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. 2020 Oct;48(10):300060520939718.
doi: 10.1177/0300060520939718.

Serum microRNA-365 suppresses non-small-cell lung cancer metastasis and invasion in patients with bone metastasis of lung cancer

Yanyan Liu  1 , Junmei Jia  1 , Bin Song  1 , Haile Qiu  1 , Gang Liang  2 , Bo Zhang  3 , Kang Wang  1
Affiliations

Serum microRNA-365 suppresses non-small-cell lung cancer metastasis and invasion in patients with bone metastasis of lung cancer

Yanyan Liu et al. J Int Med Res. 2020 Oct.

Abstract

Objective: In the present investigation, we evaluated the effects of microRNA-365 (miR-365) on non-small-cell lung cancer (NSCLC) cell metastasis and invasion in patients with bone metastasis of lung cancer.

Methods: Blood samples from patients with NSCLC and healthy controls and the A549 adenocarcinoma cell line were included in this study. Quantitative real-time PCR and microarray were performed on blood samples. The MTT assay, luciferase reporter assay, Transwell assay, ELISA, and western blot were performed to evaluate expression of associated factors.

Results: Expression of miR-365 was reduced in patients with bone metastasis of NSCLC. Downregulation of miR-365 promoted cell growth, metastasis, and invasion of NSCLC. Upregulation of miR-365 reduced cell growth, metastasis, and invasion of NSCLC. Downregulation of miR-365 induced expression of NKX homeobox-1 (NKX2-1), epidermal growth factor receptor (EGFR), phosphoinositide-3-kinase (PI3K), and p-Akt proteins in an in vitro model of NSCLC. Inhibition of NKX2-1 reduced the effects of miR-365 on cell growth, metastasis, and invasion of NSCLC. Activation of EGFR reduced the effects of miR-365 on cell growth, metastasis, and invasion of NSCLC.

Conclusions: The study established that the serum miR-365 suppresses NSCLC cell metastasis and invasion in patients with bone metastasis of lung cancer via EGFR/PI3K through NKX2-1.

Keywords: NKX homeobox-1; bone metastasis; epidermal growth factor receptor; microRNA-365; non-small-cell lung cancer; phosphoinositide-3-kinase.

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Conflict of interest statement

Declaration of conflicting interest: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Serum microRNA-365 expression in patients with bone metastasis of lung cancer. (a) Gene chip and (b) quantitative PCR for expression of microRNA-365 in the serum of patients with NSCLC; (c) overall and (d) disease-free survival associated with low or high expression of miRNA-365. **P < 0.01 compared with healthy volunteer group. Normal, healthy volunteer group; NSCLC, non-small-cell lung cancer.
Figure 2.
Figure 2.
Downregulation of miRNA-365 promoted cell growth and migration of NSCLC. (a) miRNA-365 expression, (b) cell growth, (c) LDH activity, (d, e) migration rate, (f, g) cell invasion, and (h, i) caspase-3 and caspase-9 activity levels. **P < 0.01 compared with negative group. Negative, negative mimic group; anti-365, downregulation of miR-365 group. NSCLC, non-small-cell lung cancer.
Figure 3.
Figure 3.
Upregulation of miRNA-365 reduced cell growth and migration of NSCLC. (a) miRNA-365 expression, (b) cell growth, (c) LDH activity, (d, e) migration rate, (f, g) cell invasion, and (h, i) caspase-3 and caspase-9 activity levels. **P < 0.01 compared with negative group. Negative, negative mimics group; miR-365, miR-365 overexpression group. NSCLC, non-small-cell lung cancer.
Figure 4.
Figure 4.
MiRNA-365 regulates NKX2-1 signaling in NSCLC. (a) Gene chip, (Bb representative classification of repressed genes by KEGG analysis, (c) NKX2-1 3′-UTR and miR-365 had a conservative matching area, (d) luciferase activity levels, (e, f) network analysis diagram. **P < 0.01 compared with negative group. NKX2-1, NKX homeobox-1; NSCLC, non-small-cell lung cancer; Negative, negative mimics group; miR-365, miR-365 overexpression group; WT, wild-type; MUT, mutant.
Figure 5.
Figure 5.
MiRNA-365 regulates EGFR/PI3K through NKX2-1. Expression of (a) NKX2-1, (b) EGFR, (c) PI3K, and (d) p-AKT proteins by statistical analysis and (e) western blotting assays by overexpression of miR-365; expression of (f) NKX2-1, (g) EGFR, (h) PI3K, and (i) p-AKT proteins by statistical analysis and (j) western blotting assays by downregulation of miR-365. **P < 0.01 compared with negative group. NKX2-1, NKX homeobox-1; EGFR, epidermal growth factor receptor; PI3K, phosphoinositide-3-kinase; p-AKT, phosphorylated protein kinase B; Negative, negative mimics group; miR-365, miR-365 overexpression group; anti-365, miR-365 downregulation group.
Figure 6.
Figure 6.
The inhibition of NKX2-1 reduced the effects of microRNA-365 on non-small-cell lung cancer cell metastasis and invasion. Expression of (a) NKX2-1, (b) EGFR, (c) PI3K, and (d) p-AKT proteins by statistical analysis and (e) western blotting assays, (f) cell growth, (g) LDH activity levels, (h, i) migration rate, (j, k) cell invasion, and (l, m) caspase-3 and caspase-9 activity levels. **P < 0.01 compared with negative group; ##P < 0.01 compared with miR-365 overexpression group. NKX2-1, NKX homeobox-1; EGFR, epidermal growth factor receptor; PI3K, phosphoinositide-3-kinase; p-AKT, phosphorylated protein kinase B; Negative, negative mimics group; miR-365, miR-365 overexpression group; si-NKX2-1, miR-365 and short interfering (si)-NKX2-1 overexpression group.
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