miR‑92b promotes autophagy and suppresses viability and invasion in breast cancer by targeting EZH2

Abstract

MicroRNAs (miRs) are a small non-coding RNA family with a length of 18-22 nucleotides. They are able to regulate gene expression by either triggering target messenger RNA degradation or by inhibiting mRNA translation. Enhancer of zeste homolog 2 (EZH2) is the core enzymatic subunit of polycomb repressor complex 2 and is responsible for the trimethylation of histone 3 on lysine 27 (H3K27me3); it is also able to silence a bundle of tumor suppressor genes through promoter binding. However, little is known regarding the effect of miR‑92b on cell autophagy, viability and invasion as well as how it interacts with EZH2. The present study investigated the major role of miR‑92b in the autophagy, viability and invasion of breast cancer. It was revealed that in MCF‑7 and MDA‑MB‑453 cells, the expression of miR‑92b promoted autophagy induced by starvation and rapamycin treatment. The results of in vitro experiments results demonstrated that miR‑92b inhibited breast cancer cell viability, invasion and migration. To further elucidate the regulatory mechanisms of miR‑92b in autophagy, a dual luciferase reporter assay was performed to determine whether miR‑92b targeted the EZH2 gene. The expression of miR‑92b was negatively correlated to EZH2 mRNA expression in breast cancer. Depletion of EZH2 induced phenocopied effects on miR‑92b overexpression, thereby demonstrating its importance in autophagy. These results indicated that miR‑92b may serve an important role in breast cancer in controlling autophagy, viability and invasion. The present study indicated that miR‑92b and EZH2 may serve as potential biomarkers for cancer detection and highlighted their possible therapeutic implications.

Figure 1

Overexpression of miR-92b increases autophagic activity in MCF-7 cells. (A and B) Reverse transcription-quantitative polymerase chain reaction analysis of miR-92b expression in MCF-7 cells treated with (A) STV (4 h) or (B) RAP (2.5 µM, 8 h). miR-92b expression levels were significantly increased following STV or RAP treatment. ^**P<0.01 vs. MCF 7 cells. (C and D) Immunoblotting results indicated that miR-92b increased (C) STV and (D) RAP induced conversion of LC3-I to LC3-II and SQSTM1 degradation in MCF-7 cells. (E and F) miR-92b promoted STV and RAP induced GFP-LC3 dot formation in MCF-7 cells. Detection of cytoplasmic puncta formation by soluble GFP-fused LC3 protein is a commonly used method to follow autophagy activation using microscopy (magnification, ×40). (G and H) Quantitative analysis of the experiments in (E and F). ^**P<0.01, as indicated. STV, starvation; RAP, rapamycin; miR, microRNA; LC3, light chain 3; GFP, green fluorescent protein; SQSTM1, sequestosome 1; NC, negative control.

Figure 2

Overexpression of miR-92b results in increased autophagic flux in MDA-MB-453 cells. (A) STV (4 h) and (B) RAP (2.5 µM, 8 h) induced miR-92b expression in MDA-MB-453 cells. miR-92b expression was detected by reverse transcription-quantitative polymerase chain reaction. ^**P<0.01 vs. 453 cells. (C and D) Immunoblotting results indicated that miR-92b increased (C) STV and (D) RAP induced conversion of LC3-I to LC3-II and SQSTM1 degradation in MDA-MB-453 cells. (E and F) miR-92b promoted (E) STV and (F) RAP induced GFP-LC3 dot formation in MDA-MB-453 cells (magnification, ×40). (G and H) Quantitative analysis of the experiments in (E and F). ^**P<0.01, as indicated. STV, starvation; RAP, rapamycin; miR, microRNA; LC3, light chain 3; GFP, green fluorescent protein; SQSTM1, sequestosome 1; NC, negative control; 453, MDA-MB-453 cells.

Figure 3

Effects of miR-92b on breast cancer cell migration and invasion. (A) miR-92b suppressed the migration and invasion of MCF-7 cells. However, silencing miR-92b expression promoted migration and invasion. (B) miR-92b promotes the migration and invasion ability of MDA-MB-453 cells. Scale bar = 100 µm; magnification, ×200). ^*P<0.05, ^**P<0.01 and ^***P<0.001, as indicated. miR, microRNA; NC, negative control.

Figure 4

miR-92b inhibits cancer cell proliferation. (A and B) Overexpression of miR-92b inhibited cancer cells growth. However, silencing miR-92b expression promoted cell proliferation in (A) MDA-MB-453 and (B) MCF-7 cells. The cell viability was measured from 0 to 72 h by Cell Counting Kit-8 assay. (C and D) Colony formation assays transfecting MCF-7 and MDA-MB-453 cells with miR-92b mimics or inhibitor. ^**P<0.01 and ^***P<0.001, as indicated. miR, microRNA; NC, negative control.

Figure 5

Prediction and validation of EZH2 as a direct target of miR-92b. (A) Schematic of the predicted binding site of miR-92b in EZH2 3′-UTR. A mutant EZH2 3′-UTR construct was tested in parallel. (B) The wild-type or mutant forms of EZH2 3′-UTR were co-transfected into COS7 cells with miR-92b mimic or NC RNA. Luciferase activity was measured at 48 h post-transfection. The results were normalized against Renilla luciferase values. ^**P<0.01, as indicated. (C) Negative correlation between miR-92b and EZH2 mRNA levels in breast specimens. (D) EZH2 protein expression was reduced in MCF7 cells with miR-92b overexpression in MCF7 cells. (E) EZH2 protein expression was reduced in miR-92b mimics transfected MCF7 cells. EZH2, enhancer of zeste homolog 2; miR, microRNA; STV, starvation; NC, negative control; UTR, untranslated region; WT, wild-type; MUT, mutant.

Figure 6

Silencing EZH2 expression with EZH2 shRNA promotes autophagy, and suppresses proliferation and invasion in MCF-7 cells. (A and B) Immunoblotting results indicated that shEZH2 increased (A) STV and (B) RAP induced conversion of LC3-I to LC3-II and SQSTM1 degradation. (C and D) shEZH2 promoted STV and RAP induced GFP-LC3 dot formation in MCF-7 cells (magnification, ×40). (E and F) Quantitative analyses of the experiments in panels (C and D). (G and H) shEZH2 inhibited the proliferation and invasion abilities of MCF-7 cells. Scale bars = 100 µm; magnification, ×200. ^**P<0.01, as indicated. EZH2, enhancer of zeste homolog 2; STV, starvation; RAP, rapamycin; miR, microRNA; shRNA, short hairpin RNA; LC3, light chain 3; GFP, green fluorescent protein; SQSTM1, sequestosome 1; NC, negative control.

Figure 7

EZH2 is involved in miR-92b regulated cell autophagy, proliferation and invasion. (A) Reverse transcription-quantitative polymerase chain reaction revealed the relative mRNA expression of EZH2 in MCF7 cells transfected with the pCMV6 Entry-EZH2-Myc-DDK expression plasmid or the control plasmid. (B) Western blot analyses of LC3 expression in MCF-7 cells transfected with miR-NC plus vector, miR-92b plus EZH2, miR-92b plus vector or anti-miR-92b (anti-92b) plus vector. Autophagy-associated LC3-I to LC3-II conversion (LC3 lipidation) was attenuated in anti-miR-92b plus vector overexpressing cell extracts, when compared with miR-NC plus vector as the control group. (C) Cell Counting Kit-8 analysis of MCF7 cells transfected with miR-NC plus vector, miR-92b plus EZH2 and miR-92b plus vector at the indicated times. (D) Transwell analysis of MCF-7 cells treated with miR-NC plus vector, miR-92b plus EZH2 and miR-92b plus vector, and the quantitative analysis is shown in the right-hand panel. Scale bar = 100 µm; magnification, ×200. ^**P<0.01 and ^***P<0.001, as indicated. EZH2, enhancer of zeste homolog 2; miR, microRNA; LC3, light chain 3; NC, negative control; ns, not significant.