Fig. 1.

PIK3C3 was one of the target genes of miR-338-5p in CRC. (a) QPCR was used to evaluate the miR-338-5p expression in CRC cell lines (SW480, SW620 and HCT116). The data are shown as means ± SEM (n = 5). miR-338-5p (50 nM or 100 nM) or N.C. (100 nM) was transiently transfected into HCT116 cells and analysed using QPCR. The anti-miR- 338-5p (50 nM or 100 nM) or anti-N.C. (100 nM) was transiently transfected into SW480 cells and analysed using QPCR. Data are presented as median (IQR) (n = 3) (P values were analysed using Mann-Whitney tests). (b) RNA expression of miR-338-5p target genes (SPRY2, HEMGN, NDFIP1, ID1, ADM, PPP2R5A, DDX5, SCN9A, PIK3C3, and HOXA5) was measured using RT-PCR after transfection of miR-338-5p or anti-miR-338-5p into SW480 cells. (c) The target sequence of PIK3C3 was constructed into 3´-UTR of p-miR-reporter luciferase plasmid downstream of luciferase gene. Wild-type (WT) and mutant-type (mutant) target sequences are shown. The SW480 cells were transfected with WT or mutant p-miR-report-PIK3C3 plasmid (5 μg/ml) and co-transfected with miR-338-5p, anti-miR-338-5p, N.C. or anti-N.C. (100 nM), respectively. Data are presented as means ± SEM (n = 5) (P values were analysed using Mann-Whitney tests). (d) RIP assay was performed to precipitate the Ago2 complexe from stable miR-338-5p overexpression or shGFP control cells. Western blot was used to confirm the quality of RIP. Expression of miR-338-5p and PIK3C3 RNA in Ago2 RIP fractions was measured by QPCR assay. Data are presented as median (IQR) (n = 5) (P values were analysed using Mann-Whitney tests). (e) Expression of PIK3C3 protein in CRC cell lines was evaluated using Western blotting. miR-338-5p or its N.C. was transiently transfected into HCT116 cells, and anti-miR-338-5p or anti-N.C. was transiently transfected into SW480 cells. Expression of PIK3C3 protein was assessed using Western blotting. β-actin was used as an internal control.