microRNA-29a inhibition induces Gab1 upregulation to protect OB-6 human osteoblasts from hydrogen peroxide
Highlights: • Gab1 is required for H{sub 2}O{sub 2}-induced Akt activation in OB-6 osteoblasts. • miR-29a inhibition upregulates Gab1 to protect OB-6 cells from H{sub 2}O{sub 2}. • Gab1 over-expression promotes Akt activation to inhibit H{sub 2}O{sub 2}-induced OB-6 cell death. • Gab1 is the direct and primary target of antagomiR-29a in mediating its anti-H{sub 2}O{sub 2} actions. The present study determines the role of the Gab1 in hydrogen peroxide (H{sub 2}O{sub 2})-induced death of human osteoblasts. We show that Gab1 is required for H{sub 2}O{sub 2}-induced Akt activation to promote osteoblast survival. In OB-6 human osteoblasts, Gab1 silencing (by targeted-shRNA) or complete knockout (by CRISPR-Cas9 KO plasmid) largely attenuated Akt activation by H{sub 2}O{sub 2}. Gab1-depleted OB-6 cells were more vulnerable to H{sub 2}O{sub 2}. Conversely, forced over-expression of Gab1 by an adenovirus vector increased Akt activation to protect OB-6 cells from H{sub 2}O{sub 2}. Significantly, the anti-sense of microRNA-29a (“antagomiR-29a”) induced Gab1 expression to facilitate H{sub 2}O{sub 2}-induced Akt activation, which protected OB-6 cells from apoptosis. AntagomiR-29a was however ineffective in Gab1-deficient and Akt-inhibited OB-6 cells. Forced over-expression of miR-29a induced Gab1 downregulation to inhibit H{sub 2}O{sub 2}-induced Akt activation, causing enhanced OB-6 cell death. miR-29a-induced actions were abolished by an adenovirus constitutively-active Akt1 (Ad-caAkt1) in OB-6 cells. Together, microRNA-29a inhibition induces Gab1 upregulation and Akt activation to protect OB-6 osteoblasts from H{sub 2}O{sub 2}.