miR-486-3p Suppresses Malignant Phenotypes and is Associated with Reduced WNT5B, DVL1, and β-catenin Expression in Lung Adenocarcinoma Cells

Summary

Forced miR-486-3p overexpression suppresses proliferation-associated activity, clonogenic growth, wound closure, and Matrigel traversal in A549 and H358 lung adenocarcinoma cells. These effects accompany reduced expression of WNT5B, DVL1, β-catenin, and BCL-2, indicating an in vitro association with Wnt-related changes without establishing direct targeting or pathway causality.

Abstract

Lung adenocarcinoma (LUAD) is the predominant histological subtype of non-small cell lung cancer and remains associated with substantial morbidity and mortality. Previous studies have identified tumor-suppressive functions of miR-486-3p in LUAD, but its relationship with WNT5B, DVL1, and β-catenin-related expression changes has not been clearly defined. Here, baseline miR-486-3p abundance was examined in A549 and H358 LUAD cells and compared with that in Beas-2b bronchial epithelial cells maintained under their respective routine conditions. Stable miR-486-3p-overexpressing models were generated by lentiviral transduction. Cell growth-associated activity, clonogenic growth, wound closure, and traversal of Matrigel-coated membranes were evaluated using Cell Counting Kit-8, colony-formation, wound-healing, and Transwell assays. WNT5B, DVL1, β-catenin, and BCL-2 protein abundance were assessed by Western blotting, and WNT5B, DVL1, CTNNB1, and BCL2 mRNA levels were additionally examined by RT-qPCR. Under the culture conditions used, miR-486-3p abundance was lower in A549 and H358 cells than in Beas-2b cells, and lentiviral transduction produced stable overexpression in both LUAD cell lines. Forced miR-486-3p overexpression reduced the CCK-8 signal, colony formation, wound closure, and Matrigel-coated membrane traversal and was accompanied by lower WNT5B, DVL1, total β-catenin, and BCL-2 protein abundance. RT-qPCR further showed lower WNT5B, DVL1, CTNNB1, and BCL2 transcript abundance in both LUAD cell lines after miR-486-3p overexpression. These findings support an in vitro association between forced miR-486-3p expression, attenuation of several malignant-cell readouts, and altered Wnt-related expression; however, they do not establish direct miRNA-target binding, pathway activity, or a causal linear signaling mechanism.

Introduction

Lung adenocarcinoma (LUAD) is the most common histological subtype of non-small cell lung cancer (NSCLC) and contributes substantially to lung cancer-related morbidity and mortality worldwide^1,2,3,4,5. Molecular classification and targeted therapies have improved outcomes for selected patient groups, but advanced LUAD remains difficult to treat and is biologically heterogeneous^1,2,3,4,5. Defining regulatory processes that contribute to malignant cell behavior, therefore, remains relevant to the development of testable therapeutic hypotheses.

Wnt signaling comprises β-catenin-dependent and β-catenin-independent branches that regulate proliferation, survival, polarity, and motility^6,7,8,9. WNT5B is most often associated with noncanonical signaling, although its biological effects vary with receptor and cellular context^10,11. DVL1 is a cytoplasmic signal-transduction protein shared by several Wnt branches and has been linked to β-catenin-related behavior in lung cancer¹². Accordingly, concurrent changes in WNT5B, DVL1, and β-catenin should not by themselves be interpreted as evidence for a single linear pathway.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression through sequence-dependent interactions with target transcripts^13,14,15. Because one miRNA may affect multiple RNAs, altered miRNA abundance can influence diverse tumor-associated phenotypes^{13,14,15,16,17,18}. Several miRNAs have been linked to lung cancer growth, motility, and signaling, and crosstalk between miRNAs and Wnt-related networks has been described in multiple cancer contexts^{16,17,18,19,20}.

The MIR486 locus gives rise to the mature strands miR-486-3p and miR-486-5p. miR-486-3p has shown tumor-suppressive activity in oral cancer and glioblastoma, whereas a tumor-promoting effect has been reported in cutaneous squamous cell carcinoma^{21,22,23,24,25}. miR-486-3p was selected for the present study through a hypothesis-driven strategy based on its reduced abundance in public LUAD data, its previously reported tumor-suppressive activity in LUAD, and bioinformatic predictions linking it to WNT-related candidate transcripts. Tomioka et al. demonstrated tumor-suppressive effects of both miR-486 strands in LUAD and identified GINS4 through genome-wide target screening²⁶. Therefore, the present study does not claim the first demonstration of an antitumor phenotype. Instead, it evaluates whether forced miR-486-3p expression is accompanied by changes in WNT5B, DVL1, and β-catenin abundance and by reproducible phenotypic changes in two LUAD cell lines. These analyses assess associations and do not establish direct targeting, pathway activity, or a linear WNT5B/DVL1/β-catenin mechanism.

Protocol

The reagents and the equipment used are listed in the Table of Materials.

Cell culture

Human lung adenocarcinoma (LUAD) cell lines A549 and H358, and the immortalized bronchial epithelial cell line Beas-2b were used. A549 (KRAS G12S) and H358 (KRAS G12C; STK11-deficient) were selected as commonly used LUAD models with distinct KRAS-mutant backgrounds, whereas Beas-2b was included as a non-malignant bronchial epithelial comparator. A549 and H358 cells were maintained in RPMI-1640 medium, and Beas-2b cells were maintained in DMEM; each medium contained 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. Because the tumor and epithelial comparator cell lines were maintained in different routine media, cross-cell-line baseline expression comparisons were interpreted with caution.

Lentiviral transduction and stable cell selection

Lentiviral vectors carrying the hsa-miR-486-3p precursor (LV-miR-486-3p) or a scrambled negative-control sequence (LV-NC) were used in a stable gain-of-function design. A549 and H358 cells were seeded in 6-well plates at 5 × 10⁴ cells/well and cultured overnight. The following day, cells were exposed to lentivirus at a multiplicity of infection (MOI) of 10 in the presence of 8 µg/mL polybrene. After 24 h, the viral inoculum was replaced with fresh complete medium. Transduced cells were selected with 2 µg/mL puromycin for 7 days. After selection, surviving cells were allowed to recover and were expanded for the subsequent molecular and functional assays. No miR-486-3p inhibitor arm was included because the present study was designed specifically to evaluate the effects of stable forced overexpression.

NOTE: The MOI, polybrene exposure time, and puromycin selection conditions must be kept identical between the LV-NC and LV-miR-486-3p groups to minimize treatment-related differences in cell viability.

RT-qPCR

RT-qPCR was conducted to determine miR-486-3p expression levels and to confirm the efficiency of lentiviral overexpression in A549 and H358 cells. Total RNA was isolated using TRIzol reagent in accordance with the manufacturer's instructions. RNA concentration and purity were evaluated spectrophotometrically, with an A260/A280 ratio of 1.8–2.1 considered acceptable. For mRNA analysis, 1 µg of total RNA was reverse-transcribed into cDNA using a commercial cDNA synthesis kit according to the manufacturer's protocol. For microRNA analysis, cDNA synthesis was performed using a miRNA-specific stem-loop reverse transcription primer. Quantitative PCR was carried out in a total reaction volume of 20 µL containing 10 µL of 2× SYBR Green Master Mix, 0.4 µM of each primer, and 2 µL of cDNA template. The amplification protocol consisted of an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. A melting curve analysis was subsequently performed to verify amplification specificity. U6 small nuclear RNA served as the internal control for miRNA quantification, whereas GAPDH was used as the reference gene for mRNA normalization. Relative expression levels were determined using the 2^−ΔΔCq method.

The miRNA primer sequences were as follows: stem-loop reverse-transcription primer, 5′-GTCGTATCGACTGCAGGGTCCGAGGTATTCGCAGTCGATACGAC
ATCCTG-3′; miR-486-3p forward primer, 5′-CGCGGGGCAGCTCAGTA-3′; universal reverse primer, 5′-ACTGCAGGGTCCGAGGTATT-3′; U6 forward primer, 5′-CTCGCTTCGGCAGCACA-3′; and U6 reverse primer, 5′-AACGCTTCACGAATTTGCGT-3′. Primer sequences for WNT5B, DVL1, CTNNB1, BCL2, and GAPDH, together with amplicon lengths and amplification efficiencies, are provided in Table 1. mRNA expression was normalized to GAPDH, whereas miR-486-3p expression was normalized to U6. Each experiment included three independent biological replicates, with three technical replicates per biological replicate.

Western blotting

After completion of the 7-day puromycin selection and a recovery period, cells were lysed in RIPA buffer containing protease inhibitors. Protein concentrations were determined using a BCA protein assay. Equal amounts of protein (20 µg per lane) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes by wet transfer at 300 mA for 120 min. The membranes were blocked with 5% skim milk prepared in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies against WNT5B (1:1,000), DVL1 (1:1,000), β-catenin (1:1,000), BCL-2 (0.5 µg/mL), or β-actin (1:5,000). The manufacturers and catalog numbers of all antibodies are provided in the Table of Materials. After three 10-min washes with TBST, the membranes were incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody diluted 1:5,000 for 1 h at room temperature. The membranes were then washed three times with TBST, and protein bands were visualized using a chemiluminescent substrate and imaged with a gel documentation and imaging system. Band intensities were quantified using ImageJ. The abundance of each target protein was normalized to the corresponding β-actin signal from the same biological sample. Only exposures within the linear, non-saturated detection range were used for densitometric analysis. Three independent biological experiments were performed.

Cell Counting Kit-8 assay

A549 and H358 cells stably transduced with LV-NC or LV-miR-486-3p were seeded in 96-well plates at 1.5 × 10⁴ cells/well in 100 µL of complete medium. After overnight attachment, the medium was replaced, and the 0-h absorbance was measured immediately after the addition of Cell Counting Kit-8 reagent. At 0 h, 24 h, 48 h, 72 h, and 96 h, 10 µL of reagent was added per well and incubated for 1 h at 37 °C before absorbance was read at 450 nm. For the revised time-course analysis, each group was normalized to its own 0-h value before plotting. Three independent biological experiments were performed, and technical wells within each experiment were averaged before statistical analysis.

Colony formation assay

Stably selected A549 and H358 cells were prepared as single-cell suspensions at 5 × 10² cells/mL. One milliliter of cell suspension was seeded into each well of a 6-well plate, followed by 3 mL of complete medium. Cells were cultured for 14 days, with medium replacement every 5 days. Colonies were washed with PBS, fixed with 4% paraformaldehyde for 20 min, stained with crystal violet for 20 min, washed, and air-dried. Colonies containing at least 50 cells were counted in ImageJ. Three independent biological experiments were performed.

NOTE: A uniform single-cell suspension and minimal plate disturbance during colony establishment are required to prevent artificial clustering of cells.

Cell migration assay

Stably transduced A549 and H358 cells were seeded in 6-well plates and cultured until a confluent monolayer formed. A straight scratch was generated with a sterile 10 µL pipette tip, detached cells were removed, and the same marked fields were imaged at 0 h, 24 h, 48 h, and 72 h. Wound area was measured in ImageJ, and wound closure was calculated as (area at 0 h − area at the indicated time point) / area at 0 h × 100%. Three independent biological experiments were performed. No pharmacologic proliferation blocker was used; therefore, this assay was interpreted as a measurement of wound closure rather than a migration-specific readout.

NOTE: Scratches should be generated with consistent width and pressure, and identical fields should be followed throughout the time course.

Cell invasion assay

Transwell invasion assays were performed using 24-well polycarbonate membrane inserts with an 8 µm pore size. The upper surface of each insert was coated with 100 µL of Matrigel basement membrane matrix diluted 1:8 in serum-free DMEM to an approximate final protein concentration of 1 mg/mL and incubated at 37 °C for 4 h. A549 and H358 cells were resuspended in serum-free RPMI-1640 medium at densities of 8 × 10⁵ cells/mL and 4 × 10⁵ cells/mL, respectively. A 200 µL aliquot of the cell suspension was added to the upper chamber, corresponding to 1.6 × 10⁵ A549 cells or 8 × 10⁴ H358 cells per insert. The lower chamber was filled with 500 µL of RPMI-1640 medium containing 20% fetal bovine serum as a chemoattractant. After incubation at 37 °C in a humidified atmosphere containing 5% CO₂ for 48 h, non-invading cells remaining on the upper surface of the membrane were gently removed with a cotton swab. Cells that had invaded through the membrane were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 20 min at room temperature. Five randomly selected microscopic fields were imaged for each insert, and the number of invaded cells was quantified using ImageJ. The mean cell count from the five fields was considered one value for each insert, and the insert, rather than an individual microscopic field, was treated as the experimental unit. Three independent biological experiments were performed.

NOTE: Remove the non-invading cells gently and consistently to avoid damaging the membrane or dislodging cells that have migrated to its lower surface.

Statistical analysis

Data are presented as mean ± SD, and n denotes independent biological experiments unless otherwise specified. Technical replicates or multiple microscopic fields from the same experimental unit were averaged before inferential testing. For the three-cell-line comparison, one-way analysis of variance followed by Tukey-adjusted pairwise comparisons were used. For CCK-8 and wound-closure time courses, two-way repeated-measures analysis of variance was used to evaluate treatment, time, and treatment-by-time interaction effects, followed by Šídák-adjusted comparisons at individual time points. Two-group comparisons were analyzed using two-sided unpaired Student’s t-tests. Statistical analyses and data visualization were performed using statistical analysis and graphing software. Exact p-values are reported in the figure panels or source-data tables. Statistical significance was defined as p < 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

TCGA/starBase analysis shows reduced miR-486-3p abundance in LUAD, and TargetScan identifies candidate binding sites

TCGA-derived expression data accessed through starBase v3.0 showed that miR-486-3p abundance was significantly lower in 512 lung adenocarcinoma (LUAD) samples than in 20 non-tumor lung samples (Wilcoxon rank-sum test, p = 3.2 × 10⁻⁵; Figure 1). TargetScanHuman identified a poorly conserved candidate 7mer...

Discussion

The present gain-of-function study evaluated the effects of stable, forced miR-486-3p expression in A549 and H358 lung adenocarcinoma (LUAD) cells. In both models, miR-486-3p overexpression was associated with reduced growth-associated CCK-8 signals, decreased clonogenic growth, lower wound closure, and fewer cells traversing Matrigel-coated Transwell membranes. These phenotypic changes were accompanied by reduced WNT5B, DVL1, total β-catenin, and BCL-2 protein abundance, together with lower WNT5B, DVL1...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 M Tris-HCl (pH 6.8)	Solarbio	T1020	Preparation of SDS-PAGE stacking gel
1.5 M Tris-HCl (pH 8.8)	Solarbio	T1010	Preparation of SDS-PAGE resolving gel
20× TBS buffer	Solarbio	71080	Stock buffer for western blot washing and antibody dilution
30% acrylamide/bis-acrylamide solution (29:1)	Solarbio	A1010	Preparation of SDS-PAGE gels
4% paraformaldehyde fixative	Servicebio	G1101	Fixation for colony-formation and Transwell assays
5× protein loading buffer	Beyotime	P0015	Protein sample preparation for SDS-PAGE
Ammonium persulfate	Solarbio	427A038	SDS-PAGE gel polymerization initiator
BCA Protein Assay Kit	Beyotime	P0009-1	Protein concentration determination
BCL-2 antibody	Boster Biological Technology	A00040-2	Rabbit polyclonal antibody; western blotting at 1:5,000
Cell Counting Kit-8 (CCK-8)	TargetMol	C0005	Cell growth-associated metabolic activity assay; 10 μL per 100 μL culture medium
Chemiluminescence imaging system	Bio-Rad	ChemiDoc MP	Western blot imaging
Chloroform	Servicebio	G3005	Phase separation during TRIzol-based RNA extraction
Crystal violet staining solution	Servicebio	G1014	Colony and Transwell membrane staining
DMEM	Servicebio	G4511	Routine culture medium for Beas-2b cells
DVL1 antibody	Signalway Antibody	53180	Rabbit primary antibody; western blotting at 1:5,000
Fetal bovine serum (FBS)	Biosharp	BL203B	10% (v/v) supplement for routine cell culture; 20% (v/v) in the Transwell lower chamber
GraphPad Prism 8	GraphPad Software	Version 8	Graph preparation and statistical analysis
Hieff qPCR SYBR Green Master Mix (No Rox)	Yeasen Biotechnology	11201ES08	SYBR Green-based quantitative PCR
Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus)	Yeasen Biotechnology	11141ES60	Reverse transcription for mRNA RT-qPCR
IBM SPSS Statistics	IBM	Version 22.0	Statistical analysis
ImageJ	National Institutes of Health	Version 1.53	Western blot densitometry, wound-area measurement, colony counting, and Transwell cell counting
Inverted microscope	Olympus	CKX53	Imaging of wound-healing and Transwell assays
Isopropanol	Servicebio	G3006	RNA precipitation during TRIzol-based extraction
LV-miR-486-3p and LV-NC lentiviral preparations	GenePharma	Custom synthesis	Stable miR-486-3p overexpression vector and scrambled negative-control vector
Matrigel basement membrane matrix	Corning	354234	Diluted 1:8 in serum-free DMEM; approximately 1 mg/mL final protein concentration
Methanol	Xilong Scientific	GB/T 683-2006	PVDF membrane activation
Microplate reader	BioTek	ELx800	Measurement of absorbance at 450 nm
Microvolume UV-Vis spectrophotometer	Allsheng	Nano-600	RNA concentration and A260/A280 purity assessment
Penicillin-streptomycin	Gibco, Thermo Fisher Scientific	15240062	1% (v/v) supplement for routine cell culture
Phosphate-buffered saline (PBS)	Servicebio	G0002	Cell and membrane washing
Protease Inhibitor Cocktail	APExBIO	K1007	Added to RIPA lysis buffer
Protein ladder	Biosharp	BL712A	Molecular-weight reference for western blotting
PVDF membrane	Millipore	K2MA8350E	Protein transfer membrane for western blotting
QuantStudio 5 Real-Time PCR System	Thermo Fisher Scientific	QuantStudio 5	RT-qPCR data acquisition
RIPA lysis buffer	Beyotime	P0013B	Total cellular protein extraction
RNA extraction reagent (TRIzol-type)	Servicebio	G3013	Total RNA extraction
RPMI 1640 medium	Servicebio	G4510	Routine culture medium for A549 and H358 cells and serum-free Transwell cell suspension
SDS	Solarbio	S8010	SDS-PAGE reagent
Skim milk powder	BD	2271470	5% blocking solution for western blotting
StarBase v3.0	Sun Yat-sen University	https://rnasysu.com/encori/	TCGA-derived miR-486-3p expression analysis
TargetScanHuman	Whitehead Institute for Biomedical Research	https://www.targetscan.org/vert_80/	Prediction of candidate miR-486-3p binding sites
TEMED	Sigma-Aldrich	T8090	SDS-PAGE gel polymerization catalyst
Transwell inserts, 24-well, 8 μm pore size	Corning	3428	Matrigel-coated membrane traversal assay
Trypsin	Servicebio	G4000	Cell detachment
Tween-20	Solarbio	815A043	Preparation of western blot wash buffer
Vertical electrophoresis system	Liuyi Biotechnology	DYCZ-24DN	SDS-PAGE protein separation
Western HRP substrate luminol reagent	Affinity Biosciences	KF001	Chemiluminescent western blot detection
Wet transfer system	Bio-Rad	1703930	Protein transfer to PVDF membrane
WNT5B antibody	Signalway Antibody	56808	Rabbit primary antibody; western blotting at 1:5,000
β-actin antibody	Abcam	ab8227	Loading control; western blotting at 1:5,000
β-catenin antibody	Signalway Antibody	21725	Rabbit primary antibody; western blotting at 1:5,000