RNA immunoprecipitation (RIP) assay
The RIP assay was performed using a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Bersinbio, China) according to the manufacturer’s protocol. 2×107 MG63 or U2OS cells were lysed in complete RIP lysis buffer and the cell lysates were divided into two equal parts and incubated with either 5 μg human anti-Argonaute2 (AGO2) antibody (Millipore, USA) with rotation at 4°C overnight. Magnetic beads were added to the cell lysates and incubation was continued at 4°C for 1h. The samples were then incubated with Proteinase K at 55°C for 1h. The enriched RNA was obtained using RNA Extraction Reagent (Solarbio, CHN). The purified RNA was used to detect the expression levels of the genes of interest by qRT-PCR.