Figure 6

Manipulation of Hox gene expression in periosteal stem/progenitor cells leads to cell fate changes. (A) Graphical schematic of the mammalian HoxA locus and adjacent lncRNA regulators (left) and the corresponding anatomical regions where anterior and posterior HoxA genes are preferentially expressed (right). (B,C) Using siRNA and antisense oligonucleotides (ASO) against the lncRNAs Hotairm1 and Hottip to knockdown 5′ and 3′ Hox clusters. Knockdown of Hoxa2 in hyoid periosteal stem/progenitor cells (B) and Hoxa13 and Hoxa11 in tibial periosteal stem/progenitor cells (C) using siRNA against Hotairm1 and ASOs against Hottip resulted in a change in cell fate with increased osteogenic differentiation (Osx and Collagen type I, hyoid only) and decreased chondrogenic (Sox9 and Collagen type II) and adipogenic differentiation (Ppar-gamma and FabpP4)(n = 3). (C, lower panel) Hox-deficient tibial periosteal cells, via Hottip knockdown, displayed a greater capacity to differentiate into the osteogenic lineage, as measured by alizarin red absorbance (C, lower left panel), and less adipogenic potential, as measures by Oil Red O⁺ cells/well (C, lower right panel) when compared with NT control. Abbreviations: NT, non-targeted control. *p < 0.05, **p < 0.01, ***p < 0.001.