MT1B overexpression enhances malignancy of non-small cell lung cancer cells

Abstract

Metallothioneins (MTs) are metal-binding proteins that are involved in heavy metal homeostasis and protection against oxidative stress. The MT1 family comprises several isoforms that are implicated in various diseases, including cancer. Although the dysregulated expression of MT1 isoforms has been observed in lung cancer, the specific role of MT isoform MT1B remains unclear. To investigate the role of MT1B in lung cancer progression, A549 lung cancer cells were transfected with an MT1B expression vector. In vitro assays were performed to assess cell viability, migration, invasion, and colony formation. Western blot analysis revealed increased expression of epithelial–mesenchymal transition (EMT) markers Snail, Vimentin, and N-cadherin, and decreased levels of E-cadherin, indicating EMT induction. In the xenograft model, the MT1B-transfected group formed tumors more rapidly and exhibited significantly increased tumor growth compared to the controls. In addition, RNA sequencing was performed to identify MT1B-dependent gene alterations, and Ingenuity Pathway Analysis (IPA) was applied to characterize the canonical pathways and predicted biological functions associated with these MT1B-specific genes. These findings suggest that cellular MT1B overexpression has the potential to promote lung cancer growth.

INTRODUCTION

Metallothioneins (MTs) are known to play a major role in regulating intracellular heavy metal ion homeostasis, owing to their high binding affinity for metal ions such as Zn and Cu, and also to protect cells from oxidative stress induced by reactive oxygen species (ROS). MTs are classified into four major isoforms, MT1, MT2, MT3, and MT4, among which MT1 comprises eight active isoforms (1-4). The representative active MT1 isoform MT1B has recently been reported to attenuate the progression of metabolic dysfunction-associated steatohepatitis when its expression is increased (5); however, its individual effects and precise functions remain largely unclear.

Accumulating evidence demonstrates that MTs are closely associated with multiple cancer types (2, 6, 7). However, studies directly validating MT1 expression in lung cancer are limited. In patients with non-small cell lung cancer (NSCLC), the expression of MT1/2 is not correlated with tumor malignancy; however, those with high expression of both Ki-67 and MT1/2 have significantly shorter survival times than patients with low expression (8). Moreover, the expression of MT1B, MT1F, MT1G, MT1H, and MT1X is increased in NSCLC tissues, with MT1B being particularly upregulated in both adenocarcinoma and squamous cell carcinoma specimens (9). In contrast, other studies have demonstrated decreased expression of MT1A, MT1G, MT1E, and MT2A in surgically resected lung cancer tissues; however, no data were provided regarding MT1B expression (10). Furthermore, no studies to date have elucidated the molecular mechanisms of MT1B in lung diseases, including lung cancer.

To evaluate the role of MT1B in lung cancer in the current study, we assessed cell viability, migration, invasion, and colony formation in the human alveolar carcinoma cell line A549 overexpressing MT1B. We also examined the expression of epithelial–mesenchymal transition (EMT) markers, including E-cadherin, N-cadherin, Vimentin, and Snail. To analyze tumor growth in vivo, MT1B-overexpressing A549 cells were subcutaneously injected into nude mice and tumor formation was monitored.

RESULTS

MT1B overexpression promoted cell viability and migration

To analyze the effect of MT1B on lung cancer cells, we established MT1B-overexpressing A549 cells. The overexpression of MT1B was confirmed using western blotting (Fig. 1A). The effects of MT1B overexpression on cancer cell proliferation were analyzed using cell viability, migration, and invasion assays. As shown in Fig. 1B, MT1B vector-transfected cells (MT1B cells) showed increased proliferation compared to scrambled vector-transfected cells (control cells) at each time point (24 h, 48 h, 72 h, and 96 h).

Fig. 1.

MT1B overexpression promotes proliferation and migration of A549 cells and H1299 cells. (A) Western blot analysis confirming MT1B overexpression in both cell lines transfected with an MT1B expression vector. β-actin was used as a loading control. (B) Cell viability assay showing increased proliferation of the MT1B cells compared to the control cells at 24 h and 48 h. (C) Migration assay demonstrating enhanced wound closure in MT1B-overexpressing A549 cells. (D) Migration assay demonstrating enhanced wound closure in MT1B-overexpressing H1299 cells. (E) Representation images of A549 cell migration at 6, 12, 24 h following transfection with either control vector or MT1B vector. (F) Representation images of H1299 cell migration at 6, 12, 24 h following transfection with either control vector or MT1B vector.

Overexpression of MT1B significantly enhanced the migratory ability of A549 cells. As shown in Fig. 1C-F, MT1B cells progressively migrated into the artificial wound area over time. In both A549 cells and H1299 cells, wound closure at 6 and 12 hours tended to be higher in MT1B cells than in control cells, although these differences were not statistically significant. However, at 24 hours, MT1B-overexpressing A549 cells exhibited a significantly greater wound closure (56.07%) compared with control cells (41.50%). A similar pattern was observed in H1299 cells, in which wound closure increased from 50.38% in control cells to 69.38% in MT1B cells, indicating a statistically significant enhancement of migration. These findings indicated a significant increase in migration induced by MT1B overexpression. Consistent with these observations, the quantitative analysis presented in Fig. 1C-F demonstrated a time-dependent increase in the migration of A549 cells and H1299 cells, further confirming the positive effect of MT1B on cell migration.

Cell invasion and colony growth were induced by MT1B overexpression

To examine the invasive ability of A549 cells and H1299 cells overexpressing MT1B, an invasion assay was performed using a Matrigel-coated Boyden chamber. MT1B cells for 24 h were placed in the upper chamber and the number of cells that migrated through the Matrigel-coated membrane after 24 h of incubation was observed. As shown in Fig. 2A, B, a greater level of cell invasion occurred in the MT1B cells than in the control cells. The invading cell values were quantified using ImageJ software and are shown in Fig. 2D. As shown in Fig. 2C, an invasion assay conducted in H1299 cells revealed that MT1B cells exhibited a markedly enhanced invasive capacity relative to control cells, with an increase that was substantially greater than that observed in A549 cells.

Fig. 2.

MT1B overexpression enhances invasion and colony formation of A549 cells and H1299 cells. (A, B) Representative images of Transwell invasion assays demonstrating increased invasive capacity in MT1B-overexpressing A549 and H1299 cells, respectively. (C) Colony formation assay showing enhanced clonogenic potential of MT1B cells relative to the control cells. Representative images of stained colonies are shown. (D) Quantification of invading cells from three independent experiments. Data were analyzed using ImageJ and are presented as mean ± SEM; P < 0.01 compared to the control. (E) Quantitative analysis of colony numbers indicating a significant increase in colony formation ability upon MT1B overexpression. The data represent the mean ± SEM; P < 0.05, compared to the control. (F) Western blot analysis showing the expression levels of EMT markers Snail, Vimentin, E-cadherin, and N-cadherin in A549 lung cancer cells following MT1B overexpression.

Overexpression of MT1B in A549 cells also increased colony formation. The number of colonies in the control group was 9.7, whereas that in the MT1B cells was 22.3, which was significantly higher (Fig. 2E). These results confirmed that MT1B treatment enhanced colony formation.

MT1B regulated EMT marker expression in A549 cells

EMT plays a critical role in promoting tumor cell invasion and metastasis (11). Representative markers of EMT include E-cadherin, N-cadherin, Vimentin, and Snail. In the present study, the effect of MT1B on the expression of EMT-related proteins was investigated in A549 cells using western blot analysis. MT1B expression increased the levels of Snail, Vimentin, and N-cadherin, whereas E-cadherin expression levels decreased (Fig. 2F). These findings suggest that MT1B can induce or promote EMT in A549 cells.

MT1B overexpression promoted tumor formation and growth in A549 cell-derived xenografts

To demonstrate the tumorigenic effects of MT1B-transfected A549 cells in vivo, A549 cells were injected subcutaneously into nude mice. As shown in Fig. 3A, tumors in the MT1B cells injected group (MT1B group) appeared markedly larger than those in the control cells injected group (control group) upon macroscopic examination. In the control group, tumors were observed at approximately 7 d post-injection, whereas tumors were detectable within 2 to 3 d post-injection in the MT1B group. Following excision, the tumor weights were measured and found to be higher in the MT1B group than in the control group (Fig. 3B, C).

Fig. 3.

MT1B overexpression enhances tumorigenesis and growth in A549 cell-derived xenografts. (A) Images of subcutaneous tumors formed in nude mice injected with control cells or MT1B cells. The tumors in the MT1B group were visibly larger than those in the control group. (B) The excised tumors in the MT1B group were larger than those in the control group. (C) Tumor weights were measured and demonstrate a significant increase in tumor mass in the MT1B group compared to the control group. Data are presented as the mean ± SEM; P < 0.05, versus the control.

MT1B overexpression modulates EMT-related mechanisms in A549 cells

Transcriptome profiling of A549 cells overexpressing MT1B identified ten reproducible expression changes relative to vector controls. Four genes were increased: PYURF showed the largest effect (fold change (FC): 27.846, P = 0.000), ARHGAP8 increased (FC: 2.022, P = 0.006), GH1 increased (FC: 1.986, P = 0.009), and SP140L increased (FC: 1.515, P = 0.045). Six genes were decreased: NPIPA7 (FC: 0.606, P = 0.038), CHTF8 (FC: 0.557, P = 0.013), RBM14–RBM4 readthrough (FC: 0.537, P = 0.013), PRR5–ARHGAP8 readthrough (FC: 0.479, P = 0.037), H3C14 (FC: 0.478, P = 0.006), and PIGY (FC: 0.086, P = 0.000) (Table 1). To validate the RNA-seq results, we quantified the mRNA levels of PYURF, ARHGAP8, GH1, and SP140L in control and MT1B cells using quantitative real-time PCR. The expression of GH1, ARHGAP8, and SP140L was significantly increased in MT1B cells, whereas PYURF showed a non-significant but upward trend. Consistent with the transcriptomic profiles, all four genes exhibited higher expression levels in MT1B cells compared with control cells (Supplementary Fig. 1).

Table 1.

Transcriptional alterations in MT1B-overexpressed A549 cell lines and epithelial-mesenchymal transition (EMT)-related mechanisms of corresponding genes

Gene symbol (Entrez ID)	Description	Fold change	P-value	EMT-related mechanistic verdict
PYURF (100996939)	PIGY upstream open reading frame	27.846	0.000	OXPHOS/CoQ →EMT-consistent state, but causality not established.
ARHGAP8 (23779)	Rho GTPase activating protein 8	2.022	0.006	Rac1/VAV1-driven motility/invasion →pro-EMT output.
GH1 (2688)	Growth hormone 1	1.986	0.009	GH–GHR–STAT3 activation → induces EMT transcriptional programs.
SP140L (93349)	SP140 nuclear body protein like	1.515	0.045	No specific evidence
NPIPA7 (101059938)	Nuclear pore complex interacting protein family member A7	0.606	0.038	No specific evidence
CHTF8 (54921)	Chromosome transmission fidelity factor 8	0.557	0.013	No specific evidence
RBM14-RBM4 (100526737)	RBM14-RBM4 readthrough	0.537	0.013	No specific evidence
PRR5-ARHGAP8 (553158)	PRR5-ARHGAP8 readthrough	0.479	0.037	No specific evidence
H3C14 (126961)	H3 clustered histone14	0.478	0.006	No specific evidence
PIGY (84992)	Phosphatidylinositol glycan anchor biosynthesis class Y	0.086	0.000	GPI-AP reduction → EMT-relevant raft/adhesion changes.

Predefined EMT-related labels were assigned to each gene alongside the quantitative data. These annotations describe the gene’s relevance to the pathway without suggesting causality outside of the designated category. PYURF was labeled “OXPHOS/CoQ → EMT-consistent state, but causality not established.” ARHGAP8 was labeled “Rac1/VAV1-driven motility/invasion → pro-EMT output.” GH1 was labeled “GH–GHR–STAT3 activation → induces EMT transcriptional programs.” SP140L, NPIPA7, CHTF8, RBM14–RBM4 readthrough, PRR5–ARHGAP8 readthrough, and H3C14 were labeled “No specific evidence.” PIGY was labeled “GPI-AP reduction → EMT-relevant raft/adhesion changes.” The EMT-associated genes that showed significant differential expression in MT1B-overexpressing cells were identified from the RNA-seq dataset and are summarized in Supplementary Table 1.

Compartmental signaling landscape of MT1B-overexpressing A549 cells

IPA of the RNA-seq profile from A549 cells overexpressing MT1B defined a cellular compartmental signaling architecture across extracellular, cytoplasmic, and nuclear layers. At the extracellular space, GH1 was predicted as the dominant upstream cue. In the cytoplasm, activation signatures were assigned to ERK, BCKDHA, PDK1, ARHGAP8, and a RhoGAP module, with network metrics ranking ERK as the top node and BCKDHA as the second. In the nucleus, MYC, ESR1, AR, JUNB, and MAD2L1 were prioritized in that order of influence (Fig. 4). To confirm the IPA-predicted activation of ERK, we examined phospho-ERK protein levels in MT1B-overexpressing A549 cells. Western blot analysis demonstrated that MT1B overexpression increased phospho-ERK levels compared with control cells (Supplementary Fig. 2). Taken together, the analysis indicates that an extracellular GH1 signal converges on a cytoplasmic kinase and metabolic control layer dominated by ERK and BCKDHA and extends to a nuclear transcriptional program organized by MYC, ESR1, AR, JUNB, and MAD2L1, consistent with a coordinated rather than independent set of pathway changes in the MT1B context.

Fig. 4.

Principal signaling pathway alterations in A549 cells overexpressing MT1B. This schematic depicts the molecular cascade involving key genes whose expression is altered in MT1B-overexpressing A549 cells (orange arrows, activation; blue arrows, inhibition; red, upregulated genes; green, downregulated genes).

DISCUSSION

Here, we investigated the effects of MT1B, a subtype of MT1, on the proliferation and progression of lung cancer cells. MT1B overexpression in A549 cells not only significantly enhanced cell viability and proliferation but also promoted invasiveness and colony formation. Changes in the expression of EMT marker proteins E-cadherin, N-cadherin, Vimentin, and Snail were observed in MT1B-overexpressing A549 cells. Furthermore, xenograft experiments revealed that tumors derived from subcutaneously implanted MT1B-overexpressing lung cancer cells exhibited increased tumor formation compared with those observed in the control cells. These results indicated that intracellular MT1B overexpression may facilitate the growth and progression of lung cancer.

In our previous study, we reported that the exposure of human lung epithelial cells to polyhexamethylene guanidine-phosphate (PHMG-p), a major component of humidifier disinfectants and a well-known toxicant associated with pulmonary diseases, leads to increased expression of several MT1 isoforms, including MT1B, MT1F, MT1G, and MT1H (12). In contrast, no such changes are observed following exposure to other principal humidifier disinfectant components, such as ethoxyethyl guanidine chloride (PGH), chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT), benzalkonium chloride (BKC), or sodium dichloroisocyanurate (NaDCC) (12, 13). Consistent with these findings, a previous report showed that the expression levels of MT1B, MT1F, MT1G, and MT1H are significantly increased in NSCLC tissues compared to that in non-malignant lung tissues (9). Furthermore, in long-term observational studies, in which animals were intratracheally exposed to a defined concentration of PHMG-p, lung cancer developed in more than 70% of the exposed animals (14), and even a single exposure led to the development of squamous cell carcinoma during the 52-wk observation period (15). Together, these findings suggest that the altered expression of MT1B, MT1F, MT1G, and MT1H may be directly or indirectly associated with the growth and progression of lung cancer.

Recent studies have reported that MT expression protects lungs from injury induced by air pollutants, including particulate matter (16, 17). In animal models of acute lung injury caused by exposure to heavy metals, such as nickel, loss of MT1/2 genes was associated with reduced survival and altered expression of genes related to acute lung injury (18). Cadmium inhalation markedly increases MT1 and MT2 expression in lung tissues, with elevated MT expression observed in both alveolar macrophages and alveolar type II epithelial cells (19, 20). In addition, the lungs of MT1/2-deficient animals exposed to endotoxins/lipopolysaccharide exhibit greater susceptibility to pulmonary inflammation and edema (21). However, these findings alone do not justify the conclusion that MT1 expression simply protects cells from toxic insults and maintains cellular homeostasis. Furthermore, accumulating evidence demonstrates that MT1 expression is elevated in tumor tissues of various organs (2). Thus, while MT1 may exert cytoprotective effects under physiological conditions, these findings imply that its aberrant upregulation or downregulation may have adverse effects on cellular function, thereby contributing to tumor initiation or the promotion of cancer progression.

In this study, microarray and bioinformatics analyses were performed to characterize MT1B-specific transcriptional changes. Transcriptome profiling identified four upregulated and six downregulated genes, among which four genes were associated with epithelial–mesenchymal transition (EMT) (Table 1). Notably, a significant increase in PYURF expression was observed, suggesting a close association with mitochondrial oxidative phosphorylation (OXPHOS) metabolism (22, 23). Indeed, cancer progression and EMT activation have been reported to be tightly linked to OXPHOS (24). In addition, upregulation of ARHGAP8 has been shown to cooperate with BPGAP1 to activate the Vav1–Rac1 axis, thereby promoting cancer cell migration (25). Increased expression of GH1 further implies activation of the GH–GHR–STAT3 axis, which may drive EMT (26). Conversely, downregulation of PIGY is predicted to impair glycosylphosphatidylinositol biosynthesis, potentially contributing to EMT-related transcriptional alterations (27). IPA revealed RHOGDI signaling, HOTAIR regulatory pathway, and histone modification signaling as the major canonical pathways, with predicted biological functions associated with solid tumor invasion and chemotherapy resistance (Fig. 4). Therefore, MT1B expression may function as a key regulator that not only induces genes activating EMT but also promotes the plasticity and invasiveness of lung cancer cells.

There remain unresolved questions regarding how increased MT1B expression influences lung cancer growth and progression within the complex and interdependent molecular systems of lung tissue. In this study, we demonstrated the induced proliferation and progression of lung cancer cells by intracellular MT1B overexpression. However, multiple MT1 isoforms interact with each other, and thus, the potential synergistic or antagonistic effects of these isoforms warrant further investigation. In addition, the specific responses of MT1 isoforms to distinct environmental toxicants should be examined. It is important to determine how the disruption of expression homeostasis caused by aberrant MT1 regulation contributes to the development of pulmonary diseases and lung cancer.

MATERIALS AND METHODS

Cell culture

The lung carcinoma cell lines A549 and H1299 were purchased from the Korean Cell Line Bank (Seoul, Korea). The A549 cells and H1299 were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO, Thermo Fisher Scientific, Grand Island, NY, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO, Thermo Fisher Scientific, Grand Island, NY, USA). The cells were cultured in a 37°C humidified incubator with 5% CO₂.

Cell transfection

A549 cells and H1299 were seeded into six-well plates at a density of 1 × 10⁶ cells/well 24 h prior to transfection. A549 cells and H1299 were transfected with the MT1B human tagged ORF clone vector (1 μg; OriGene Technologies, Rockville, MD, USA) or a non-targeting scrambled control vector (1 μg; OriGene Technologies), which was used as a negative control. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, the cells were changed in 2  ml of serum-free RPMI 1640 medium 6 h after transfection. Then, the cells were incubated for an additional 18 h in RPMI 1640 medium supplemented with 10% FBS.

Western blotting

To extract total protein, control cells or MT1B cells were lysed using a pre-cooled (4°C) radioimmunoprecipitation assay (RIPA) lysis buffer (ATTO, Tokyo, Japan) for 30 min on ice. The lysates were then centrifuged at 12,000 rpm for 20 min at 4°C to remove cell debris. Protein concentrations were quantified using a Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were separated by 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (ATTO). Membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 30 min at room temperature, followed by overnight incubation at 4°C with primary antibodies. The following primary antibodies were used: rabbit anti-MT1B (LSBio, 1:1000), rabbit anti-Snail (Abcam, Cambridge, UK, 1:1000), rabbit anti-Vimentin (Abcam, 1:1000), rabbit anti-E-cadherin (Sigma-Aldrich, Saint Louis, MO, USA, 1:1000), rabbit anti-N-cadherin (Abcam, 1:1000). After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Mouse anti-β-actin (Santa Cruz Biotechnology, CA, USA, 1:2000) was used as a loading control. Protein bands were visualized using the ChemiDoc^TM Imaging System (Bio-Rad Laboratories, Tuas, Singapore).

Cell viability assay

Control cells or MT1B cells were seeded at a density of 6 × 10⁴ cells per well in 96-well plates and incubated for 24 h, 48 h, 72 h, and 96 h at 37°C in a humidified atmosphere containing 5% CO₂. Cell viability was assessed using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) in accordance with the manufacturer’s instructions. Briefly, 10 μl of CCK-8 solution was added to each well, followed by incubation for 4 h at 37°C in a 5% CO₂ humidified incubator. The absorbance of each well was then measured at 450 nm using a SpectraMax M2e microplate spectrophotometer (Bucher Biotec, Basel, Switzerland).

Migration assay

Control cells or MT1B cells were seeded at a density of 5 × 10⁶ cells/ml, and 200 μl of the cell suspension was added to each SPL Scar^TM Block (cat. #201935; SPL Life Sciences, Pocheon, Korea). After 24 h of cell seeding, artificial wounds were generated by removing the block using sterile forceps. At specific time points after wound creation (0, 6, 12, and 24 h), cells were washed twice with phosphate-buffered saline (PBS). The cells within the wound area were counted under ×50 magnification and recorded in a blinded manner. Each dish was counted three times to ensure accuracy.

Invasion assay

The invasive ability of cells was determined using an SPLInsert^TM Hanging culture system (SPL, Pocheon, Korea). Matrigel (Corning, Corning, NY, USA) was diluted to 200 μg/ml in serum-free RPMI 1640 medium. The upper chambers were coated with 100 μl of the diluted Matrigel and allowed to dry in a 37°C oven for 45 min. Control cells or MT1B cells were seeded into the Matrigel-coated upper chambers at a density of 5 × 10⁵ cells/ml (200 μl/well). The lower chambers were filled with 500 μl of RPMI 1640 medium containing 10% FBS and incubated in a humidified atmosphere with 5% CO₂ at 37°C for 24 h. Non-invading cells on the upper surface of the membrane were gently removed using a cotton swab, and invading cells on the lower surface were then fixed with 4% formaldehyde in PBS and stained with 2% crystal violet. Invading cells were visualized under a light microscope at ×50 magnification.

Colony-formation assay

Control cells or MT1B cells were seeded into 6-well plates at a density of 5 × 10² cells/ml and cultured for 12 d at 37°C in a humidified incubator with 5% CO₂. After incubation, the cell colonies were gently washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The colonies were then stained with a 0.1% (w/v) crystal violet solution for 15 min and washed twice with PBS. The number of colonies was counted and recorded.

Animals

Seven-week-old male BALB/c nude mice were purchased from Orient Bio (Seongnam, Gyeonggi-do, Korea). Mice were acclimated for 1 week under the following standard conditions: temperature 22-25°C, relative humidity 40-60%, and a 12-h light–dark cycle. Mice were provided with pelleted food (Purina, Seongnam, Korea) and purified tap water. All experiments were performed at Korea University Hospital in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). The experimental protocol was approved by the Institutional Animal Care and Use Committee of Korea University Medical Center (KOREA-2025-0068).

Subcutaneous implantation of tumor cells

For subcutaneous injections, nude mice were randomly divided into groups assigned to either control group or MT1B group (n = four mice/group). A549 cells (1 × 10⁷), transfected with scrambled or MT1B vector, were administered either by subcutaneous injection into the dorsal region of the mice. Tumor growth in the subcutaneous injection group was monitored for 21 d, after which time the tumors were excised and weighed.

RNA isolation

Total RNA was extracted from control cells or MT1B cells using either Trizol reagent (Invitrogen) or the Maxwell^Ⓡ RSC miRNA kit (Promega^TM Corporation). RNA quality was assessed with the Agilent 4200 TapeStation System (Agilent Technologies, Amstelveen, The Netherlands), and RNA concentrations were measured using the Invitrogen Qubit 4 or Qubit Flex fluorometers (Thermo Fisher Scientific, DE, USA).

Library preparation and sequencing

mRNA was isolated utilizing the Poly(A) RNA Selection Kit (LEXOGEN, Inc., Austria). Libraries were then prepared from the purified mRNA using the CORALL RNA-Seq V2 Library Prep Kit (LEXOGEN, Inc., Austria). The process of cDNA synthesis and fragmentation was carried out following the manufacturer’s instructions. Indexing was performed with the Lexogen UDI 12 nt Unique Dual Indexing V2 system. PCR amplification was used to enrich the libraries. Library size distribution was assessed with the Agilent 4200 TapeStation System or the Agilent 2100 Bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands). Library quantification was conducted via qPCR using the KAPA Library Quantification Kit (Kapa Biosystems, MA, USA), following the manufacturer’s recommended protocol. Sequencing was performed as paired-end 100 bp reads on the NovaSeq 6000 platform (Illumina, Inc., USA).

Data analysis

Raw sequencing data quality was evaluated using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapter sequences and low-quality reads were trimmed using Fastp (28). The cleaned reads were aligned to the reference genome with STAR (29), and transcript quantification was performed using Salmon (30). Read counts were normalized using the TMM and CPM methods via the Python “conorm” package (https://gitlab.com/georgy.m/conorm). Downstream data mining and visualization were conducted with ExDEGA software (Ebiogen Inc., South Korea). Canonical pathways and biological functions were analyzed using the IPA software (Qiagen, MD, USA).

Quantitative real-time PCR

Quantitative real-time PCR was conducted in triplicate using a 25-μl reaction mixture containing 2× SYBR^Ⓡ Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Reactions were run on an Applied Biosystems real-time PCR system (Foster City, CA, USA).

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± standard error of the mean (SEM). Two-way analysis of variance (ANOVA) was used to analyze the cell viability and migration rate data. For comparisons between the two groups in the invasion assays, colony formation assays, and tumor weight analysis, an unpaired two-tailed Student’s t-test was employed. Statistical significance was set at P < 0.05.