Effect of suppressor of cytokine signaling 3 on osteogenic differentiation of mouse calvarial osteoblast precursors

doi:10.3969/j.issn.1674-8115.2026.07.004

Abstract

Abstract:

Objective ·To construct a stable suppressor of cytokine signaling 3 (Socs3)-knockdown MC3T3-E1 mouse calvarial osteoblast precursor cell line and to investigate the role of SOCS3 in the osteogenic differentiation of calvarial osteoblast precursors. Methods ·The expression levels of SOCS3 in MC3T3-E1 cells after osteogenic induction were detected by real-time quantitative PCR (RT-qPCR) and Western blotting. Two pairs of short hairpin RNAs (shRNAs) specifically targeting the Socs3 gene were designed, and lentiviral vectors for Socs3 knockdown were constructed. MC3T3‑E1 cells were then infected with the packaged lentiviruses, followed by puromycin screening to obtain MC3T3‑E1 cell lines with stable Socs3 knockdown. Knockdown efficiency was verified by RT-qPCR and Western blotting. The effect of SOCS3 on the proliferation capacity of MC3T3-E1 cells was assessed using a CCK-8 assay. After osteogenic differentiation for 7 d, alkaline phosphatase (ALP) staining and RT‑qPCR detection of osteogenesis‑related genes were performed. After 14 d of osteogenic differentiation, alizarin red S (ARS) staining was performed to assess the osteogenic differentiation capacity of the stable knockdown cell lines. Results ·RT-qPCR and Western blotting showed that both Socs3 mRNA and SOCS3 protein levels were elevated at 4 d and 7 d after osteogenic induction compared with those before induction, and the levels at 7 d were higher than those at 4 d (both P<0.001). In MC3T3-E1 stable cell lines infected with shSocs3-1 and shSocs3-2 lentivirus, the expression levels of Socs3 mRNA and SOCS3 protein were significantly inhibited (P<0.001), indicating the successful establishment of stable Socs3-knockdown MC3T3-E1 cell lines. The CCK-8 assay showed that Socs3 knockdown inhibited the proliferation capacity of MC3T3-E1 cells at 1 d, 4 d, and 7 d of culture (P<0.001). ALP staining demonstrated reduced ALP activity in the Socs3-knockdown MC3T3-E1 cell lines after 7 d of osteogenic induction (P<0.001). ARS staining indicated that Socs3 knockdown inhibited mineralized nodule formation in MC3T3-E1 cells (P<0.001). RT-qPCR detection of osteogenic marker genes indicated that the expression levels of runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), osterix (Osx), α1 type Ⅰ collagen (Col1a1), secreted phosphoprotein 1 (Spp1), and bone γ-carboxyglutamate protein (Bglap) were significantly reduced in the Socs3-knockdown group (all P<0.001). Conclusion ·SOCS3 expression increases during the osteogenic differentiation of MC3T3-E1 cells. Knockdown of Socs3 can inhibit both the proliferation and osteogenic differentiation capacities of MC3T3-E1 cells.

Key words: suppressor of cytokine signaling 3 (SOCS3), MC3T3-E1 cell, calvarial osteoblast precursor, osteogenic differentiation

CLC Number:

R329.2

Cui Yiwen, Sun Siyuan, Huang Zihan, Dai Qinggang, Jiang Lingyong. Effect of suppressor of cytokine signaling 3 on osteogenic differentiation of mouse calvarial osteoblast precursors[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026, 46(7): 857-867.

Figures/Tables 7

TrendMD

References 36

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Gene	Direction	Primer sequence (5′→3′)
shcon	Forward	GATCCGTTCTCCGAACGTGTCACGTAATTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTC
	Reverse	AATTGAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGAATTACGTGACACGTTCGGAGAACG
shSocs3-1	Forward	GATCCGCTTCGACTGTGTACTCAAGCTGGTCTCGAGACCAGCTTGAGTACACAGTCGAAGCTTTTTTG
	Reverse	AATTCAAAAAAGCTTCGACTGTGTACTCAAGCTGGTCTCGAGACCAGCTTGAGTACACAGTCGAAGCG
shSocs3-2	Forward	GATCCGCCTGGATCAGTATGATGCTCCACTTCTCGAGAAGTGGAGCATCATACTGATCCAGGTTTTTTG
	Reverse	AATTCAAAAAACCTGGATCAGTATGATGCTCCACTTCTCGAGAAGTGGAGCATCATACTGATCCAGG

Gene	Direction	Primer sequence (5′→3′)
shcon	Forward	GATCCGTTCTCCGAACGTGTCACGTAATTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTC
	Reverse	AATTGAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGAATTACGTGACACGTTCGGAGAACG
shSocs3-1	Forward	GATCCGCTTCGACTGTGTACTCAAGCTGGTCTCGAGACCAGCTTGAGTACACAGTCGAAGCTTTTTTG
	Reverse	AATTCAAAAAAGCTTCGACTGTGTACTCAAGCTGGTCTCGAGACCAGCTTGAGTACACAGTCGAAGCG
shSocs3-2	Forward	GATCCGCCTGGATCAGTATGATGCTCCACTTCTCGAGAAGTGGAGCATCATACTGATCCAGGTTTTTTG
	Reverse	AATTCAAAAAACCTGGATCAGTATGATGCTCCACTTCTCGAGAAGTGGAGCATCATACTGATCCAGG

Gene	Direction	Primer sequence (5'→3')
Hprt	Forward	GTTAAGCAGTACAGCCCCAAA
	Reverse	AGGGCATATCCAACAACAAACTT
Socs3	Forward	ATGGTCACCCACAGCAAGTTT
	Reverse	TCCAGTAGAATCCGCTCTCCT
Runx2	Forward	AACGATCTGAGATTTGTGGGC
	Reverse	CCTGCGTGGGATTTCTTGGTT
Alp	Forward	CCAACTCTTTTGTGCCAGAGA
	Reverse	GGCTACATTGGTGTTGAGCTTTT
Osx	Forward	GGCTTTTCTGCGGCAAGAGGTT
	Reverse	CGCTGATGTTTGCTCAAGTGGTC
Col1a1	Forward	CCTCAGGGTATTGCTGGACAAC
	Reverse	CAGAAGGACCTTGTTTGCCAGG
Spp1	Forward	GCCTGTTTGGCATTGCCTCCTC
	Reverse	CACAGCATTCTGTGGCGCAAGG
Bglap	Forward	GAACAGACAAGTCCCACACAGC
	Reverse	TCAGCAGAGTGAGCAGAAAGAT

Gene	Direction	Primer sequence (5'→3')
Hprt	Forward	GTTAAGCAGTACAGCCCCAAA
	Reverse	AGGGCATATCCAACAACAAACTT
Socs3	Forward	ATGGTCACCCACAGCAAGTTT
	Reverse	TCCAGTAGAATCCGCTCTCCT
Runx2	Forward	AACGATCTGAGATTTGTGGGC
	Reverse	CCTGCGTGGGATTTCTTGGTT
Alp	Forward	CCAACTCTTTTGTGCCAGAGA
	Reverse	GGCTACATTGGTGTTGAGCTTTT
Osx	Forward	GGCTTTTCTGCGGCAAGAGGTT
	Reverse	CGCTGATGTTTGCTCAAGTGGTC
Col1a1	Forward	CCTCAGGGTATTGCTGGACAAC
	Reverse	CAGAAGGACCTTGTTTGCCAGG
Spp1	Forward	GCCTGTTTGGCATTGCCTCCTC
	Reverse	CACAGCATTCTGTGGCGCAAGG
Bglap	Forward	GAACAGACAAGTCCCACACAGC
	Reverse	TCAGCAGAGTGAGCAGAAAGAT