Investigating the role of exosomal microRNA-5703 in modulating tumor-associated endothelial cells in lung cancer

Sampling from patients

A cohort of patients with lung cancer was meticulously selected using stringent criteria. Each patient underwent an exhaustive pre-operative clinical evaluation encompassing chest radiography, computed tomography, and pulmonary function test for the accurate diagnosis of lung carcinoma and precise delineation of tumor dimensions and locations. Apart from diagnostic modalities, an in-depth collection of patients’ clinical attributes was undertaken. The data encompassed age, gender, metastatic status, tumor dimension, and clinical stage. On the confirmation of lung cancer diagnosis, patients were apprised in detail regarding the study’s objectives and methodologies. The study was conducted in compliance with the Declaration of Helsinki. Informed consent was obtained from all study participants, and ethics approval was obtained from the Ethics Committee of the Second People’s Hospital of Yibin (no. 2023-162-01). During surgical intervention, paired tissue specimens, cancer tissues (n = 20), and adjacent normal tissues (n = 20) were harvested. Blood samples were drawn from each patient or healthy individual (n = 20), and serum samples were isolated through centrifugation. Serum exosomes were isolated using an exosome isolation kit (4478360, Invitrogen, Massachusetts, USA). As for the tissues, potential RNA degradation was prevented by immediately immersing the specimens in an RNA preservation solution (R0118, Beyotime, Shanghai, China). Serum blood samples were harvested, aliquoted, and stored at −80°C. Patient data for the tissue samples are shown in Table 1.

Cell culture and co-culture

Human bronchial epithelial cell line (Bronchial Epithelial Cell Line 2B [BEAS2B], Delf-10130) and 95D cell line (Delf-10140) were newly purchased from the Hefei Wanwu Biotechnology Co., LTD; non-small-cell lung cancer cell lines A549 (1101HUM-PUMC000002), (Human lung cancer cell line 827, 1101HUM-PUMC000478), (National Cancer Institute-H1299, 1101HUM-PUMC000469), and (National Cancer Institute-H1650, 1101HUM-PUMC000251); lung squamous carcinoma cell line (Squamous Cell Carcinoma-MES 1, 1101HUM-PUMC000262); and small-cell lung cancer cell line (National Cancer Institute-H1688, 3101HUMTCHu154) were purchased from the National Infrastructure of Cell Line Resource of China (Beijing, China). A certificate of analysis confirming cell line identity through short tandem repeat profiling and confirming lack of mycoplasma contamination was obtained. The cells were cultured under 37°C in an environment with saturated humidity and 5% Carbon dioxide (CO₂) in a Roswell Park Memorial Institute (RPMI) 1640 medium (C0893, Beyotime, Shanghai, China) enriched with 10% Fetal Bovine Serum (FBS; C0252, Beyotime, Shanghai, China) and 100 U/mL penicillin–streptomycin (C0222, Beyotime, Shanghai, China). Appropriate nutrition and pH balance were ensured by replacing the medium every 2–3 days.

Interactions between lung cancer and endothelial cells were evaluated with a Transwell chamber co-culture system (3422, Corning, New York, USA). Lung cancer cells were seeded in the bottom chamber, and TAECs were positioned in the top chamber in a ratio of 5:1. Afterward, the chambers were incubated under standard culture conditions: temperature of 37°C, 100% humidity, and atmosphere containing 5% CO₂.

TAEC isolation

TAECs were isolated from resected lung carcinoma specimens obtained immediately after surgical removal.^[20] The specimens were meticulously minced and enzymatically dissociated in an RPMI 1640 medium enriched with 0.1% collagenase IV (C5138, Sigma–Aldrich, Missouri, USA) at 37°C for 1 h. The resultant cellular suspension was sequentially filtered through a series of graded meshes, and stromal and aggregated materials were discarded. TAECs were enriched using anti-CD34 monoclonal antibodies (ab81289, Abcam, Massachusetts, USA) bound to magnetic beads, and isolation was carried out with a magnetic cell sorting (MACS) system (Miltenyi Biotech, Bergisch Gladbach, Germany). Diphtheria toxin (500 ng/mL, Calbiochem, San Diego, CA, USA) was added to the TAEC cultures to eliminate remaining human tumor cells. After 24 h, dead cells were aspirated, and non-endothelial tumor cells were completely removed. The TAECs were further purified using a second MACS step, and Fluorescein Isothiocyanate-labeled BS1-B4 Lectin (FITC-BS1-B4 lectin) was used to eliminate contaminating stromal cells and enhance the purity of the endothelial cells. The purity of the isolated TAECs was confirmed through flow cytometric (FCM) analysis. Markers, such as FITC-BS1-B4, were used to ensure that the cell population was primarily composed of endothelial cells. After isolation, the TAECs were tested and found negative for mycoplasma contamination. The TAECs were grown in endothelial growth medium-2 (CC-3162, Lonza Group Ltd., Basel, Switzerland), which was enhanced with 10% FBS and 100 U/mL penicillin–streptomycin. The cultured TAECs were then passaged at subconfluency, and experiments were conducted using cells from passages 1–6 to ensure cellular fidelity.

Exosome extraction and characterization detection

After tumor cells were transfected with miR-5703 mimics, inhibitors, and negative control (NC) (GenePharma, Suzhou, China), the culture supernatant was collected and retained after 10 min of centrifugation at ×350 g. Then, the supernatant was centrifuged again (×2000 g for 30 min). The supernatant was mixed with an exosome extraction working solution (C10130-1, Ribo Bio, Guangzhou, China) and left overnight at 4°C. The next day, the samples were centrifuged, and the lower sediment was retained. The exosomes were reconstituted in an optimal volume of Phosphate-Buffered Saline (PBS) and transferred to a cuvette. Exosome particle size analysis was carried out using a nanoparticle size analyzer (N30E, Xiamen Fuliu Biological Technology Co., Ltd, Xiamen, China). Exosome markers (CD81, CD63, and Alix) and negative markers (calnexin and α-tubulin) were detected by Western blotting. In addition, CD81 (ab79559, Abcam, Massachusetts, USA) was confirmed by FCM.^[21] The method for isolating exosomes from patient sera was the same as that used for isolating exosomes from cell supernatants.

Exosomes were isolated and then fixed using 100 μL of 2.5% glutaraldehyde (G1102, Servicebio, Wuhan, China) and kept at 4°C. An exosome suspension (5 μL) was gently placed on a copper grid, where the exosomes were allowed to adhere for 20 min at room temperature. Subsequently, the grids were rinsed with PBS and then fixed with 1% glutaraldehyde for 5 min. The samples were rinsed 10 times with double- distilled water and negatively stained with 4% uranyl acetate (Hubei Xingyinhe Chemical Co., Ltd. Wuhan, China) for 5 min. Any excess staining solution was carefully dabbed off with filter paper, and the samples were allowed to dry at ambient temperature. The exosomes were visualized with a transmission electron microscope (H-7000; Hitachi, Ltd., Tokyo, Japan). The reagents, including the culture medium, FBS, and liquid used in this experiment, were exosome free.

Transwell assay

TAECs were seeded into six-well plates at a density of 2 × 10⁵ cells per well and cultured until they reached roughly 70% confluency. The isolated exosomes were then added to the TAECs at a concentration of 50 μg/mL, and the culture was maintained for 24–48 h at 37°C in a 5% CO₂ atmosphere. Subsequently, a cell suspension consisting of either endothelial cells or lung cancer cells was prepared at a concentration of 1 × 10⁶ cells/mL. This cell suspension was then added to the Transwell migration chamber (3428, Corning, New York, USA), typically dispensing 500 μL per well. The lower chamber was either seeded with an alternate cell type (TAECs or lung cancer cells) or left cell free. The Transwell chamber was incubated at 37°C with 100% humidity. After 48 h of incubation, the upper chamber was collected. Cells that did not migrate from the bottom of the inner chamber were scraped off, retaining only the cells that had migrated through the membrane. The cells were visualized through underwent 10 min 2% crystal violet staining, which were subsequently counted and imaged under a microscope. The migration behavior of the cells was then analyzed. To assess the invasive capability of lung cancer cells, we added a layer of Matrigel matrix (C0372, Beyotime, Shanghai, China) to the bottom of the Transwell chamber. On the basis of the number of cells that penetrated this matrix, the invasive potential of the cells was evaluated.

Cell proliferation assay

Cell viability was determined using a cell counting kit 8 (CCK8) kit (CCK-8, Dojindo, Kumamoto, Japan) and 5-ethynyl-2'-deoxyuridine (EdU) staining. After the cells were transferred with miRNA mimics, inhibitors, or plasmids, the cells were prepared for cell viability detection with the CCK8 kit or EdU staining.

Cell apoptosis detection

Hoechst: Initially, cover slips were sterilized in 70% ethanol for at least 5 min and then air-dried or rinsed 3 times with PBS. After washing with the cell culture medium, cover slips were placed in six-well plates, and the cells were seeded and grown to 50–80% confluency. To measure cell apoptosis, we replaced the medium with 0.5 mL of fixative. After fixation, the cover slips were washed twice with PBS for 3 min each. The cells were then stained with 0.5 mL of Hoechst 33258 dye (C1011, Beyotime, Shanghai, China) for 5 min, agitated, and washed twice. Subsequently, the cells were stained with 0.5 mL of DAPI staining solution (C1002, Beyotime, Shanghai, China) for 5 min and washed twice. An antifade mounting medium was applied for microscopy. The stained cell nuclei appeared blue under a fluorescence microscope at excitation and emission wavelengths of approximately 350 and 460 nm, respectively.

Immunofluorescence assay

After co-cultivation with lung cancer cells, the expression of α-smooth muscle actin (α-SMA) and ING4 in endothelial cells was evaluated through immunofluorescence. Fixed with 4% paraformaldehyde for 15 min. Then, the cells were infiltrated with 0.1% Triton X-100 for 5 min and blocked with 1% BSA at room temperature for 1 h. Primary antibodies targeting ING4 (1:200, ab113425, Abcam, Massachusetts, USA) and (α-SMA, 1:100, ab7817, Abcam, Massachusetts, USA) were applied to the cells, which were incubated overnight. Then, the cells were incubated with suitable secondary antibodies (1:1000, ab150077, Abcam, Massachusetts, USA). Finally, the cells were stained with DAPI (C1002, Beyotime, Shanghai, China) and examined using a confocal laser scanning microscope (OLS5000, Olympus, Tokyo, Japan). The expression levels of ING4 and α-SMA in the endothelial cells were examined using images.

Tube formation assay

Endothelial cells, on reaching 80% confluency, were washed with PBS once or twice and then treated with trypsin (P4201, Beyotime, Shanghai, China). They were then incubated at 37°C for 2–3 min. To neutralize trypsin, a fresh complete medium was introduced, and the cells were resuspended to yield a single-cell suspension. The cell density was adjusted to 1 × 10⁶ cells/mL. Subsequently, 100 μL of the cell suspension with 1 × 10⁵ cells/mL was plated into a six-well plate and cultured overnight at 37°C in a 5% CO₂ atmosphere. The next day, after specific treatments, the cells were incubated for 48 h. Concurrently, Matrigel was thawed overnight at 4°C. All materials, including pipettes and tips, were prechilled. With a cold pipette tip, 100 μL of Matrigel was added to a prechilled 48-well plate, and even distribution in the wells was ensured. After the application of Matrigel to the plate, the plate was incubated for 40 min at 37°C for the solidification of the Matrigel matrix. Subsequently, the cells were counted, and their density was adjusted to 3 × 10⁵ cells/mL. The Matrigel-coated 48-well plate was washed with PBS 3 times. The volume of 200 μL of the cell suspension was 6 × 10⁴ cells. The suspension was dispensed into each well until the density was 8 × 10⁴ cells per well. The plate was then maintained at 37°C in an atmosphere containing 5% CO₂. After 5 h, tube formation was inspected using an inverted microscope, and images were obtained for subsequent analysis.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from the collected clinical tissues and cell lines with TRIzol reagent (15596026, ThermoFisher, Illinois, USA). A reverse transcription kit (G3335-50, Servicebio, Wuhan, China) was used in synthesizing complementary DNA (cDNA). For qRT-PCR detection, a reaction mixture consisting of cDNA, SYBR Green fluorescent dye and specific primers was prepared using SYBR Green I Master Mix on a 7900 sequence detector (Applied Biosystems, Foster City, CA, USA). The conditions for qRT- PCR were as follows: Initial denaturation step at 95°C for 10 min for 40 cycles. Each cycle consisted of a denaturation phase at 95°C for 15 s and an annealing/extension phase at 60°C for 1 min. The data were analyzed using the 2^−ΔΔCT method.^[22] For miRNAs, U6 served as the internal reference. As for mRNAs, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized as the internal reference. The primer sequences in RT-PCR are provided in Table S1.

Western blot

Protein lysates from tissues or cells were prepared using a radio-immunoprecipitation assay buffer (G2002, Servicebio, Wuhan, China) containing protease and phosphatase inhibitors (G2006, Servicebio, Wuhan, China). Protein concentrations were measured with a bicinchoninic acid assay. Equal amounts of the proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (G6015, Servicebio, Wuhan, China). These were incubated overnight at 4°C with primary antibodies against E-cadherin (1:1000; ab227639; Abcam), N-cadherin (1:1000; ab76011; Abcam), vimentin (1:1000; EPR3776; Abcam), Matrix Metalloproteinase-2 (MMP2, 1:1000; ab92536; Abcam), Matrix Metalloproteinase-9 (MMP9, 1:1000; ab76003; Abcam), and GAPDH (1:1000; ab8245; Abcam). After incubation with primary antibodies, the membranes were washed and treated with Horseradish Peroxidase -linked secondary antibodies (1:5000, ab205718, Abcam, Massachusetts, USA), and bands were detected using an enhanced chemiluminescence system (G2020, Servicebio, Wuhan, China). Band intensities were quantified, and expression was normalized to that of GAPDH.

In vivo model

Breeders’ Association of Laboratory Animals, California nude mice (6–8 weeks old, approximately 20 g, n = 20) were purchased from Animal Center of Tianjin Medical University. They were subcutaneously injected with 5 × 10⁶ H1299 cells each. The mice were divided into four groups (n = 5) and received intratumoral injections every 3 days: 100 μL PBS (the blank group), H1299-derived exosomes (the control group), exosomes with miR-5703 inhibitor NC (the NC group), and exosomes with miR-5703 inhibitor (the inhibitors group). Tumor volumes were measured weekly with the formula V = width² × length/2 (mm³). On day 28, mice were euthanized after 1% pentobarbital sodium anesthesia (50 mg/kg). Tumors were collected, weighed, and prepared for histological analysis. For hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunohistochemistry, the tissues were fixed in 4% paraformaldehyde. For quantitative PCR (qPCR) and Western blot, the samples were stored at −80°C. Ethics approval was obtained from the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital (approval No. 2023078). All animal experiments were conducted in strict accordance with the guidelines for the care and use of laboratory animals established the Second People’s Hospital of Yibin. All procedures were performed under appropriate anesthesia, and all animals received humane care throughout the duration of the study.

H&E staining

The tissues were fixed in paraformaldehyde, embedded in paraffin, and sectioned continuously (4 μm). After dehydration and dewaxing, the sections were sequentially stained with H&E (G1076, Servicebio, Wuhan, China), mounted with neutral resin, and examined under a bright- field microscope (Eclipse 80i, Nikon, Tokyo, Japan) for histopathological analysis.

Immunohistochemistry

After dehydration and dewaxing, the tissue sections were subjected to antigen retrieval and peroxidase blocking. After blocking with serum, the sections were incubated overnight with the following primary antibodies: E-cadherin (1: 100, ab227639; Abcam), N-cadherin (1: 50, ab76011, Abcam), Ki67 (1:500, ab15580; Abcam), and CD34 (1:200, ab81289; Abcam). Then, the sections were incubated with secondary antibodies for 1 h (1:5000, ab205718, Abcam). After developing with 3,3’-Diaminobenzidine (G1212, Servicebio, Wuhan, China), the sections were counterstained with hematoxylin, mounted with neutral resin, and examined under a bright-field microscope (Eclipse 80i, Nikon, Tokyo, Japan) for the assessment of protein expression levels and localization.

TUNEL assay

After dehydration and dewaxing, the tissue sections were treated with proteinase K (MK1012-100, Boster Biotechnology, Wuhan, China). After thorough washing with PBS, a TUNEL assay reagent consisting of Terminal Deoxynucleotidyl Transferase enzyme, fluorescence labeling solution, and TUNEL detection solution (MK1012-100, Boster Biotechnology, Wuhan, China) was applied. The sections were then incubated at 37°C for 60 min, extensively washed with PBS, and finally mounted using an antifade mounting medium to prevent fluorescence quenching. The sections were then observed with a fluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan). The excitation and emission wavelengths of Cy3 were 550 and 570 nm, respectively, resulting in red fluorescence.

Luciferase assay

For ING4 and has-miR-5703 luciferase assay, the cells were co-transfected with 50 nM miR-5703 mimics and 200 ng of a pmirGLO-ING4 vector or corresponding mutant type. A dual luciferase assay kit (Promega, WI, USA) was used for dual luciferase assay. At 48 h post-transfection, cells were collected, and Renilla luciferase activity was assessed. Results were assessed as the ratios of Firefly luciferase activity to Renilla luciferase activity.

Data collection and bioinformatics analysis

To explore the potential mechanisms of circulating exosomal biomarkers in the plasma of patients with lung cancer, we selected the GEO Series 198959 (GSE198959) dataset to analyze the differentially expressed miRNAs in the Exos of patients with recurrent lung cancer. At an adjusted P < 0.05 and |log2 (Fold Change)| of >1, highly expressed miRNAs were screened for subsequent validation. Kaplan–Meier curves were generated using Kaplan–Meier Plotter (KMplot, www.kmplot.com).^[23] The gene expression data and relapse- free and Overall Survival information in the KMplot program were downloaded from Gene Expression Omnibus (GEO), European Genome-phenome Archive, and The Cancer Genome Atlas.

Data analysis

Data were presented as mean ± Standard deviation. The Statistical Package for the Social Sciences 19.0 (IBM, Armonk, NY, USA) was used for statistical analysis. Analysis of variance and Turkey’s post hoc tests were used for multiple- group comparisons, and t-test was utilized for two-group comparisons. P < 0.05 indicated statistical significance. Image editing was conducted using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA).

Expression characteristics of miR-5703 in lung cancer

In a bid to identify potential factors in lung cancer, a study was conducted based on the GEO dataset database, using the GSE198959 dataset. Five miRNAs, namely, miR-4286, miR-6869, miR-630, miR-5703, and miR- 7641, were identified. By performing qPCR detection on clinical lung cancer tissues (T) and peri-cancerous tissues, it was observed that the expression levels of the miRNAs significantly varied in the lung cancer tissues (P < 0.0001), [Figure 1a].

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Figure 1:

Expression profiling and clinical relevance of microRNA-5703 (miR-5703) in lung cancer. (a) Quantitative polymerase chain reaction detection of the relative expression levels of miR-4286, miR-6869, miR-630, miR-5703, and miR-7641 in lung cancer tissues compared with peri-cancerous tissues. Significant difference in the expression of each microRNA was found between the groups. (b) Kaplan– Meier survival analysis revealed the significant correlations of miR-4286, miR-6869, miR-630, miR-5703, and miR-7641 expression levels to patient prognosis. n = 20, ^✶✶✶✶P < 0.0001 versus peri-cancerous tissues. Pt: peri-cancerous tissues; T: lung cancer tissues

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Further, employing the Kaplan–Meier Plotter platform (http://kmplot.com/analysis/index.php?p=background), the correlation of miR-4286, miR-6869, miR-630, miR-5703, and miR-7641 with lung cancer patients was respectively analyzed. Results revealed a significant correlation between miR-5703 and patient prognosis (P = 0.0011), [Figure 1b].

Expression characteristics of miR-5703 in exosomes of patients with lung cancer

Exosomes were isolated from the serum samples of healthy individuals and patients with lung cancer for characterization and detection. Electron microscopy observation showed that the exosomes were approximately 100 nm in size [Figure 2a]. Western blotting revealed the presence of CD81, CD63, and Alix and the absence of calnexin and α-tubulin within the exosomes [Figure 2b]. FCM tests showed a clear CD81-positive marker in the exosomes [Figure 2c].

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Figure 2:

Characterization and microRNA-5703 (miR-5703) quantification of exosomes derived from serum of lung cancer patients and healthy individuals. (a) Electron microscopy image showing exosomes of approximately 100 nm in size, n = 3. (b) Western blotting results of specific exosome markers CD81, CD63, and Alix and negative markers calnexin and α-tubulin, n = 3. Pt: healthy individuals; T: lung cancer patients. (c) Flow cytometry test showing the CD81-positive marker within the exosomes, n = 3. (d) Particle size analysis of the extracted exosomes showing diameters ranging from 50 nm to 120 nm, n = 3. (e) Quantitative polymerase chain reaction results showing significantly higher levels of miR-5703 in the serum exosomes of patients with lung cancer compared with the healthy individuals, n = 20. ^✶✶✶✶P < 0.0001 versus health control. SS-A: Side scatter-area

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Subsequent analysis of particle size revealed that the diameters of the isolated exosomes varied from 50 nm to 120 nm, and no significant difference in diameter was observed between the exosomes obtained from the healthy individuals and those from patients with lung cancer [Figure 2d]. However, qPCR results showed that the concentration of miR-5703 in serum exosomes from the patients with lung cancer was significantly elevated compared with that in serum exosomes from the healthy individuals (P < 0.0001), [Figure 2e].

Biological role of miR-5703 in lung cancer cells

To explore the biological role of miR-5703 in lung cancer, the experiment incorporated various lung cancer cell lines and a non-tumorigenic cell line (BEAS2B). The highest expression levels of miR-5703 were observed in the 95-D and H1299 cell lines [Figure 3a]. Thus, these cell lines were selected for further experiments.

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Figure 3:

Functional analysis of microRNA-5703 (miR-5703) in 95-D and H1299 lung cancer cell lines. (a) Relative miR-5703 expression levels in different lung cancer cell lines and non-tumor cell line Bronchial Epithelial Cell Line 2B as detected by quantitative polymerase chain reaction (qPCR).SK-MES: Squamous Cell Carcinoma- MES; NCI-H1688: National Cancer Institute- H1688. (b) miR-5703 expression in the 95-D and H1299 cell lines after treatment with miR-5703 inhibitors or mimics, as demonstrated by quantitative polymerase chain reaction. (c and d) Cell migration (c) and invasion (d) assays showing the effects of miR-5703 expression modulation in the 95-D and H1299 cells. Scale: 50 μm. (e and f)5-ethynyl-2'-deoxyuridine staining (e, scale: 20 μm) and CCK8 kit method tests. DAPI: 4’,6-Diamidino-2-phenylindole, a commonly used fluorescent dye for staining the nucleus. (f) demonstrating changes in cell proliferation and vitality respective to different treatments. (g) Western blot results showing altered the expression of E-cadherin, N-cadherin, vimentin, MMP2, and MMP9 in the cells treated with the miR-5703 inhibitors or mimics. MMP2: Matrix Metalloproteinase-2; MMP9: Matrix Metalloproteinase-9; NC: Negative Control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. n = 3, ^✶P < 0.05, ^✶✶P < 0.01, ^✶✶✶P < 0.001 versus negative control.

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In the 95-D and H1299 cells, the expression of miR-5703 was manipulated. As shown in Figure 3b, the inhibitors led to a significant decrease in miR-5703 expression (P < 0.01), whereas the mimics caused a dramatic increase in miR- 5703 expression (P < 0.001), [Figure 3b]. Subsequently, the malignant behavior of the cells was assessed. As shown in Figure 3c and d, the inhibitors led to a significant decrease in 95-D and H1299 cell migration and invasion (P < 0.05), whereas the mimics led to an increase in migration and invasion (P < 0.05), [Figure 3c and d]. The results of EdU staining and CCK8 kit on the 95-D and H1299 cells showed that cell proliferation was significantly inhibited and promoted, respectively (P < 0.05), [Figures 3e and f, S1a].

Western blot results showed that the inhibitors enhanced the expression of E-cadherin and suppressed the expression of N-cadherin, vimentin, MMP2, and MMP9. Meanwhile, cells treated with the mimics showed reduced E-cadherin and increased N-cadherin, vimentin, MMP2, and MMP9 expression levels [Figure 3g].

Consistent with the FCM results, Hoechst staining results showed that lung cancer cells treated with the inhibitors had increased number of apoptosis cells (P < 0.05), [Figure 4a and b]. Conversely, after the cells were treated with the mimics, apoptosis rate decreased significantly (P < 0.05), [Figure 4a and b]. These results suggested that miR-5703 promoted the malignant biological behavior of the lung cancer cells. Notably, after treatment with the miR-5703 inhibitors and mimics, the exosomes showed a significant decrease (P < 0.01) or increase (P < 0.001) in miR-5703 abundance, respectively [Figure 5].

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Figure 4:

Impact of microRNA-5703 (miR-5703) modulation on apoptosis in lung cancer cells. (a) Hoechst staining images showing increased apoptosis in lung cancer cells treated with the miR-5703 inhibitors and decreased apoptosis after treatment with mimics. White arrows indicate apoptotic cells. Scale: 20 μm. (b) Flow cytometry results reflecting those of Hoechst staining showing change in apoptosis levels after the treatments. n = 3, ^✶P < 0.05, ^✶✶P < 0.01. NC: Negative Control.

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Figure 5:

Modulation of microRNA-5703 (miR-5703) levels in exosomes derived from lung cancer cells after treatment with inhibitors and mimics. Quantitative polymerase chain reaction results showing a significant decrease in miR-5703 expression in exosomes derived from lung cancer cells treated with the inhibitors and significant increase after treatment with mimics. n = 3, ^✶✶P < 0.01, ^✶✶✶P < 0.001. NC: Negative Control.

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Effect of lung cancer cell–derived exosomes on TAECs

TAECs isolated from lung cancer tissues were CD34 positive according to the immunofluorescence [Figure 6a] and FCM results, and the positive ratio exceeded 95% [Figure 6b].

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Figure 6:

Influence of microRNA-5703 (miR-5703) modulation in lung cancer cells on tumor-associated endothelial cells (TAECs). (a) Immunofluorescence visualization (scale: 20 μm) and (b) flow cytometry quantification revealing >90% CD34-positive ratio in isolated TAECs from lung cancer tissues. (c-d) Western blot (c) and immunofluorescence (d, scale: 20 μm) displayed change in α-smooth muscle actin (α-SMA) expression in endothelial cells upon co-culture with lung cancer cells treated with miR-5703 inhibitors or mimics. GADPH: glyceraldehyde-3-phosphate dehydrogenase. (e-f) Variation in the angiogenesis (e) and migration (f, scale: 50 μm) of endothelial cells after co-cultivation. (g) Western blot analysis of Claudin-5, Occludin and ZO-1 protein expression in endothelial cells co-cultured with lung cancer cells treated with inhibitors or mimics. n = 3, ^✶P < 0.05, ^✶✶P < 0.01 versus negative control. NC: Negative Control.

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The co-cultivation of TAECs and lung cancer cells were performed. Notable changes in α-SMA expression in TAECs were observed. Western blot and immunofluorescence results showed that α-SMA expression was attenuated in the TAECs co-cultured with inhibitor-treated lung cancer cells. By contrast, endothelial cells co-cultured with lung cancer cells treated with the mimics showed rapid increase in α-SMA expression (P < 0.01), [Figure 6c]; (P < 0.05), [Figures 6d and S1b]. The results showed that TAECs co-cultured with lung cancer cells treated with inhibitors showed inhibited tube formation and migration. Conversely, lung cancer cells treated with mimics promoted TAECs tube formation and migration abilities (P < 0.05), [Figure 6e and f]. The elevated expression levels of claudin-5, occludin, and ZO-1 were observed in the TAECs co-cultured with inhibitor-treated lung cancer cells, and expression in TAECs was suppressed after co-culturing with mimic-treated lung cancer cells. Surprisingly, in H1299, ZO-1 expression was reversely reduced under inhibitor treatment (P < 0.05), [Figure 6g].

Lung cancer cell-derived exosomal miR-5703 promoted TAEC EndMT and angiogenesis and inhibited cell adjunction

To elucidate the effect of exosomal miR-5703 on the biological functions of TAECs, the exosomes derived from lung cancer cells were isolated, and then co-cultured with TAECs. Concurrently, to assess the direct impact of exosome uptake on miR-5703 levels in TAECs, the TAECs were treated with Cytochalasin D, an inhibitor known to block exosome uptake, as a NC to mimic the absence of exosomes. As shown in Figure 7a, PKH67-labeled exosomes were taken up by the TAECs. Exosomes derived from lung cancer cells significantly up-regulated miR-5703 levels in the TAECs (P < 0.001), but treatment with cytochalasin D reversed this effect (P < 0.001). No significant difference in miR-5703 level was observed between these cells and the cells in the blank group, indicating the effective inhibition of exosome uptake [Figure 7b].

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Figure 7:

Lung cancer cell-derived exosomes encapsulating microRNA-5703 (miR-5703) regulate EndMT, angiogenic ability, and cell adjunction in tumor-associated endothelial cells (TAECs). (a) Confocal microscopy images Ashowing PKH67-labeled exosome uptake by TAECs after co-culturing. Scale: 20 μm. (b) Quantitative polymerase chain reaction analysis revealing increased miR-5703 levels in TAECs co-cultured with exosomes or treated with cytochalasin D. (c) Migration assay demonstrating the enhanced migratory capacity of TAECs. Scale: 50 μm. (d) α-smooth muscle actin expression was measured through immunofluorescence. Scale: 20 μm. (e) The angiogenesis of TAECs was evaluated using tube formation assay. ^✶P < 0.05, ^✶✶P < 0.01, ^✶✶✶P < 0.001. EXOs: exosomes; NC: Negative Control.

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Further, results showed that exosome-induced migration (P < 0.001) and tube formation in TAECs, along with the inhibition of α-SMA expression, can be abolished by exosomes from inhibitor-treated lung cancer cells (P < 0.001) and partially abolished by exosome uptake inhibitors [Figure 7c-e]. Specifically, exosome-treated TAECs exhibited diminished E-cadherin, claudin-5, occludin, and Zonula Occludens-1(ZO-1) expression levels, and elevated N-cadherin and vimentin expression can be reversed through incubation with exosomes from inhibitor-treated lung cancer cells or through cytochalasin D treatment [Figure S2].

miR-5703 directly targets ING4

The targets of miR-5703 were predicted using the TargetScan database (https://www.targetscan.org/vert_80/). Using the WEB-Based Gene SeT AnaLysis Toolkit (https://www.webgestalt.org/), we found that the target gene set was associated with angiogenesis and lung cancer [Figure S3]. After the target set (cumulative weighted context++ score < −0.6, Total context++ score < −0.8) was screened, potential miR-5703 targets were obtained [Table S2]. Subsequently, the expression of each gene and prognosis was evaluated using the Gene Expression Profiling Interactive Analysis database (http://gepia.cancer-pku.cn/index.html). ING4 was determined as the target of miR-5703 for subsequent experiments [Figures S4, S5, and 8a].

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Figure 8:

Identification of the target genes of microRNA-5703 (miR-5703) and correlation analysis. (a) Inhibitor of growth family, member 4 (ING4) was screened as the target of miR-5703. SPATA: Spermatogenesis-Associated Transcript; SCN: Sodium Channel; CBL: Casitas B-lineage Lymphoma; RHO: Rhodopsin; ZNF133: Zinc Finger Protein 133. (b) The expression feature of ING4 in the clinical tissues of patients with lung cancer. (c) Binding site information for miR-5703 and ING4 as provided by the TargetScan database. (d) Verification of the relationship between miR-5703 and ING4 in 293T cells. WT: Wild Type; MUT: Mutant. (e and f) miR-5703-mediated suppression of ING4 expression observed on miR-5703 messenger RNA (e) and ING4 protein (f) levels. n = 3, ^✶P < 0.05, ^✶✶P < 0.01, ^✶✶✶P < 0.001. NC: Negative Control; Para-ca: adjacent tissues; Ca: Lung cancer tissues.

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The difference in ING4 expression between lung cancer and adjacent tissues was detected through qPCR [Figure 8b]. ING4 expression in cancer tissues was significantly lower than in the adjacent tissues (P = 0.0094), [Figure 8b]. The TargetScan database provided binding site information for miR-5703 and ING4 [Figure 8c]. Then, whether miR-5703 can directly interact with ING4 in 293T cells, which was determined with dual-luciferase reporter assays. miR-5703 overexpression significantly reduced luciferase activity [Figure 8d], and the transfection of miR-5703 mimic mediated the suppression of ING4 protein expression [Figure 8e and f].

Modulation of the biological function of TAECs by miR- 5703 through ING4

To determine the biological effects of miR-5703 in endothelial cells and explore the latent mechanism of miR-5703/ING4 interactions, we overexpressed miR-5703 and reinstated ING4 expression. As corroborated by the immunofluorescence and Western Blot results, mimic treatment subdued ING4 expression. Concurrently, after transfection with the ING4 overexpression vector, rapid increase in the expression of ING4 was observed [Figure 9a and b].

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Figure 9:

Biological role of microRNA-5703 (miR-5703) in endothelial cells and functional investigation of miR-5703/Inhibitor of growth family, member 4 (ING4) interactions. (a and b) Results from immunofluorescence (a, scale: 20 μm) and Western blot (b) showing the effect of mimic treatment on ING4 expression. After transfection with the ING4 overexpression vector, an increase in ING4 expression was observed. (c and d) Immunofluorescence (c, Scale: 20 μm) and Western blot (d) illustrating increased α-smooth muscle actin levels in the endothelial cells after mimic treatment and reduced level after ING4 overexpression. (e) Transwell experiments displaying an increase in endothelial cell migration after mimic treatment and the reversal of this effect by ING4 overexpression. Scale: 50 μm. (f) Increased angiogenesis (blood vessel formation) in endothelial cells after mimic treatment and the reversal of this effect by ING4 overexpression. (g) Western blot showing the reduced expression of claudin-5, occludin, and ZO-1 after mimic treatment and restoration of their expression after ING4 overexpression. n = 3, ^✶P < 0.05, ^✶✶P < 0.01. NC: Negative Control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; α-SMA: α-smooth muscle actin; ZO-1: Zonula Occludens-1.

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The immunofluorescence and Western blot results also showed a heightened level of α-SMA in the endothelial cells treated with the mimics. By contrast, the overexpression of ING4 led to a reduction in the expression of α-SMA [Figure 9c and d]. In addition, as depicted in Figure 9e, the mimics promoted TAEC migration (P < 0.01) and tube formation and inhibited cell junctions, resulting in decreased expression of claudin-5, occludin, and ZO-1. However, the overexpression of ING4 reversed this mimics-induced effect (P < 0.01, [Figure 9e-g]).

miR-5703-loaded exosomes enhanced tumor growth and tumor microvasculature and impaired endothelial cell barrier function through ING4

To investigate the effect of exosomes encapsulated with miR-5703 on tumor growth in vivo, exosomes derived from lung cancer cells were administrated to animals, with miR-5703 inhibitors injected intratumorally as a remedial measure. As demonstrated in Figure 10, exosomes from lung cancer cells promoted tumor volume and mass growth (P < 0.05, [Figures 10a-d]). However, treatment with miR-5703 inhibitors significantly inhibited exosome-induced enhancement in tumor mass and volume (P < 0.01, [Figure 10a-d]). H&E staining revealed no significant histological changes in the tumor tissues after exosome treatment, but miR-5703 inhibitors led to areas of apoptosis [Figure 10e].

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Figure 10:

Exosomal microRNA-5703 (miR-5703) and its inhibition affect tumor growth and protein expression. (a-d) Exosome treatment increased tumor volume and mass, but this effect was reversed by miR-5703 inhibitors. ^✶P < 0.05, ^✶✶P < 0.01. (e) Hematoxylin and eosin staining (H&E) showed apoptotic regions after miR-5703 inhibitor treatment. Green dotted lines highlighted the areas of tissue apoptosis and necrosis. (f-i) Immunohistochemical analysis revealed the altered expression of Cluster of Differentiation 34 (CD34) (f), N-cadherin (N-Cad, g), E-cadherin (E-Cad, h), and Kiloelectron Volts 67 (Ki-67). (i), in response to treatments. (j) Terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) indicated enhanced apoptosis with miR-5703 inhibitors. (k) Western blot analysis demonstrated changes in the expression of claudin-5, Zonula Occludens-1 (ZO-1), occludin, and Inhibitor of growth family, member 4 due to exosome and inhibitor treatments. MMP2: Matrix Metalloproteinase-2; MMP9: Matrix Metalloproteinase-9; ZO-1: Zonula Occludens-1; ING4: INhibitor of Growth 4; GADPH: glyceraldehyde-3-phosphate dehydrogenase. Scale: 50 μm. n = 5, ^✶P < 0.05, ^✶✶P < 0.01 versus Blank; #P < 0.05, ##P < 0.01 versus Exo-negative control. NC: Negative Control.

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In addition, the expression of CD34, N-cadherin, and Ki-67 increased in the tumor tissues of the exosome-injected groups (including the inhibitor NC group) compared with the blank group, whereas E-cadherin expression decreased. Treatment with miR-5703 inhibitors significantly reduced the expression of CD34, N-cadherin, and Ki-67, while up-regulating E-cadherin [Figures 10f-i and S6a-d]. Moreover, exosome treatment did not alter TUNEL signaling in the tissues, which was enhanced by miR-5703 inhibitor treatment [Figure 10j]. Similarly, the results of Western blot analysis confirmed the immunohistochemistry findings for E-cadherin and N-cadherin expression. Furthermore, it was observed that exosome treatment decreased the expression of the intercellular junction proteins claudin-5, ZO-1, occludin, and ING4, while miR-5703 inhibitor treatment increased their expression levels [Figure 10k]. H&E staining showed that the cell nuclei of the adjacent noncancerous tissues were of the same shape, the nuclear staining was uniform and regular, and there were no significant pathological changes, while the cell atypia of the cancerous tissues was obvious, the tissue structure was disordered, and the growth pattern was invasive [Figure S7a]. The expression of CD34 and ING4 was determined using immunohistochemistry. CD34 was more extensively expressed in the cancerous tissues than in adjacent noncancerous tissues, whereas ING4 expression was down-regulated in the cancerous tissues [Figure S7b, c].