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. 2023 Nov 15;34:102079. doi: 10.1016/j.omtn.2023.102079

Figure 6.

Figure 6

CM-miR-143 inhibited CRC cell proliferation by downregulating MARCKS in vitro

(A) Schematic of putative miR-143 seed sequence binding at the MARCKS 3ʹ UTR region (WT) and MUT sequence of the putative binding site. Red letters are binding sites and mutation sequences used for luciferase assays in MUT. (B) Am-miR-143 significantly inhibited luciferase activity in WT. Activities were completely recovered to the control levels in MUT. Data are presented as mean ± SEM (∗p < 0.05; n = 6). ns, not significant. (C) Cell viability at 72 h after transfection of each miRNA (10 nM) with lipofectamine RNAiMAX in the DLD-1 or HT-29 cells. CM-miR-143 significantly suppressed cell viability in both cell lines compared with control miRNA or Am-miR-143. Data are presented as mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 3). (D) Western blotting to evaluate p-MARCKS, MARCKS, p-AKT, and AKT protein expression at 72 h posttransfection in DLD-1 or HT-29 cells. All of the proteins were significantly downregulated in the CM-miR-143 group. β-Actin was used as an internal control. (E) Cell viability at 72 h after siRNA-MARCKS transfection (5 nM) in DLD-1 or HT-29 cells. Compared with control siRNA, siRNA-MARCKS 1 and 2 significantly suppressed cell growth in both cell lines. Data are presented as mean ± SEM (∗∗∗p < 0.001; n = 3). (F) Western blotting to evaluate p-MARCKS, MARCKS, p-AKT, and AKT protein expression at 72 h after transfection in DLD-1 or HT-29 cells. p-MARCKS and MARCKS were downregulated after transfection of siRNA-MARCKS 1 or 2, whereas p-AKT and AKT were not downregulated. β-Actin was used as an internal control. si-MARCKS1, si1; si-MARCKS2, si2.