Abstract
Osteoporotic patients often suffer from bone fracture but its healing is compromised due to impaired osteogenesis potential of bone marrow-derived mesenchymal stem cells (BMSCs). Here we aimed to exploit adipose-derived stem cells from ovariectomized rats (OVX-ASCs) for bone healing. We unraveled that OVX-ASCs highly expressed miR-214 and identified 2 miR-214 targets: CTNNB1 (β-catenin) and TAB2. We demonstrated that miR-214 targeting of these two genes blocked the Wnt pathway, led to preferable adipogenesis and hindered osteogenesis. As a result, OVX-ASCs implantation into OVX rats failed to heal critical-size metaphyseal bone defects. We further engineered the OVX-ASCs with a novel Cre/loxP-based hybrid baculovirus vector that conferred prolonged expression of miR-214 sponge. Gene delivery for miR-214 sponge expression successfully downregulated miR-214 levels, activated the Wnt pathway, upregulated osteogenic factors β-catenin/Runx2, downregulated adipogenic factors PPAR-γ and C/EBP-α, shifted the differentiation propensity towards osteogenic lineage, enhanced the osteogenesis of co-cultured OVX-BMSCs, elevated BMP7/osteoprotegerin secretion and hindered exosomal miR-214/osteopontin release. Consequently, implanting the miR-214 sponge-expressing OVX-ASCs tremendously improved bone healing in OVX rats. Co-expression of miR-214 sponge and BMP2 further synergized the OVX-ASCs-mediated bone regeneration in OVX rats. This study implicates the potential of suppressing miR-214 by baculovirus-mediated gene delivery in osteoporotic ASCs for regenerative medicine.
Highlights
MicroRNA are small RNAs that regulate cellular events by binding to the 3′-untranslated region (UTR) of target mRNAs10 and a number of miRNAs have been correlated with bone marrow-derived mesenchymal stem cells (BMSCs) differentiation and osteoporosis
We have developed Cre/loxP-based hybrid baculovirus (BV) vector system comprising two BV: one expressing Cre recombinase and the other substrate BV harboring the transgene cassette flanked by loxP sites[18]
Using ASCs isolated from OVX rats (OVX-ASCs), here we investigated the miR-214 expression level, adipogenesis/osteogenesis preference, molecular pathway and how miR-214 affects the differentiation of surrounding OVX-BMSCs in vitro and bone healing in OVX rats
Summary
MicroRNA (miRNA) are small RNAs that regulate cellular events by binding to the 3′-untranslated region (UTR) of target mRNAs10 and a number of miRNAs have been correlated with BMSCs differentiation and osteoporosis. ASCs are inferior to BMSCs in osteogenic differentiation capability, often resulting in delayed or incomplete repair of large bone defects[16,17] To overcome this problem, we have developed Cre/loxP-based hybrid baculovirus (BV) vector system comprising two BV: one expressing Cre recombinase and the other substrate BV harboring the transgene cassette flanked by loxP sites[18]. After co-transduction of ASCs with the two BV, the expressed Cre recognizes the loxP sequences, excises the transgene cassette off the substrate BV genome, thereby leading to the formation of episomal DNA minicircle encompassing the transgene within the cells Such hybrid BV system enables sustained transgene expression[19] and improves ASCs differentiation and bone healing in vivo[12]. We further exploited the Cre/loxP-based BV persistently expressing miR-214 sponge to engineer OVX-ASCs, so as to knockdown intracellular and exosomal miR-214 levels, reverse the differentiation preference, substantiate osteogenesis and ameliorate bone healing in OVX rats
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