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MicroRNA-202 maintains spermatogonial stem cells by inhibiting cell cycle regulators and RNA binding proteins

ABSTRACT: As miRNAs regulate gene expression either by inhibiting translation or by initiating mRNA degradation, we performed RNA sequencing to identify genes regulated by miR-202, using a Cas9/double gRNAs mediated and doxycycline induced knockout approach.

ORGANISM(S): Mus musculus

PROVIDER: GSE99160 | GEO | 2017/05/23

SECONDARY ACCESSION(S): PRJNA387452

REPOSITORIES: GEO

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Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP). Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4M-BM-0C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 M-NM-<l of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4M-BM-0C. Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells. Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.

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MicroRNA-202 maintains spermatogonial stem cells by inhibiting cell cycle regulators and RNA binding proteins

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