Figure 9.

miR-338-3p directly targeted SOX4 mRNA in TPC-1 and IHH-4 cell lines

(A) The complementary pairing between wild-type miR-338-3p and 3′ UTR of SOX4 is illustrated based on a TargetScan Human 7.2 algorithm. The mutated type of the SOX4 3′ UTR represents a mismatched pairing with wild-type miR-338-3p in three positions. (B) Relative luciferase activity was detected by a luciferase reporter assay in TPC-1 and IHH-4 cells. ∗∗p < 0.01, compared with miR-NC group, in which group the cells were transfected with only the transfection reagent. (C) The transfection efficiency of miR-338-3p mimics was determined by RT-PCR in TPC-1 and IHH-4 cells. U6 was used as the reference. (D) The expression of SOX4 mRNA was determined by qRT-PCR in TPC-1 and IHH-4 cells transfected with miRNA mimic, SOX4 overexpression plasmids, or co-transfected with both. GAPDH was used the reference gene. ∗∗p < 0.01, versus control; ^##p < 0.01, compared with the mimic group. (E) The relative expression of SOX4 protein was detected by western blotting in TPC-1 and IHH-4 cell lines transfected with miRNA mimic, SOX4 overexpression plasmids, or co-transfected with both. GAPDH was used as the reference. ∗∗p < 0.01, versus control; ^##p < 0.01, compared with the mimic group. (F) The expression of SOX4 protein in both cell lines transfected with circ_0001018 shRNA, miR-338-3p inhibitor, or both was detected using the immunoblotting method. ∗∗p < 0.01, versus control; ^##p < 0.01, versus the sh-circRNA group. n = 3, repetition = 3. Data indicate mean ± SD. Statistical analysis was done using one-way ANOVA except for the luciferase assay, in which the Student’s t test was used. OE, overexpression; mimic, miR-338-3p mimic; sh-circRNA, circ_0001018 shRNA; inhibitor, miR-338-3p inhibitor.