HOXA10敲低抑制绵羊骨髓间充质干细胞成骨分化并促进其成脂分化

doi:10.12160/j.issn.1672-5190.2026.03.002

摘要/Abstract

摘要： [目的] 探究同源盒A10（homeobox, HOXA10）转录因子对绵羊骨髓间充质干细胞（BMSCs）成骨-成脂分化平衡的调控作用及其分子机制。[方法] 采用qRT-PCR检测HOXA10 mRNA在绵羊BMSCs成骨和成脂诱导过程中的动态表达;构建HOXA10敲低载体,通过茜素红S染色、油红O染色评价HOXA10敲低对成骨、成脂分化表型的影响,通过qRT-PCR和Western blot检测HOXA10敲低对成骨、成脂标志基因表达的影响;结合JASPAR数据库分析、AlphaFold 3结构预测及双荧光素酶报告实验,探究HOXA10对LPL和FABP4启动子的调控机制。[结果] ①HOXA10 mRNA相对表达量在成骨诱导中先升后降,在成脂诱导中自第2天起持续下调。②HOXA10敲低后,HOXA10 mRNA和HOXA10 蛋白表达均极显著降低（P<0.01）。③成骨分化方面,HOXA10敲低使矿化水平极显著降低（P<0.01）,成骨标志基因RUNX2（P<0.01）、ALPL（P<0.05）和OPN（P<0.05）的mRNA及RUNX2、ALPL蛋白表达均极显著下调（均P<0.01）;成脂分化方面,HOXA10敲低使脂质积累极显著升高（P<0.01）,成脂标志基因PPARG（P<0.05）、LPL（P<0.01）和FABP4（P<0.01）的mRNA及LPL、FABP4蛋白表达均极显著上调（P<0.01）。④JASPAR数据库在LPL和FABP4启动子区各预测出3个和6个潜在HOXA10结合位点。AlphaFold 3建模显示HOXA10同源异型结构域可与LPL PB1和FABP4 PB3形成稳定结合界面。双荧光素酶报告实验证实,HOXA10敲低后LPL和FABP4启动子活性均极显著升高（P<0.01）。[结论] HOXA10可能通过抑制LPL和FABP4启动子转录活性调控绵羊BMSCs的成骨-成脂分化平衡;敲低HOXA10可抑制成骨、促进成脂,为间充质干细胞相关疾病机制研究、绵羊生物医学应用及绵羊种质改良提供了理论依据。

关键词: 绵羊, HOXA10基因, 骨髓间充质干细胞, 成骨分化, 成脂分化

Abstract: [Objective] To investigate the regulatory effect of homeobox A10（HOXA10） transcription factor on the balance of osteogenic and adipogenic differentiation of sheep bone marrow mesenchymal stem cells （BMSCs） and its molecular mechanism. [Methods] The dynamic expression of HOXA10 mRNA in sheep BMSCs during osteogenic and adipogenic induction was detected by qRT-PCR. The HOXA10 knockdown vector was constructed, and the effects of HOXA10 knockdown on osteogenic and adipogenic differentiation phenotypes were evaluated by Alizarin Red S staining and Oil Red O staining. The effects of HOXA10 knockdown on the expression of osteogenic and adipogenic marker genes were detected by qRT-PCR and Western blot. Combined with JASPAR database analysis, AlphaFold 3 structure prediction and dual-luciferase reporter assays, the regulatory mechanism of HOXA10 on LPL and FABP4 promoters was explored. [Results] ①The relative expression of HOXA10 mRNA initially increased and then decreased in osteogenic induction, and continued to decrease from the second day in adipogenic induction. ②After HOXA10 knockdown, the expression of HOXA10 mRNA and HOXA10 protein were extremely significantly decreased （P<0.01）. ③In terms of osteogenic differentiation, HOXA10 knockdown extremely significantly reduced the mineralization level （P<0.01）, and the mRNA expression of osteogenic marker genes RUNX2 （P<0.01）, ALPL （P<0.05） and OPN （P<0.05） and the protein expression of RUNX2 and ALPL were extremely significantly down-regulated （P<0.01）. In terms of adipogenic differentiation, HOXA10 knockdown extremely significantly increased lipid accumulation （P<0.01）, and the mRNA expression of adipogenic marker genes PPARG （P<0.05）, LPL （P<0.01） and FABP4 （P<0.01） and the protein expression of LPL and FABP4 were extremely significantly up-regulated （P<0.01）. ④The JASPAR database predicted three and six potential HOXA10 binding sites in the LPL and FABP4 promoter regions, respectively. AlphaFold 3 modeling showed that HOXA10 homeodomain could form stable binding interfaces with LPL PB1 and FABP4 PB3. The dual-luciferase reporter assays confirmed that the LPL and FABP4 promoter activities were extremely significantly increased after HOXA10 knockdown （P<0.01）. [Conclusion] HOXA10 may regulate the balance of osteogenic and adipogenic differentiation of sheep BMSCs by inhibiting the transcriptional activity of LPL and FABP4 promoters. HOXA10 knockdown can inhibit osteogenesis and promote adipogenesis, which provides a theoretical basis for the study of mesenchymal stem cell-related disease mechanism, sheep biomedical application and sheep germplasm improvement.

Key words: sheep, HOXA10 gene, bone marrow mesenchymal stem cells, osteogenic differentiation, adipogenic differentiation

中图分类号:

马敏, 贺丽娜, 张熠聪, 郭田雨. HOXA10敲低抑制绵羊骨髓间充质干细胞成骨分化并促进其成脂分化[J]. 畜牧与饲料科学, 2026, 47(3): 9-22.

MA Min, HE Lina, ZHANG Yicong, GUO Tianyu. HOXA10 Knockdown Inhibits Osteogenic Differentiation and Promotes Adipogenic Differentiation of Sheep Bone Marrow Mesenchymal Stem Cells[J]. Animal Husbandry and Feed Science, 2026, 47(3): 9-22.

参考文献

[1] PEARSON J C,LEMONS D,MCGINNIS W.Modulating Hox gene functions during animal body patterning[J].Nature Reviews Genetics,2005,6(12):893-904.
[2] RIJLI F M,CHAMBON P.Genetic interactions of Hox genes in limb development:learning from compound mutants[J].Current Opinion in Genetics and Development,1997,7(4):481-487.
[3] KRUMLAUF R.Hox genes in vertebrate development[J].Cell,1994,78(2):191-201.
[4] ABATE-SHEN C.Deregulated homeobox gene expression in cancer:cause or consequence?[J].Nature Reviews Cancer,2002,2(10):777-785.
[5] LI C J,CHENG P,LIANG M K,et al.microRNA-188 regulates age-related switch between osteoblast and adipocyte differentiation[J].The Journal of Clinical Investigation,2015,125(4):1509-1522.
[6] LIAO L,YANG X,SU X,et al.Redundant miR-3077-5p and miR-705 mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow[J].Cell Death and Disease,2013,4(4):e600.
[7] MOERMAN E J,TENG K,LIPSCHITZ D A,et al.Aging activates adipogenic and suppresses osteogenic programs in mesenchymal marrow stroma/stem cells:the role of PPAR-gamma2 transcription factor and TGF-beta/BMP signaling pathways[J].Aging Cell,2004,3(6):379-389.
[8] CHEN Q,SHOU P,ZHENG C,et al.Fate decision of mesenchymal stem cells:adipocytes or osteoblasts?[J].Cell Death and Differentiation,2016,23(7):1128-1139.
[9] XIONG Z,SONG Y,WU J F,et al.Bidirectional regulation factor of bone marrow mesenchymal stromal cells differentiation:a focus on bone-fat balance in osteoporosis[J].Stem Cell Research and Therapy,2025,17(1):14.
[10] HASSAN M Q,TARE R,LEE S H,et al.HOXA10 controls osteoblastogenesis by directly activating bone regulatory and phenotypic genes[J].Molecular and Cellular Biology,2007,27(9):3337-3352.
[11] GORDON J A R,HASSAN M Q,SAINI S,et al.Pbx1 represses osteoblastogenesis by blocking Hoxa10-mediated recruitment of chromatin remodeling factors[J].Molecular and Cellular Biology,2010,30(14):3531-3541.
[12] CHEN J R,ZHANG J,LAZARENKO O P,et al.Inhibition of fetal bone development through epigenetic down-regulation of HOXA10 in obese rats fed high-fat diet[J].FASEB Journal,2012,26(3):1131-1141.
[13] HE L X,FENG X,HU C L,et al.HOXA9 gene inhibits proliferation and differentiation and promotes apoptosis of bovine preadipocytes[J].BMC Genomics,2024,25(1):358.
[14] YAN X,ZHU M J,DODSON M V,et al.Developmental programming of fetal skeletal muscle and adipose tissue development[J].Journal of Genomics,2013,1:29-38.
[15] PURBANTORO S D,TAEPHATTHANASAGON T,PURWANINGRUM M,et al.Trends of regenerative tissue engineering for oral and maxillofacial reconstruction in veterinary medicine[J].Frontiers in Veterinary Science,2024,11:1325559.
[16] NARASIMHA R B,SHREYA S,JAYABAL V A,et al.Stem cell therapy for diseases of livestock animals:an in-depth review[J].Veterinary Sciences,2025,12(1):67.
[17] SHU C C,DART A,BELL R,et al.Efficacy of administered mesenchymal stem cells in the initiation and co-ordination of repair processes by resident disc cells in an ovine (Ovis aries) large destabilizing lesion model of experimental disc degeneration[J].Jor Spine,2018,1(4):e1037.
[18] ATHIEL Y,CARIOT L,JOUANNIC J M,et al.Safety and efficacy of human umbilical cord-derived mesenchymal stromal cells in fetal ovine myelomeningocele repair[J].Stem Cell Research and Therapy,2024,15(1):444.
[19] HERNAIZ A,COBETA P,MARÍN B,et al.Susceptibility of ovine bone marrow-derived mesenchymal stem cell spheroids to scrapie prion infection[J].Animals,2023,13(6):1043.
[20] VERONESI F,MARTINI L,GIAVARESI G,et al.Bone regenerative medicine:metatarsus defects in sheep to evaluate new therapeutic strategies for human long bone defect.A systematic review[J].Injury,2020,51(7):1457-1467.
[21] LIU Z Z,WANG W,GAO J F,et al.Isolation,culture,and induced multiple differentiation of Mongolian sheep bone marrow-derived mesenchymal stem cells[J].In Vitro Cellular and Developmental Biology - Animal,2014,50(5):464-474.
[22] CZERNIK M,FIDANZA A,SARDI M,et al.Differentiation potential and GFP labeling of sheep bone marrow-derived mesenchymal stem cells[J].Journal of Cellular Biochemistry,2013,114(1):134-143.
[23] RENTSCH C,HESS R,RENTSCH B,et al.Ovine bone marrow mesenchymal stem cells:isolation and characterization of the cells and their osteogenic differentiation potential on embroidered and surface-modified polycaprolactone-co-lactide scaffolds[J].In Vitro Cellular and Developmental Biology Animal,2010,46(7):624-634.
[24] MUSIC E,FUTREGA K,DORAN M R.Sheep as a model for evaluating mesenchymal stem/stromal cell (MSC)-based chondral defect repair[J].Osteoarthritis and Cartilage,2018,26(6):730-740.
[25] GROMOLAK S,KRAWCZENKO A,ANTOŃCZYK A,et al.Biological characteristics and osteogenic differentiation of ovine bone marrow derived mesenchymal stem cells stimulated with FGF-2 and BMP-2[J].International Journal of Molecular Sciences,2020,21(24):9726.
[26] RAULUSEVICIUTE I,RIUDAVETS-PUIG R,BLANC-MATHIEU R,et al.JASPAR 2024:20th anniversary of the open-access database of transcription factor binding profiles[J].Nucleic Acids Research,2024,52(D1):D174-D182.
[27] ABRAMSON J,ADLER J,DUNGER J,et al.Accurate structure prediction of biomolecular interactions with AlphaFold 3[J].Nature,2024,630(8016):493-500.
[28] DOMINICI M,LE BLANC K,MUELLER I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement[J].Cytotherapy,2006,8(4):315-317.
[29] GREGORY C A,GRADY GUNN W,PEISTER A,et al.An Alizarin red-based assay of mineralization by adherent cells in culture:comparison with cetylpyridinium chloride extraction[J].Analytical Biochemistry,2004,329(1):77-84.
[30] HUANG C Y,LIN W L.High-throughput quantification of osteogenesis in WJ-MSCs using Alizarin Red S,von Kossa,and Picro-Sirius red[J].Future Science OA,2025,11(1):2567169.
[31] RAMÍREZ-ZACARÍAS J L,CASTRO-MUÑOZLEDO F,KURI-HARCUCH W.Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O[J].Histochemistry,1992,97(6):493-497.
[32] WANG H,ECKEL R H.Lipoprotein lipase:from gene to obesity[J].American Journal of Physiology Endocrinology and Metabolism,2009,297(2):E271-E288.
[33] HOTAMISLIGIL G S,BERNLOHR D A.Metabolic functions of FABPs:mechanisms and therapeutic implications[J].Nature Reviews Endocrinology,2015,11(10):592-605.
[34] ROSEN E D,MACDOUGALD O A.Adipocyte differentiation from the inside out[J].Nature Reviews Molecular Cell Biology,2006,7(12):885-896.
[35] ZHANG X Y,LIU C Y,KONG Y Y,et al.Effects of intramuscular fat on meat quality and its regulation mechanism in Tan sheep[J].Frontiers in Nutrition,2022,9:908355.
[36] LAN M X,QIN Q,XIE Y C,et al.Construction of CeRNA networks of lncRNA and miRNA associated with intramuscular fat deposition in Ujumqin sheep[J].Frontiers in Veterinary Science,2025,12:1559727.
[37] AL-MUTHEFFER E A,REINWALD Y,EL HAJ A J.Donor variability of ovine bone marrow derived mesenchymal stem cell - implications for cell therapy[J].International Journal of Veterinary Science and Medicine,2023,11(1):23-37.
[38] BUSTIN S A,BENES V,GARSON J A,et al.The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments[J].Clinical Chemistry,2009,55(4):611-622.
[39] QUIROZ F G,POSADA O M,GALLEGO-PEREZ D,et al.Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage[J].Cytotechnology,2010,62(2):109-120.
[40] JACOBI A,RAUH J,BERNSTEIN P,et al.Comparative analysis of reference gene stability in human mesenchymal stromal cells during osteogenic differentiation[J].Biotechnology Progress,2013,29(4):1034-1042.
[41] ZHANG J,TANG H J,ZHANG Y Q,et al.Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation[J].International Journal of Molecular Medicine,2014,33(5):1209-1218.
[42] RAGNI E,VIGANÒ M,REBULLA P,et al.What is beyond a qRT-PCR study on mesenchymal stem cell differentiation properties:how to choose the most reliable housekeeping genes[J].Journal of Cellular and Molecular Medicine,2013,17(1):168-180.
[43] FERGUSON B S,NAM H,HOPKINS R G,et al.Impact of reference gene selection for target gene normalization on experimental outcome using real-time qRT-PCR in adipocytes[J].PLoS One,2010,5(12):e15208.
[44] VANDESOMPELE J,DE PRETER K,PATTYN F,et al.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J].Genome Biology,2002,3(7):research0034.1-reserch0034.11.
[45] ANDERSEN C L,JENSEN J L,ØRNTOFT T F.Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets[J].Cancer Research,2004,64(15):5245-5250.
[46] SCHULZE F,MALHAN D,EL KHASSAWNA T,et al.A tissue-based approach to selection of reference genes for quantitative real-time PCR in a sheep osteoporosis model[J].BMC Genomics,2017,18(1):975.
[47] FRANKO N,VRŠCAJ L A,ZORE T,et al.TBP,PPIA,YWHAZ and EF1A1 are the most stably expressed genes during osteogenic differentiation[J].International Journal of Molecular Sciences,2022,23(8):4257.
[48] RIVEROLL A L,SKYBA-LEWIN S,LYNN K D,et al.Selection and validation of reference genes for gene expression studies in an equine adipose-derived mesenchymal stem cell differentiation model by proteome analysis and reverse-transcriptase quantitative real-time PCR[J].Genes,2023,14(3):673.