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. 2021 Feb 11;13(4):756. doi: 10.3390/cancers13040756

Figure 4.

Figure 4

MiR-582-5p directly targets the 3’UTRs of NCKAP1 and PIP5K1C. (A) Venn diagram shows an integrated analysis of two gene lists, namely predicted miR-582-5p targets and regulators of actin cytoskeleton, which were derived from TargetScan and Wikipathways, respectively. Nine candidate genes were identified. (B,C) RT-qPCR analysis was performed to examine the changes in relative mRNA levels of the nine candidate genes post-transfection of miR-582-5p mimics in H1299 (B) and H661 (C) cells. The ΔCT of examined genes were normalized to that of 18SrRNA in order to determine the relative expression. (D) Western blot reflects the changes in protein levels of NCKAP1 and PIP5K1C in H1299 and H661 cells upon overexpression of miR-582-5p. (E) Dual luciferase assays were performed to evaluate the predicted binding sites of miR-582-5p to eight sites on NCKAP1 3’ UTR (NCKAP1-1—NCKAP1-8) and to two sites on PIP5K1C 3’UTR (PIP5K1C-1—PIP5K1C-2). Firefly luciferase readouts were normalized to the corresponding Renilla luciferase readouts to evaluate relative luciferase activity. (F–H) The wildtype (WT) and mutant (Mut) miR-582-5p binding sites on the 3’UTRs of NCKAP1 (F,G) and PIP5K1C (H) were constructed into a pmiR-Glo reporter plasmid followed by co-transfection of the aforementioned plasmids and microRNA mimics. Firefly luciferase readout was normalised to the corresponding Renilla luciferase readout. (I,J) Cell proliferation kinetics was quantified following the siRNA-mediated knockdown of NCKAP1 (I) and PIP5K1C (J). (K,L) Cell apoptosis was measured using Caspase-3/7 Glo upon the knockdowns of NCKAP1 (K) and PIP5K1C (L) in H1299 cells. The readouts of Caspase-3/7 Glo were normalized against that of CellTiter Glo. (M,N) RT-qPCR analysis was conducted upon siRNA-mediated knockdowns of NCKAP1 (M) and PIP5K1C (N) to examine the relative mRNA levels of NCKAP1, PIP4K1C, and the YAP/TAZ transcriptional targets, ANKRD1, CTGF, and CYR61 in H1299 cells. (O) A schematic working model illustrates the potential negative feedback loop between miR-582-5p and YAP/TAZ. YAP/TAZ represses pri-miR-582 gene expression but is eventually required for the maintenance of mature miR-582-5p expression. MiR-582-5p directly inhibits the expression of NCKAP1 and PIP5K1C and attenuates YAP/TAZ activity, potentially by compromising F-actin. Results are presented as the mean of three or five replicates as indicated with error bars representing standard deviation. Statistical analysis was conducted using Student’s t-test with * p < 0.05, ** p < 0.01, *** p < 0.001.