Fig 1: Circ_HIPK3 regulated the expression of ULK1, a target of miR-148a-3p, by targeting miR-148a-3p. (A) The binding sites between ULK1 3'UTR and miR-148a-3p were analyzed by starbase. (B and C) The relationship between ULK1 and miR-148a-3p was validated by dual-luciferase reporter assay (n=3, ANOVA). (D) The expression of miR-148a-3p in RBE and HCCC-9810 cells after miR-148a-3p or anti-miR-148a-3p transfection was measured by qPCR (n=3, ANOVA). (E and F) The expression of ULK1 in RBE and HCCC-9810 cells after miR-148a-3p or anti-miR-148a-3p transfection was measured by qPCR and Western blot (n=3, ANOVA). (G and H) The expression of ULK1 in CCA tumor tissues (n=42) and normal tissues (n=42) was measured by qPCR and Western blot (Student’s t-test). (I and J) The correlation between ULK1 expression and miR-148a-3p expression or circ_HIPK3 expression in CCA tissues was analyzed by Pearson correlation coefficient. (K and L) The expression of ULK1 in HIBEpiC, RBE and HCCC-9810 cells was measured by qPCR and Western blot (n=3, ANOVA). (M and N) The expression of ULK1 in RBE and HCCC-9810 cells transfected with si-circ_HIPK3, si-NC, si-circ_HIPK3+anti-miR-148a-3p or si-circ_HIPK3+anti-miR-NC was measured by qPCR and Western blot (n=3, ANOVA). *P<0.05.
Fig 2: MiR-148a-3p inhibited RBE and HCCC-9810 cell proliferation, migration and invasion by targeting ULK1. (A and B) The expression of ULK1 in RBE and HCCC-9810 cells transfected with miR-148a-3p, miR-NC, miR-148a-3p+ULK1 or miR-148a-3p+pcDNA was measured by qPCR and Western blot (n=3, ANOVA). (C and D) Cell proliferation in these transfected cells was detected by MTT assay (n=3, ANOVA). (E and F) Cell cycle in these transfected cells was monitored using flow cytometry assay (n=3, ANOVA). (G) Cell proliferation in these transfected cells was also assessed by colony formation assay (n=3, ANOVA). (H) Cell apoptosis in these transfected cells was monitored by flow cytometry assay (n=3, ANOVA). (I and J) Cell migration and cell invasion in these transfected cells were monitored using transwell assay (n=3, ANOVA). (K and L) The protein levels of Snail and E-cadherin in these transfected cells were quantified by Western blot (n=3, ANOVA). *P<0.05.
Fig 3: Circ_HIPK3 knockdown inhibited tumor growth in vivo. (A) HCCC-9810 cells harboring sh-circ_HIPK3 or sh-NC were injected into nude mice, and tumor volume was measured every 5 days from 10th day post-injection (ANOVA). (B) Tumor weight was measured after 30 days (Student’s t-test). (C) The expression of circ_HIPK3 in the excised tissues was detected by qPCR (Student’s t-test). (D) The expression of miR-148a-3p in the excised tissues was detected by qPCR (Student’s t-test). (E and F) The expression of ULK1 in the excised tissues was detected by qPCR and Western blot (Student’s t-test). *P<0.05.
Supplier Page from Abcam for Anti-ULK1 (phospho S467) antibody [EPR6153(2)-118]