Oncogenic microRNA-411 promotes lung carcinogenesis by directly targeting suppressor genes SPRY4 and TXNIP - PubMed (original) (raw)
Oncogenic microRNA-411 promotes lung carcinogenesis by directly targeting suppressor genes SPRY4 and TXNIP
Caiyan Zhang et al. Oncogene. 2019 Mar.
Abstract
Lung cancer is one of the most common malignant diseases globally, composed of non-small cell lung cancer (NSCLC, 85%) and small cell lung cancer (SCLC, 15%). MicroRNAs (miRNAs) are single-stranded noncoding RNAs having important roles in lung cancer development. miR-411-5p/3p were reported to be increased significantly in human NSCLC tissues and cell lines. Moreover, miR-411-5p/3p overexpression could accelerate cell proliferation and migration, and impede cell apoptosis in NSCLC cell lines. Mechanically, SPRY4 is confirmed a direct target of miR-411-5p/3p. Furthermore, our findings showed that miR-411-5p/3p promoted lung tumor growth in vivo, decreased SPRY4 expression dramatically, and induced EGFR, AKT signaling activation, as well as epithelial-mesenchymal transition (EMT) simultaneously in tumor tissues. In addition, we showed that miR-411-5p also targeted tumor suppressor TXNIP, involved in regulating positively cell cycle progress in SPC-A1 cells rather than in H1299. Whether cell specificity of low TXNIP mRNA level in H1299 is responsible for the different response to cell cycle between H1299 and SPC-A1 would need further explorations. Collectively, these results suggest that miR-411-5p/3p are required for NSCLC development by suppressing SPRY4 and TXNIP; thus, the miR-411-SPRY4-AKT axis might act as a promising target for lung cancer therapy clinically.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
Fig. 1
MiR-411 expression was upregulated in NSCLC. a, b Relative miR-411-5p/3p expression in NSCLC and corresponding paracancerous lung tissues (n = 33). c, d MiR-411-5p/3p expression in NSCLC cell lines A549, SPC-A1, H1299, PC-9, and 95-D. HBE cells was the normal control. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
miR-411-5p/3p promote cell proliferation and impeded apoptosis in NSCLC cell lines. a–d miR-411-5p/3p expression was upregulated in pLenti-miR-411 H1299 and SPC-A1 cells compared to pLenti cells. e, f Cell proliferation of pLenti/pLenti-miR-411 H1299 and SPC-A1 was assessed by CCK-8 assay. g, h Flow cytometry was used to analyze the apoptosis of pLenti/pLenti-miR-411 H1299 and SPC-A1 cells. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
MiR-411 induce cell migration in NSCLC cell lines. a, b Wound-healing assay of pLenti/pLenti-miR-411 H1299 and SPC-A1 cells. The relative position of cells was recorded at 0 h and 48 h, respectively. c, d Transwell assay of pLenti/pLenti-miR-411 H1299 and SPC-A1 cells. The migrated cells were fixed, stained, and photographed 48 h post incubation and then the cell numbers were counted. *P < 0.05, **P < 0.01
Fig. 4
miR-411-5p/3p inhibition induces apoptosis and suppressed migration in NSCLC cell lines. a Relative expression of miR-411-5p in H1299 and SPC-A1 cells with transfection of NC inhibitor (NC inh) and miR-411-5p inhibitor (miR-411-5p inh). b Relative expression of miR-411-3p in H1299 cells with transfection of NC inh miR-411-3p inhibitor (miR-411-3p inh). c–g The effect of miR-411-5p/3p inhibition on proliferation (c–e), apoptosis (f), and migration (g) in H1299 and SPC-A1 cells. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 5
miR-411-5p/3p directly target SPRY4. a Six candidate genes were obtained by miRWalk 2.0 plus two sets of microarray data (GSE51852 and GSE19188). b The six candidate genes, SPRY4, DUSP1, CYBRD1, SHANK2, PGR, and RUNX1T1. c SPRY4 was associated with longer overall patient survival. d The seed regions of miR-411-5p/3p of SPRY4 3′-UTR and 3′-mUTR. e The luciferase activity of HEK293T cells co-transfected with NC mimic or miR-411-5p/3p mimic, and pGL3-SPRY4-3′-UTR (SPRY4 Wild) or pGL3-SPRY4-3′-mUTR (SPRY4 Mut). f–h SPRY4 protein expression (f, g) and mRNA expression (h) were determined by western blotting and qRT-PCR, respectively, in pLenti/pLenti-miR-411 H1299 and SPC-A1 cells. i SPRY4 mRNA expression was measured by qRT-PCR in patient tissues. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
siSPRY4 effects on cell proliferation and migration in NSCLC. a, b siSPRY4 downregulated SPRY4 at mRNA (a) and protein (b) levels in H1299 and SPC-A1 cells, as determined by qRT-PCR and western blotting, respectively. c–f siSPRY4 effects on cell proliferation (c, d), migration (e), and apoptosis (f) in H1299 and SPC-A1 cells. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 7
SPRY4 rescues the miR-411-5p/3p effects in H1299 cells. a, b SPRY4 mRNA and protein level in pLenti/pLenti-miR-411 H1299 cells transfected with pcDNA3.1 or pcDNA3.1-SPRY4. c Cell proliferation of pLenti-miR-411 H1299 with transfection of pcDNA3.1-SPRY4 was rescued compared with pLenti cells of pcDNA3.1. d Cell apoptosis rates of pLenti-miR-411 H1299 with transfection of pcDNA3.1-SPRY4 were analyzed by flow cytometry. The inhibition of miR-411 on H1299 apoptosis was rescued by pcDNA3.1-SPRY4. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 8
TXNIP is a direct target of miR-411-5p. a TXNIP mRNA expression in A549, SPC-A1, H1299, PC-9, and 95-D cells. HBE cell line was normal control. b TXNIP mRNA expression in NSCLC tissue samples and corresponding non-tumor tissues (n = 33). c TXNIP protein level was determined in pLenti/pLenti-miR-411 H1299 and SPC-A1 cells by western blotting. d The seed regions of miR-411-5p of TXNIP 3′-UTR and 3′-mUTR. e The luciferase activity of HEK293T cells co-transfected with NC mimic or miR-411-5p mimic and pGL3-TXNIP-3′-UTR (TXNIP Wild) or pGL3-TXNIP-3′-mUTR (TXNIP Mut). **P < 0.01, ***P < 0.001
Fig. 9
miR-411-5p/3p overexpression promotes tumor growth in vivo. a pLenti/pLenti-miR-411 H1299 cells were subcutaneously implanted into SCID mice with volumes of tumor recorded weekly. b, c Mice tumors were weighed (b) and photoed (c) 8 weeks post injection. d miR-411-5p/3p expression was measured in mice tumors by qRT-PCR. e SPRY4, E-cadherin, and N-cadherin protein levels were detected in mice tumors by western blotting. f HE staining (original magnifications × 200) of tumors and SPRY4, Ki67, E-cadherin, and N-cadherin protein levels were determined by immunohistochemistry. *P < 0.05
Fig. 10
A diagram of the functions of miR-411 in NSCLC
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