Introduction
Lung cancer is an extremely lethal disease worldwide [
1]. Lung adenocarcinoma (LUAD), accounting for approximately 40% of all lung cancers, is the most common cancer [
2]. Although important progress has been made in drug therapy, especially targeted drugs and immune check point inhibitors, the 5-year survival rate of LUAD is merely 15% [
3]. The main reason for this is that the mechanisms involved in LUAD remain unclear, so to improve the survival rate, it is critical to investigate the potential mechanisms of LUAD.
Investigators have paid attention to junctional adhesion molecules (JAMs) in recent years. JAMs belong to the immunoglobulin (Ig) superfamily, consisting of junctional adhesion molecule-A (JAM-A), junctional adhesion molecule-B (JAM-B), junctional adhesion molecule-C (JAM-C), junctional adhesion molecule-4 (JAM-4) and junctional adhesion molecule-like (JAML); among them, JAM-A is the most studied molecule. Multiple studies had shown that overexpression (OE) of JAM-A induced tumorigenesis in cancers such as lung cancer, nasopharyngeal cancer and oral squamous cell carcinoma [
4‐
6]. Moreover, a tumor-suppressive role of JAM-A was observed in pancreatic cancer and renal cell carcinoma [
7,
8]. Interestingly, both tumorigenesis and tumor-suppressive roles of JAM-A were reported in breast cancer [
9,
10]. Based on these studies, the role of JAM-A in carcinogenesis is complex and diverse in different cancers [
11]. However, there have been few studies on JAML, especially on the relationship between JAML and LUAD.
As a newly reported JAM, JAML was first reported in 2003, and consists of one transmembrane fragment, two extracellular Ig-like domains, and a cytoplasmic tail. Some T cells, monocytes, neutrophils and other multiple cell types can express JAML. JAML can enhance the adhesion and migration ability of endothelial/epithelial cells, thereby influencing inflammatory reactions [
12‐
15]. JAML also plays key roles in wound healing and atherosclerosis occurrence [
16,
17]. In gastric cancer, overexpressed JAML facilitated the proliferation and migration of gastric cancer cells [
18]. Gastric adenocarcinoma represents 95% of gastric cancer cases. Both LUAD and gastric adenocarcinoma originate from epithelial tissues, indicating that there may be common or similar mechanisms in the pathogenesis between the two diseases. Recently, the role of JAML was reported in LUAD, they found that JAML expression was decreased in LUAD and that JAML expression was correlated with immune infiltrates [
19,
20], but most of their conclusions were based on online databases, and the role of JAML may be very complex in cancers. The actual role of JAML in LUAD is still unclear.
In our present study, the role of JAML in LUAD was elucidated. Initially, we investigated the expression of JAML in LUAD, followed by functional experiments both in vitro and in vivo. We also explored the underlying mechanisms by which JAML affects tumor progression in LUAD. Our results reveal the critical role of JAML in the progression of LUAD.
Materials and methods
Human LUAD samples and cell lines
All tumor and peritumor tissues were surgically resected from LUAD patients at the Second Hospital of Shandong University (Jinan, China). Each patient had an independent pathological review-confirmed diagnosis of LUAD. All studies were performed under supervision and approved by the Ethics Committee of the Second Hospital of Shandong University (KYLL-2021(LW)085), and adhered to the Declaration of Helsinki. Informed consent was obtained from human subjects. The privacy rights of human subjects must always be observed.
The LUAD cell lines, including A549, H1299, PC9 and H1975, were purchased from Procell Life Science & Technology Co. Ltd. (Wuhan, China). All LUAD cells were cultured in complete medium (RPMI 1640 for A549, H1299, H1975 or DMEM for PC9, plus 10% FBS). Cells were cultured in a 37 °C incubator with a humidified 5% CO2 atmosphere.
RNA extraction and qRT-PCR
Snap-frozen tissues were homogenized and extracted using TRIzol
™ Reagent (Ambion, 155596-026) following the manufacturer’s instructions. The concentration and purity of the RNA were determined by a DeNovix spectrophotometer. cDNA was synthesized by Hiscript
® III RT SuperMix for qPCR (Vazyme, R323-01). Real-time PCR was performed on a Bio-Rad CFX Connect
™ Real-Time System using a SYBR Green Premix Pro Taq HS qPCR Kit (ACCURATE BIOLOGY). Fold change was calculated as 2
–ΔΔCT. PCR parameter settings were shown in Table
1. The melting curve data were collected to ensure PCR specificities. The primers used for PCR were shown in Table
2.
Table 1
PCR parameter settings
95 °C | 30 s | |
95 °C | 10 s | 45 cycles |
59 °C | 30 s |
Melting Curve 65 °C, to 95 °C, increment 0.5 °C |
Table 2
Primer sequences for PCR are as follows
HOMO-JAML-F | 5′-AGAGCACGCCAAGGACGAATA-3′ |
HOMO-JAML-R | 5′-GGAGCAGGAGAGAGCCATCAT-3′ |
HOMO-GAPDH-138F | 5′-GCACCGTCAAGGCTGAGAAC-3′ |
HOMO-GAPDH-138R | 5′-TGGTGAAGACGCCAGTGGA-3′ |
Western blotting
Total proteins were extracted with RIPA buffer (Solarbio, #R0020) which added protease inhibitors (Solarbio, #P0100) and phosphatase inhibitors (Solarbio, #P1260). The lysates were separated on SDS-PAGE gels under constant voltage. After gel electrophoresis, the proteins were immediately trans-blotted to a Millipore PVDF membrane (0.45 µm) under constant current. 5% nonfat dry milk dissolved in TBST was used for blocking the blots for 1 h at room temperature (RT), and thus the blots were incubated with primary antibodies overnight at 4 °C. Next day, secondary antibodies were incubated with the blots for 1 h after extensive washing with TBST. All blots were detected by ECL reagent (Millipore) using a Tanon System for visualization after washing. β-actin expression was used to quantify the data. ImageJ software was used for the Western blotting analysis (version 5.2.1).
Immunohistochemistry (IHC)
Slides (4 μm) were dewaxed in transparency agent and rehydrated in ethanol prior to antigen retrieval. Antigen retrieval was conducted by steaming slides in 1X EDTA buffer (Beyotime, #P0085) for 15 min by microwave irradiation (500 W). Endogenous peroxidase was quenched by treating samples with 3% H2O2 for 10 min. After the above steps, 5% goat serum (BOSTER, #16K03A09) was added onto slides for blocking, 30 min later, followed by staining in primary antibodies overnight at 4 °C. After a 1 h rewarming phase, secondary antibody was applied prior to DAB treatment (Zhongshanjinqiao, #ZLI-9108) for 1 h at RT. Hematoxylin was used for counterstain, then slides rinsed in running water for 5 min and differentiated with 1% hydrochloric acid alcohol for 2 s until the nuclei returned to blue. The tissues were dehydrated with graded ethanol and mounted with neutral resin (BOSTER, AR0038). Images were photographed by inverted microscopy and analyzed using IPP software.
Immunofluorescence assay
Immunofluorescence assay was carried out with cells grown on coverslips (NEST, 801009). Adherent cells were fixed with 4% paraformaldehyde (Solarbio, #P1110) at RT for 30 min. The slides were incubated with 0.1% Triton X-100 (Solarbio, T8200) for 20 min for permeabilization immediately following 5% BSA (Solarbio, SW3015) for 30 min for blocking. The slides were incubated with primary antibody overnight at 4 °C after washing 3 times in PBS. A diluted fluorescent-labeled secondary antibody (BOSTER, BA1127) was incubated with slides for 2 h at 37 °C after 3 washes with PBS. DAPI (Solarbio, # C0065) was used for DNA staining. After extensive washing, the slides were mounted in fluorescence-quenching glycerol (Beyotime, P0126) and visualized by fluorescence microscopy (Nikon, Ti2).
Antibodies
The antibodies and dilution ratios used for Western blotting, IHC and immunofluorescence assays are listed in Table
3.
Table 3
The antibodies and dilution ratios used for experiments
JAML | abcam | ab183714 | 1:1000 | Western blotting |
β-Actin | CST | 3700 | 1:1000 | Western blotting |
MMP2 | abcam | ab92536 | 1:1000 | Western blotting |
MMP9 | abcam | ab76003 | 1:1000 | Western blotting |
β-Catenin | abcam | ab32572 | 1:5000 | Western blotting |
cyclin D1 | abcam | ab226977 | 1:1000 | Western blotting |
Bcl2 | abcam | ab32124 | 1:1000 | Western blotting |
Bax | abcam | ab32503 | 1:1000 | Western blotting |
Survivin | abcam | ab76424 | 1:5000 | Western blotting |
E-cadherin | Affinity | AF0131 | 1:1000 | Western blotting |
N-cadherin | abcam | ab76011 | 1:5000 | Western blotting |
Vimentin | abcam | ab92547 | 1:1000 | Western blotting |
Anti-mouse IgG, HRP-linked Antibody | CST | 7076 | 1:3000 | Western blotting |
Anti-rabbit IgG, HRP-linked Antibody | CST | 7074 | 1:3000 | Western blotting |
JAML | Proteintech | 21302–1 | 1:200 | IHC |
Ki-67 | CST | 9449 | 1:400 | IHC |
JAML | Proteintech | 21302–1 | 1:200 | Immunofluorescence |
DyLight 488 Conjugated AffiniPure Goat Anti-rabbit IgG | BOSTER | BA1127 | 1:1000 | Immunofluorescence |
SiRNA and plasmid transfection experiment
SiRNAs and plasmids were obtained from GenePharma (Shanghai, China) and used for gene silencing or overexpression. The siRNA sequences are shown in Table
4. SiRNAs or plasmids were transiently transfected into LUAD cells by jetPRIME transfection reagent (Polyplus, France). The transfection efficiency was validated after 48–72 h of transfection through Western blotting. After 48 h, the transfected cells were collected for in vitro functional experiments except for in the colony formation assay.
Table 4
SiRNAs sequences used in JAML knockdown experiment
Negative control siRNA | sense sequence | 5′-UUCUCCGAAGGUGUCACGUTT-3′ |
antisense sequence | 5′-ACGUGACACGUUCGGAGAATT-3′ |
siJAML1 siRNA | sense sequence | 5′-GGAAUUGUCUGUGCCACAATT-3′ |
antisense sequence | 5′-UUGUGGCACAGACAAUUCCTT-3′ |
siJAML132 siRNA | sense sequence | 5′-GAGCACAGAAGACAAAUGUTT-3′ |
antisense sequence | 5′-ACAUUUGUCUUCUGUGCUCTT-3′ |
siJAML272 siRNA | sense sequence | 5′-GGGACAUCUUAUGCAAUGATT-3′ |
antisense sequence | 5′-UCAUUGCAUAAGAUGUCCCTT-3′ |
Lentivirus transfection experiment
The lentiviruses were obtained from GeneChem (Shanghai, China). Lentivirus sequences used for JAML knockdown are listed in Table
5. After transfected with lentivirus (multiplicity of infection, MOI: 10 for A549 and 20 for PC9) and selected with puromycin (2 mg/ml) for 7 days, stable cell lines were harvested. HitransG (Genechem, Shanghai, China) was used for better lentivirus infection. The transfection efficiency was analyzed by Western blotting. The stable cell lines were collected for colony formation assays and xenograft tumor models.
Table 5
The lentivirus sequences used in JAML knockdown experiment
JAML- | NM_153206 | gaAGACTAATCCAGAGATAAA | 30.1184 | 31.58% |
RNAi_ |
(102663) |
JAML- | NM_153206 | ccCTGTTCTGATATTGATCGT | 30.1184 | 36.84% |
RNAi_ |
(102664) |
JAML- | NM_153206 | gtATTTCGTTACTACCACAAA | 30.1184 | 31.58% |
RNAi_ |
(102665) |
CCK8 and EdU assays
For the CCK8 assay, 100 μl of cell suspension containing 1500 cells was inoculated in 96-well plates, followed by culturing in a cell incubator. Twenty-four hours after seeding, the cell viability was measured using CCK8 reagent (MedChemExpress, #HY-K0301) following the manufacturer’s protocol. This was considered as Day 0. The proliferation rates at Day 0, 1, 2, 3 and 4 after seeding were detected. At each time point, 100 μl CCK-8 containing medium without FBS was replaced into every well for measurement 1 h later. OD450 was measured on a microplate reader (Bio–Rad Laboratories).
For the EdU assay, cells were precultured in medium containing 10 μmol/L EdU (RiboBio, #C10310) in 96-well plates for 2 h. Then, the EdU-containing medium was removed. Cells were fixed with 4% paraformaldehyde at RT for 30 min, and washed twice with PBS. After 0.5% Triton-100 for 20 min for permeabilization, Apollo and Hoechst dyes were used according to the manufacturer’s protocol. The number of Hoechst-stained cells (blue-fluorescent, total cells) and EdU-positive cells (red-fluorescent) in each field was recorded under a Nikon Eclipse Ti2 microscope. The percentage of EdU-positive cells was calculated using the formula below: Relative EdU rate % = (EdU-positive cells/total cells) × 100%.
A total of 5 × 102 cells were independently inoculated into six-well plates after resuspension. The plates were incubated in a 37 °C incubator for 2 weeks. Every 3 days, the complete medium was replaced in per well. On Day 15, visible colonies (diameter > 0.5 mm) were stained with 0.1% crystal violet (Solarbio, #G106) overnight after fixing with 4% paraformaldehyde for 20 min. After washing 3 times and air drying, monoclonal colonies were photographed by camera.
Wound healing assay
LUAD cells used for wound healing assay were cultured in six-well plates and grown to 100% confluency. Artificial homogeneous wounds were made with a 200 μl pipette tip. After washing twice with PBS to remove debris, FBS-free medium was added to six-well plates. At 0 h and after incubation for an appropriate time in the cell incubator, the wounded areas were photographed under microscopy. The data were quantified using the following formula: Migration Rate % = (initial scratch area − scratch area at time t)/initial scratch area × 100%.
Cell Transwell assays
8.0-µm transwell chambers (Corning, USA) in 24-well plates were used for transwell assays. A total of 5 × 104 cells suspended in 200 μl FBS-free medium were seeded evenly onto Matrigel (Corning, 356234)-coated or Matrigel-uncoated top chambers for the invasion or migration assay, and 700 μl medium (1640 or DMEM plus 20% FBS) was added to the lower chambers. The plates were then returned to a 37 °C incubator for 24 h. Cotton swabs were used to wiped off non-migrated and non-invaded cells on the top layer, the migrated and invaded cells on the bottom of the chambers were fixed with 4% paraformaldehyde, subsequently stained with 0.1% crystal violet. Three random fields of stained cells were photographed by microscope (Nexcope).
Cell cycle assay
Cells were harvested with 0.05% trypsin at 70–80% cell density and then washed twice with precooled PBS. The cell pellet was fixed with 70% cold ethanol at − 20 °C overnight. Within 3 days, the fixed cells were washed twice with precooled PBS, and resuspended in PBS containing propidium iodide (PI) (40 μg/ml, BB-4104) plus RNAase A (250 μg/ml, BB-4104). The mixtures were under a 37 °C water bath protected from light for 30 min before analysis. The distribution of cell cycle was detected by flow cytometry (BD FACSCalibur). Mod-Fit software (Verity Software House) was used to analyze different cell cycle phases.
Cell apoptosis assay
Cells were grown to 70–80% confluency before experiments, and collected with EDTA-free trypsin. After washing twice with precooled PBS, the cell pellet was resuspended in 1X binding buffer, and then collected 1 × 105 cells, 5 μl PI and 5 μl Annexin V-FITC (Meilunbio, MA0220-2) were added into binding buffer. The samples protected from light for 15 min at RT. Flow cytometry was carried out to record the apoptosis rate, thus the data were analyzed using FlowJo software.
Xenograft transplantation in vivo
The animal study was approved by Ethics Committee of the Second Hospital of Shandong University (KYLL-2021(LW)086). All animal experiments complied with the ARRIVE guidelines. All experiments were handled according to U.K. legislation (1986) Animals (Scientific Procedures) Act. 4 weeks old female athymic nude mice (BALB/c mice) were acquired from Vital River Company (Beijing, China). Animals were randomly assigned to each group before start. Lentivirus-transfected LUAD cell lines were resuspended on ice with precooled PBS. A total of 1 × 107 cells were subcutaneously injected into right armpit areas of mice for tumorigenesis. At the end point, the tumors were excised, followed by preparation for IHC staining. The tumor volume was calculated with the following formula: volume (mm3) = 1/2 (length (mm) × width2 (mm2)).
Statistical analysis
GraphPad Prism version 6 (GraphPad Software, USA) was used for data statistical analysis and drawing graphics. The data are expressed as the mean ± SEM for all groups. A P value of < 0.05 was considered statistically significant.
Discussion
The highlight of our study is demonstration of the tumorigenic role of JAML in LUAD both in vivo and in vitro, as well as elucidation of the potential mechanisms in regulating the progression of LUAD. Our studies show that the expression of JAML is elevated in LUAD and that overexpression of JAML is positively related to pT and pTNM (Fig.
1, Table
6). We discovered that JAML plays an oncogenic role in LUAD. Reduction of JAML expression was associated with repression the abilities of proliferation, migration and invasion in LUAD cells. Correspondingly, overexpression of JAML was associated with increasion in proliferation, migration, and invasion in LUAD cells (Figs.
2,
3). JAML knockdown also mediated cell cycle arrest in G
0/G
1 phase and promoted cell apoptosis (Fig.
4). To in-depth clarify the actual function of JAML in tumorigenesis in vivo, a subcutaneous xenograft tumor model in nude mice with LUAD cells was established. The results show that overexpression of JAML significantly increased the xenograft tumor volume and vice versa (Fig.
5). Thus, we aimed to explore the possible potential mechanisms to explain how JAML plays an oncogenic role in LUAD. First, we discovered the effects of JAML on the MMPs, apoptotic-related proteins and hallmarks of EMT, the results demonstrated that silencing of JAML concomitant with a reduction in MMP2, MMP9, Bcl2, N-cadherin and vimentin, and an increase in Bax and E-cadherin and vice versa (Fig.
6). The Wnt/β-catenin pathway is a major pathway that can participate in complex malignant biological behaviors in various tumors. Next, the changes in key proteins of the pathway were analyzed in detail. It was found that the silencing of JAML was associated with a decrease in β-catenin, survivin and cyclin D1 and vice versa (Fig.
7). Rescue experiments showed that CHIR99021 could rescue the inhibition of EMT and MMP2, MMP9 expression caused by JAML knockdown in A549 cells (Fig.
8). EdU and transwell assays also confirmed that CHIR99021 could rescue the repression of proliferation, migration and invasion caused by JAML knockdown in A549 cells (Fig.
8). These results suggest that JAML exerts most of functions via the Wnt/β-catenin pathway. JAML may be a predictive biomarker and novel therapeutic target in LUAD.
Previous studies have shown that JAML promotes tumor progression in gastric cancer in vitro [
18]. Since LUAD and most gastric cancers originate from epithelial cells, they may have a similar pathogenesis. Recent studies noted that the expression of JAML was decresed in LUAD [
19,
20], which was different from our results. However, there were no sufficient experimental supports to confirm their conclusions. The main reason for this difference might be that most of their conclusions were obtained from online databases. As we know that tumor purity of surgical tumor specimens are often impure, the nontumor cells, such as immune cells and stromal cells dilute the tumor purity and influence on the genomic analysis of tumour samples [
21,
22]. In addition, such findings may be also attributed to racial/ethnic differences.
Anti-apoptotic properties are common features of most cancers. Bcl2 family members consist of antiapoptotic proteins, such as Bcl2 and BclXL, and proapoptotic proteins, such as Bax and Bad [
23]. Survivin can suppress apoptosis and promote cell division in various cancers [
24,
25]. Our studies showed that overexpression of JAML tends to inhibit apoptosis through upregulation of Bcl2 and survivin and downregulation of Bax.
During the tumor progression and metastasis stages, EMT is a key process by which epithelial cells become detached since they lose their polarity due to the reduction of their expression of E-cadherin and increase in mesenchymal characteristics due to increased expression of N-cadherin and vimentin [
26‐
30]. Thus, elucidating the underlying EMT mechanism is critical for tumor progression research. The Wnt/β-catenin pathway accounts for a large proportion of EMT among many signaling pathways [
31]. β-catenin plays a central role in tumorigenesis in the Wnt/β-catenin signaling pathway [
32,
33]. Our studies found that JAML is associated with EMT. Both EMT phenotype and MMP2, MMP9 expression could be rescued by an activator specific for Wnt/β-catenin. JAML exerts most of functions by activiting the Wnt/β-catenin signaling pathway.
The shortcomings of our study should be acknowledged. First, we did not have prognostic information for LUAD patients. As a result, the prognostic value of JAML in LUAD is still unknown. Second, although we propose that JAML may affect the Wnt/β-catenin pathway, multiple mechanisms are likely to govern JAML-mediated oncogenic functions and may act on any given gene, and the exact detailed mechanism by which JAML regulates the Wnt/β-catenin pathway was not identified in our study. Third, JAML is considered to be associated with immune infiltration. However, the experiments in this study did not involve immune-related aspects. Thus, further research on JAML is also needed.
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