Long non-coding RNA linc00665 promotes lung adenocarcinoma progression and functions as ceRNA to regulate AKR1B10-ERK signaling by sponging miR-98

Abstract

Long non-coding RNAs (lncRNAs) are frequently dysregulated in multiple malignancies, demonstrating their potential oncogenic or tumor-suppressive roles in tumorigenesis. Herein, we reported the identification of a novel lncRNA, linc00665 (ENST00000590622), which was markedly upregulated in lung adenocarcinoma (LUAD) tissues and might serve as an independent predictor for poor prognosis. Functional assays indicated that linc00665 reinforced LUAD cell proliferation and metastasis in vitro and in vivo. Mechanistically, transcription factor SP1 induced the transcription of linc00665 in LUAD cells, which exerted its oncogenic role by functioning as competing endogenous RNA (ceRNA) for miR-98 and subsequently activating downstream AKR1B10-ERK signaling pathway. Together, our study elucidates oncogenic roles of linc00665-miR98-AKR1B10 axis in LUAD tumorigenesis, which may serve as potential diagnostic biomarkers and therapeutic targets.

Fig. 1. Identification of dysregulated lncRNAs in LUAD.

a Cluster analysis of differentially expressed lncRNAs from microarray dataset GSE27262. b Relative expression levels of lncRNA candidates quantified by qRT-PCR in clinical samples. c Relative linc00665 expression in LUAD from TCGA database. In all, 533 LUAD tumor tissues and 59 normal tissues, respectively. d Linc00665 expression data from TCGA was analyzed by Cancer RNA-seq Nexus. LUAD lung adenocarcinoma, TCGA The Cancer Genome Atlas, qRT-PCR quantitative real-time PCR

Fig. 2. Expression of linc00665 in LUAD tissues and cells.

a Linc00665 expression was significantly higher in LUAD specimens than in adjacent normal tissues. b–d Linc00665 expression was significantly higher in patients with big tumor size, advanced TNM stage and lymph node metastasis. Linc00665 expression was examined by qRT-PCR and normalized to GAPDH expression. e, f Kaplan–Meier survival analysis of overall and recurrence-free survival according to linc00665 expression in 80 LUAD patients. g Abundance of linc00665 in NSCLC cell lines relative to that in normal human bronchial epithelial cells (16HBE) as determined by qRT-PCR. h Subcellular location of linc00665 in A549 and H1299 cells. U6 and GAPDH acted as nucleus and cytoplasm marker, respectively. i RNA FISH assay to confirm subcellular location of linc00665 in A549 cells. U6 and 18S acted as nucleus and cytoplasm marker, respectively. LUAD lung adenocarcinoma, NSCLC non-small cell lung cancer, qRT-PCR quantitative real-time PCR; *p < 0.05, **p < 0.01

Fig. 3. Effects of linc00665 on LUAD cell proliferation, migration, invasion, and EMT process in vitro.

a Validation of siRNA knockdown and overexpression vector efficiency in A549 and H1299 cells as determined by qRT-PCR. b CCK-8 proliferation assays in A549 and H1299 cells after transfection with linc00665 siRNA or overexpression plasmid. c Representative images of colony formation assays in A549 and H1299 cells after transfection, with the media changed and siRNA transfection repeated every 3 days. d Representative images of wound-healing assays at indicated times after scratching in A549 and H1299 cells. e, f Representative images of transwell migration and invasion assays in A549 and H1299 cells after transfection. g Quantified bar charts of migrated or invaded cell numbers. h Expression of E-cadherin, Vimentin, N-cadherin and GAPDH protein in A549 and H1299 cells after transfection with linc00665 siRNA. LUAD lung adenocarcinoma, NC negative control, qRT-PCR quantitative real-time PCR; *p < 0.05

Fig. 4. Knockdown of linc00665 promotes cell cycle arrest and induces cell apoptosis in vitro.

a, b Flow cytometric analysis of cell cycle distributions in A549 and H1299 cells after transfection with linc00665 siRNA. c, d Flow cytometric analysis of apoptosis in A549 and H1299 cells after transfection. e Expression of Bcl-2, Bax, Caspase-3, PARP, and GAPDH protein in A549 and H1299 cells after transfection with linc00665 siRNA. NC negative control; *p < 0.05, **p < 0.01

Fig. 5. Downregulation of linc00665 suppressed LUAD tumor growth and metastasis in vivo.

a Representative images of tumors collected from mice. b Tumor volume curve of mice upon shRNA-NC or shRNA-Linc00665 treatment. c Tumor weights were represented. d Relative linc00665 expression in xenograft tumors was detected by qRT-PCR, normalized to GAPDH. e Histopathology of xenograft tumors. The tumor sections were under HE staining and immunohistochemical staining using antibodies against Ki-67. f Ki-67 index calculated as the percentage of Ki-67-positive cells. g Representative images of lung metastasis and HE staining of sections. The metastasis was marked with box. Scale bar = 200 μm; NC negative control, HE hematoxylin and eosin, qRT-PCR quantitative real-time PCR; **p < 0.05, ***p < 0.01

Fig. 6. Identification of AKR1B10 as a downstream target of linc00665 in LUAD cells.

a RNA transcriptome sequencing analysis was performed to analyze gene expression profiling in A549 cells following linc00665 knockdown. b Gene ontology analysis for all genes with altered expressions. c The altered mRNA levels of genes were selectively confirmed by qRT-PCR in A549 cells following linc00665 knockdown. d mRNA level of AKR1B10 was confirmed by qRT-PCR in A549 and H1299 cells following linc00665 knockdown or overexpression. e Protein level of AKR1B10 was confirmed by western blot in A549 and H1299 cells following linc00665 knockdown or overexpression. f Correlation between linc00665 and AKR1B10 expression in 80 LUAD samples. g, h Representative images and quantification of transwell invasion assays in A549 and H1299 cells after transfection with linc00665 overexpression plasmid or AKR1B10 siRNA. i CCK-8 proliferation assays in A549 and H1299 cells after transfection with linc00665 overexpression plasmid or AKR1B10 siRNA. j Expression of AKR1B10, ERK, p-ERK, MMP2, Vimentin, and GAPDH protein in A549 and H1299 cells after transfection with linc00665 overexpression plasmid or AKR1B10 siRNA. Original uncropped western images are shown in Supplementary Figure 7. NC negative control, LUAD, lung adenocarcinoma, qRT-PCR quantitative real-time PCR; *p < 0.05, **p < 0.01

Fig. 7. Linc00665 upregulates AKR1B10 by competitively binding to miR-98.

a Correlation between miR-98 and linc00665 or AKR1B10 expression in 80 LUAD samples. b Relative miR-98 expression in A549 and H1299 cells following linc00665 knockdown or overexpression. c Relative linc00665 or AKR1B10 expression in A549 and H1299 cells after transfection with miR-98 mimics. d Putative binding sites of miR-98 to linc00665/AKR1B10, and schematic of wild-type and mutant pmirGLO-linc00665/pmirGLO-AKR1B10 constructs. e miR-98 mimics or mimics NC and pmirGLO-linc00665-WT or pmirGLO-linc00665-MUT were co-transfected into 293T, A549, and H1299 cells. Luciferase activity was detected 24 h after transfection using the dual-luciferase assay. f miR-98 mimics or mimics NC and pmirGLO-AKR1B10-WT or pmirGLO-AKR1B10-MUT were co-transfected into 293T, A549, and H1299 cells. Luciferase activity was detected 24 h after transfection using the dual-luciferase assay. g, h Biotin-labeled RNA pull down assays were performed to confirm the binding of linc00665 with miR-98 in A549 and H1299 cells. A significant amount of linc00665 and miR-98 were observed in linc00665-probe pulled down pellets. i Expression of AKR1B10 and GAPDH protein in A549 and H1299 cells after transfection with miR-98 mimics, miR-98 inhibitors, or linc00665 overexpression plasmid. NC negative control, NS not significant, LUAD, lung adenocarcinoma; *p < 0.05, **p < 0.01, ***p<0.001.

Fig. 8. Expression of linc00665 was upregulated by transcription factor SP1.

a, b Validation of SP1 siRNA knockdown and overexpression vector efficiency in A549 cells as determined by qRT-PCR and western Blot. c Relative linc00665 expression in A549 cells after transfected with SP1 siRNA or overexpression plasmid. d ChIP assay was used to detect the binding of SP1 protein to linc00665 promoter in A549 and H1299 cells. e, f The sequence –42 to –33-bp upstream of linc00665 was detected in SP1 immunoprecipitates, which was further enhanced upon overexpressing SP1 in A549 and H1299 cells, as determined by ChIP assay. g Expression levels of SP1 mRNA in the 80 paired LUAD tissues by qRT-PCR. h Correlation between SP1 and linc00665 expression in 80 LUAD samples. i Schematic diagram of linc00665-based regulatory mechanism in LUAD cells. NC negative control, NS not significant, LUAD, lung adenocarcinoma, ChIP chromatin immunoprecipitation, qRT-PCR quantitative real-time PCR; **p < 0.01