Cancer Res Treat.  2018 Jul;50(3):936-949. 10.4143/crt.2017.302.

MicroRNA-373 Inhibits Cell Proliferation and Invasion via Targeting BRF2 in Human Non-small Cell Lung Cancer A549 Cell Line

• Wang L
• Qu J
• Zhou L
• Liao F
• Wang J

• KMID: 2417882
• DOI: http://doi.org/10.4143/crt.2017.302

Abstract

PURPOSE
The purpose of this study was to investigate the biological role and mechanism of miR-373 targeting of TFIIB-related factor 2 (BRF2) in the regulation of non-small cell lung cancer (NSCLC) cells.
MATERIALS AND METHODS
miRNA microarray chip analysis of four paired NSCLC and adjacent non-tumor tissues was performed. Quantitative real-time polymerase chain reaction (qRT-PCR) andwestern blotting were used to detect the expression levels of miR-373 and BRF2 in NSCLC tissues and cell lines. The dual-luciferase reporter method was performed to determine if BRF2 is a target of miR-373. MTT, wound-healing, Transwell, and flow cytometric assays were conducted to examine the proliferation, migration, invasion, and cell cycle progression of NSCLC A549 cells, respectively; western blotting was used to detect the expression of epithelial-mesenchymal transition (EMT)-related proteins.
RESULTS
The miRNA microarray chip analysis demonstrated that miR-373 was down-regulated in NSCLC tissues, and this result was confirmed by qRT-PCR. Additionally, miR-373 was confirmed to target BRF2. Moreover, miR-373 expression was inversely correlated with BRF2 expression in NSCLC tissues and cell lines; both miR-373 down-regulation and BRF2 up-regulation were strongly associated with the clinicopathological features and prognosis of NSCLC patients. In vitro, overexpression of miR-373 markedly inhibited cell proliferation, migration, and invasion; up-regulated the expression of E-cadherin; and down-regulated the expression of N-cadherin and Snail in A549 cell. Knockdown BRF2 by siRNA resulted in effects similar to those caused by overexpression of miR-373.
CONCLUSION
MiR-373 is decreased in NSCLC, and overexpression of miR-373 can suppress cell EMT, and inhibit the proliferation, migration, and invasion of NSCLC A549 cells by targeting BRF2.

Keyword

miR-373; BRF2; Non-small cell lung carcinoma; Cell proliferation; Cell migration inhibition; Epithelial-mesenchymal transition

MeSH Terms

Blotting, Western
Cadherins
Carcinoma, Non-Small-Cell Lung*
Cell Cycle
Cell Line*
Cell Migration Inhibition
Cell Proliferation*
Down-Regulation
Epithelial-Mesenchymal Transition
Humans*
In Vitro Techniques
Methods
MicroRNAs
Prognosis
Real-Time Polymerase Chain Reaction
RNA, Small Interfering
Snails
Up-Regulation
Cadherins
MicroRNAs
RNA, Small Interfering

Figure

• 

  Fig. 1. miRNA microarray chip analysis and luciferase assay. (A) miRNA microarray chip analysis was performed to identify miRNAs in tumor tissues of five non-small cell lung cancer patients and their corresponding non-tumor lung tissues. (B) The predicted miR-373 binding site on the BRF2 3′-untranslated region (3′-UTR) and the corresponding mutant binding site are shown. (C) Relative activities of luciferase reporters encoding the BRF2 3′-UTR variants co-transfected either the with miR-373 mimic or negative control (NC) oligonucleotides in NSCLC A549 cells. ****p < 0.001 compared with the other three groups.

• 

  Fig. 2. The expression levels of miR-373 and BRF2 in non-small cell lung cancer (NSCLC) tissues and adjacent normal tissues. (A, B) Quantitative real-time polymerase chain reaction was used to detect the relative expression levels of miR-373 (A) and BRF2 (B) in 92 paired NSCLC tissues and adjacent non-tumor tissues. (C, D) Western blotting was performed to measure the relative protein expression level of BRF2 in 92 paired NSCLC tissues and adjacent non-tumor tissues. *p < 0.05 compared with adjacent tissues.

• 

  Fig. 3. The Kaplan-Meier curves of the relative expression levels of miR-373 (A) and BRF2 (B) in patients with non-small cell lung cancer.

• 

  Fig. 4. The expression levels of miR-373 and BRF2 in different non-small cell lung cancer (NSCLC) cell lines and in a normal lung bronchus epithelial cell line. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression of miR-373 in NSCLC cell lines and in normal lung bronchus epithelial cell line. ****p < 0.001 compared with cells from the human bronchial epithelial cell line; ††† p < 0.005, compared with A549 cells. (B) qRT-PCR was used to detect the relative expression levels of miR-373 and BRF2 in each transfected group. ****p < 0.001, ***p < 0.005, **p < 0.01 compared with the Mock and negative control (NC) groups; †††† p < 0.001, †† p < 0.01 compared with the miR-373 mimic group; ‡‡‡‡ p < 0.001 compared with the miR-373 inhibitor group. (C, D) Western blotting was performed to measure the relative protein expression of BRF2 in each transfected group. **p < 0.01, *p < 0.05 compared with the Mock and NC groups; †††† p < 0.001, † p < 0.05 compared with the miR-373 mimic group; ‡‡ p < 0.01 compared with the miR-373 inhibitor group. Each sample was analyzed in triplicate.

• 

  Fig. 5. Effects of miR-373 targeting of BRF2 on the proliferation and cell cycle progression of non-small cell lung cancer (NSCLC) A549 cells as determined using MTT and flow cytometric assays, respectively. (A) MTT assay was performed to examine NSCLC A549 cell proliferation at 24, 48, and 72 hours after transfection. (B) Flow cytometry assay analysis of cell cycle progression. Each sample was analyzed in triplicate. OD, optical density; NC, negative control.

• 

  Fig. 6. Effects of miR-373 targeting of BRF2 on the migration and invasion of non-small cell lung cancer A549 cells as determined by using wound-healing and Transwell assays, respectively. (A) The effect of miR-373 on the cell migration ability of A549 cells was verified by wound-healing experiments. (B) The effect of miR-373 on the cell invasion ability of A549 cells was verified by Transwell experiments. ****p < 0.001, **p < 0.01, *p < 0.05 compared with the Mock and negative control (NC) groups; ††††p < 0.001 compared with the miR-373 mimic group; ‡‡‡‡p < 0.001, ‡‡‡p < 0.005 compared with the miR-373 inhibitor group. Each sample was analyzed in triplicate.

• 

  Fig. 7. Effects of miR-373 targeting of BRF2 on the relative protein expression levels of N-cadherin (A, B), E-cadherin (A, C), and Snail (A, D) in non-small cell lung cancer A549 cells as determined by western blotting. ****p < 0.001 compared with the Mock and negative control (NC) groups; †††† p < 0.001 compared with the miR-373 mimic group; ‡‡‡‡ p < 0.001 compared with the miR-373 inhibitor group. Each sample was analyzed in triplicate.

Reference

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