Fig. 7

Identification of miRNA binding to circPTK2 that regulates the proliferation, migration and invasion in HCC cells. (A) RIP assays validated the combination of circPTK2 with anti-AGO2 in HCC-LM3 and MHCC 97-H cells (Mean ± SD, n = 3, ***p < 0.001). (B) Venn plot showed the miRNAs combination to AKR1C3 predicted by miRDB, Targetscan, and mirDIP. (C) miRNAs combination sites with circPTK2 forecast by miRanda v3.3a. (D) Spearman correlation analyses showing the relationships of miR-379-5p and AKR1C3 (left), and circPTK2 (right) in HCC tissues (n = 72). (E) Biotin-labeled miRNA pull-down assay confirmed the interaction of miR-379-5p with circPTK2 and AKR1C3 (Mean ± SD, n = 3, ***p < 0.001). (F) The co-location of miR-379-5p and circPTK2 in HCC cells judged by FISH. (G) Luciferase reporter assay detected the luciferase activity in HEK-293T cells co-transfected with miRNA-379-5p mimic (or mimic-NC) and circPTK2-wt (or circPTK2-mut) luciferase reporter vectors. Mean ± SD, n = 3, ***p < 0.001. (H) Cell viability of HCC-LM3 and MHCC 97-H cells transfected with sh-circPTK2 alone or co-transfected with miR-379-5p inhibitor, measured by CCK8(Mean ± SD, n = 6, ***p < 0.001). (I) EdU assays estimates the DNA replication capacity of HCC-LM3 and MHCC 97-H cells transfected with sh-circPTK2 alone or co-transfected with miR-379-5p inhibitor. Mean ± SD, n = 3, **p < 0.01, ***p < 0.001, scale bar: 100 μm. (J,K) The cells migration of HCC-LM3 and MHCC 97-H co-transfected with sh-circPTK2 and miR-379-5p inhibitor, assessed by scratch-healing experiments (J) and transwell migration assays (K). Mean ± SD, n = 3, ***p < 0.001, scale bar:100 μm. (L) The invasion ability of HCC-LM3 and MHCC 97-H transfected with sh-circPTK2 and miR-379-5p inhibitor, measured by transwell invasion assays. Mean ± SD, n = 3, ***p < 0.001.