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. 2026 Sep 23;19:609640. doi: 10.2147/OTT.S609640

Reduced Exosomal miR-22 Promotes Cisplatin Resistance and Migration in Non-Small Cell Lung Cancer Cells Through MDM4 Upregulation

Peng Wang 1, Jie Jiao 1, Lijun Yu 1, Rigetu Zhao 1,✉
PMCID: PMC13616163  PMID: 42802834

Abstract

Background

Cisplatin resistance remains a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Exosome-mediated transfer of microRNAs contributes to tumor progression and chemotherapy resistance. However, the role of exosomal miR-22 in cisplatin resistance and its relationship with mouse double minute 4 homolog (MDM4)-regulated p53 signaling remain unclear.

Methods

Parental A549 cells and cisplatin-resistant A549/CDDP cells were used as a wild-type p53 NSCLC cell model. Exosomes were isolated from conditioned medium and characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Exosome uptake was evaluated by PKH26 labeling with a dye-only control. Cell viability, migration, and apoptosis were assessed using Cell Counting Kit-8, Transwell, and Annexin V-FITC/PI assays, respectively. A dual-luciferase reporter assay and miR-22/MDM4 gain- and loss-of-function experiments were performed to investigate the underlying mechanism. The p-p53/p53 ratio and the expression of p21, cleaved caspase-3, and γ-H2AX were determined by Western blotting.

Results

A549/CDDP-derived exosomes contained significantly less miR-22 than A549-derived exosomes. Both exosome preparations were efficiently internalized by recipient cells, whereas the processed PKH26 dye-only control showed negligible fluorescence. A549/CDDP-derived exosomes increased cell viability and migration under cisplatin exposure, whereas A549-derived exosomes enhanced cisplatin sensitivity. MiR-22 directly targeted the 3′-untranslated region of MDM4 and suppressed MDM4 expression. MDM4 silencing inhibited cell viability and migration, whereas MDM4 overexpression partially reversed the suppressive effects of miR-22. Furthermore, miR-22 increased the p-p53/p53 ratio, p21, cleaved caspase-3, and γ-H2AX expression and promoted apoptosis; these effects were attenuated by MDM4 overexpression. MDM4 silencing also restored p53-associated DNA-damage and apoptotic signaling suppressed by A549/CDDP-derived exosomes.

Conclusion

In the wild-type p53 A549/A549-CDDP model, reduced exosomal miR-22 contributes to cisplatin resistance through MDM4-associated suppression of p53-related DNA-damage and apoptotic responses. These findings identify the exosomal miR-22/MDM4 axis as a candidate mechanism of cisplatin resistance that requires further validation in additional NSCLC models, animal studies, and patient-derived samples.

Keywords: non-small cell lung cancer, exosomes, miR-22, MDM4, p53, cisplatin resistance

Introduction

Lung cancer is one of the most common types of cancer worldwide.1 Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases.2 With advances in targeted therapy and immunotherapy, the treatment of NSCLC has become increasingly dependent on disease stage, molecular alterations and immune characteristics. Platinum-doublet chemotherapy, using cisplatin (CDDP) or carboplatin, remains an important component of NSCLC treatment and is frequently combined with immune checkpoint inhibitors in patients without actionable driver alterations. However, intrinsic or acquired resistance to platinum-based chemotherapy continues to limit treatment efficacy.3 Understanding the mechanisms underlying CDDP resistance is therefore important for improving therapeutic responses in NSCLC.

In recent years, the potential roles of exosomes in cancer diagnosis and treatment have gained increasing attention. Exosomes are small membrane-enclosed vesicles, typically 30–150 nm in diameter, that are released by various cell types, including stem cells, immune cells and cancer cells, into the extracellular environment.4,5 They carry a wide range of bioactive molecules, including proteins, lipids and nucleic acids, and can transfer these cargos to recipient cells, thereby modulating tumor progression and drug resistance.6 MicroRNAs (miRNAs) are an important class of exosomal cargo and have been extensively investigated in cancer biology. These small non-coding RNAs regulate the expression of different target mRNAs via binding to their 3’-untranslated region (3’-UTR), thus resulting in translational repression or mRNA degradation.7 Dysregulated miRNAs participate in multiple biological processes, including cancer initiation, progression and sensitivity to chemotherapy.8,9

The miR-22 gene is an evolutionarily conserved miRNA located on chromosome 17p13.10 Aberrant miR-22 expression has been reported in several types of cancer, including prostate cancer, esophageal squamous cell carcinoma, breast cancer, and gastric cancer.11–14 Depending on the cellular and molecular context, miR-22 may exert either tumor-suppressive or tumor-promoting effects. Previous studies have demonstrated that miR-22 participates in the regulation of chemotherapy responsiveness in osteosarcoma,15,16 thus affecting CDDP sensitivity via regulating osteosarcoma cell proliferation, invasion, and metastasis.17 However, whether miR-22 is differentially carried by exosomes released from CDDP-resistant NSCLC cells and whether exosome-mediated transfer of miR-22 influences the CDDP response of recipient cells remain unclear.

Mouse double minute 4 homolog (MDM4) is an important negative regulator of the tumor suppressor p53. MDM4 can interact with p53 and suppress its transcriptional activity, thereby attenuating p53-dependent cell-cycle arrest and apoptosis following cellular stress.18 This regulatory relationship may be relevant to CDDP resistance because CDDP induces DNA damage and activates p53-associated cellular responses. MDM4 is aberrantly expressed in several malignancies and can be regulated by upstream miRNAs.19 Nevertheless, whether MDM4 is directly regulated by miR-22 in NSCLC cells and whether the miR-22/MDM4 axis affects CDDP sensitivity through p53-associated DNA-damage and apoptotic signaling remain unknown.

In the present study, exosomes were isolated from the culture supernatants of parental A549 cells and CDDP-resistant A549/CDDP cells. The effects of exosome-mediated miR-22 transfer on cell viability, migration and CDDP sensitivity were evaluated. A dual-luciferase reporter assay and MDM4 gain- and loss-of-function experiments were performed to determine whether MDM4 acted as a functional downstream mediator of miR-22. Furthermore, because A549 cells harbor wild-type TP53, p53 phosphorylation, p21 expression, DNA-damage signaling and apoptosis were examined to further clarify the mechanism. The present study aimed to determine whether reduced exosomal miR-22 contributes to CDDP resistance in the p53-competent A549/A549-CDDP model by increasing MDM4 expression and suppressing p53-associated DNA-damage and apoptotic responses.

Materials and Methods

Cell Culture and Establishment of CDDP-Resistant Cells

The human A549 NSCLC cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). A549 cells harbor wild-type TP53 and were used as a p53-competent NSCLC cell model. Cells were cultured in DMEM supplemented with 10% FBS (both from HyClone; Cytiva), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified incubator containing 5% CO2. To establish the A549/CDDP model, parental A549 cells were exposed to 2 μM cisplatin (CDDP) for 24 h and subsequently cultured in drug-free medium until recovery. Before subsequent experiments, the cells were maintained in CDDP-free medium for at least 7 days. The resistant phenotype was confirmed using Cell Counting Kit-8 (CCK-8) dose–response assays. The half-maximal inhibitory concentration (IC50) was calculated by nonlinear regression, and the resistance index was calculated as the IC50 of A549/CDDP cells divided by that of parental A549 cells. Unless otherwise indicated, experiments involving A549/CDDP cells were performed in the absence of CDDP.

Cell Transfection

MiR-22 mimic, mimic negative control (mimic NC), miR-22 inhibitor, inhibitor negative control (inhibitor NC), small interfering RNA targeting MDM4 (si-MDM4), and the corresponding siRNA negative control (si-NC) were designed and synthesized by Shanghai GenePharma Co., Ltd. The pcDNA3.1-MDM4 plasmid was used to overexpress MDM4, and the empty pcDNA3.1 vector served as the corresponding control. A549 or A549/CDDP cells were transfected with 250 pmol miRNA or siRNA using Lipofectamine® 2000 (Nanjing Enhancer Bio-Technology Co., Ltd.) according to the manufacturer’s instructions. Plasmid transfection was performed using the same reagent according to the manufacturer’s protocol. After approximately 5 h, the transfection medium was replaced with complete culture medium, and the cells were cultured for an additional 24–48 h. Transfection efficiency was evaluated by RT-qPCR and Western blotting. A Cy3-labeled miR-22 mimic was used in the exosome-mediated miR-22 transfer experiments.

Exosome-Mediated Transfer of Cy3-Labeled miR-22

To evaluate exosome-mediated miR-22 transfer, donor A549 cells were transfected with Cy3-labeled miR-22 mimic or mimic NC. After transfection, the culture medium was replaced with complete medium containing exosome-depleted FBS, and the conditioned medium was collected for exosome isolation. The isolated exosome fractions were subsequently added to recipient A549 cells. Where indicated, donor cells transfected with Cy3-labeled miR-22 mimic were treated with the exosome-release inhibitor GW4869 before conditioned-medium collection. Exosome fractions obtained from equal numbers of donor cells were applied to recipient cells. Following incubation, recipient cells were washed with PBS, stained with Hoechst and examined by fluorescence microscopy. The expression of miR-22 and the response to CDDP were subsequently evaluated.

RT-qPCR Analysis

Total RNA was extracted from cells or isolated exosomes using TRIzol® reagent (Nanjing Enhancer Bio-Technology Co., Ltd.) and reverse transcribed into cDNA using the PrimeScript RT kit (Takara Bio, Inc). RT-qPCR was performed to determine the relative expression levels of miR-22 and MDM4. Cellular and exosomal miR-22 expression was normalized to U6, whereas MDM4 mRNA expression was normalized to GAPDH. Relative expression was calculated using the 2−ΔΔCq method. The primer sequences were as follows: GAPDH forward, 5′-CGGACCAATACGACCAAATCCG-3′ and reverse, 5′-AGCCACATCGCTCAGACACC-3′; miR-22 forward, 5′-GCTACTCGAGTCCCCCGCTCATCTGA-3′ and reverse, 5′-GCCAAGCTTCGAGGGGGAGCAAATCAC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; and MDM4 forward, 5′-AGGTACGACCAAAACTGCCG-3′ and reverse, 5′-CTGCACTTTGCTTCAGTTGGT-3′. Primer specificity was confirmed by melting-curve analysis.

Western Blot Analysis

Total proteins were extracted from cells or exosome preparations using RIPA lysis buffer (Beijing Solarbio Science and Technology Co., Ltd.) and quantified using a BCA protein assay kit (Thermo Fisher Scientific, Inc). Equal amounts of protein were separated by 10% SDS-PAGE (cat. no. 1200; Beijing Sunlight Biotechnology Co., Ltd.) and transferred onto 0.45-μm nitrocellulose membranes (MilliporeSigma). The membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 2 h at room temperature and incubated overnight at 4°C with primary antibodies against MDM4, CD63, CD81, tumor susceptibility gene 101 (TSG101), Calnexin and GAPDH. The newly examined proteins were detected using antibodies against cleaved caspase-3 (Proteintech Group, Inc.; cat. no. 25128-1-AP; 1:1,000), p21 (Proteintech Group, Inc.; cat. no. 27296-1-AP; 1:1,000), phospho-p53 (Ser15; Proteintech Group, Inc.; cat. no. 28961-1-AP; 1:1,000), total p53 (Proteintech Group, Inc.; cat. no. 10442-1-AP; 1:5,000), and phospho-Histone H2A.X (Ser139; γ-H2AX; Proteintech Group, Inc.; cat. no. 83307-2-RR; 1:5,000). Following washing, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies for 50 min at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence and quantified using ImageJ software. MDM4, p21, cleaved caspase-3 and γ-H2AX expression was normalized to GAPDH, whereas p-p53 expression was normalized to total p53.

Dual Luciferase Reporter Assay

The wild-type (WT) or mutant (MUT) MDM4 3′-UTR containing the predicted miR-22 binding region was cloned into the pGL6-miR vector (Beyotime Institute of Biotechnology). A549 cells were seeded into 12-well plates at a density of 5×104 cells/well and cultured for 24 h. The cells were subsequently co-transfected with the WT or MUT reporter construct and miR-22 mimic or mimic NC using Lipofectamine 2000. Luciferase activity was measured 24 h after transfection using a dual-luciferase reporter assay kit (Beyotime Institute of Biotechnology). Firefly luciferase activity was normalized to Renilla luciferase activity. The MUT construct contained nucleotide substitutions within the predicted miR-22 binding site of the MDM4 3′-UTR.

Cell Viability Assay

Cell viability was evaluated using a CCK-8 assay according to the manufacturer’s instructions. Briefly, A549 or A549/CDDP cells were seeded into 96-well plates at a density of 2×104 cells/well and cultured for 12–24 h. Following the indicated transfections and exosome treatments, the cells were exposed to CDDP as specified in the individual experiments. For dose–response analysis, cells were treated with 0, 1, 10, 30 or 60 μM CDDP for 24 h. Subsequently, 10 μL CCK-8 solution was added to each well, and the cells were incubated for 2 h at 37°C. Absorbance was measured at 450 nm using an Epoch microplate reader (Bio-Tek Instruments, Inc). Cell viability was calculated relative to the corresponding untreated control. Dose–response curves were fitted by nonlinear regression to determine IC50 values.

Transwell Migration Assay

Cell migration was evaluated using 24-well Transwell chambers with 8-μm pore-size membranes (Corning, Inc). Briefly, 1×104 cells suspended in serum-free medium were added to the uncoated upper chamber, and medium containing 10% FBS was added to the lower chamber. Following incubation for 24 h, non-migrated cells on the upper surface of the membrane were removed using a cotton swab. Cells that had migrated to the lower membrane surface were fixed, stained and photographed under an inverted microscope. Migrated cells were counted in randomly selected microscopic fields by investigators blinded to the group allocation.

Isolation of Exosomes from Culture Medium

A549 and A549/CDDP cells were cultured until they reached 70–80% confluence. The culture medium was then replaced with complete DMEM containing 10% exosome-depleted FBS, and the cells were cultured for an additional 48 h. The conditioned medium was sequentially centrifuged at 300×g for 10 min, 2,000×g for 20 min and 10,000×g for 30 min to remove cells, cellular debris and larger extracellular vesicles, respectively. The supernatant was subsequently filtered through a 0.22-μm membrane and ultracentrifuged at 100,000×g for 70 min at 4°C. The resulting pellet was resuspended in PBS and washed by a second ultracentrifugation at 100,000×g for 70 min. The final exosome pellet was resuspended in PBS and used immediately or stored at −80°C. Exosomes derived from A549 and A549/CDDP cells were designated A549-exo and A549/CDDP-exo, respectively. For the original functional co-culture assays, exosomes were added at a protein concentration of 50 μg/mL. For the newly added comparative signaling experiments, A549-exo and A549/CDDP-exo were normalized to equivalent particle numbers based on nanoparticle tracking analysis.

Nanoparticle Tracking Analysis

The size distribution and particle concentration of A549-exo and A549/CDDP-exo were determined using a NanoSight NS300 instrument (Malvern Panalytical, Malvern, UK). Exosome suspensions were diluted in particle-free PBS to an appropriate concentration and analyzed according to the manufacturer’s instructions. The mean particle diameter, modal diameter and particle concentration were recorded. Three independently isolated exosome preparations from each group were analyzed.

Transmission Electron Microscopy (TEM)

Exosomes were fixed in 2% glutaraldehyde, followed by post-fixation in 1% osmium tetroxide. Ultrathin sections were prepared and stained with 10% uranyl acetate and 1% lead citrate. The ultrastructure of exosomes was observed under a Hitachi H-7000 transmission electron microscope (Hitachi, Ltd).

Fluorescent Labeling of Exosomes

A549-exo and A549/CDDP-exo were labeled using the PKH26 lipophilic dye (cat. no. MINI26-1KT; MilliporeSigma). Briefly, 25 μL exosome suspension was diluted in 1 mL Diluent C, and 4 μL PKH26 dye was separately diluted in 1 mL Diluent C. The two solutions were mixed and incubated for 3 min. The labeling reaction was terminated by adding 2 mL FBS. Labeled exosomes were collected by ultracentrifugation at 100,000×g for 70 min and washed with PBS to remove unincorporated dye. For the dye-only control, PBS without exosomes was subjected to the same PKH26 labeling, termination, ultracentrifugation and washing procedures. Recipient A549 cells were assigned to the Control, dye-only, A549-exo and A549/CDDP-exo groups and incubated with the corresponding preparations for 4 h. The cells were then washed thoroughly with PBS, stained with Hoechst and examined by confocal microscopy. Intracellular PKH26 fluorescence intensity was quantified using ImageJ software under identical image-acquisition settings.

MDM4 Rescue and p53-Associated Signaling Experiments

To evaluate the role of MDM4 in miR-22-mediated p53 signaling, A549/CDDP cells were assigned to the mimic NC + Vector, miR-22 mimic + Vector and miR-22 mimic + OE-MDM4 groups. To evaluate the effects of resistant-cell-derived exosomes, cells were assigned to the Control, A549-exo, A549/CDDP-exo, A549/CDDP-exo + si-NC and A549/CDDP-exo + si-MDM4 groups. At 24–48 h after transfection and exosome treatment, cells were exposed to 10 μM CDDP for 24 h. MDM4, p53, p-p53, p21, cleaved caspase-3 and γ-H2AX expression was subsequently determined by Western blotting, and apoptosis was evaluated by flow cytometry where indicated.

Flow Cytometric Analysis of Apoptosis

Cell apoptosis was evaluated using a FITC Annexin V Apoptosis Detection Kit I (cat. no. 556547; BD Biosciences) according to the manufacturer’s instructions. Following the indicated treatments, cells were collected, washed twice with cold PBS and resuspended in 1× binding buffer. Approximately 1×105 cells were incubated with FITC Annexin V and propidium iodide for 15 min at room temperature in the dark and analyzed using a BD Accuri C6 Plus flow cytometer (BD Biosciences). Annexin V-FITC-positive/PI-negative cells were defined as early apoptotic cells, whereas Annexin V-FITC-positive/PI-positive cells were defined as late apoptotic cells. Total apoptosis was calculated as the sum of the early and late apoptotic populations.

Statistical Analysis

All experiments were independently repeated three times using separate cell cultures or exosome preparations. For CCK-8 assays, each biological replicate contained at least three technical replicate wells. Data are presented as the mean ± standard deviation. Statistical analyses were performed using SPSS version 22.0 (IBM Corp) and GraphPad Prism software. Normality and homogeneity of variance were assessed using the Shapiro–Wilk and Levene tests, respectively. Comparisons between two groups were performed using an unpaired two-tailed Student’s t-test. Comparisons among three or more groups were performed using one-way analysis of variance followed by Tukey’s post hoc test when the assumptions of normality and equal variance were satisfied. Dose–response curves were fitted by nonlinear regression, and IC50 values were compared based on independently fitted biological replicates. Exact P values were reported where possible; otherwise, statistical significance was defined as P<0.05.

Results

Exosome-Mediated Transfer of miR-22 Enhances CDDP Sensitivity in A549 Cells

To evaluate the exosome-mediated transfer and functional effects of miR-22, donor A549 cells were transfected with Cy3-labeled miR-22 mimic or negative control (NC), and the resulting exosome fractions were subsequently applied to recipient A549 cells. Strong cytoplasmic Cy3 fluorescence was observed in recipient cells treated with exosomes derived from miR-22 mimic-transfected donor cells, whereas weaker fluorescence was detected in the NC group (Figure 1A). Consistently, miR-22 expression was significantly higher in the miR-22 mimic group than in the NC group (Figure 1B), confirming the successful exosome-mediated transfer of miR-22. Following exosome treatment, recipient A549 cells were exposed to increasing concentrations of CDDP for 24 h. Cell viability was lower in the miR-22 mimic group than in the NC group, particularly at higher CDDP concentrations, indicating that exosome-delivered miR-22 enhanced CDDP sensitivity (Figure 1C). To further determine whether miR-22 transfer depended on exosome release, donor cells were treated with GW4869 before exosome collection. GW4869 treatment markedly reduced Cy3 fluorescence and miR-22 expression in recipient cells (Figure 1D and E). Correspondingly, the sensitizing effect of miR-22 on CDDP-induced cytotoxicity was partially attenuated by GW4869 (Figure 1F). These results indicated that the intercellular transfer and CDDP-sensitizing effects of miR-22 were, at least in part, dependent on exosome release.

Figure 1.

Fluorescence images and graphs show miR-22 expression and cell viability in A549 cells with treatments. The image A shows fluorescence images of A549 cells treated with NC and miR-22 mimic, labeled with Hoechst and Cy3 and their merged view. Image B is a bar graph showing miR-22 expression levels, with higher expression in the miR-22 mimic group compared to NC, marked by two asterisks. Image C is a line graph depicting cell viability percentages at various cisplatin concentrations, showing reduced viability in the miR-22 mimic group. Image D shows fluorescence images of cells treated with miR-22 mimic and miR-22 mimic plus GW4869, labeled with Hoechst and Cy3 and their merged view. Image E is a bar graph showing miR-22 expression, significantly lower in the miR-22 mimic plus GW4869 group, marked by three asterisks. Image F is a line graph of cell viability percentages, showing higher viability in the miR-22 mimic plus GW4869 group at increasing cisplatin concentrations, marked by three asterisks.

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Exosome-mediated transfer of miR-22 enhances cisplatin sensitivity in A549 cells. A549 donor cells were transfected with Cy3-labeled miR-22 mimic or the corresponding negative control, and the isolated exosomes were subsequently applied to recipient A549 cells. (A) Representative fluorescence images showing Cy3-labeled miR-22 uptake by recipient cells. (B) Relative miR-22 expression in recipient cells following exosome treatment. (C) Cell viability following exposure to increasing concentrations of cisplatin for 24 h. (D) Representative fluorescence images of recipient cells treated with exosomes from miR-22 mimic-transfected donor cells in the presence or absence of GW4869. GW4869 was applied to donor cells before exosome isolation. (E) Relative miR-22 expression following the indicated treatments. (F) Cell viability following cisplatin exposure. Data are presented as the mean ± SD from three independent biological experiments. **P<0.01 and ***P<0.001. CDDP, cisplatin; NC, negative control.

Characterization and Uptake of Exosomes Derived from A549 and A549/CDDP Cells

Exosomes were isolated from the conditioned media of A549 and A549/CDDP cells. TEM revealed rounded or cup-shaped vesicles with typical exosome-like morphology in both preparations (Figure 2A). Nanoparticle tracking analysis showed that A549-exo and A549/CDDP-exo exhibited comparable particle-size distributions, with mean diameters of approximately 90–100 nm. No significant differences in mean particle diameter or particle concentration were observed between the two groups (Figure 2B). In addition, Western blot analysis further confirmed the identity of the isolated vesicles: canonical exosomal markers CD63, CD81, and TSG101 were abundantly expressed, whereas the endoplasmic reticulum protein calnexin was undetectable, indicating minimal cellular contamination (Figure 2C). PKH26-labeled A549-exo and A549/CDDP-exo produced prominent intracellular red fluorescence after incubation with recipient A549 cells. In contrast, the untreated control and processed dye-only groups showed negligible fluorescence (Figure 2D). Quantitative analysis confirmed that PKH26 fluorescence intensity was significantly higher in both exosome-treated groups than in the dye-only group, whereas no significant difference was observed between the A549-exo and A549/CDDP-exo groups (Figure 2E). These findings indicated that the fluorescence signals were attributable predominantly to exosome uptake rather than free-dye aggregation. RT-qPCR further showed that miR-22 expression was significantly lower in A549/CDDP-exo than in A549-exo (Figure 2F).

Figure 2.

Exosome analysis of A549 and A549/CDDP cells: microscopy, tracking, fluorescence data. The image A shows transmission electron microscopy of exosomes from A549 and A549/CDDP cells. The image B presents nanoparticle tracking analysis profiles for A549-exo and A549/CDDP-exo, with graphs showing exosome diameter and concentration. The image C displays Western blot analysis of exosomal markers CD63, CD81, TSG101 and the negative marker Calnexin. The image D includes fluorescence images of untreated A549 cells and those incubated with PKH26 dye-only, PKH26-labeled A549-derived exosomes, or PKH26-labeled A549/CDDP-derived exosomes, with nuclei counterstained with Hoechst. The image E shows quantitative analysis of PKH26 fluorescence intensity, indicating significant differences between groups. The image F illustrates relative miR-22 expression in A549-derived and A549/CDDP-derived exosomes, with significant differences noted. Scale bars and statistical significance are indicated in the images.

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Characterization and cellular uptake of exosomes derived from A549 and A549/CDDP cells. (A) Representative transmission electron microscopy images of exosomes isolated from A549 and A549/CDDP cells. Scale bar, 200 nm. (B) Representative nanoparticle tracking analysis profiles and quantitative analysis of particle size and concentration in A549-derived exosomes and A549/CDDP-derived exosomes. (C) Western blot analysis of the exosomal markers CD63, CD81 and TSG101 and the negative marker Calnexin in cell lysates and exosome preparations. (D) Representative fluorescence images of untreated A549 cells and A549 cells incubated for 4 h with the processed PKH26 dye-only preparation, PKH26-labeled A549-derived exosomes or PKH26-labeled A549/CDDP-derived exosomes. Nuclei were counterstained with Hoechst. (E) Quantitative analysis of intracellular PKH26 fluorescence intensity. (F) Relative miR-22 expression in A549-derived and A549/CDDP-derived exosomes, as determined by reverse transcription-quantitative PCR. Data are presented as the mean ± SD from three independent biological experiments (n=3). Two-group comparisons were performed using an unpaired two-tailed Student’s t-test, and multiple-group comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. ***P<0.001; ns, not significant.

A549/CDDP-Derived Exosomes Promote Migration and Reduce CDDP Sensitivity in Recipient Cells

The CDDP-resistant phenotype of A549/CDDP cells was first evaluated using CCK-8 dose–response assays. A549/CDDP cells maintained higher viability than parental A549 cells across increasing CDDP concentrations. Nonlinear regression analysis yielded an IC50 of approximately 14 μM for A549/CDDP cells and approximately 2 μM for parental A549 cells, corresponding to a resistance index of approximately 7 (Figure 3A). Treatment with A549-exo significantly reduced the migration of recipient cells, whereas A549/CDDP-exo increased migration compared with the control group (Figure 3B). Similarly, cells treated with A549-exo exhibited lower viability during CDDP exposure, whereas cells treated with A549/CDDP-exo retained greater viability (Figure 3C). RT-qPCR showed that miR-22 expression was increased in cells treated with A549-exo and reduced in those treated with A549/CDDP-exo relative to the untreated A549/CDDP group (Figure 3D). These results suggested that the functional differences between the two exosome preparations were associated with their different miR-22 contents.

Figure 3.

Mixed plots of cisplatin response, migration images with bar chart, viability curves and miR-22 bars. Image A: Left graph shows cell viability vs CDDP concentration (0-60 micro M). Two curves: A549 and A549/CDDP. Right bar chart shows IC50 (0-20 micro M) for A549 (~2) and A549/CDDP (~14) with three asterisks. Image B: Micrographs labeled NC, A549 exo, A549/CDDP exo, plus bar chart. Cell count: NC (~65), A549 exo (~45), A549/CDDP exo (~90) with two asterisks. Image C: Line graph shows cell viability vs concentration (0, 1, 10, 30, 60 micro M). Curves: NC (1.0 to 0.4), A549 exo (1.0 to 0.2), A549/CDDP exo (1.0 to 0.5). Image D: Bar chart shows miR 22 expression (0.0 to 2.0) for A549/CDDP (1.0), A549 exo (1.6 with two asterisks), A549/CDDP exo (0.6 with three asterisks).

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A549/CDDP-derived exosomes promote cell migration and reduce cisplatin sensitivity. (A) Cisplatin dose-response curves and half-maximal inhibitory concentration values of parental A549 and A549/CDDP cells following treatment with increasing concentrations of cisplatin for 24 h. Half-maximal inhibitory concentration values were estimated using nonlinear regression analysis. (B) Representative images and quantitative analysis of Transwell migration in recipient cells treated with the negative control, A549-derived exosomes or A549/CDDP-derived exosomes. Scale bar, 50 µm. (C) Cell viability of recipient cells following the indicated exosome treatments and cisplatin exposure. (D) Relative miR-22 expression in recipient cells following treatment with A549-derived or A549/CDDP-derived exosomes, as determined by reverse transcription-quantitative PCR. Data are presented as the mean ± SD from three independent biological experiments (n=3). Group comparisons in panels B–D were performed using one-way ANOVA followed by Tukey’s post hoc test. **P<0.01 and ***P<0.001.

MiR-22 Directly Targets MDM4 and Mediates the Effects of A549-Derived Exosomes

Bioinformatic prediction identified a putative miR-22 binding sequence within the 3′-UTR of MDM4. Co-transfection with miR-22 mimic significantly reduced the luciferase activity of the wild-type MDM4 3′-UTR reporter but did not significantly affect the mutant reporter, confirming that MDM4 was a direct target of miR-22 (Figure 4A). To determine whether miR-22 mediated the functional effects of A549-exo, A549/CDDP cells were treated with a miR-22 inhibitor in the presence or absence of A549-exo or A549/CDDP-exo. MiR-22 inhibition increased cell viability under CDDP exposure, whereas A549-exo partially attenuated this effect. In contrast, A549/CDDP-exo did not produce a comparable inhibitory effect (Figure 4B). Transwell assays showed a similar pattern: miR-22 inhibition promoted migration, A549-exo reduced the inhibitor-induced increase in migration, and A549/CDDP-exo maintained a comparatively high migratory phenotype (Figure 4C). RT-qPCR and Western blotting further demonstrated that miR-22 inhibition increased MDM4 mRNA and protein expression, whereas treatment with A549-exo reduced MDM4 expression (Figure 4D and E). Together, these results supported miR-22 as a functional component of A549-derived exosomes that suppresses MDM4 expression.

Figure 4.

Five-part figure showing miR-22 effects on MDM4 expression, cell viability, migration, mRNA and protein levels. The figure consists of five parts: A) Predicted miR-22 binding sequence within MDM4 wild-type and mutant sequences, with a graph showing relative luciferase activity for wild-type (WT) and mutant (MUT) MDM4, indicating significant reduction in WT with miR-22. B) Bar graph of relative cell viability for A549/CDDP cells treated with inhibitor NC, miR-22 inhibitor and exosomes, showing increased viability with miR-22 inhibition. C) Images and graph of Transwell migration assay, showing increased cell count with miR-22 inhibition and A549/CDDP-exo treatment. D) Bar graph of MDM4 mRNA expression, showing increased levels with miR-22 inhibition and A549/CDDP-exo treatment. E) Western blot and graph of MDM4 protein expression normalized to GAPDH, showing increased levels with miR-22 inhibition and A549/CDDP-exo treatment. Statistical significance is indicated with asterisks: P<0.05, P<0.01, P<0.001.

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miR-22 directly targets MDM4 and regulates cisplatin resistance and migration through MDM4. (A) Predicted miR-22-binding sequence within the wild-type MDM4 3′-untranslated region and the corresponding mutant sequence, together with the results of the dual-luciferase reporter assay. Cells were co-transfected with wild-type or mutant MDM4 reporter constructs and miR-22 mimic or the corresponding negative control. Firefly luciferase activity was normalized to Renilla luciferase activity. (B) Cell viability of A549/CDDP cells transfected with inhibitor NC or miR-22 inhibitor and subsequently treated with A549-derived or A549/CDDP-derived exosomes as indicated under cisplatin exposure. (C) Representative images and quantitative analysis of Transwell migration following the indicated treatments. Scale bar, 50 µm. (D) Relative MDM4 mRNA expression following the indicated treatments, as determined by reverse transcription-quantitative PCR. (E) Representative Western blot images and quantitative analysis of MDM4 protein expression. MDM4 protein expression was normalized to GAPDH. Data are presented as the mean ± SD from three independent biological experiments (n=3). *P<0.05, **P<0.01 and ***P<0.001.

MDM4 Mediates the Effects of Exosomal miR-22 on CDDP Resistance and Migration

To determine whether MDM4 functionally mediated the effects of exosomal miR-22, MDM4 was first silenced in A549/CDDP cells before exosome treatment. Compared with si-NC, si-MDM4 significantly reduced cell viability and migration. These inhibitory effects were further enhanced by A549-exo, whereas A549/CDDP-exo partially restored cell viability and migration in MDM4-silenced cells (Figure 5A and B). In a separate set of experiments, A549-exo significantly reduced cell viability and migration compared with A549/CDDP-exo. Addition of the miR-22 inhibitor to A549/CDDP-exo-treated cells did not further increase cell viability significantly but markedly enhanced cell migration, whereas MDM4 silencing reduced both phenotypes (Figure 5C and D). A549-exo increased miR-22 expression, whereas the miR-22 inhibitor significantly reduced miR-22 levels. MDM4 silencing did not significantly alter miR-22 expression in A549/CDDP-exo-treated cells (Figure 5E). Conversely, A549-exo reducd MDM4 mRNA and protein expression, miR-22 inhibition increased MDM4 expression, and si-MDM4 effectively suppressed MDM4 expression (Figure 5F).

Figure 5.

Six-panel infographic: MDM4 silencing and exosomes alter A549/CDDP proliferation, migration, miR-22 and MDM4. The image A showing a bar chart of cell proliferation. X-axis labels: si-NC, si-MDM4, si-MDM4 plus A549-exo, si-MDM4 plus A549/CDDP-exo. Y-axis label: cell proliferation, scale 0.0 to 1.0. Bars show si-NC highest near 0.8; si-MDM4 lower near 0.6; si-MDM4 plus A549-exo lower near 0.5; si-MDM4 plus A549/CDDP-exo higher near 0.7. Significance marks include double asterisk. The image B showing four Transwell migration micrographs labeled si-NC, si-MDM4, si-MDM4 plus A549-exo and si-MDM4 plus A549/CDDP-exo, plus a bar chart. X-axis labels match the four groups. Y-axis label: cell count, scale 0 to 100. Bars show si-NC highest near 75; si-MDM4 near 35; si-MDM4 plus A549-exo near 20; si-MDM4 plus A549/CDDP-exo near 65. Significance marks include triple asterisk and double asterisk. The image C showing a bar chart of cell proliferation. X-axis labels: A549/CDDP-exo, A549-exo, inhibitor plus A549/CDDP-exo, si-MDM4 plus A549/CDDP-exo. Y-axis label: cell proliferation, scale 0.0 to 1.0. Bars show A549/CDDP-exo near 0.7; A549-exo near 0.45; inhibitor plus A549/CDDP-exo near 0.65; si-MDM4 plus A549/CDDP-exo near 0.3. Significance marks include ns, double asterisk and triple asterisk. The image D showing four Transwell migration micrographs labeled A549/CDDP-exo, A549-exo, inhibitor plus A549/CDDP-exo and si-MDM4 plus A549/CDDP-exo, plus a bar chart. X-axis labels match the four groups. Y-axis label: cell count, scale 0 to 120. Bars show A549/CDDP-exo near 75; A549-exo near 45; inhibitor plus A549/CDDP-exo near 90; si-MDM4 plus A549/CDDP-exo near 55. Significance marks include single asterisk and double asterisk. The image E showing two bar charts. Left chart x-axis labels: A549/CDDP-exo, A549-exo, inhibitor plus A549/CDDP-exo, si-MDM4 plus A549/CDDP-exo; y-axis label: miR-22 expression, scale 0.0 to 3.0. Bars show A549/CDDP-exo near 1.0; A549-exo near 2.0; inhibitor plus A549/CDDP-exo near 0.4; si-MDM4 plus A549/CDDP-exo near 1.2. Significance marks include ns, double asterisk and triple asterisk. Right chart x-axis labels match; y-axis label: MDM4 mRNA expression, scale 0.0 to 2.0. Bars show A549/CDDP-exo near 0.9; A549-exo near 0.5; inhibitor plus A549/CDDP-exo near 1.2; si-MDM4 plus A549/CDDP-exo near 0.2. Significance marks include double asterisk and triple asterisk. The image F showing a Western blot labeled MDM4 and GAPDH with size labels 70 kDa and 36 kDa, plus a bar chart. Bar chart y-axis label: MDM4/GAPDH, scale 0.0 to 1.5. X-axis labels: A549/CDDP-exo, A549-exo, inhibitor plus A549/CDDP-exo, si-MDM4 plus A549/CDDP-exo. Bars show A549/CDDP-exo near 0.8; A549-exo near 0.55; inhibitor plus A549/CDDP-exo near 1.1; si-MDM4 plus A549/CDDP-exo near 0.25. Significance marks include double asterisk and triple asterisk.

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MDM4 contributes to exosome-mediated cisplatin resistance and migration in A549/CDDP cells. (A) Cell viability of A549/CDDP cells transfected with si-NC or si-MDM4 and treated with A549-derived or A549/CDDP-derived exosomes under cisplatin exposure. (B) Representative images and quantitative analysis of Transwell migration following the indicated treatments. (C) Cell viability of A549/CDDP cells treated with A549-derived exosomes, A549/CDDP-derived exosomes, miR-22 inhibitor or si-MDM4 as indicated. (D) Representative images and quantitative analysis of Transwell migration in the corresponding groups. (E) Relative miR-22 expression following the indicated treatments, as determined by reverse transcription-quantitative PCR. (F) Relative MDM4 mRNA expression and representative Western blot images with quantitative analysis of MDM4 protein expression. MDM4 protein expression was normalized to GAPDH. Data are presented as the mean ± SD from three independent biological experiments (n=3). Multiple-group comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. *P<0.05, **P<0.01 and ***P<0.001; ns, not significant.

To further verify the functional role of MDM4, A549/CDDP cells were co-transfected with miR-22 mimic and an MDM4-overexpression plasmid. MiR-22 overexpression reduced cell viability and migration, whereas MDM4 restoration partially reversed these effects (Figure 6A and C). RT-qPCR and Western blotting confirmed that miR-22 suppressed MDM4 expression and that OE-MDM4 effectively restored its mRNA and protein levels without reducing miR-22 expression (Figure 6B and D). These findings further supported MDM4 as a functional downstream mediator of miR-22.

Figure 6.

A four-part scientific figure shows miR-22 lowers viability and migration, partly restored by OE-MDM4. Image A: Bar chart of cell viability. Groups: mimic NC + Vector, miR-22 mimic + Vector, miR-22 mimic + OE-MDM4. Highest viability in mimic NC + Vector, lowest in miR-22 mimic + Vector, intermediate in miR-22 mimic + OE-MDM4. Significance: *** and **. Image B: Two bar charts. Left: MDM4 mRNA expression, lowest in miR-22 mimic + Vector, highest in miR-22 mimic + OE-MDM4. Right: miR-22 expression, lowest in mimic NC + Vector, higher in miR-22 mimic + Vector, slightly lower in miR-22 mimic + OE-MDM4. Significance: ***, **, *. Image C: Transwell migration micrographs and bar chart. Fewer migrated cells in miR-22 mimic + Vector than mimic NC + Vector, more in miR-22 mimic + OE-MDM4 than miR-22 mimic + Vector. Significance: **. Image D: Western blot, rows: MDM4, GAPDH; lanes: mimic NC + Vector, miR-22 mimic + Vector, miR-22 mimic + OE-MDM4. MDM4 size: 70 kDa, GAPDH: 36 kDa. Bar chart MDM4/GAPDH ratio, lowest in miR-22 mimic + Vector, highest in miR-22 mimic + OE-MDM4. Significance: **, ***.

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MDM4 overexpression partially reverses the suppressive effects of miR-22 in A549/CDDP cells. (A) Cell viability following the indicated treatments under cisplatin exposure. (B) Relative MDM4 mRNA and miR-22 expression, as determined by reverse transcription-quantitative PCR. (C) Representative images and quantitative analysis of Transwell migration. (D) Representative Western blot images and quantitative analysis of MDM4 protein expression. MDM4 protein expression was normalized to GAPDH. Data are presented as the mean ± SD from three independent biological experiments (n=3). Multiple-group comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. *P<0.05, **P<0.01 and ***P<0.001.

MDM4 Mediates miR-22-Induced p53-Associated DNA Damage and Apoptosis

Because MDM4 is a negative regulator of p53, we next examined whether the functional effects of the miR-22/MDM4 axis were associated with p53-related DNA-damage and apoptotic signaling. Compared with the mimic NC + Vector group, miR-22 overexpression significantly increased the p-p53/p53 ratio and the expression levels of p21, cleaved caspase-3 and γ-H2AX. MDM4 overexpression significantly attenuated these changes (Figure 7A and B). Flow cytometric analysis further showed that miR-22 overexpression increased total apoptosis, whereas restoration of MDM4 partially reduced the apoptotic rate (Figure 7C). The effects of resistant-cell-derived exosomes were subsequently examined. Compared with A549-exo, A549/CDDP-exo increased MDM4 expression and reduced p21, cleaved caspase-3 and γ-H2AX expression. Addition of si-NC did not significantly alter these effects, whereas MDM4 silencing markedly reduced MDM4 expression and restored the downstream p53-associated DNA-damage and apoptotic proteins (Figure 7D and E). The p-p53/p53 ratio was lower in the A549/CDDP-exo group than in the A549-exo group and showed an upward trend following MDM4 silencing. These findings indicated that reduced exosomal miR-22 attenuated p53-associated DNA-damage and apoptotic responses through increased MDM4 expression.

Figure 7.

Mixed plots of protein expression and apoptosis across mimic, miR 22 mimic and MDM4 conditions. The image A showing Western blot bands for cleaved caspase 3, p21, phosphorylated p53, total p53, gamma H2AX and glyceraldehyde 3 phosphate dehydrogenase across three lanes labeled, mimic negative control plus vector, miR 22 mimic plus vector and miR 22 mimic plus overexpression MDM4. Molecular masses at right read 17 kilodalton, 21 kilodalton, 53 kilodalton, 53 kilodalton, 15 kilodalton and 36 kilodalton. The image B showing four bar charts. Chart 1 y axis, cleaved caspase 3 over glyceraldehyde 3 phosphate dehydrogenase, from 0 to 2.0; x axis groups are mimic negative control plus vector, miR 22 mimic plus vector, miR 22 mimic plus overexpression MDM4. Bars are about 1.0, 1.6 and 1.1. Chart 2 y axis, p21 over glyceraldehyde 3 phosphate dehydrogenase, 0 to 2.5; bars about 1.0, 2.1, 1.6. Chart 3 y axis, phosphorylated p53 over total p53, 0 to 1.5; bars about 1.0, 1.3, 1.1. Chart 4 y axis, gamma H2AX over glyceraldehyde 3 phosphate dehydrogenase, 0 to 2.5; bars about 1.0, 2.0, 1.4. The image C showing three flow cytometry scatter plots with x axis, Annexin V and y axis, propidium iodide. Each plot is divided into four quadrants with printed percentages. Left plot labeled mimic negative control plus vector shows about 1.34, 3.44, 10.08 and 85.13 percent. Middle plot labeled miR 22 mimic plus vector shows about 4.46, 6.81, 23.05 and 65.68 percent. Right plot labeled miR 22 mimic plus overexpression MDM4 shows about 2.91, 4.67, 18.26 and 74.16 percent. A bar chart at right has y axis, apoptotic cell ratio percent, from 0 to 40; x axis groups match the three conditions; bars are about 13, 31 and 21. The image D showing Western blot bands for MDM4, cleaved caspase 3, p21, phosphorylated p53, total p53, gamma H2AX and glyceraldehyde 3 phosphate dehydrogenase across six lanes labeled, control, A549 exosomes, A549 cisplatin exosomes, A549 cisplatin exosomes plus small interfering negative control and A549 cisplatin exosomes plus small interfering MDM4. Molecular masses at right include 70 kilodalton for MDM4, then 17, 21, 53, 53, 15 and 36 kilodalton. The image E showing five bar charts with x axis groups, control, A549 exosomes, A549 cisplatin exosomes, A549 cisplatin exosomes plus small interfering negative control and A549 cisplatin exosomes plus small interfering MDM4. Chart 1 y axis, MDM4 over glyceraldehyde 3 phosphate dehydrogenase, 0 to 1.5; bars about 1.0, 0.6, 1.3, 1.35, 0.45. Chart 2 y axis, cleaved caspase 3 over glyceraldehyde 3 phosphate dehydrogenase, 0 to 2.5; bars about 1.0, 2.1, 0.55, 0.6, 1.8. Chart 3 y axis, p21 over glyceraldehyde 3 phosphate dehydrogenase, 0 to 2.0; bars about 1.0, 1.6, 0.45, 0.45, 1.2. Chart 4 y axis, phosphorylated p53 over total p53, 0 to 1.5; bars about 1.0, 1.1, 0.75, 0.75, 1.05. Chart 5 y axis, gamma H2AX over glyceraldehyde 3 phosphate dehydrogenase, 0 to 2.0; bars about 1.0, 1.7, 0.7, 0.7, 1.2.

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MDM4 mediates the effects of miR-22 on p53-associated DNA-damage and apoptotic responses in A549/CDDP cells. (A) Representative Western blot images of cleaved caspase-3, p21, phosphorylated p53, total p53, γ-H2AX and GAPDH. (B) Quantitative analysis of cleaved caspase-3/GAPDH, p21/GAPDH, phosphorylated p53/total p53 and γ-H2AX/GAPDH. (C) Representative Annexin V-FITC/propidium iodide flow cytometry plots and quantitative analysis of total apoptotic cells. Total apoptosis was calculated as the sum of the early and late apoptotic cell populations. (D) Representative Western blot images of MDM4, cleaved caspase-3, p21, phosphorylated p53, total p53, γ-H2AX and GAPDH. (E) Quantitative analysis of MDM4/GAPDH, cleaved caspase −3/GAPDH, p21/GAPDH, phosphorylated p53/total p53 and γ-H2AX/GAPDH. Data are presented as the mean ± SD from three independent biological experiments (n=3). Multiple-group comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. *P<0.05, **P<0.01 and ***P<0.001; ns, not significant.

Discussion

NSCLC remains a leading cause of cancer-related mortality worldwide.20 Contemporary systemic treatment for advanced NSCLC is selected according to histological and molecular characteristics and commonly incorporates immune checkpoint inhibitor-based regimens with or without platinum-doublet chemotherapy,2,3 whereas routine postoperative radiotherapy is not considered standard after complete resection of pN2 disease.21 CDDP exerts its antitumor effects primarily by inducing DNA damage, which activates cell-cycle arrest and apoptotic responses.22,23 However, intrinsic or acquired resistance substantially limits the efficacy of CDDP-based treatment, particularly in patients with recurrent or advanced disease.24 In the present study, A549/CDDP-derived exosomes contained reduced levels of miR-22 and promoted CDDP resistance through MDM4-associated suppression of p53-related DNA-damage and apoptotic responses.

Emerging evidence has suggested that miRNAs play critical roles in tumor progression and chemotherapy response in several types of cancer.25 In the present study, restoration of miR-22 reduced cell viability and migration and enhanced the response of A549 cells to CDDP, whereas miR-22 inhibition attenuated the effects of A549-derived exosomes. Consistent with the broader involvement of miRNAs in platinum resistance, Xiao et al26 reported that miR-2467 promoted CDDP resistance and malignant phenotypes in NSCLC cells. The present findings extend this concept by identifying miR-22 as a suppressive regulator of CDDP resistance in the p53-competent A549/A549-CDDP model. Importantly, miR-22 levels were lower in exosomes derived from A549/CDDP cells than in those derived from parental A549 cells, and the functional experiments linked this reduction to increased MDM4 expression and reduced CDDP sensitivity. The principal advance of the present study is therefore not merely the association of miR-22 with chemotherapy response, but the identification of reduced exosomal miR-22 as an intercellular signal connected to the MDM4/p53 pathway. The gain- and loss-of-function experiments further showed that MDM4 mediated a substantial proportion of the effects of miR-22 on cell viability, migration, DNA-damage signaling and apoptosis. Rather than suggesting that miR-22 is superior to previously reported exosomal miRNAs, these findings identify it as an additional mechanistically defined component of the multifactorial network underlying CDDP resistance.

MDM4 is an important negative regulator of p53 and is aberrantly expressed in multiple malignancies, where it can support tumor-cell survival by limiting p53-dependent stress responses.27 In NSCLC, MDM4 has been implicated in tumor progression and can be regulated by several miRNAs, including miR-1205 and miR-34a-5p.28,29 In the present study, MDM4 expression was higher in cells exposed to A549/CDDP-derived exosomes than in those exposed to A549-derived exosomes and varied inversely with miR-22 expression. MiR-22 inhibition increased MDM4 expression, whereas MDM4 silencing reduced cell viability and migration under CDDP exposure. Similarly, Wang et al30 reported that miR-1307 overexpression could sensitize breast cancer cells to CDDP via regulating MDM4. The loss- and gain-of-function experiments in the present study provided stronger evidence for this relationship: MDM4 silencing suppressed the resistant phenotype, whereas MDM4 overexpression partially restored the cell viability and migration reduced by miR-22 overexpression. Notably, miR-22 remained highly expressed following MDM4 restoration, indicating that the rescue effect resulted from manipulation of the downstream target rather than loss of miR-22 overexpression. The newly added mechanistic experiments further connected this regulatory relationship to p53-associated signaling in the wild-type TP53 A549 background. MiR-22 overexpression increased the p-p53/p53 ratio, p21, cleaved caspase-3 and γ-H2AX expression and promoted apoptosis, whereas MDM4 overexpression attenuated these effects. Conversely, MDM4 silencing restored p21, cleaved caspase-3 and γ-H2AX expression suppressed by A549/CDDP-derived exosomes and produced a trend toward recovery of the p-p53/p53 ratio. Together, these findings support a model in which reduced exosomal miR-22 permits MDM4 accumulation, thereby attenuating p53-associated DNA-damage and apoptotic responses and promoting CDDP resistance. However, miR-22 can regulate multiple target genes, and the partial rather than complete rescue observed after MDM4 manipulation indicates that the phenotypes identified here are unlikely to depend exclusively on a single miR-22/MDM4 pathway.

Previous studies have shown that exosomes derived from CDDP-resistant NSCLC cells exhibit distinct miRNA profiles and can transmit resistance-associated signals to recipient cells. Exosomal miRNAs, including miR-100-5p and miR-425-3p, have been implicated in CDDP resistance in NSCLC, supporting the possibility that multiple extracellular miRNAs may act in parallel rather than through a single dominant pathway.31,32 CDDP resistance is a multifactorial process involving altered drug accumulation and detoxification, enhanced DNA-damage repair, impaired apoptosis and activation of adaptive survival pathways.33,34 The A549/CDDP model used in the present study was generated following a 24-h exposure to 2 μM CDDP and subsequent recovery and may therefore reflect an early drug-tolerant adaptation rather than the stable acquired resistance produced by prolonged stepwise drug selection. Within this experimental context, the lower miR-22 content of A549/CDDP-derived exosomes was associated with increased MDM4 expression, greater cell viability and migration and reduced p53-associated DNA-damage and apoptotic responses during CDDP exposure. These findings support reduced exosomal miR-22 as one component of the resistant phenotype, but do not exclude simultaneous contributions from altered drug transport, detoxification, DNA repair or other exosomal cargos. Because cisplatin and carboplatin both induce platinum-associated DNA damage, the miR-22/MDM4/p53 axis may also influence carboplatin responsiveness; however, this possibility requires direct experimental validation, and extrapolation to oxaliplatin is even less certain because its cellular effects and clinical applications differ from those of cisplatin. Paclitaxel- or pemetrexed-containing platinum regimens combined with immune checkpoint inhibitors remain important first-line treatment options for metastatic squamous and nonsquamous NSCLC without actionable driver alterations, respectively.35,36 However, whether the miR-22/MDM4 axis influences responses to these regimens remains unknown. Given that platinum resistance in lung cancer involves altered drug transport, enhanced DNA repair, apoptotic escape and non-coding RNA regulation, further studies are required to determine whether this axis affects contemporary chemoimmunotherapy responses.37 Future studies should therefore determine whether miR-22/MDM4 cooperates with other exosomal cargos and established resistance mechanisms and whether its effects extend to additional platinum agents and contemporary combination treatments.

Several limitations of the present study should be acknowledged. First, all experiments were performed using the A549/A549-CDDP cell pair, and the wild-type TP53 background of A549 cells limits the generalization of the findings to TP53-mutant or TP53-null NSCLC models. Second, the A549/CDDP cells were generated following a relatively short CDDP exposure and recovery period, which may represent early drug tolerance rather than the stable acquired resistance produced by prolonged stepwise selection. Third, the study was conducted exclusively in vitro and did not include xenograft or orthotopic models, limiting assessment of the effects of the exosomal miR-22/MDM4 axis within the tumor microenvironment and in vivo treatment response. In addition, patient-derived exosomal miR-22, tumor MDM4 expression and their associations with platinum response or clinical outcome were not evaluated using clinical specimens or publicly available datasets. Recent clinical and bioinformatic studies have identified epigenetic and metabolism-related biomarkers, including ABCG1 hypermethylation and sideroflexin family members, as potentially relevant to the diagnosis, prognosis and immune characteristics of NSCLC, further highlighting the importance of validating candidate molecular mechanisms in patient cohorts and public datasets.38,39 Finally, miR-22 is likely to regulate additional target genes, and the partial rescue observed after MDM4 manipulation indicates that the functional effects identified here are not mediated exclusively through MDM4. Future studies using long-term resistant models, additional NSCLC cell lines with different TP53 backgrounds, animal models and patient-derived samples are therefore required to determine the broader biological and clinical relevance of this mechanism.

In summary, the present study identified reduced miR-22 content in exosomes derived from A549/CDDP cells and linked this alteration to increased MDM4 expression, reduced p53-associated DNA-damage and apoptotic signaling, and enhanced CDDP resistance. The direct interaction between miR-22 and MDM4 was supported by the dual-luciferase reporter assay, while MDM4 loss- and gain-of-function experiments provided functional evidence for this regulatory relationship. These findings extend the current understanding of exosome-mediated platinum resistance by defining a miR-22/MDM4/p53-associated mechanism in the wild-type TP53 A549/A549-CDDP model.

Conclusion

Reduced exosomal miR-22 contributed to CDDP resistance in the wild-type TP53 A549/A549-CDDP model by increasing MDM4 expression and attenuating p53-associated DNA-damage and apoptotic responses. Restoration of miR-22 or silencing of MDM4 increased CDDP sensitivity, whereas MDM4 overexpression partially reversed the effects of miR-22. These findings identify the exosomal miR-22/MDM4 axis as a candidate mechanism of CDDP resistance that requires validation in additional NSCLC models, animal studies and patient-derived samples.

Funding Statement

This work was supported by the Science and Technology Program of the Joint Fund of Scientific Research for the Public Hospitals of Inner Mongolia Academy of Medical Sciences (Grant No. 2023GLLH0005).

Data Sharing Statement

The datasets generated and/or analyzed during the present study are available from the corresponding author upon reasonable request.

Ethics Approval and Consent to Participate

The ethics statement is not applicable.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no competing interests.

References

  • 1.Hendriks LEL, Remon J, Faivre-Finn C, et al. Non-small-cell lung cancer. Nat Rev Dis Primers. 2024;10(1):71. doi: 10.1038/s41572-024-00551-9 [DOI] [PubMed] [Google Scholar]
  • 2.Meyer ML, Fitzgerald BG, Paz-Ares L, et al. New promises and challenges in the treatment of advanced non-small-cell lung cancer. Lancet. 2024;404(10454):803–18. doi: 10.1016/s0140-6736(24)01029-8 [DOI] [PubMed] [Google Scholar]
  • 3.Riely GJ, Wood DE, Aisner DL, et al. Non-small cell lung cancer, version 4.2026, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2026;24(4). doi: 10.6004/jnccn.2026.0017 [DOI] [PubMed] [Google Scholar]
  • 4.Lee KWA, Chan LKW, Hung LC, Phoebe LKW, Park Y, Yi KH. Clinical applications of exosomes: a critical review. Int J Mol Sci. 2024;25(14). doi: 10.3390/ijms25147794 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.He C, Zheng S, Luo Y, Wang B. Exosome theranostics: biology and translational medicine. Theranostics. 2018;8(1):237–255. doi: 10.7150/thno.21945 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Sun Z, Du T, Yang G, Sun Y, Xiao X. Identification of exosome-related genes in NSCLC via integrated bioinformatics and machine learning analysis. Sci Rep. 2025;15(1):22962. doi: 10.1038/s41598-025-04485-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Farazi TA, Spitzer JI, Morozov P, Tuschl T. miRNAs in human cancer. J Pathol. 2011;223(2):102–115. doi: 10.1002/path.2806 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet. 2005;37(7):766–770. doi: 10.1038/ng1590 [DOI] [PubMed] [Google Scholar]
  • 9.Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet. 2009;10(10):704–714. doi: 10.1038/nrg2634 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science. 2001;294(5543):853–858. doi: 10.1126/science.1064921 [DOI] [PubMed] [Google Scholar]
  • 11.Knyazev EN, Samatov TR, Fomicheva KA, Nyushko KM, Alekseev BY, Shkurnikov MY. MicroRNA hsa-miR-4674 in hemolysis-free blood plasma is associated with distant metastases of prostatic cancer. Bull Exp Biol Med. 2016;161(1):112–115. doi: 10.1007/s10517-016-3358-6 [DOI] [PubMed] [Google Scholar]
  • 12.Yang C, Ning S, Li Z, Qin X, Xu W. miR-22 is down-regulated in esophageal squamous cell carcinoma and inhibits cell migration and invasion. Cancer Cell Int. 2014;14(1):138. doi: 10.1186/s12935-014-0138-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Damavandi Z, Torkashvand S, Vasei M, Soltani BM, Tavallaei M, Mowla SJ. Aberrant expression of breast development-related MicroRNAs, miR-22, miR-132, and miR-212, in breast tumor tissues. J Breast Cancer. 2016;19(2):148–155. doi: 10.4048/jbc.2016.19.2.148 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Tang Y, Liu X, Su B, et al. microRNA-22 acts as a metastasis suppressor by targeting metadherin in gastric cancer. Mol Med Rep. 2015;11(1):454–460. doi: 10.3892/mmr.2014.2682 [DOI] [PubMed] [Google Scholar]
  • 15.Gougelet A, Pissaloux D, Besse A, et al. Micro-RNA profiles in osteosarcoma as a predictive tool for ifosfamide response. Int J Cancer. 2011;129(3):680–690. doi: 10.1002/ijc.25715 [DOI] [PubMed] [Google Scholar]
  • 16.Torreggiani E, Roncuzzi L, Perut F, Zini N, Baldini N. Multimodal transfer of MDR by exosomes in human osteosarcoma. Int J Oncol. 2016;49(1):189–196. doi: 10.3892/ijo.2016.3509 [DOI] [PubMed] [Google Scholar]
  • 17.Zhou X, Natino D, Zhai X, Gao Z, He X. MicroRNA‑22 inhibits the proliferation and migration, and increases the cisplatin sensitivity, of osteosarcoma cells. Mol Med Rep. 2018;17(5):7209–7217. doi: 10.3892/mmr.2018.8790 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Bardot B, Toledo F. Targeting MDM4 Splicing in Cancers. Genes. 2017;8(2). doi: 10.3390/genes8020082 [DOI] [Google Scholar]
  • 19.Jiang K, Sun F, Zhu J, Luo G, Ban Y, Zhang P. miR-33a inhibits cell growth in renal cancer by downregulation of MDM4 expression. Mol Genet Genomic Med. 2019;7(8):e833. doi: 10.1002/mgg3.833 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Schabath MB, Cote ML. Cancer progress and priorities: lung cancer. Cancer Epidemiol Biomarkers Prev. 2019;28(10):1563–1579. doi: 10.1158/1055-9965.Epi-19-0221 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Le Pechoux C, Pourel N, Barlesi F, et al. Postoperative radiotherapy versus no postoperative radiotherapy in patients with completely resected non-small-cell lung cancer and proven mediastinal N2 involvement (Lung ART): an open-label, randomised, Phase 3 trial. Lancet Oncol. 2022;23(1):104–114. doi: 10.1016/s1470-2045(21)00606-9 [DOI] [PubMed] [Google Scholar]
  • 22.Cisplatin GS. The first metal based anticancer drug. Bioorg Chem. 2019;88:102925. doi: 10.1016/j.bioorg.2019.102925 [DOI] [PubMed] [Google Scholar]
  • 23.Hanif M, Hartinger CG. Anticancer metallodrugs: where is the next cisplatin? Future Med Chem. 2018;10(6):615–617. doi: 10.4155/fmc-2017-0317 [DOI] [PubMed] [Google Scholar]
  • 24.Rybak LP, Mukherjea D, Ramkumar V. Mechanisms of cisplatin-induced ototoxicity and prevention. Semin Hear. 2019;40(2):197–204. doi: 10.1055/s-0039-1684048 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Xie L, Jing R, Qi J, Lin Z, Ju S. Drug resistance-related microRNAs in hematological malignancies: translating basic evidence into therapeutic strategies. Blood Rev. 2015;29(1):33–44. doi: 10.1016/j.blre.2014.09.005 [DOI] [PubMed] [Google Scholar]
  • 26.Xiao S, Sun L, Ruan B, et al. Long non-coding RNA LINC01224 promotes progression and cisplatin resistance in non-small lung cancer by sponging miR-2467. Pulm Pharmacol Ther. 2021;70:102070. doi: 10.1016/j.pupt.2021.102070 [DOI] [PubMed] [Google Scholar]
  • 27.Xiong S, Pant V, Zhang Y, et al. The p53 inhibitor Mdm4 cooperates with multiple genetic lesions in tumourigenesis. J Pathol. 2017;241(4):501–510. doi: 10.1002/path.4854 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Yan H, Chen X, Li Y, et al. MiR-1205 functions as a tumor suppressor by disconnecting the synergy between KRAS and MDM4/E2F1 in non-small cell lung cancer. Am J Cancer Res. 2019;9(2):312–329. [PMC free article] [PubMed] [Google Scholar]
  • 29.Jiang ZQ, Li MH, Qin YM, Jiang HY, Zhang X, Wu MH. Luteolin inhibits tumorigenesis and induces apoptosis of non-small cell lung cancer cells via regulation of MicroRNA-34a-5p. Int J Mol Sci. 2018;19(2):447. doi: 10.3390/ijms19020447 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Wang X, Zhu J. Mir-1307 regulates cisplatin resistance by targeting Mdm4 in breast cancer expressing wild type P53. Thorac Cancer. 2018;9(6):676–683. doi: 10.1111/1759-7714.12607 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Qin X, Yu S, Zhou L, et al. Cisplatin-resistant lung cancer cell–derived exosomes increase cisplatin resistance of recipient cells in exosomal miR-100–5p-dependent manner. Int J Nanomed. 2017;12:3721–3733. doi: 10.2147/ijn.S131516 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Ma Y, Yuwen D, Chen J, et al. Exosomal transfer of cisplatin-induced miR-425-3p confers cisplatin resistance in NSCLC through activating autophagy. Int J Nanomed. 2019;14:8121–8132. doi: 10.2147/ijn.S221383 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Duan M, Leng S, Mao P. Cisplatin in the era of PARP inhibitors and immunotherapy. Pharmacol Ther. 2024;258:108642. doi: 10.1016/j.pharmthera.2024.108642 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Fu R, Zhao B, Chen M, et al. Moving beyond cisplatin resistance: mechanisms, challenges, and prospects for overcoming recurrence in clinical cancer therapy. Med Oncol. 2023;41(1):9. doi: 10.1007/s12032-023-02237-w [DOI] [PubMed] [Google Scholar]
  • 35.Owen DH, Halmos B, Puri S, et al. Therapy for stage IV non-small cell lung cancer without driver alterations: ASCO living guideline, version 2025.1. J Clin Oncol. 2025;43(24):e45–e58. doi: 10.1200/jco-25-01062 [DOI] [PubMed] [Google Scholar]
  • 36.Jeon H, Wang S, Song J, Gill H, Cheng H. Update 2025: management of non‑small-cell lung cancer. Lung. 2025;203(1):53. doi: 10.1007/s00408-025-00801-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Yu X, Jia L, Tang Q, Zhou Q, Wang G, Wang S. Regulation of cisplatin resistance in lung cancer by epigenetic mechanisms. Clin Clin Epigenet. 2025;17(1):145. doi: 10.1186/s13148-025-01961-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Tran TO, Lam LHT, Le NQK. Hyper-methylation of ABCG1 as an epigenetics biomarker in non-small cell lung cancer. Funct Integr Genomics. 2023;23(3):256. doi: 10.1007/s10142-023-01185-y [DOI] [PubMed] [Google Scholar]
  • 39.Dang HH, HDK T, Nguyen TTT, et al. Prospective role and immunotherapeutic targets of sideroflexin protein family in lung adenocarcinoma: evidence from bioinformatics validation. Funct Integr Genomics. 2022;22(5):1057–1072. doi: 10.1007/s10142-022-00883-3 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated and/or analyzed during the present study are available from the corresponding author upon reasonable request.

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