LncRNA LINC00511 promotes COL1A1-mediated proliferation and metastasis by sponging miR-126-5p/miR-218-5p in lung adenocarcinoma

Abstract

Background: Lung adenocarcinoma (LUAD) is currently the leading cause of cancer-related death worldwide. Long noncoding RNAs (lncRNAs) play key roles in tumor occurrence and development as crucial cancer regulators. The present study aimed to explore the molecular mechanism and regulatory network of Linc00511 in LUAD and to identify new potential therapeutic targets for LUAD.

Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to determine the relative Linc00511 levels in LUAD tissues and cells. The proliferation, apoptosis, migration, and invasion abilities of LUAD cells were assessed by a Cell Counting Kit-8 (CCK-8) assay, a colony formation assay, flow cytometry, and a Transwell assay. Changes in hsa_miR-126-5p, hsa_miR-218-5p, and COL1A1 expression were analyzed using western blotting and RT-qPCR. Targeted binding between miR-126-5p/miR-218-5p and Linc00511 or COL1A1 was verified with a luciferase reporter system and confirmed by an RNA pulldown assay. The participation of the PI3K/AKT signaling pathway was confirmed via western blotting. Xenograft animal experiments were performed to detect the impact of Linc00511 on LUAD tumor growth in vivo.

Results: In the present work, we observed that Linc00511 was upregulated in LUAD tissues and cells. Loss/gain-of-function experiments indicated that knockdown of Linc00511 significantly inhibited LUAD cell proliferation, migration and invasion and promoted LUAD cell apoptosis, whereas overexpression of Linc00511 showed the opposite effects. In addition, we determined that Linc00511 promoted COL1A1-mediated cell proliferation and cell motility by sponging miR-126-5p and miR-218-5p. Moreover, Linc00511 activated the PI3K/AKT signaling pathway through upregulation of COL1A1. Finally, silencing of Linc00511 inhibited LUAD tumor growth in vivo.

Conclusions: Linc00511 acts as a competing endogenous RNA to regulate COL1A1 by targeting miR-126-5p and miR-218-5p, thereby promoting the proliferation and invasion of LUAD cells.

Keywords: COL1A1; Linc00511; Lung adenocarcinoma; Proliferation/metastasis; miR-126-5p; miR-218-5p.

Fig. 1

Linc00511 is upregulated in lung adenocarcinoma tissues and cells. A The relative expression of Linc00511 in lung adenocarcinoma tissues and adjacent normal tissues (n = 35) was measured by RT–qPCR. B RT–qPCR was used to measure the relative Linc00511 levels in BEAS-2B, A549 and PC9 cells. *P < 0.05, **P < 0.01, ***P < 0.001 versus normal or BEAS-2B cells

Fig. 2

Linc00511 downregulation inhibits LUAD cell proliferation, migration, and invasion and promotes LUAD cell apoptosis. A The Linc00511 knockdown efficiency in A549 and PC9 cells was determined by RT–qPCR. The proliferation and viability of A549 and PC9 cells after silencing Linc00511 was evaluated by CCK-8 (B) and colony formation assays (C). D The impact of Linc00511 silencing on A549 and PC9 cell apoptosis was assessed by flow cytometric analysis. Transwell assays were used to verify the changes in the migratory (E) and invasive (F) abilities of A549 and PC9 cells after Linc00511 knockdown. *P < 0.05, **P < 0.01, ***P < 0.001 versus si-nc, Scale bars, 50 μm

Fig. 3

Linc00511 overexpression enhances the proliferation, migration and invasion of LUAD cells. A The relative expression of Linc00511 in A549 and PC9 cells transfected with oe-nc and oe-lin00511 was determined by RT–qPCR. B–E The proliferation ability of A549 and PC9 cells after overexpression of Linc00511 was examined by a CCK-8 assay (B, C) and a colony formation assay (D, E). F–I Transwell assays were performed to determine the changes in the migratory (F, G) and invasive (H, I) abilities of A549 and PC9 cells after Linc00511 overexpression. **P < 0.01, ***P < 0.001 versus oe-nc

Fig. 4

Linc00511 interacts with miR-126-5p and miR-218-5p. A After Linc00511 knockdown, the levels of nine differentially expressed miRNAs in lung adenocarcinoma were determined by RT–qPCR. B The potential targeting sequences of miR-126-5p and miR-128-5p in Linc00511 were predicted, and a schematic of the wild-type and mutant Linc00511 constructs is shown. C Dual luciferase reporter vectors were used to test whether Linc00511 directly binds to miR-126-5p and miR-218-5p. Luciferase activity was measured in A549 cells cotransfected with Linc00511-WT or Linc00511-MUT and the miR-126-5p mimic or miR-218-5p mimic. D An RNA pulldown assay revealed the interactions of Linc00511 with miR-126-5p and miR-218-5p. E Measurement of miR-126-5p and miR-218-5p mRNA levels in BEAS-2B, A549, and PC9 cells. **P < 0.01, ***P < 0.001 versus the si-nc, miR-NC, BEAS-2B, or Biotin-nc group

Fig. 5

Linc00511 regulates COL1A1 expression by targeting miR-126-5p or miR-218-5p. A The Venn diagram shows the overlapping genes coregulated by miR-126-5p and miR-218-5p that were predicted by the TargetScan and starBase databases. B Gene ontology (GO) enrichment analysis of miR-126-5p and miR-218-5p target genes. C Prediction of miR-126-5p and miR-218-5p binding sites in the COL1A1 mRNA 3’-UTR. D Dual luciferase reporter vectors were used to verify the interactions of miR-126-5p and miR-218-5p with COL1A1. E An RNA pulldown assay confirmed the relationships between COL1A1 and both miR-126-5p and miR-218-5p. F The relative COL1A1 expression levels were examined in BEAS-2B, A549 and PC9 cells by RT–qPCR. G COL1A1 mRNA and H protein expression in A549 cells transfected with si-linc00511, anti-nc + si-linc00511, anti-miR-218-5p + si-linc00511, or anti-miR-126-5p + si-linc00511. **P < 0.01, ***P < 0.001 versus the miR-NC, BEAS-2B or Biotin-nc group; ^#P < 0.05 versus the si-linc00511 + anti-nc group

Fig. 6

Linc00511 promotes the proliferation, migration and invasion of lung adenocarcinoma cells by regulating COL1A1 expression. A Measurement of the COL1A1 overexpression efficiency by RT–qPCR in A549 and PC9 cells. B–F Cell proliferation (B), colony formation (C), apoptosis (D), migration (E) and invasion (F) were evaluated in A549 and PC9 cells transfected with si-nc, si-Linc00511, si-Linc00511 + vector or si-Linc00511 + COL1A1. G The PI3K, phosphorylated PI3K (p-PI3K), AKT, and phosphorylated AKT (p-AKT) levels were determined by western blot analysis in LUAD cells with Linc00511 silencing or forced COL1A1 expression. * P < 0.05, ** P < 0.01, ***P < 0.001 versus the si-nc group; ^# P < 0.05, ^## P < 0.01, ^###P < 0.001 versus the si-linc00511 + vector group

Fig. 7

Knockdown of Linc00511 expression inhibits tumor growth in vivo. A Representative images of tumors in mice injected with Ad-sh-NC/A549, Ad-sh-linc00511 + Ad-NC/A549 and Ad-sh-linc00511 + Ad-COL1A1/A549. B Tumor growth curves of mice after injection of Ad-sh-NC/A549, Ad-sh-linc00511 + Ad-NC/A549 and Ad-sh-linc00511 + Ad-COL1A1/A549. C Tumor weights were measured at the experimental endpoint. ^##P < 0.01, ^###P < 0.001 versus the Ad-sh-linc00511 + Ad-NC group; **P < 0.01, ***P < 0.001 versus the Ad-sh-NC group