Fig. 5

Promoter hypermethylation silenced miR-424-5p in clinical iCCA. A Promoter CpG sites methylation degree of miR-424 in 10 pairs of iCCA tissues samples. **p < 0.01, using two-tailed paired Student's t test. B The association of miR-424-5p with promoter methylation degree of miR-424 in 10 pairs of iCCA samples was assessed by Pearson correlation test. C, D Relationships between DNA methyltransferases (DNMTs) and miR-424 family members in 8 pairs of iCCA samples from TCGA were assessed by Pearson correlation test. Numbers in heatmaps denote Pearson coefficient (C) or statistical significance (D). E COL12A1 in the whole cell lysate of HuCCT1 or CCLP1 cells after treated with the indicated dose of decitabine for 48 h was evaluated by immunoblotting. F CpG sites methylation degree of miR-424 in HuCCT1 or CCLP1 cells after treated with 10/20 μmol/mL decitabine for 48 h was determined by targeted bisulfite sequencing. Decitabine was dissolved with phosphate-buffered saline (Vector). G, H MiR-424-5p and COL12A1 expression in HuCCT1 or CCLP1 cells after treated with 10/20 μmol/mL decitabine for 48 h was determined by RT-PCR, respectively. ****p < 0.0001, using one-way analysis of variance (ANOVA). I Decitabine attenuated the growth of subcutaneous xenograft tumors. NSG mice (n = 5 mice in each group) were treated with 2 mg/kg decitabine (5 times weekly) by tail vein injection. Tumor weights in the two groups were analyzed. COL12A1 in xenograft tumors from the indicated groups was assessed by immunoblotting. *p < 0.05, using two-tailed unpaired Student's t test. Experiments were in triplicates. iCCA intrahepatic cholangiocarcinoma, RT-PCR real-time PCR, TCGA The Cancer Genome Atlas