression of BMP2 and S100 was higher in the β-TCP+ siRNA-NLK transfection hBMSCs group than in the other groups. Conclusion: Gene silencing of NLK by siRNA promoted the ability of neural differentiation of hBMSCs in vitro and promoted neuralized tissue engineered bone formation in subcutaneous ectopic osteogenic models in vivo in nude mice.

Key words: Small interfering RNA, NLK gene, Neural-differentiation, Bone tissue engineering

CLC Number: 

  • R783.3

Table 1

Quantitative real-time PCR primer sequence"

Gene Forward primer (5′-3′) Reverse primer (5′-3′)
GADPH CGACAGTCAGCCGCATCTT CCAATACGACCAAATCCGTTG
NLK CCAACCTCCACACATTGACTATT ACTTTGACATGATCTGAGCTGAG
TUBB3 AGTATCCCGACCGCATCA CATCCGTGTTCTCCACCAG
MAP2 ACCCTCTTCATCCTCCCTGT ACCTCACTGGACCTCAGCAC
NES CTCTGACCTGTCAGAAGAAT CCCACTTTCTTCCTCATCTG
NG2 CACGGCTCTGACCGACATAG CCCAGCCCTCTACGACAGT
CGRP ATGGGCTTCCAAAAGTTCTC TTAGTTGGCATTCTGGGGCATG
S100 TGAAGAAATCCGAACTGAAGGA GAATTCCTGGAAGTCACATTCG

Figure 1

The efficiency of NLK gene silencing by siRNA, and the expression of gene and protein in NLK-knockdown hBMSCs A, gene expression of NLK after gene silencing by siRNA as determined by qPCR; B, protein expression of NLK in NLK-knockdown hBMSCs; C, expression of neuronal markers TUBB3, MAP2, S100, NES, NG2 and CGRP in NLK-knockdown hBMSCs were elevated as determined by qPCR; D, Western blot analysis of the neuronal proteins TUBB3 and MAP2 were increased in the NLK-knockdown hBMSCs comparing with NC. * P < 0.05; * * P < 0.01; * * * P < 0.001; NLK, Nemo-like kinase; si, small interfering RNA; NC, negative control; GADPH, glyceraldehyde-3-phosphate dehydrogenase; TUBB3, class Ⅲ β-tubulin; MAP2, microtubule-associated protein 2; S100, soluble protein-100; NES, nestin; NG2, NG2 proteoglycan; CGRP, calcitonin gene-related peptide; siRNA, small interfering RNA; qPCR, quantitative real-time polymerase chain reaction; hBMSCs, human mesenchymal stem cells."

Figure 2

Immunofluorescence detection in NLK-knockdown hBMSCs Immunofluorescence detection of TUBB3 (A) and GFAP (B) proteins in hBMSCs were stronger in si-NLK group than si-NC group. * P < 0.05; * * P < 0.01; Mean gray value = integrated density/area; DAPI, 4', 6-diamidino-2'-phenylindole; TUBB3, class Ⅲ β-tubulin; GFAP, glial fibrillary acidic protein; hBMSCs, human mesenchymal stem cells; si, small interfering RNA; NC, negative control; NLK, Nemo-like kinase."

Figure 3

Efficiency of NLK overexpression, and the expression of gene and protein in OE-NLK hBMSCs A, gene expression of NLK in OE-NLK hBMSCs as determined by qPCR; B, protein expression of NLK in OE-NLK group; C, expression of neuronal markers TUBB3, MAP2, S100 and NG2 in OE-NLK hBMSCs were reduced as determined by qPCR; D, Western blot analysis of TUBB3 was reduced in the OE-NLK group comparing with vector. * P < 0.05; * * P < 0.01; * * * P < 0.001; NLK, Nemo-like kinase; Vector, empty vector; OE-NLK, over-expression NLK; GADPH, glyceraldehyde-3-phosphate dehydrogenase; TUBB3, class Ⅲ β-tubulin; MAP2, microtubule-associated protein 2; S100, soluble protein-100; NG2, NG2 proteoglycan; qPCR, quantitative real-time polymerase chain reaction; hBMSCs, human mesenchymal stem cells."

Figure 4

Histological observations of subcutaneous ectopic osteogenesis specimens in nude mice 4 weeks after operation A, hematoxylin-eosin staining; B, Masson' s trichrome staining. β-TCP, β-tricalcium phosphate; hBMSCs, human mesenchymal stem cells; si, small interfering RNA; NC, negative control; NLK, Nemo-like kinase."

Figure 5

Immunohistochemical staining of subcutaneous ectopic osteogenesis specimens in nude mice 4 weeks after operation Microscopic observation (A) and statistical results (B) showed that immunohistochemistry detection of BMP2 proteins were stronger in β-TCP+ si-NLK hBMSCs group than the other three groups. * * * P < 0.001; β-TCP, β-tricalcium phosphate; hBMSCs, human mesenchymal stem cells; si, small interfering RNA; NC, negative control; NLK, Nemo-like kinase; BMP2, bone morphogenetic protein 2."

Figure 6

Immunofluorescent staining of subcutaneous ectopic osteogenesis specimens in nude mice 4 weeks after operation Immunofluorescence detection of S100 proteins were stronger in β-TCP+ si-NLK hBMSCs group than the other three groups. DAPI, 4', 6-diamidino-2'-phenylindole; S100, soluble protein-100; β-TCP, β-tricalcium phosphate; hBMSCs, human mesenchymal stem cells; si, small interfering RNA; NC, negative control; NLK, Nemo-like kinase."

1 Grässel SG . The role of peripheral nerve fibers and their neurotransmitters in cartilage and bone physiology and pathophysiology[J]. Arthritis Res Ther, 2014, 16 (6): 485.
doi: 10.1186/s13075-014-0485-1
2 Maryanovich M , Takeishi S , Frenette PS . Neural regulation of bone and bone marrow[J]. Cold Spring Harb Perspect Med, 2018, 8 (9): a031344.
doi: 10.1101/cshperspect.a031344
3 Chen H , Hu B , Lv X , et al. Prostaglandin E2 mediates sensory nerve regulation of bone homeostasis[J]. Nat Commun, 2019, 10 (1): 181.
doi: 10.1038/s41467-018-08097-7
4 Li Z , Meyers CA , Chang L , et al. Fracture repair requires TrkA signaling by skeletal sensory nerves[J]. J Clin Invest, 2019, 129 (12): 5137- 5150.
doi: 10.1172/JCI128428
5 Wu Y , Jing D , Ouyang H , et al. Pre-implanted sensory nerve could enhance the neurotization in tissue-engineered bone graft[J]. Tissue Eng Part A, 2015, 21 (15/16): 2241- 2249.
6 Zhang Y , Xu J , Ye CR , et al. Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats[J]. Nat Med, 2016, 22 (10): 1160- 1169.
doi: 10.1038/nm.4162
7 Liu Q , Lei L , Yu T , et al. Effect of brain-derived neurotrophic factor on the neurogenesis and osteogenesis in bone engineering[J]. Tissue Eng Part A, 2018, 24 (15/16): 1283- 1292.
8 覃俊君, 尹东, 裴国献, 等. 植入单纯血管束、感觉神经束的组织工程骨修复大段骨缺损[J]. 中国组织工程研究, 2017, 21 (8): 1161- 1166.
9 陈思圆, 王簕, 江汕, 等. 植入含感觉神经束和血管束组织工程骨修复股骨大段骨缺损: 降钙素基因相关肽Ⅰ型受体的中长期表达[J]. 中国组织工程研究, 2017, 21 (6): 848- 853.
10 Yu B , Zhou S , Wang Y , et al. miR-221 and miR-222 promote Schwann cell proliferation and migration by targeting LASS2 after sciatic nerve injury[J]. J Cell Sci, 2012, 125 (Pt 11): 2675- 2683.
11 Song J , Li X , Li Y , et al. Biodegradable and biocompatible cationic polymer delivering microRNA-221/222 promotes nerve regeneration after sciatic nerve crush[J]. Int J Nanomedicine, 2017, 12, 4195- 4208.
doi: 10.2147/IJN.S132190
12 Lei L , Liu Z , Yuan P , et al. Injectable colloidal hydrogel with mesoporous silica nanoparticles for sustained co-release of micro-RNA-222 and aspirin to achieve innervated bone regeneration in rat mandibular defects[J]. J Mater Chem B, 2019, 7 (16): 2722- 2735.
doi: 10.1039/C9TB00025A
13 Ishitani T , Ishitani S . Nemo-like kinase, a multifaceted cell signaling regulator[J]. Cell Signal, 2013, 25 (1): 190- 197.
doi: 10.1016/j.cellsig.2012.09.017
14 Nifuji A , Ideno H , Ohyama Y , et al. Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation[J]. Exp Cell Res, 2010, 316 (7): 1127- 1136.
doi: 10.1016/j.yexcr.2010.01.023
15 Zanotti S , Canalis E . Nemo-like kinase inhibits osteoblastogenesis by suppressing bone morphogenetic protein and WNT canonical signaling[J]. J Cell Biochem, 2012, 113 (2): 449- 456.
doi: 10.1002/jcb.23365
16 Fakhr E , Zare F , Teimoori-Toolabi L . Precise and efficient siRNA design: A key point in competent gene silencing[J]. Cancer Gene Ther, 2016, 23 (4): 73- 82.
doi: 10.1038/cgt.2016.4
17 Sun X , Peng X , Cao Y , et al. ADNP promotes neural differentiation by modulating Wnt/β-catenin signaling[J]. Nat Commun, 2020, 11 (1): 2984.
doi: 10.1038/s41467-020-16799-0
18 Tawk M , Makoukji J , Belle M , et al. Wnt/beta-catenin signaling is an essential and direct driver of myelin gene expression and myelinogenesis[J]. J Neurosci, 2011, 31 (10): 3729- 3742.
doi: 10.1523/JNEUROSCI.4270-10.2011
19 Kodama D , Hirai T , Kondo H , et al. Bidirectional communication between sensory neurons and osteoblasts in an in vitro coculture system[J]. FEBS Lett, 2017, 591 (3): 527- 539.
doi: 10.1002/1873-3468.12561
20 Burbach JP . Neuropeptides from concept to online database[J]. Eur J Pharmacol, 2010, 626 (1): 27- 48.
doi: 10.1016/j.ejphar.2009.10.015
21 龙域丰, 朱古力, 易伟宏, 等. 神经肽类物质在骨代谢与骨再生中的调控作用[J]. 中华实验外科杂志, 2020, 37 (11): 2131- 2136.
doi: 10.3760/cma.j.cn421213-20191020-00747
22 He H , Chai J , Zhang S , et al. CGRP may regulate bone metabolism through stimulating osteoblast differentiation and inhibiting osteoclast formation[J]. Mol Med Rep, 2016, 13 (5): 3977- 3984.
doi: 10.3892/mmr.2016.5023
23 Chen J , Liu W , Zhao J , et al. Gelatin microspheres containing calcitonin gene-related peptide or substance P repair bone defects in osteoporotic rabbits[J]. Biotechnol Lett, 2017, 39 (3): 465- 472.
doi: 10.1007/s10529-016-2263-4
24 Niedermair T , Kuhn V , Doranehgard F , et al. Absence of substance P and the sympathetic nervous system impact on bone structure and chondrocyte differentiation in an adult model of endochondral ossification[J]. Matrix Biol, 2014, 9 (38): 22- 35.
25 Song L , Jin D , Wu JQ , et al. Neuropeptide Y stimulates osteoblastic differentiation and VEGF expression of bone marrow mesenchymal stem cells related to canonical Wnt signaling activating in vitro[J]. Neuropeptides, 2016, 56, 105- 113.
doi: 10.1016/j.npep.2015.12.008
26 Zhang S , Li J , Jiang H , et al. Dorsal root ganglion maintains stemness of bone marrow mesenchymal stem cells by enhancing autophagy through the AMPK/mTOR pathway in a coculture system[J]. Stem Cells Int, 2018, 2018, 8478953.
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