Figure 3. The relationship between long noncoding RNA LINC00963 and tumor suppressor miR-542-3p. (A) The quality of LINC00963 in DU 145 cytoplasmic and nuclear fractions. Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and U6 snRNA in purified nuclear fractions were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). (B) RIP experiments were performed in DU 145 cells, and the coprecipitated RNA was subjected to RT-qPCR for LINC00963. The fold enrichment of LINC00963 in argonaute 2 (Ago2) RIP is higher relative to that of its matching immunoglobulin (IgG) control. (C) The luciferase reporter plasmid (RLuc-LINC00963) was co-transfected into 293T cells with the 6 miRNA-coding plasmids. (D, E) The luciferase reporter plasmid containing wild-type (WT) or mutant (Mut) LINC00963 (D) was co-transfected into 293T cells with miR-542-3p or with an empty plasmid vector (E). (F) RNA levels in Ago2 immunoprecipitates are presented as fold enrichment relative to IgG immunoprecipitates. (G) RT-qPCR analysis of miR-542-3p expression in DU 145 and PC3 cells transfected with empty lentivirus vector or SH-LINC00963 Lentivirus. (H, I) Correlation of LINC00963 and miR-542-3p in CRPC tissues from TRAMP (H) and ProbCre/Ptenfl/fl mice (I). Mean ± SEM, *P<0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.