| Mesenchymal stem cells(MSCs),a stem cell population discovered from various tissues such as bone marrow,fat,muscle,lung,liver,are multipotent cells able to differentiating into multiple cell types,such as bone,fat and muscle.As common progenitor cells of osteoblasts and adipocytes,MSCs are delicately balanced for their differentiation commitment.Multiple transcription factors have been demonstrated to be critical for the differentiation of MSCs to adipocytes or osteoblasts.Numerous in vitro investigations have validated that bone-induction factors inhibit adipogenesis,and,conversely,fat-induction factors hinder osteogenesis.HOXA10 is a transcriptional regulator belonging to the HOX family transcription factors which is known to be involved in the genetic control of development,proliferation,apoptosis,differentiation,and oncogenesis.Sheep is an important farm animal that provides meat for human consumption,of which the normal development of the spine and other bones and the content of intramuscular fat(IMF)are important factors affecting their carcass weight and meat quality,respectively.However,to date,few studies have been devoted to investigating the role of HOXA10 in sheep.Therefore,the aim of this study was to investigate the role of HOXA10 in sheep MSCs and the potential mechanism underlying its function for adipogenic and osteogenic differentiation.Our study is composed of two sections.In the first section,we respectively isolated sBMSCs and sMDSCs from three-month-old sheep fetal bone marrow and skeletal muscles,and then investigate the effect of HOXA10 overexpression on cell proliferation,apoptosis,osteogenic and adipogenic differentiation in sMDSCs which exhibited lower HOXA10 expression level than sBMSCs.The sBMSCs and sMDSCs were obtained from three-month-old sheep fetal bone marrow and skeletal muscles,respectively.They exhibited a typical fibroblast-like morphology,positively expressed MSCs positive markers CD44,CD90 and CD105 but negatively expressed negative markers CD34,CD45 and Desmin.Compared to the sheep fetal fibroblasts(sFFs),the pluripotency and self-renewal associated factors,including OCT4,NANOG,AKP and TERT,were significantly highly expressed in sBMSCs and sMDSCs,whereas SOX2 expression showed little difference.Under appropriate in vitro conditions,sBMSCs and sMDSCs were able to differentiate into osteocytes and adipocytes.All these results indicated that our isolated sBMSCs and sMDSCs were MSCs with self-renewal,adipogenic and osteogenic differentiation capabilities,and can be used in subsequent experiments.Quantitative RT-PCR was used to quantitate the HOXA10 expression level in sBMSCs and sMDSCs.Because of lower HOXA10 expression,sMDSCs were transfected with HOXA10-overexpressing vector,and then used to study the role of HOXA10 in cell proliferation,apoptosis,osteogenic and adipogenic differentiation.In sMDSCs,HOXA10 overexpression significantly promoted cell proliferation,and upregulated the cell percentage in S phase and G2/M phase.Besides,the protein levels of PCNA,Cyclin A,Cyclin D2,CDK4 were significantly up-regulated whereas p57 were significantly down-regulated,indicating that HOXA10 might promoted cell proliferation and induced cell cycle by impairing the repression effect of p57 on Cyclin D-CDK4.Moreover,HOXA10 overexpression significantly inhibited the apoptosis of sMDSCs,up-regulated the expression of apoptosis inhibitor BCL2 and down-regulated the expression of Cleaved CASPASE3 and pro-apoptotic factor BAX.The dual luciferase activity assay proved that HOXA10 directly up-regulated BCL2 promoter activity,which might be involved in its effect on apoptosis of sMDSCs.Additionally,HOXA10 overexpression activated PI3K/AKT/mTOR/p70S6 K pathway,which might also be involved in its promotion of proliferation and inhibition of apoptosis.Previous studies have demonstrated that HOXA10 controls osteogenesis by directly activating bone regulatory and phenotypic genes,including RUNX2,OCN,and ALPL.Our study validated that HOXA10 overexpression significantly up-regulated RUNX2,OPN,and ALPL mRNA and protein levels,and promoted the osteogenic differentiation of sMDSCs.For adipogenesis,we found that HOXA10 overexpression significantly reduced the lipid accumulation of sMDSCs,up-regulated FABP4 but down-regulated LPL mRNA and protein levels.Using JASPAR database,we predicted that the FABP4 and LPL promoter regions may harbor six and three potential HOXA10 binding sites,respectively.Subsequent dual luciferase activity experiments validated that HOXA10 directly up-regulated FABP4 but down-regulated LPL promoter activity.We reasoned that HOXA10 may suppress adipogenic and lipid accumulation in sMDSCs,at least in part,by directly enhancing FABP4 expression and reducing LPL expression.In the second section of study,to further elucidate the role of HOXA10 on sMDSCs,sheep HOXA10 was respectively knocked out using CRISPR/Cas9 technology and overexpressed using pcDNA3.1(+),and then studied by comparative transcriptome analysis.The sgRNA targeting sites were designed and analyzed with online software tools E-CRISPR and Cas-OFFinder.T7E1 cleavage assay was performed to investigate the targeting efficiency and off-target mutations of the sgRNAs.As a result,a pair of sgRNAs targeting sheep HOXA10 exon 1were selected.Genotypes of monoclonal cell lines were validated by PCR amplification and sequencing.The results showed that the HOXA10 locus in different monoclonal cell lines was mutated on the same position,indicating that dual sgRNAs induced a much higher CRISPR/Cas9 targeting efficiency compared to single sgRNA.Specially,the 74-bp homozygous deletions in monoclonal cell line HOXA10-KO-1 stretched from nucleotides 34 to 107,resulting in a premature stop codon and loss of the homeodomain.On the other hand,stable HOXA10-overexpressing cell lines and their controls were selected by G418 culture and then validated by qRT-PCR and western blotting.Three biological replicates of total RNA were prepared from cell lines HOXA10-KO-1(hereafter HOXA10-KO),WT,HOXA10-OV-4(hereafter HOXA10-OV),and Contr-4(hereafter Contr).Transcriptome sequencing and gene expression profiles were analyzed in two groups:(I)HOXA10-KO vs.WT and(II)HOXA10-OV vs.Contr.Differentially expressed genes(DEGs)of both groups were selected based on ≥ 2.0-fold-change and q < 0.05.Through comparative analysis of the two groups,we identified 172 potential HOXA10 targets,including 42 positive-regulated and 130 negative-regulated genes.The expression levels of the 22 selected genes were consistent between the qRT-PCR and transcriptome analysis,indicating that the transcriptome data are effective and reliable.GO-term and KEGG pathway enrichment analyses were performed to further annotate the functions of the candidate target genes of sheep HOXA10.In biological process terms,cellular process,biological regulation,metabolic process,and developmental process were the most represented categories,indicating a role for HOXA10 in regulating transcription,molecular function,and signaling.Moreover,we filtered out 27 significantly enriched KEGG pathways with p ≤ 0.05.These pathways provide valuable information for investigating the mechanisms by which HOXA10 regulates differentiation,development,and other biological processes.One of HOXA10 potential targets,NLRP3,attracted our attention.Based on the consensus binding sequence of HOXA10,JASPAR database prediction showed that there were three potential HOXA10 binding sites in the NLRP3 promoter.Subsequent dual luciferase activity experiments proved that the promoter activity of NLRP3 was directly down-regulated by HOXA10.NLRP3 is an intracellular sensor that can be activated by various factors and then formed inflammasomes with apoptotic speck protein(ASC)and procaspase-1.In this study,LPS/PA treatment was used to activate NLRP3 inflammasome in sMDSCs.It was confirmed that activation of NLRP3 inflammasome were able to impair the bone-induction and fat-inhibition effect of HOXA10 on sMDSCs,suggesting a novel regulatory mechanism of HOXA10 on osteogenic and adipogenic differentiation. |
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