LncRNA B4GALT1-AS1 promotes non-small cell lung cancer cell growth via increasing ZEB1 level by sponging miR-144-3p

Abstract

Background: Long noncoding RNAs (lncRNAs) are emerging as key players in the development and progression of cancer. Several malignancies involve dysregulated long noncoding ribonucleic acids (lncRNAs) in non-small cell lung cancer cell growth and their aggressive phenotypes. LncRNA B4GALT1-AS1 is important in the advancement of various malignancies, although its contribution to non-small cell lung cancer (NSCLC) remains unexplored.

Methods: LncRNA B4GALT1-AS1 in NSCLC tissues was detected and further validated in a cohort of non-small cell lung cancer tissues. The effects of lncRNA B4GALT1-AS1 on proliferation were determined by in vitro experiments. The B4GALT1-AS1-miR-144-3p-ZEB1 axis was assessed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Furthermore, the mechanism of B4GALT1-AS1 was investigated using loss-of-function assays in vitro.

Results: We showed significant upregulation of B4GALT1-AS1 in cell lines and tissues of NSCLC. B4GALT1-AS1 knockdown impeded the in vitro proliferation-related characteristics of the NSCLC cells. The demonstration of the binding capacity of B4GALT1-AS1 and miR-144-3p was predicted by bioinformatics and luciferase reporter activity assay. The B4GALT1-AS1 and miR-144-3p interaction was shown by using rescue experiments. NSCLC has a positive association with its target, zinc finger e-box binding homeobox 1 (ZEB1).

Conclusions: In summary, the progression of NSCLC was facilitated by lncRNA B4GALT1-AS1 via interaction with miR-144-3p and positive regulation of ZEB1 expression.

Keywords: B4GALT1-AS1; Zinc finger e-box binding homeobox 1 (ZEB1); metastasis; miR-144-3p; non-small cell lung cancer (NSCLC).

Figure 1

Upregulation of B4GALT1-AS1 in NSCLC. (A) The level of B4GALT1-AS1 in NSCLC tissues and equivalent normal tissues (n=36) was estimated through qRT-PCR assay; (B) detection of B4GALT1-AS1 level in NSCLC metastatic (n=14) and non-metastatic tissues (n=22) by qRT-PCR; (C) representative correlation between expression of B4GALT1-AS1 and stages I (n=20) or II–III (n=16); (D) qRT-PCR estimation of B4GALT1-AS1 level in NSCLC cell lines and BEAS-2B cell. *, P<0.05; **, P<0.01; ***, P<0.001. NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Figure 2

The growth and metastatic- traits of NSCLC cells are promoted by B4GALT1-AS1. (A) Knockdown efficiency assessment by qRT-PCR in the A549 and H1299 cell lines after siB4GALT1-AS1 transfection. The internal control for the detection of B4GALT1-AS1 expression was GAPDH; (B) cell viability assessment through MTT assay after transfecting A549 and H1299 cells with siB4GALT1-AS1; (C,D) assessment of the level of KI67 and PCNA mRNA level in NSCLC cell with knockdown of B4GALT1-AS1. *, P<0.05; ***, P<0.001. NSCLC, non-small cell lung cancer.

Figure 3

LncRNA B4GALT1-AS1 binds with miR-144-3p in NSCLC cell. (A) The binding sites between miR-144-3p and WT-B4GALT1-AS1 (B4GALT1-AS1 wide-type) or MUT-B4GALT1-AS1 (the mutated type) are shown; (B) detection of the transcript levels of miR-144-3p by qRT-PCR; (C) dual-luciferase reporter assays to determine the activity of luciferase in 293T cells transfected with miR-144-3p as well as luciferase reporters with WT-B4GALT1-AS1 or MUT-B4GALT1-AS1; (D) precipitation of ZEB1, and the miR-144-3p complex in AGO2. Detection of the transcript levels of miR-144-3p and ZEB1 by qRT-PCR; (E) qRT-PCR detection of miR-144-3p expression in NSCLC and equivalent normal tissues; (F) evaluation of B4GALT1-AS1, and miR-144-3p correlation. The endogenous controls U6 and GAPDH for the detection of miR-144-3p and B4GALT1-AS1. *, P<0.05; ***, P<0.001; ^#, P>0.05. NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Figure 4

The NSCLC cell aggressiveness is regulated by B4GALT1-AS1 via miR-144-3p/ZEB1 axis modulation. (A) The sites of binding between miR-144-3p and WT-ZEB1 (ZEB1 wide-type) or MUT-ZEB1 (the mutated type) are shown; (B) Luciferase reporter assay to assess luciferase activities of HEK293 cells co-transfected with luciferase reporters containing ZEB1 3'-UTR WT or ZEB1 3'-UTR MUT and miR-144-3p mimics; (C) qRT-PCR to determine the ZEB1 levels in miR-144-3p mimics or NC mimics-transfected A549 and H1299 cells; (D) qRT-PCR to determine the miR-144-3p levels in miR-144-3p inhibitor or NC inhibitor-transfected A549 and H1299 cells; (E) qRT-PCR analysis to estimate ZEB1 levels in indicated treated A549 and H1299 cells. *, P<0.05; ***, P<0.001; ^#, P>0.05. NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Figure 5

A diagram that lncRNA B4GALT1-AS1 promotes the NSCLC cell metastasis via increasing ZEB1 level by sponging miR-144-3p. NSCLC, non-small cell lung cancer.