Extraction and Analysis of mRNA and miRNA from Cells and Cartilage

In this study, mRNA was isolated from cultured cells and knee articular cartilage tissues. The cultured cells were rinsed with PBS and lysed in RNA-Solv^® Reagent (Omega Bio-tek, Norcross, GA, USA). The knee cartilage samples were placed in paired RNase-Free 1.5 EP tubes with four ground beads (5 mm in diameter) and frozen with liquid nitrogen. Subsequently, the tissues were pulverized and homogenized using Tissuelyser-24 (Jingxin, Shanghai, China). The TissueLyser was operated twice for 30 s at 45 Hz. The above tissue powder (50–100 mg) was lysed in Omega RNA-Solv^® Reagent and RNA was isolated using the E.Z.N.A.^® Total RNA Kit I (Omega Bio-tek) according to manufacturer’s protocol. MiRNA levels were extracted using a miRNA Isolation Kit (Ambion). RNA was stored at − 80 °C. Reverse transcription was performed using 1.0 µg total RNA and then used to prepare cDNA using miRNA and HiFiScript cDNA kits (CWBIO, Beijing, China), which were used to investigate the expression of miRNA and mRNA, respectively. All qPCRs were performed in a 20 µL volume using appropriate primers (1 µL; Sangon Biotech, Shanghai, China), cDNA (1 µL), and a ROX-containing UltraSYBR Mixture (CWBIO) with an ABI 7500 Sequencing Detection instrument (Applied Biosystems, CA, USA). The thermocycler settings were as follows: 40 cycles of 95 °C for 5 s and 60 °C for 24 s. U6 was used as an internal control for microRNA, whereas β-actin served as the control for messenger RNA. The cycle threshold (Ct) values were collected and normalized to the level of U6 or β-actin, with three samples per group. The relative mRNA level of each target gene was calculated by using the 2^−ΔΔCt method. Primer sequences are shown in Table 1.Table 1

Primer sequences for qPCR

Gene	Primers
MiR-760	Forward: UUCUCCGAACGUGUCACGUTT Reverse: ACGUGACACGUUCGGAGAATT
MMP3	Forward: AGTCTTCCAATCCTACTGTTGCT Reverse: TCCCCGTCACCTCCAATCC
MMP13	Forward: ACTGAGAGGCTCCGAGAAATG Reverse: GAACCCCGCATCTTGGCTT
ADAMTS4	Forward: GAGGAGGAGATCGTGTTTCCA Reverse: CCAGCTCTAGTAGCAGCGTC
COL2A1	Forward: TGGACGATCAGGCGAAACC Reverse: GCTGCGGATGCTCTCAATCT
Aggrecan	Forward: ACTCTGGGTTTTCGTGACTCT Reverse: ACACTCAGCGAGTTGTCATGG
HBEGF	Forward: ATCGTGGGGCTTCTCATGTTT Reverse: TTAGTCATGCCCAACTTCACTTT
CBL	Forward: TGGTGCGGTTGTGTCAGAAC Reverse: GGTAGGTATCTGGTAGCAGGTC
CAMK2G	Forward: ACCCGTTTCACCGACGACTA Reverse: CTCCTGCGTGGAGGTTTTCTT
MAP2K1	Forward: CAATGGCGGTGTGGTGTTC Reverse: GATTGCGGGTTTGATCTCCAG
ADCY1	Forward: AGGCACGACAATGTGAGCATC Reverse: TTCATCGAACTTGCCGAAGAG
RPS6KA3	Forward: CGCTGAGAATGGACAGCAAAT Reverse: TCCAAATGATCCCTGCCCTAAT
U6	Forward: CTCGCTTCGGCAGCACA Reverse: AACGCTTCACGAATTTGCGT
β-actin	Forward: AGATGTGGATCAGCAAGCAG Reverse: GCGCAAGTTAGGTTTTGTCA