FIGURE 8.

Upregulation of DNMT1, 3A, and 3B dictated promoter CpG sites’ hypermethylation inversely correlated with miR-29c-3p expression in clinical iCCA. (A) Luciferase activity in HuCCT1 cells after cotransfected with the putative promoter of miR-29c or promoter-less vector (Control), respectively. **p < 0.01 using a 2-tailed unpaired Student t test. (B) CpG sites’ methylation pattern of the miR-29c promoter region in 10 pairs of iCCA tissue samples. The dot represents the methylation level of each CpG site in each group. ****p < 0.0001 using a 2-tailed paired Student t test. (C) The relationship between miR-29c-3p and the promoter methylation degree of miR-29c in 10 pairs of iCCA samples was determined by the Pearson correlation test. (D) The expression patterns of DNMT1, 3A, and 3B in 10 pairs of iCCA samples were evaluated by immunoblotting and RT-PCR, respectively. Up represents that DNMTs’ expression level in the tumor is higher than that in the paired nontumor sample in each patient. (E) CpG sites’ methylation degree of miR-29c and the expression levels of miR-29c-3p in HuCCT1 and CCLP1 cells after being treated with 10/20 μmol/mL decitabine for 48 hours were determined by targeted bisulfite sequencing and RT-PCR, respectively. ns, p ≥ 0.05, ***p < 0.001, and ****p < 0.0001 using a 2-tailed unpaired Student t test. (F) Decitabine inhibited the growth of already developed subcutaneous xenograft tumors. 2 mg/kg decitabine was administrated by tail vein or i.p. injection at day 53 to day 58 into NSG mice (n = 5 in each group). Tumor weights in the different groups were analyzed. MLF1 and miR-29c-3p in xenograft tumors from the indicated groups were assessed by immunoblotting or RT-PCR, respectively. *p < 0.05, **p < 0.01, and ****p < 0.0001 using 2-tailed unpaired Student t test. Experiments were triplicated. Abbreviations: P, paired nontumor tissue; RT-PCR, real-time PCR; T, tumor tissue.