Research Paper Volume 12, Issue 14 pp 14418—14433

Figure 4. SPINK1 enhances Enz resistance and metastasis via miR-5089-5p inhibition. (A) Venn diagram (left panel) of SPINK1-targeting miRNAs predicted by miRmap and Targetscan. Putative miR-5089-5p binding sites on the 3′-UTR of SPINK1 (right panel). (B) qRT-PCR results showing differential expression of miR-5089-5p in C4-2R/C4-2B-R compared to C4-2/C4-2B cells. (C) Luciferase reporter construct with the wild-type or mutated SPINK1 3’UTR downstream of the firefly luciferase reporter gene (left panel). Luciferase reporter activity in HEK293T cells co-transfected with the respective constructs along with miR-5089-5p mimics (right panel). (D) SPINK1 mRNA levels in cells treated with the miR-5089-5p inhibitor and miR-5089-5p mimic. (E, F) The migration abilities of +/ shSPINK1 and +/ miR325p inhibitor C4-2R cells (upper panels), and +/ oeSPINK1 and +/ miR-5089-5p mimic C4-2 cells (lower panels) in the wound healing assay. Percentage of migrating cells are on the right panels. (G, H) The invasiveness of +/ shSPINK1 and +/ miR325p inhibitor C4-2R cells (upper panels), and +/ oeSPINK1 and +/ miR-5089-5p mimic C4-2 cells (lower panels) in transwell assay. Percentage of invading cells are on the right panels. (I) ARv7 protein levels in +/oeSPINK1 and +/miR-5089-5p mimic C4-2 cells (upper panels), and +/ shSPINK1 and +/ miR325p inhibitor C4-2R cells (lower panels). Relative protein levels are shown on the right. (J) ARv7 mRNA levels in +/oeSPINK1 and +/miR-5089-5p mimic C4-2 cells (upper panels), and +/ shSPINK1 and +/ miR325p inhibitor C4-2R cells (lower panels).