Long Noncoding RNA LINC01006 Facilitates Cell Proliferation, Migration, and Epithelial-Mesenchymal Transition in Lung Adenocarcinoma via Targeting the MicroRNA 129-2-3p/CTNNB1 Axis and Activating Wnt/β-Catenin Signaling Pathway

Abstract

Lung adenocarcinoma (LUAD) is a common type of malignancy of lung cancers. Long intergenic noncoding RNAs (lincRNAs) have emerged as crucial regulators of various cancers, including LUAD. LINC01006 is a newly discovered long noncoding RNA (lncRNA) whose function in LUAD remains to be explored. This study is to explore the role of LINC01006 in LUAD. Quantitative real-time PCR (RT-qPCR) analysis and Western blotting were used to determine the expression levels and protein levels, respectively. Functional assays and animal experiments investigated the role of LINC01006 both in vivo and in vitro. Moreover, TOP/FOP assay was performed to detect the activation of the Wnt/β-catenin signaling pathway. The interaction between LINC01006 and microRNA 29-2-3-p (miR-29-2-3-p)/catenin beta 1 (CTNNB1) was explored by RNA binding protein immunoprecipitation (RIP), RNA pulldown, luciferase reporter assays, and rescue experiments. According to the results, LINC01006 was highly expressed in LUAD tissues and cell lines. LINC01006 knockdown significantly suppressed cell proliferative, migratory, and epithelial-mesenchymal transition (EMT) capacities and tumor development. Moreover, LINC01006 enhanced CTNNB1 via sequestering miR-129-2-3p and activated the Wnt/β-catenin pathway in LUAD. Overall, LINC01006 promotes LUAD development via activating the Wnt/β-catenin pathway, implying that LINC01006 might be a promising biomarker for LUAD treatment.

Keywords: CTNNB1; LINC01006; LUAD; Wnt/β-catenin pathway; miR-129-2-3p.

FIG 1

LINC01006 promotes cell proliferation, migration, and EMT in LUAD. (A) The expression of LINC01006 in healthy lung tissues and LUAD tumor tissues was analyzed by examining the GEPIA database (http://gepia2.cancer-pku.cn/#index). (B) LINC01006 expression in human healthy lung epithelial cell line (BEAS-2B) and LUAD cell lines (PC9, H1650, H1975, and A549) was evaluated by RT-qPCR. (C) RT-qPCR was applied to assess the knockdown efficiency of sh-LINC01006#1, sh-LINC01006#2, and sh-LINC01006#3. (D) Colony formation assay was implemented to investigate the proliferative capacity of LUAD cells. (E) Cell migration before and after LINC01006 knockdown was examined by transwell assay. (F) The protein levels of epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin and vimentin) were analyzed by Western blotting. *, P < 0.05; **, P < 0.01.

FIG 2

Depletion of LINC01006 inhibits LUAD tumor growth in vivo. (A) Tumor growth curve displayed the tumor volume and tumor growth rate. (B) Tumor weight in sh-NC and sh-LINC01006#2 groups was assessed. **, P < 0.01.

FIG 3

Downregulation of LINC01006 inactivates the Wnt/β-catenin signaling in LUAD cells. (A) Key proteins (β-catenin, p65, p-p65, AKT, and p-AKT) in common signaling pathways were measured by Western blot assay when LINC01006 was knocked down; GAPDH served as the internal reference. (B) Western blot assay was performed to assess the expression of protein markers (β-catenin, c-myc, and cyclin D1) in the Wnt/β-catenin signaling pathway. β-Actin was used as the internal reference. (C) The TOP/FOP flash assay was conducted to measure the activity of Wnt/β-catenin signaling pathway in H1975 and A549 cells transfected with sh-NC or sh-LINC01006#2. **, P < 0.01.

FIG 4

LINC01006 acts as a sponge for miR-129-2-3p to upregulate CTNNB1. (A) Subcellular fractionation was performed to locate LINC01006 in LUAD cells. (B) The effect of LINC01006 downregulation on the relative expression level of CTNNB1 was evaluated by RT-qPCR analysis. (C) Four potential miRNAs binding to LINC01006 and CTNNB1 were discovered with the application of bioinformatics. (D) miR-129-2-3p was screened out as the miRNA that could bind to both LINC01006 and CTNNB1 through RNA pulldown assay. (E) The expression of miR-129-2-3p in healthy lung epithelial cell line (BEAS-2B) and LUAD cell lines (PC9, H1650, H1975, and A549) was examined. (F) RIP assay was implemented to demonstrate the coexistence of LINC01006, miR-129-2-3p, and CTNNB1 in RNA-induced silencing complexes (RISCs). (G) RIP assays researching the enrichment of LINC01006 were carried out when miR-129-2-3p was overexpressed or downregulated in LUAD cells. (H) RIP assays researching the enrichment of CTNNB1 were done when the expression of LINC01006 or miR-129-2-3p was inhibited in LUAD cells. (I) Luciferase reporter assay was carried out to verify the interaction between miR-129-2-3p and LINC01006 or CTNNB1 in HEK-293T or LUAD cells. (J) RT-qPCR was performed to detect the relative expression level of LINC01006 after either the overexpression or downregulation of miR-129-2-3p. (K) Copy numbers of LINC01006, miR-129-2-3p, and CTNNB1 in H1975 and A549 cells were measured by RT-qPCR. **, P < 0.01.

FIG 5

LINC01006 promotes proliferation, migration, and EMT of LUAD cells by acting as a sponge for miR-129-2-3p. (A) Colony formation assay was used to evaluate A549 cell proliferation. (B) Cell migration was appraised by transwell assay. (C) Western blotting was conducted to analyze EMT-related protein levels in A549 cells. (D) The mRNA and protein levels of CTNNB1 were evaluated by RT-qPCR and Western blot analyses. (E) Colony formation assay was used to evaluate A549 cell proliferation in the presence of wild-type and mutant LINC01006 after the transfection of sh-LINC01006#2. (F) The migratory ability of A549 cells was appraised by transwell assay in the presence of wild-type and mutant LINC01006 after the transfection of sh-LINC01006#2. (G) Western blotting was performed to analyze EMT-related protein (E-cadherin, N-cadherin, and vimentin) levels in different groups. **, P < 0.01.

FIG 6

LINC01006 facilitates proliferation, migration, and EMT of LUAD cells via enhancing CTNNB1 to activate the Wnt/β-catenin pathway. (A) Colony formation assay was carried out to evaluate the proliferative ability of A549 cells in different groups: pcDNA3.1, pcDNA3.1/CTNNB1, sh-NC, sh-LINC01006#2, sh-LINC01006#2 plus CTNNB1-WT, sh-LINC01006#2 plus CTNNB1-Mut, sh-LINC01006#2 plus CTNNB1-WT plus miR-129-2-3p-mimics and sh-LINC01006#2 plus CTNNB1-Mut plus miR-129-2-3p-mimics. (B) Transwell assay was performed to assess cell migration in the above different groups. (C) E-cadherin, N-cadherin, and vimentin protein levels in the above different groups were measured via Western blotting. (D) The protein levels of β-catenin, c-myc, and cyclin D1 in Wnt/β-catenin signaling pathways in the above different groups were measured by Western blotting. **, P < 0.01.