Mengdi Li, Lei Lei, Zhongning Liu, Jian Li, Ting Jiang
{"title":"[Gene silencing of Nemo-like kinase promotes neuralized tissue engineered bone regeneration].","authors":"Mengdi Li, Lei Lei, Zhongning Liu, Jian Li, Ting Jiang","doi":"","DOIUrl":null,"url":null,"abstract":"<p><strong>Objective: </strong>To identify the role of gene silencing or overexpression of Nemo-like kinase (<i>NLK</i>) during the process of neural differentiation of human mesenchymal stem cells (hBMSCs), and to explore the effect of <i>NLK</i> downregulation by transfection of small interfering RNA (siRNA) on promoting neuralized tissue engineered bone regeneration.</p><p><strong>Methods: </strong><i>NLK</i>-knockdown hBMSCs were established by transfection of siRNA (the experimental group was transfected with siRNA silencing the <i>NLK</i> gene, the control group was transfected with control siRNA and labeled as negative control group), and <i>NLK</i>-overexpression hBMSCs were established using lentivirus vector transfection technique (the experimental group was infected with lentivirus overexpressing the <i>NLK</i> 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-<i>NLK</i> 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 <i>t</i> test.</p><p><strong>Results: </strong>After gene silencing of <i>NLK</i> by siRNA in hBMSCs, neural-related genes, including the class Ⅲ β-tubulin (<i>TUBB3</i>), microtubule association protein-2 (<i>MAP2</i>), soluble protein-100 (<i>S100</i>), nestin (<i>NES</i>), NG2 proteoglycan (<i>NG2</i>) and calcitonin gene-related peptide (<i>CGRP</i>), were increased significantly in <i>NLK</i>-knockdown hBMSCs compared with the negative control group(<i>P</i> < 0.05), and the expression levels of TUBB3 and MAP2 of the <i>NLK</i> silencing group were also increased. Oppositely, after <i>NLK</i> was overexpressed using lentivirus vector transfection technique, <i>TUBB3</i>, <i>MAP2</i>, <i>S100</i> and <i>NG2</i> were significantly decreased in <i>NLK</i>-overexpression hBMSCs compared with the empty vector group (<i>P</i> < 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-<i>NLK</i> transfection hBMSCs group compared with the other three groups, and the expression of BMP2 and S100 was higher in the β-TCP+ siRNA-<i>NLK</i> transfection hBMSCs group than in the other groups.</p><p><strong>Conclusion: </strong>Gene silencing of <i>NLK</i> by siRNA promoted the ability of neural differentiation of hBMSCs <i>in vitro</i> and promoted neuralized tissue engineered bone formation in subcutaneous ectopic osteogenic models <i>in vivo</i> in nude mice.</p>","PeriodicalId":8790,"journal":{"name":"北京大学学报(医学版)","volume":"57 2","pages":"227-236"},"PeriodicalIF":0.0000,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11992439/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"北京大学学报(医学版)","FirstCategoryId":"3","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Medicine","Score":null,"Total":0}
引用次数: 0
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.
期刊介绍:
Beijing Da Xue Xue Bao Yi Xue Ban / Journal of Peking University (Health Sciences), established in 1959, is a national academic journal sponsored by Peking University, and its former name is Journal of Beijing Medical University. The coverage of the Journal includes basic medical sciences, clinical medicine, oral medicine, surgery, public health and epidemiology, pharmacology and pharmacy. Over the last few years, the Journal has published articles and reports covering major topics in the different special issues (e.g. research on disease genome, theory of drug withdrawal, mechanism and prevention of cardiovascular and cerebrovascular diseases, stomatology, orthopaedic, public health, urology and reproductive medicine). All the topics involve latest advances in medical sciences, hot topics in specific specialties, and prevention and treatment of major diseases.
The Journal has been indexed and abstracted by PubMed Central (PMC), MEDLINE/PubMed, EBSCO, Embase, Scopus, Chemical Abstracts (CA), Western Pacific Region Index Medicus (WPR), JSTChina, and almost all the Chinese sciences and technical index systems, including Chinese Science and Technology Paper Citation Database (CSTPCD), Chinese Science Citation Database (CSCD), China BioMedical Bibliographic Database (CBM), CMCI, Chinese Biological Abstracts, China National Academic Magazine Data-Base (CNKI), Wanfang Data (ChinaInfo), etc.