Quantitative real-time PCR analysis
The level of miR-195-5p after transfection and mRNA expressions of osteogenic-related genes runt-related transcription factor 2 (Runx2) and osteopontin (OPN) during osteogenic differentiation were evaluated by real-time quantitative reverse transcription PCR (RT-qPCR) as previously described (Li et al. , 2018). The primer sequences used were as follows: Runx2, forward 5’-CAGACACAATCCTCCCCACC-3’, and reverse 5’-GCCAGAGGCAGAAGTCAGAG-3’; OPN, forward 5’-TCAAGGTCATCCCAGTTGCC-3’, and reverse 5’-GACTCATGGCTGGTCTTCCC-3’; β-actin, forward 5’-CTCTGTGTGGATTGGTGGCT-3’, and reverse 5’-CGCAGCTCAGTAACAGTCCG-3’; miRNA-195-5p, forward 5’- CGTTATCCTAGCAGCACAGAAAT-3’, and reverse 5’-TATGGTTTTGACGACTGTGTGAT-3’; and U6, forward 5’-CAGCACATATACTAAAATTGGAACG-3’, and reverse 5’-ACGAATTTGCGTGTCATCC-3’. Each sample was tested in triplicate and the relative gene expressions were calculated using the 2-ΔΔCT method, with β-actin or U6 for normalization.