Background MicroRNA has been also shown to mediate regulation of embryonic stem cell (ESC) division, as well as differentiation in adipocyte, cardiac, neural, and hematopoietic lineages. Compared with these examples examining microRNAs in ESC, relatively few studies have identified microRNA expression patterns in differentiating MSC or specific pathways that could be targeted.
Objectives In this study, we tried microRNA microarray analysis with total RNAs from PTHrP treated BMSCs. Four novel microRNAs were detected by microarray analysis, we conformed that one of them can increase the chondrogenic potential of BMSCs in the lentiviral transduction with the miRNA.
Methods We performed experiments as follows for this study: miRNA microarray, miRNA overexpression in hBMSCs, Chondrogenic induction of lentiviral transduced hBMSCs, 3′UTR reporter assay, Western blotting, Realtime PCR analysis
Results Four novel microRNAs were detected by microarray analysis, we confirmed that one of them can increase the chondrogenic potential of MSCs. In chondrogenic induction of PTHrP-treated hBMSCs, 9 novel miRNAs were up- and down-regulated. 4 miRNAs of them were up-regulated in PTHrP treatment. miR-7, 8 and 9 expression patterns were gradually increased for 21 days. In case of miR-im6, expression pattern was increased by 7 days and then decreased at 21 days in spite of long-term treatment of PTHrP. Each up-regulated miRNA was subcloned into lenti-viral expression vector and successfully overexpressed in hBMSCs by lentiviral infection at MOI=10. Amount of GAG/DNA measured in cultured lentiviral transduced pellets after 4 weeks of culture. GAG/DNA content of lenti-miR-im6 transduced hBMSCs was higher than other groups treated with TGF-β. From safranin-O staining of each chondrogenic hMSC pellet, staining intensity of lenti-miR-im6 transduced hBMSCs was also stronger than TGF-β-treated positive control, similar to PTHrP-treated positive control. Overexpression of miR-im6 in chondrogenic induction of hMSCs enhanced the expressions of chondrogenic markers (Type II collagen, SOX9) and suppressed the expression of hypertrophic markers (ALP, Type X collagen). Desert hedgehog (DHH), Wnt6, and Wnt9b were screened as target genes to miR-im6 in this pathway. PTCH2, WNT6 and WNT9B of these proteins also were down-regulated in lenti-miR-im6- transduced and chondrogenesis-induced hMSCs pellets. Luciferase activities were significantly decreased in Hela cells which were transformed with luciferase reporter vectors harboring PTCH1 or WNT6 3’UTR. This result demonstrates that PTCH1 and WNT6 are target genes of has-miR-im6. We are currently studying about another target gene (KLF10) of miR-im6, can be key regulator in novel PTHrP mediated chondrocyte hypertrophy blocking pathway.
Conclusions Our results show that miR-im6 acts as a positive regulator of chondrogenic differentiation as well as a hypertrophy blocker in BMSCs by decreasing the ALP expression through unknown mechanism. To our knowledge, this is the first report showing that a specific microRNA, miR-im6, plays a crucial role in the course of chondrogenesis.
Disclosure of Interest None Declared
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