Long non-coding RNA linc00673 regulated non-small cell lung cancer proliferation, migration, invasion and epithelial mesenchymal transition by sponging miR-150-5p

Abstract

Background: The function of a new long non-coding RNA linc00673 remains unclear. While identified as an oncogenic player in non-small cell lung cancer (NSCLC), linc00673 was found to be anti-oncogenic in pancreatic ductal adenocarcinoma (PDAC). However whether linc00673 regulated malignancy and epithelial mesenchymal transition (EMT) has not been characterized.

Methods: Cell proliferation was assessed using CCK-8 and EdU assays, and cell migration and invasion were assessed using scratch assays and transwell invasion assays. Epithelial mesenchymal transition was examined using western blot, qRT-PCR and immunofluorescence staining. Interaction between miRNA and linc00673 was determined using luciferase reporter assays. In vivo experiments were performed to assess tumor formation. In addition, the expression data of NSCLC specimens of TCGA and patient survival data were utilized to explore the prognostic significance of linc00673.

Results: In the present study, we found high linc00673 expression was associated with poor prognosis of NSCLC patients. In vitro experiments showed linc00673 knockdown reversed TGF-β induced EMT, and miR-150-5p was predicted to target linc00673 through bioinformatics tools. Overexpression of miR-150-5p suppressed lin00673's expression while inhibition of miR-150-5p led to significant upregulation of lin00673, suggesting that linc00673 could be negatively regulated by miR-150-5p, which was further confirmed by the inverse correlation between linc00673 and miR-150-5p in NSCLC patients' specimen. Furthermore, we proved that miR-150-5p could directly target linc00673 through luciferase assay, so linc00673 could sponge miR-150-5p and modulate the expression of a key EMT regulator ZEB1 indirectly. In addition, miR-150-5p inhibition abrogated linc00673 silence mediated proliferation, migration, invasion and EMT suppressing effect. Moreover, the inhibition of linc00673 significantly attenuated the tumorigenesis ability of A549 cells in vivo.

Conclusions: We validated linc00673 as a novel oncogenic lncRNA and demonstrated the molecular mechanism by which it promotes NSCLC, which will advance our understanding of its clinical significance.

Keywords: Competing endogenous RNA; Epithelial mesenchymal transition; Non-small cell lung cancer; linc00673; miR-150-5p.

Fig. 1

Linc00673 is associated with poor survival in NSCLC patients. a. Association of linc00673 expression with overall survival of NSCLC patients, red: expression above upper quartile, blue: expression below upper quartile. b. Linc00673 expression in 6 NSCLC cell lines and 1 human bronchial epithelial cell line as determined by qRT-PCR

Fig. 2

Effects of linc00673 on lung cancer cell apoptosis, viability, migration and invasion. a. Validation of siRNA knockdown efficiency in A549 cells as determined by qRT-PCR. b. Validation of siRNA knockdown efficiency in H1975 cells as determined by qRT-PCR. c. Validation of pcDNA3.1-linc00673 over-expression vector in H1703 cells as determined by qRT-PCR. d. Flow cytometric analysis of apoptosis in si-NC or si-L3 transfected A549 and H1975 cells. e. Expression of PARP and pro-Caspase 3 in si-NC or si-L3 transfected A549 cells as determined by western blot. f. CCK-8 proliferation assay in si-NC or si-L3/si-L5 transfected A549 and H1975 cells. g. EdU proliferation assay in si-NC or si-L3/si-L5 transfected A549 and H1975 cells. h. Wound scratch assay in si-NC or si-L3/si-L5 transfected A549 and H1975 cells and in pcDNA3.1-linc00673 transfected H1703 cells. i. Transwell invasion assay in si-NC or si-L3/si-L5 transfected A549 and H1975 cells and in pcDNA3.1-linc00673 transfected H1703 cells. j. Tumor growth in NOD/SCID mice with tail vein injection of linc00673-shRNA or sh-nc transfected A549 cells. Error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005

Fig. 3

Linc00673 was required for epithelial mesenchymal transition. a. Expression of Vimentin, N-cadherin, Snail, ZEB1 and E-cadherin in TGF-β treated A549 cells as determined by western blot. b. Expression of Vimentin and E-cadherin in TGF-β receptor antagonist SB431542 and TGF-β treated A549 cells as determined by western blot. c. Expression of linc00673 in TGF-β and SB431542 treated A549 cells as determined by qRT-PCR. d. Expression of linc00673 in TGF-β and SB431542 treated H1975 cells as determined by qRT-PCR. e. Expression of EMT markers in si-NC or si-L3/si-L5 transfected followed by TGF-β treated A549 and H1975 cells as determined by western blot. f. mRNA level of EMT markers in si-NC or si-L3 transfected followed by TGF-β treated A549 and H1975 cells as determined by qRT-PCR. g. Immunofluorescence staining of Vimentin expression in si-NC or si-L3 transfected followed by TGF-β treated A549 and H1975 cells. Error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, #p < 0.05, ##p < 0.01, ###p < 0.005

Fig. 4

Reciprocal correlation between linc00673 and miR-150-5p. a. The predicted binding sites of miR-150-5p to the linc00673 sequence. b. Association of miR-150-5p expression with overall survival of NSCLC patients, red: expression above upper quartile, blue: expression below upper quartile. c. Negative correlation between linc00673 and miR-150-5p expression in NSCLC patients. d. Association of linc00673 and miR-150-5p expression with pathologic stage in NSCLC patients. “Prophase” stands for stage I/II and “advanced” stands for stage III/IV. e. MiR-150-5p expression in 6 NSCLC cell lines and 1 human bronchial epithelial cell line as determined by qRT-PCR. f. Expression of linc00673 in miR-150-5p mimics transfected A549 cells as determined by qRT-PCR. g. Expression of linc00673 in miR-150-5p mimics transfected H1975 cells as determined by qRT-PCR. h. Expression of miR-150-5p in TGF-β treated A549 cells as determined by qRT-PCR. i. Expression of miR-150-5p in TGF-β treated H1975 cells as determined by qRT-PCR. Error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005

Fig. 5

Linc00673 acted as a ceRNA by sponging miR-150-5p and regulated ZEB1 expression indirectly. a. 293 T cells were co-transfected with miR-150-5p mimics or mimics NC and pmirGLO-ZEB1–3’UTR-WT or pmirGLO-ZEB1–3’UTR-MUT. Luciferase activity was detected 24 h after transfection using the dual luciferase assay. b. A549 cells were co-transfected with miR-150-5p mimics or mimics NC and pmirGLO-ZEB1–3’UTR-WT or pmirGLO-ZEB1–3’UTR-MUT. Luciferase activity was detected 24 h after transfection using the dual luciferase assay. c. Predictive binding sites of miR-150-5p to linc00673 and schematic of wild-type and mutant pmirGLO-linc00673 constructs. d. 293 T cells were co-transfected with miR-150-5p mimics or mimics NC and pmirGLO-linc00673-WT or pmirGLO-linc00673-MUT. Luciferase activity was detected 24 h after transfection using the dual luciferase assay. e. A549 and H1975 cells were co-transfected with miR-150-5p mimics or mimics NC and pmirGLO-linc00673-WT or pmirGLO-linc00673-MUT. Luciferase activity was detected 24 h after transfection using the dual luciferase assay. Error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005

Fig. 6

Inhibition of miR-150-5p reversed linc00673 silence mediated suppressing proliferation, migration, invasion and EMT effect. a. Expression of linc00673 in miR-150-5p inhibitors and si-L3 transfected A549 cells as determined by qRT-PCR. b. Expression of linc00673 in miR-150-5p inhibitors and si-L3 transfected H1975 cells as determined by qRT-PCR. c. Expression of EMT markers in miR-150-5p inhibitors and si-L3 transfected A549 cells as determined by western blot. d. Expression of EMT markers in miR-150-5p inhibitors and si-L3 transfected H1975 cells as determined by western blot. e. Wound scratch assay in miR-150-5p inhibitors and si-L3 transfected A549 and H1975 cells. f. Transwell invasion assay in miR-150-5p inhibitors and si-L3 transfected A549 and H1975 cells. g. EdU proliferation assay in miR-150-5p inhibitors and si-L3 transfected A549 and H1975 cells. Error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005

Fig. 7

Schematic plot showed linc00673 acted as a miRNA sponge to regulate ZEB1 expression indirectly