miR-181d regulates RAD51 expression to mediate temozolomide-induced cross-resistance to ionizing radiation in glioblastoma
Description
RAD51 is regulated by miR-181d To identify downstream targets of miR-181d, mRNAs were extracted and profiled after transfection of non-targeting miRNA (miR-NT) or miR-181d mimic, to determine mRNA expression down-regulated by miR-181d. In parallel, we identified mRNAs that exhibited preferential binding to transfected Bi-miR-181d over Bi-miR-NT. The top 10% mRNAs from these two screens (all of which scored higher than MGMT, a bona fide miR-181d target) were cross-referenced, identifying 260 common mRNA candidates (Figure 1A, left panel) that were subsequently subjected to Ingenuity Pathway Analysis (Qiagen). The five top-ranking pathways implicated by the mRNA candidates. Of note, previously identified miR-181d-regulated genes (MGMT, K-Ras, BCL-2, EGR1, and TNF-alpha) play crucial roles in these pathways. The top 10 putative miR-181d targets emerging from our screen. The highest-ranking pathway from this analysis involved genes regulating DNA repair, as evidenced by the presence of RAD51, FANCA, and FANCC. These genes encode proteins that play distinct yet interconnected roles in HR medicated DNA repair. Method To identify miR-181d silenced mRNAs, RNA was extracted 48 h after transfection of a miR-181d mimic (40 nM). To identify mRNA bound to Bi-miR-181d, mRNA was isolated 48 h after transfection of a Bi-miR-181d (40 nM) was affinity purified by streptavidin magnetic beads. miR-181d-silenced or -bound mRNA was profiled using an Affymetrix HG-U133+PM microarray by the Beth Israel Deaconess Medical Center (BIDMC) Bioinformatics and Systems Biology Core.
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Institutions
- University of Minnesota