Clinical samples

A total of 30 pairs of NSCLC tumor tissues and adjacent normal tissues were collected from NSCLC patients at the First Affiliated Hospital of Gannan Medical University between 2016 to 2020. All patients have signed the informed consent. This study was approved by the First Affiliated Hospital of Gannan Medical University (No. 87655). Animal experiments was in accordance with ARRIVE guidelines. All tissue samples were stored in -80 °C before use. Ethical approval numbers: No.43922.

Cell culture

Human bone marrow mesenchymal stem cells (BMSCs), and NSCLC cell lines A549 and H1299 were purchased from American Type Culture Collection (MD, USA). Cells were grown in DMEM/F12 medium containing 10% FBS (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) in a 37 °C humid environment with 5% CO2.

Characterization of hBMSCs and exosomes

The hBMSCs at passages 4–6 were cultured overnight, and the cellular morphology was examined under an inverted phase contrast microscope. The hBMSCs-derived exosomes (Exo) were isolated via ultra-high-speed centrifugation as previously reported [16, 17]. In brief, hBMSCs at passages 4–6 were cultured in 6-well plates with a cell density of 5 × 105 cells per well, totaling 12 ml of serum-free medium. After that, the supernatant was harvested when changing fresh medium. The supernatant was centrifuged at 12,000 r/min for 40 min to remove cell residues. Then exosome extraction reagent was added into the supernatant and incubated at 4 °C overnight. Finally, ultra-high-speed centrifugation was performed, and the precipitate was re-suspended in PBS as the exosomes and stored at − 80 °C. The concentration of exosomes were measured by BCA method. The morphology of exosomes was examined by a transmission electron microscope, and the exosomes-related surface markers (Tsg101, CD63 and Calnexin) were detected by Western blot analysis.

Transfection

The 100 nM miR-145-5p mimics and inhibitors, along with their respective control miR-NC and inhibitor NC, and si-ROX9, si-NC were obtained from Shanghai GenePharma Co. Ltd. These molecules were then transfected into A549, and H1299 cells using Lipofectamine 2000 reagent (Invitrogen).

Preparation of Exo-miR-145-5p mimics and other Exos

First, 5 × 106 hBMSCs per dish were seeded into 10 cm dishes (total 6 dishes). The next day, miR-NC, miR-145-5p mimics, inhibitor NC, miR-145-5p inhibitor, miR-145-5p inhibitor + si-SOX9 and miR-145-5p inhibitor + si-NC were transfected into hBMSCs using 60 µL Lipofectamine 2000 reagent (Invitrogen). After incubating these cells for 48 h, the medium was changed with serum-free medium for 24 h. Next, we collected the supernatant and extracted exosome following the steps mentioned above. The relative miR-145-5p and SOX9 mRNA expression were calculated by qRT-PCR to verify these molecules were loaded.

Exosome treatment

To investigate its role, cells were transfected with hBMSCs-Exo carrying different molecules, and labeled as Exo-miR-NC, Exo-miR-145-5p mimics, Exo-inhibitor NC, Exo-miR-145-5p inhibitor, Exo-miR-145-5p inhibitor + si-SOX9, and Exo-miR-145-5p inhibitor + si-NC. Subsequently, 50 μg of hBMSCs-derived exosomes with these molecules were added to the cell cultures of A549 or H1299, and incubated for two days. After this incubation period, the cells were utilized for other functional assays.

Cell proliferation

Cell viabilities were assessed using the CCK8 kit (Dojindo, Rockville, MD, USA) as previously reported [18]. The optical density at 450 nm at different time points were detected using a spectrophotometer. For EdU staining, the BeyoClick EdU Cell Proliferation Kit (Beyotime Biotechnology, Shanghai, China) was used. Briefly, the transfected cells were incubated with 10 μM Edu for 2 h, followed by the incubation of DAPI solution for nuclei. Staining images were observed under a fluorescence microscope. EdU positive cells (proliferating cells) % = EdU positive cells/total cells × 100% from three random visual fields.

Transwell assay

An 8-μm well size Transwell chamber with well inserts (Corning, N.Y., USA) was used for invasion assay. To promote cell invasion, 100 μL of 50 mg/L Matrigel (1:40 dilution) was coated onto the upper surface of chamber’s bottom membrane. A cell suspension containing 2 × 105 cells in 100 μL was added to the upper chamber, while the lower chamber was filled with 600 µL of DMEM medium containing 20% FBS. After 24 h, cells were fixed by methanol for 10 min and stained with 0.1% crystal violet for imaging and counting under five random fields using optical microscope with 400 × magnification. The five random fields were obtained from the left, right, up, down and center of the chamber. The cell number was showed by mean ± standard deviation and compared by Student's t test. Cell migration assay was performed the same as invasion assay without coating the Matrigel.

Apoptosis analysis

A total of 1 × 105 cells were seeded into 6-well plates and cultured overnight, and the supernatant was removed by centrifugation. According to the Annexin VFITC cell apoptosis detection kit (Biovision, USA), cell pellets were incubated with 500 μL loading buffer, 5 μL Annexin V-FITC and 10 μL propidium iodide (PI) solution for 20 min. Cell apoptosis was evaluated with the flow cytometry (BD Biosciences). Apoptosis was quantified following Annexin-V/PI staining % were only quadrants Q2/Q3 combined.

qRT-PCR

Total RNAs were isolated using TRIzol reagent (Invitrogen). The cDNA was synthesized using the specific Reverse Transcription Kit (Applied Biosystems). PCR reactions were performed using SYBR green Super-mix (Thermo Fisher) on an ABI 7500 PCR detection system. Gene expression was quantified the 2–ΔΔCT method. GAPDH/U6 was used as the normalization control. Primer sequences were: miR-145-5p forward 5′-ACACTCCAGCTGGGTCCCTAAGGACCCTTTT-3′ and reverse 5- CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGGTCAA-3′; and SOX9 forward 5′- AGCGAACGCACATCAAGAC-3′ and reverse 5′-CTGTAGGCGATCTGTTGGGG-3′. U6 F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH F: 5′-TCCCATCACCATCTTCCA-3′, R: 5′-CATCACGCCACAGTTTTCC-3′.

Western blot analysis

Total proteins were extracted using RIPA buffer. Approximately 50 µg of protein sample per lane was separated by 12% SDS-PAGE and subsequently transferred onto PVDF membranes, followed by incubation with the appropriate primary antibodies CD63 (1:1,000) (ab134045, abcam), Tsg101 (1:1,000) (ab125011, abcam), Calnexin (1:1,000) (ab227310, abcam), and SOX9 (1:1,000) (ab185966, abcam) at 4 °C for overnight. The next day, the membrane was subjected to HRP-conjugated secondary antibody (1:10,000) for 1 h. All antibodies were purchased from Abcam. The protein bands were visualized using a chemiluminescence reagent, and data analysis was conducted using Image J software.

Luciferase reporter assay

The wild type and mutant sequences of the SOX9 3′-UTR, containing binding sites of miR-145-5p, were cloned downstream of the pmirGLO reporter vector (that it is a SOX9 3'UTR luciferase reporter vector). The H1299 cells were co-transfected with luciferase reporter vector, miR mimic or miR-NC for two days. The relative luciferase activity was measured using a Dual-Luciferase reporter assay system and subsequently normalized to Renilla activity.

RNA pull-down assay

The RNA of patient samples were extracted using MolPure® Cell/Tissue Total RNA Kit (19221ES50, YESEN). Biotin labelled and biotin non-labelled SOX9 probes (Bio-SOX9 and Bio-NC) were synthesize by Shanghai GenePharma Co., Ltd. H1299 cells were lysed and the whole lysates were incubated with probe-coated M-280 streptavidin-magnetic beads (Invitrogen) at 4 °C overnight. Then RNAs were extracted, and the expression of miR-145-5p was detected using qRT-PCR analysis.

Tumor xenograft in vivo model

A total of 25 male BALB/c nude mice which lack mature T cells through the thymus (6 weeks old) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. were used to establish the in vivo model. Each mouse was subcutaneously injected with 1 × 106 A549 cells. Once the mean tumor volume reached 100 mm3, all mice were randomly divided into five groups (each group consisted of 5 mice): Control group (PBS injection), Exo-miR inhibitor-NC group, Exo-miR inhibtor group, Exo miR-145-5p inhibitor + si-NC group, and Exo miR-145-5p inhibitor + si-SOX9 group. Approximately 200 μg/mouse hBMSCs-derived Exo was intratumorally injected into mice every two days for a total of 10 times. Four weeks later, when the longest tumour diameter reached 1.5 cm, the mice were euthanized using asphyxiation in a CO2 chamber, and tumors were weighted. Tumor volume was determined using the formula (length × width2)/2. This study was approved by First Affiliated Hospital of Gannan Medical University (No. 76395).

Statistical analysis

Data were presented as the mean ± standard deviation (SD), and each experiment was repeated for three independent times. Student's t test was used to conduct comparison between two groups. One-way-ANOVA followed by Tukey’s multiple comparisons test was applied for multi-group comparison. Statistical significance was set at p < 0.05.