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Journal of Peking University (Health Sciences) ›› 2025, Vol. 57 ›› Issue (1): 7-12. doi: 10.19723/j.issn.1671-167X.2025.01.002

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Knockdown of NPTX1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells

Ting SHUAI1, Yanyan GUO1, Chunping LIN2, Xiaomei HOU1,*(), Chanyuan JIN1,*()   

  1. 1. Second Clinical Division, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digi-tal Medical Devices & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
    2. Department of Stomatology, Shengli Clinical Medical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou University Affiliated Provincial Hospital, Fuzhou 350001, China
  • Received:2024-09-12 Online:2025-02-18 Published:2025-01-25
  • Contact: Xiaomei HOU, Chanyuan JIN E-mail:houxiaomei1108@163.com;jinchanyuanjcy@bjmu.edu.cn
  • Supported by:
    the Young Elite Scientists Sponsorship Program by China Association for Science and Technology(YESS20220554);Beijing Young Elite Scientists Sponsorship Program by Beijing Association for Science and Technology(BYESS2024297)

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Abstract:

Objective: To initially investigate the function of neuronal pentraxin 1 (NPTX1) gene on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: hBMSCs were induced to undergo osteogenic differentiation, and then RNA was collected at different time points, namely 0, 3, 7, 10 and 14 d. The mRNA expression levels of key genes related with osteogenic differentiation, including runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and NPTX1, were detected on the basis of quantitative real-time polymerase chain reaction (qPCR) technology. In order to establish a stable NPTX1-knockdown hBMSCs cell line, NPTX1 shRNA lentivirus was constructed and used to infect hBMSCs. ALP staining, alizarin red (AR) staining, and qPCR were employed to assess the impact of NPTX1-knockdown on the osteogenic differentiation ability of hBMSCs. Results: The results showed that during the osteogenic differentiation of hBMSCs in vitro, the mRNA expression levels of osteogenic genes RUNX2, ALP and OCN significantly increased compared with 0 d, while NPTX1 expression decreased markedly (P < 0.01) as the osteogenic induction period exten-ded. At 72 h post-infection with lentivirus, the result of qPCR indicated that the knockdown efficiency of NPTX1 was over 60%. After knocking down NPTX1 in hBMSCs, RNA was extracted from both the NPTX1-knockdown group (sh NPTX1 group) and the control group (shNC group) cultured in regular proliferation medium. The results of qPCR showed that the expression levels of osteogenic-related genes RUNX2 and osterix (OSX) were significantly higher in the sh NPTX1 group compared with the shNC group (P < 0.01). ALP staining revealed a significantly deeper coloration in the sh NPTX1 group than in the shNC group at the end of 7 d of osteogenic induction. AR staining demonstrated a marked increase in mineralized nodules in the sh NPTX1 group compared with the shNC group at the end of 14 d of osteogenic induction. Conclusion: NPTX1 exerts a modulatory role in the osteogenic differentiation of hBMSCs, and its knockdown has been found to enhance the osteogenic differentiation of hBMSCs. This finding implies that NPTX1 could potentially serve as a therapeutic target for the treatment of osteogenic abnormalities, including osteoporosis.

Key words: Bone marrow mesenchymal stem cells, NPTX1 gene, Osteogenic differentiation

CLC Number: 

  • R34

Table 1

Primer sequences used for qPCR"

Gene nameForward primer sequences (5' to 3')Reverse primer sequences (5' to 3')
NPTX1CAGCGAGCTCGAGAAAGGTCATCCGCAGTGGGAATGTGAGC
GAPDHCGGACCAATACGACCAAATCCGAGCCACATCGCTCAGACACC
RUNX2TCTTAGAACAAATTCTGCCCTTTTGCTTTGGTCTTGAAATCACA
ALPGACCTCCTCGGAAGACACTCTGAAGGGCTTCTTGTCTGTG
OCNAGCAAAGGTGCAGCCTTTGTGCGCCTGGGTCTCTTCACT
OSXCCTCCTCAGCTCACCTTCTCGTTGGGAGCCCAAATAGAAA

Table 2

shRNA sequences of lentiviruses"

shRNA nameshRNA sequences (5' to 3')
shNCTTCTCCGAACGTGTCACGT
shNPTX1GGATCTGCTGCAGAGCAAGAT

Figure 1

Expression level of NPTX1 and osteogenesis-related genes after osteogenic induction in hBMSCs A, the mRNA expression level of NPTX1 after 0, 3, 7, 14 d of osteoge-nic induction in hBMSCs; B, the mRNA expression level of RUNX2 after 0, 3, 7, 14 d of osteogenic induction in hBMSCs; C, the mRNA expression level of ALP after 0, 3, 7, 14 d of osteogenic induction in hBMSCs; D, the mRNA expression level of OCN after 0, 3, 7, 14 d of osteogenic induction in hBMSCs. ** P < 0.01, vs. 0 d. hBMSCs, human bone marrow mesenchymal stem cells. The other abbreviation as in Table 1."

Figure 2

Transfection efficiency of NPTX1 was examined by qPCR ** P < 0.01, vs. shNC group. NPTX1, neuronal pentraxin 1; NC, negative control; qPCR, quantitative real-time polymerase chain reaction."

Figure 3

Results after osteogenic induction in NPTX1-knockdown hBMSCs A, mRNA expression level of RUNX2 under PM conditions in NPTX1-knockdown hBMSCs and shNC hBMSCs; B, mRNA expression level of OSX under PM conditions in NPTX1-knockdown hBMSCs and shNC hBMSCs; C, ALP staining results after osteogenic induction in NPTX1-knockdown hBMSCs. ** P < 0.01, vs. shNC group. PM, proliferation medium; OM, osteogenic medium; hBMSCs, human bone marrow mesenchymal stem cells; NC, negative control. The other abbreviation as in Table 1."

Figure 4

Results of AR staining after osteogenic induction in NPTX1-knockdown hBMSCs A, results of AR staining after osteogenic induction in NPTX1-knockdown hBMSCs; B, quantitative results of AR staining after osteogenic induction in NPTX1-knockdown hBMSCs. ** P < 0.01, vs. shNC group. PM, proliferation medium; OM, adipogenic medium; AR, alizarin red; NPTX1, neuronal pentraxin 1; NC, negative control."

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