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Excessive MiR-365b-5p Maturation via N6-methyladenosine Regulates Chondrocyte Apoptosis and Phenotype by NCL/MiR-365b-5p/FHL1 Axis in Osteoarthritis

ABSTRACT: RNA pull-down assays were performed as the protocol of the PureBinding RNA-Protein pull-down Kit (Geneseed, Guangzhou, China). Specific biotinylated probes that hybridize to target pri-miR-365 were obtained from Ribobio. The probes (200pmol) targeting pri-miR-365 or control probes and RNA samples of chondrocytes were added to the pull-down system. After the mixture of RNA and probes was denaturated at 85°C for 5 minutes, and hybridized at room temperature for 2 hours, 100 µl streptavidin-coated magnetic beads were added to the mixture. Non-specifically bound RNAs were removed by hybridization, capture, and washing. The proteins that interacted with pri-miR-365 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, pri-miR-365-interacting bands were subjected to mass spectrometry (Beijing Genomics Institute, Shenzhen, China). The result was gained to establish a proteomic library.

ORGANISM(S): Homo Sapiens

SUBMITTER: Ming Fu

PROVIDER: PXD038257 | iProX | Mon Nov 21 00:00:00 GMT 2022

REPOSITORIES: iProX

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