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Journal of Peking University (Health Sciences) ›› 2024, Vol. 56 ›› Issue (1): 9-16. doi: 10.19723/j.issn.1671-167X.2024.01.003

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Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells

Yuan PAN1,Hang GU1,Han XIAO1,Lijun ZHAO1,Yiman TANG2,*(),Wenshu GE1,*()   

  1. 1. Department of General Dentistry Ⅱ, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomate-rials and Digital Medical Devices, Beijing 100081, China
    2. Fourth Clinical Division, Peking University School and Hospital of Stomatology, Beijing 100025, China
  • Received:2023-10-08 Online:2024-02-18 Published:2024-02-06
  • Contact: Yiman TANG,Wenshu GE E-mail:yimantang@bjmu.edu.cn;wenshuge@bjmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(82071089);National Natural Science Foundation of China(82001013)

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Abstract:

Objective: To explore the effect of ubiquitin-specific protease 42 (USP42) on osteogenic differentiation of human adipose-derived stem cells (hASCs) in vivo and in vitro. Methods: A combination of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy. Alkaline phosphatase (ALP) staining and quantification, alizarin red S (ARS) staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group (knockdown group and overexpression group) and the control group. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of osteogenesis related genes in the experimental group and control group, and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group. Nude mice ectopic implantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo. Results: The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group, and those in overexpression group were significantly higher than those in control group. After 7 days of osteogenic induction, the ALP activity in the knockdown group was significantly higher than that in the control group, and ALP activity in overexpression group was significantly lower than that in control group. After 14 days of osteogenic induction, ARS staining was significantly deeper in the knockdown group than in the control group, and significantly lighter in overexpression group than in the control group. The results of qRT-PCR showed that the mRNA expression levels of ALP, osterix (OSX) and collagen type Ⅰ (COLⅠ) in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction, and those in overexpression group were significantly lower than those in control group. The results of Western blotting showed that the expression levels of runt-related transcription factor 2 (RUNX2), OSX and COLⅠ in the knockout group were significantly higher than those in the control group at 14 days after osteogenic induction, while the expression levels of RUNX2, OSX and COLⅠ in the overexpression group were significantly lower than those in the control group. Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group. Conclusion: Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vivo, and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs, and USP42 can provide a potential therapeutic target for bone tissue engineering.

Key words: Ubiquitin-specific proteases, Human adipose-derived stem cells, Cell differentiation, Bone and bones, Regenerative medicine

CLC Number: 

  • R318

Table 1

Sequences of experimentally relevant qRT-PCR primers"

Gene Forward primer (5′ to 3′) Reverse primer (5′ to 3′)
GAPDH GGAGCGAGATCCCTCCAAAAT GGCTGTTGTCATACTTCTCATGG
USP42 AATCTTCAGACCCATCAGCCT AGAACCTGCATCCATGTCTCC
ALP ATGGGATGGGTGTCTCCACA CCACGAAGGGGAACTTGTC
OSX CCTCTGCGGGACTCAACAAC TAAAGGGGGCTGGATAAGCAT
COLⅠ TGGTCCCAAGGGTAACAGCG AACACCAACAGGGCCAGGCT

Figure 1

Expression of green fluorescent protein in hASCs after 72 h transfection with lentivirus in knockdown group (shUSP42-1, shUSP42-2) and control group (shNC) shNC, short hairpin RNA normal control; shUSP42, short hairpin RNA ubiquitin-specific protease 42; hASCs, human adipose-derived stem cells."

Figure 2

Expression of green fluorescent protein in hASCs after 72 h transfection with lentivirus in overexpression group (WT-USP42) and control group (Vector) WT-USP42, wildtype ubiquitin-specific protease 42; hASCs, human adipose-derived stem cells."

Figure 3

Detection of knockdown efficiency of USP42 by qRT-PCR and Western blotting USP42, ubiquitin-specific protease 42; shNC, short hairpin RNA normal control; shUSP42, short hairpin RNA USP42; GAP, glyceraldehyde-3-phosphate; qRT-PCR, quantitative reverse transcription PCR."

Figure 4

Detection of overexpression efficiency of USP42 by qRT-PCR and Western blotting USP42, ubiquitin-specific protease 42; WT-USP42, wildtype USP42; GAP, glyceraldehyde-3-phosphate; qRT-PCR, quantitative reverse transcription PCR."

Figure 5

ALP staining and quantification of activity at 7 days of osteogenic induction after USP42 knockdown by hASCs shNC, short hairpin RNA normal control; USP42, ubiquitin-specific protease 42; shUSP42, short hairpin RNA USP42; PM, proliferation medium; OM, osteogenic medium; ALP, alkaline phosphatase; hASCs, human adipose-derived stem cells."

Figure 6

ARS staining and quantification at 14 days of osteogenic induction after USP42 knockdown by hASCs shNC, short hairpin RNA normal control; USP42, ubiquitin-specific protease 42; shUSP42, short hairpin RNA USP42; PM, proliferation medium; OM, osteogenic medium; ARS, alizarin red S; hASCs, human adipose-derived stem cells."

Figure 7

mRNA expression levels of ALP, OSX, and COLⅠ at 14 days of osteogenic induction after USP42 knockdown by hASCs PM, proliferation medium; OM, osteogenic medium; shNC, short hairpin RNA normal control; USP42, ubiquitin-specific protease 42; shUSP42, short hairpin RNA USP42; ALP, alkaline phosphatase; OSX, osterix; COLⅠ, collagen type Ⅰ; hASCs, human adipose-derived stem cells."

Figure 8

Protein expression levels of RUNX2, OSX, and COLⅠ at 14 days of osteogenic induction after USP42 knockdown by hASCs shNC, short hairpin RNA normal control; USP42, ubiquitin-specific protease 42; shUSP42, short hairpin RNA USP42; RUNX2, runt-related transcription factor 2; GAP, glyceraldehyde-3-phosphate; COLⅠ, collagen type Ⅰ; OSX, osterix; hASCs, human adipose-derived stem cells."

Figure 9

ALP staining and quantification of activity at 7 d of osteogenic induction after hASCs overexpression of USP42 USP42, ubiquitin-specific protease 42; WT-USP42, wildtype USP42; PM, proliferation medium; OM, osteogenic medium; ALP, alkaline phosphatase; hASCs, human adipose-derived stem cells."

Figure 10

ARS staining and quantification at 14 d of osteogenic induction after hASCs overexpression of USP42 USP42, ubiquitin-specific protease 42; WT-USP42, wildtype USP42; PM, proliferation medium; OM, osteogenic medium; ARS, alizarin red S; hASCs, human adipose-derived stem cells."

Figure 11

mRNA expression levels of ALP, OSX, and COLⅠ at 14 d of osteogenic induction after hASCs overexpression of USP42 USP42, ubiquitin-specific protease 42; WT-USP42, wildtype USP42; PM, proliferation medium; OM, osteogenic medium; ALP, alkaline phosphatase; OSX, osterix; COLⅠ, collagen type Ⅰ; hASCs, human adipose-derived stem cells."

Figure 12

Protein expression levels of RUNX2, OSX, and COLⅠ at 14 d of osteogenic induction after hASCs overexpression of USP42 USP42, ubiquitin-specific protease 42; WT-USP42, wildtype USP42; RUNX2, runt-related transcription factor 2; GAP, glyceraldehyde-3-phosphate; COLⅠ, collagen type Ⅰ; OSX, osterix; hASCs, human adipose-derived stem cells."

Figure 13

Hematoxylin-eosin staining (×200) and bone morphometrics analysis shNC, short hairpin RNA normal control; shUSP42, short hairpin RNA ubiquitin-specific protease 42."

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