Mutual regulation between microRNA-373 and methyl-CpG-binding domain protein 2 in hilar cholangiocarcinoma

Figure 1 Expression of miR-373 and the association with clinicopathological factors in patients with hilar cholangiocarcinoma. A: Representative expression of miR-373 decreased in tumor and in QBC₉₃₉ detected by RT-PCR (^aP < 0.05 vs control); B: Taqman microRNA assay of miR-373 displayed 2.94-fold down-regulation in tumor group (^bP < 0.01 vs control); C: Relationship between miR-373 expression and overall survival in the patients with hilar cholangiocarcinoma. The median overall survival time was 16.3 mo and 29.7 mo in low- and high- miR-373 group, respectively ^cP < 0.05 vs high-group; D: Kaplan-Meier disease-free survival, the median disease-free survival time were 11.2 mo, 23.4 mo in low- and high- miR-373 group, respectively (^eP < 0.05 vs high-group).

Figure 2 Methylation of miR-373 promoter-associated CpG island in hilar cholangiocarcinoma. A: Top panel, a schematic drawing of the putative CpG island in the 5′-flank region of miR-373 gene; Bottom panel, sequence information of miR-373 gene including partial 5′-flank region. The transcriptional start site (TSS) in boldface uppercase indicated by black arrow, 26 CpG dinucleotides are underlined and italic, pre-miR-373 sequences are capitalized; B: DNA methylation detected by methylation-specific polymerase chain reaction (MSP). Five representative cases were shown to indicate homozygous methylation (cases 1, 3), heterozygous methylation (cases 4, 5) and unmethylated (case 2), respectively. Lanes labeled M and U denote products amplified by primers recognizing methylated and unmethylated sequences; C: Percent of methylated reference (PMR) of miR-373 promoter-associated CpG island in tumor group is significant higher than control (^bP < 0.01 vs control); D: Relationship between CpG island methylation and miR-373 expression in hilar cholangiocarcinoma. A reverse correlation between PMR values and miR-373 expression could be observed (high ΔC_T value indicated low expression) excluding 3 extra samples displayed high PMR values and high mRNA expression(samples 14, 18 and 44).

Figure 3 Methylation of CpG island regulates the expression of miR-373. A: Promoter luciferase reporter gene assay for miR-373. Prior CpG methylated resulted in dramatic decrease of luciferase activity of pGL4-m373-prom compared with pGL4-u373-prom (^bP < 0.01 vs control); B: miR-373 expression was reactivated by epigenetic reagents of 5’-Aza-2-CdR or combination with trichostatin A (TSA). ^dP < 0.01 vs untreated QBC₉₃₉.

Figure 4 Methyl-CpG binding domain proteins expression and enrichment in fragment of promoter-associated CpG island. A: Expression methyl-CpG binding domain proteins (MBPs) in hilar cholangiocarcinoma. Compared to control, 2.9-fold increase of methyl-CpG-binding domain protein (MBD)2 was found while no difference of MBD1 and Mecp2 were detected; B: Chromatin immunoprecipitation (ChIP)-polymerase chain reaction analysis showed selective enrichment of MBD2 at region of CpG island; C: Correlation between MBD2 enrichment and different frequency of CpG island methylation. Remarkable difference was observed between super-/hyper-methylation and stand-/hypo-methylation group (^aP < 0.05, ^bP < 0.01 vs control). GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 5 Enrichment of methyl-CpG-binding domain protein 2 at CpG island region is essential for methylation-mediated silencing of miR-373 gene. A: Enrichment of methyl-CpG-binding domain protein (MBD)2 in fragment of CpG island in HEK-m373-prom stable cells showed 6.2-fold increase compared to control and no significant change was detected in HEK-u373-prom cells; B: MBD2 enrichment in MBD2-siRNA QBC₉₃₉ cells presented dramatically deduction compared to QBC₉₃₉ cells; C: Knock-down of MBD2 induced a increase of miR-373 expression in QBC₉₃₉ cells; D: Epigenetic treatment of QBC₉₃₉ cell with 5-Aza-CdR or combination with trichostatin A (TSA) eliminated the recruitment MBD2 at the region of miR-373 promoter-associated CpG island. ^bP < 0.01 vs background.

Figure 6 miR-373 negatively regulates methyl-CpG-binding domain protein expression through binding to three prime untranslated region. A: miRNA target prediction screened one computative miR-373 binding site at methyl-CpG-binding domain protein (MBD)2-three prime untranslated region (3’UTR); B: 3’UTR luciferase reporter assay showed a reduction of relative luciferase activity of wild-type MBD2 3’UTR by pre-miR-373 in HEK293 cells; C: Exogenous miR-373 down-regulates MBD2 protein in QBC₉₃₉; D: Epigenetic treatment of QBC₉₃₉ cells inhibites MBD2 protein following reactivation of miR-373 (Figure 3A). ^aP < 0.05, ^bP < 0.01 vs background. TSA: Trichostatin A; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 7 Dual regulation between miR-373 and methyl-CpG-binding domain protein 2. miR-373 is one direct transcriptional target and negative regulator to methyl-CpG-binding domain protein (MBD)2 through a feedback loop of CpG methylation. SAM: S-adenosylmethionine; DNMTs: DNA methyltransferases.