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. 2021 Feb 17;13(4):6055-6065.
doi: 10.18632/aging.202549. Epub 2021 Feb 17.

miR-29b restrains cholangiocarcinoma progression by relieving DNMT3B-mediated repression of CDKN2B expression

Kun Cao  1 , Bo Li  1 , Ye-Wei Zhang  1 , Hui Song  2 , Yi-Gang Chen  1 , Yong-Jun Gong  1 , Hai-Yang Li  1 , Shi Zuo  1
Affiliations

miR-29b restrains cholangiocarcinoma progression by relieving DNMT3B-mediated repression of CDKN2B expression

Kun Cao et al. Aging (Albany NY). .

Abstract

Numerous studies have reported the important role of microRNAs (miRNAs) in human cancers. Although abnormal miR-29b expression has been linked to tumorigenesis in several cancers, its role in cholangiocarcinoma remains largely unknown. We found that miR-29b expression is frequently downregulated in human cholangiocarcinoma QBC939 cells and in clinical tumor samples. In cholangiocarcinoma patients, low miR-29b expression predicts poor overall survival. Overexpression of miR-29b in QBC939 cells inhibited proliferation, induced G1 phase cycle arrest, and promoted apoptosis. Methylation-specific PCR (MSP) analysis revealed a decreased methylation imprint at the promoter of the cell cycle inhibitor gene CDKN2B in cells overexpressing miR-29b. After identifying the DNA methyltransferase DNMT3B as a putative miR-29b target, luciferase reporter assays confirmed a suppressive effect of miR-29b on DNMT3B expression. Accordingly, we detected an inverse correlation between miR-29b and DNMT3B expression in clinical cholangiocarcinoma specimens. In QBC939 cells, DNMT3B overexpression promoted proliferation and inhibited apoptosis. DNMT3B silencing, in turn, led to increased CDKN2B expression. We also observed significant growth arrest in subcutaneous tumors formed in nude mice by QBC939 cells overexpressing miR-29b. These findings suggest miR-29b functions as a tumor suppressor in cholangiocarcinoma by relieving DNMT3B-mediated repression of CDKN2B expression.

Keywords: CDKN2B; DNMT3B; cholangiocarcinoma; methylation; miR-29b.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
miR-29b downregulation is associated with poor overall survival in cholangiocarcinoma. (A) Relative miR-29b expression in 30 patients with cholangiocarcinoma after normalization to adjacent, normal tissue expression (B) miR-29b expression data for 30 cholangiocarcinoma and 20 non-tumor specimens. (C) Kaplan-Meier curves for cholangiocarcinoma patients with low and high expression of miR-29b. (D) Relative expression of miR-29b in the human cholangiocarcinoma cell line QBC939 and in human intrahepatic biliary epithelial cells (HIBEC). The values presented are means ± SD. *P<0.05 and **P<0.01 compared to the normal tissues or HIBEC cells, as determined by analysis of Student’s t-test.
Figure 2
Figure 2
Overexpression of miR-29b suppresses proliferation and induces cell cycle arrest and apoptosis in cholangiocarcinoma cells. (A) Top: Representative fluorescence images of untransfected (CON), LV-miR-NC-transfected (NC), and LV-miR-29b-transfected (miR-29b) QBC939 cells (original magnification: 100×). Bottom: Relative miR-29b expression detected by qRT-PCR. (B) MTT assay results showing the time-course effect of miR-29b overexpression on the proliferation of QBC939 cells. (C) Representative images from colony formation assays. (D) Relative colony formation percentage. (E) Cell cycle distribution was subjected by flow cytometry. (F) Quantified histograms display the effect of miR-29b overexpression on cell cycle distribution. (G) Flow cytometry plots illustrating apoptosis in Annexin V/PI-stained QBC939 cells. (H) Quantified histograms display the effect of miR-29b overexpression on the apoptosis of QBC939 cells. The values presented are means ± SD. *P<0.05 and **P<0.01compared to the negative control group, as determined by analysis one-way variance (ANOVA), followed by the repeated measures.
Figure 3
Figure 3
miR-29b reduces CDKN2B promoter methylation and increases CDKN2B expression by directly targeting DNMT3B. Effect of miR-29b overexpression on CDKN2B mRNA (A) and protein (B) levels in QBC939 cells. (C) MSP analysis of CDKN2B gene promoter methylation changes in QBC939 cells transfected with LV-miR-29b or LV-miR-NC. (D) Schematic representation of the putative miR-29b binding site in the DNMT3B 3’UTR. (E, F) Effect of miR-29b overexpression on DNMT3B mRNA (E) and protein (F) levels. (G) Dual-luciferase reporter assay results showing reduced WT-DNMT3B promoter-driven luciferase activity in QBC939 cells transfected with miR-29b mimics. (H) Relative expression of DNMT3B protein in QBC939 cells. (I) Relative expression of DNMT3B mRNA in clinical cholangiocarcinoma specimens. (J) Pearson correlation analysis between miR-29b and DNMT3B expression in cholangiocarcinoma specimens. The values presented are means ± SD. *P<0.05 compared to the negative control group, as determined by analysis of one-way variance (ANOVA), followed by the repeated measures.
Figure 4
Figure 4
DNMT3B overexpression promotes proliferation and inhibits cell cycle arrest and apoptosis in cholangiocarcinoma cells. Analysis of DNMT3B mRNA (A) and protein (B) levels in QBC939 cells transfected with LV-DNMT3B or LV-control. (C–E) Effect of DNMT3B overexpression on QBC939 cell colony formation (C and D) and proliferation (E). (F) Cell cycle distribution was subjected by flow cytometry. (G) Quantified histograms display the effect of DNMT3B overexpression on cell cycle distribution. (H) Relative CDKN2B gene promoter methylation level in QBC939 cells transfected with LV-DNMT3B or LV-control. (I) Flow cytometry plots illustrating apoptosis in Annexin V/PI-stained QBC939 cells. (J) Quantified histograms display the effect of DNMT3B overexpression on the apoptosis of QBC939 cells. (K–L) Effect of DNMT3B knockdown on CDKN2B mRNA (K) and protein (L) levels. The values presented are means ± SD. *P<0.05 compared to the negative control group, as determined by analysis of one-way variance (ANOVA), followed by the repeated measures.
Figure 5
Figure 5
miR-29b overexpression inhibits cholangiocarcinoma tumorigenesis in vivo. (A) Representative images of subcutaneous tumors excised from nude mice (day 63 post-inoculation) and tumor volume curve. Measurements began on day 15 post-implantation and were repeated every 7 days until sacrifice. (B) Tumor weight at sacrifice. (C) Representative images of immunohistochemical analysis of DNMT3B and CDKN2B expression in excised tumors. (D–G) Corresponding semiquantitative expression analyses of DNMT3B and CDKN2B. DNMT3B mRNA (D) and protein (E) levels. CDKN2B mRNA (F) and protein (G) levels. Scale bars = 100 μm. The values presented are means ± SD. *P<0.05 compared to the negative control group, as determined by analysis of one-way variance (ANOVA), followed by the repeated measures.
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