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. Novel microRNA-im6 were detected by microarray analysis, we conformed that miR-im6 can increase the chondrogenic potential and inhibit the hypertrophy of BMSC through down-regulation of transcription factor SPB-X.
Methods Design of miRNA microarray experiment using human bone marrow mesenchymal stem cells, chondrogenesis of PTHrP-treated BMSCs, lenti-viral transduction, analysis of hedgehog signalling in BMSCs overexpressing miR-im6, analysis of hedgehog signalling in MSCs isolated from SPB-X knock-out mouse.
Results Overexpression of miR-im6 enhances the chondrogenic potential and inhibits hypertrophy of hBMSCs: GAG/DNA content of lenti-miR-im6 transduced hBMSCs was higher than other groups in treatment of TGF-β. In safranin-O staining of each chondrogenic hBMSC pellet, staining intensity of lenti-miR-im6 transduced hBMSCs was also stronger than TGF-β treated positive control, like PTHrP-treated positive control. Western blotting results showed overexpression of miR-im6 in chondrogenic induction of hBMSCs enhances the expressions of chondrogenic markers and suppresses the expression of hypertrophic markers.
MiR-im6 inhibits major molecules of hedgehog signal and wnt signal pathway: We confirmed mRNA and protein levels using Rq-PCR and Western blotting when miR-im6 was overexpressed in hBMSCs chondrogenic pellets after 4 weeks. Rq-PCR results show that Ihh and ptch1 initiating hedgehog signal pathway were down regulated with SPB-X, simultaneously. Their protein levels were decreased in miR-im6 transduced hBMSCs when compared with control. We also confirmed that the down-regulation of hedgehog signals by miR-im6 can suppress expression of wnt signal molecules (Wnt3a, Wnt7a, Wnt6, Wnt9b and β-catenin). On the contrary, indian hedgehog was transiently up-regulated by overexpression of SPB-X in hBMSCs.
Transcription factor SPB-X and Wnt6 are target genes of hsa-miR-im6: Luciferase activities were significantly decreased in Hela cells was co-transfected with luciferase reporter vectors harboring SPB-X or WNT6 3'UTR and hsa-miR-im6 mimic. This result demonstrates that SPB-X and WNT6 are target genes of hsa-miR-im6.
SPB-X knockout induce down-regulation of indian hedgehog: We investigated SPB K/O effects using mouse MSCs isolated from SPB K/O mouse. GAG/DNA ratio and safranin-O staining results show that chondrogenic potentials of SPB K/O MSC is similar to that of wild type MSC. However, we confirmed that expressions of IHH, PTCH1, WNT6 and WNT9b were suppressed in chondrogenic pellet of SPB K/O MSC when compared with that of wild type MSC.
Conclusions Our results show that miR-im6 acts as a positive regulator of chondrogenic differentiation as well as a hypertrophy blocker in BMSCs by direct decreasing the transcription factor SBP-X and WNT expression in PTHrP-IHH negative feedback inhibition pathway.
Acknowledgements This study was supported by a grant from the National Research Foundation of Korea (2009-0092196 and 2012R1A1A2040926).
Disclosure of Interest None declared