Specimen collection and immunohistochemistry (IHC)

This study included a total of 93 lung cancer cases, 32 normal lung tissue samples for IHC (from 2018 to 2020), 32 pairs of fresh non-small-cell lung cancer tissues and their adjacent tissues for western blotting (2020), collected from the Department of Pathology, the First Hospital of China Medical University, 44 cases of adenocarcinoma, and 49 cases of squamous cell carcinoma, defined according to the 2021 edition of WHO lung cancer histological classification standards [18]; the average age of patients with lung cancer was 60 years. Using the UICC/AJCC TNM staging criteria (2023) [19], we categorized 61 cases as stages I and II and 32 cases as stage III cancer. Follow-up for all patients was considered from the date of surgery to the end of the follow-up period or the date of death attributed to recurrence or metastasis.

The polyclonal rabbit-derived ZMIZ2 antibody (HPA040716, 1:50; Sigma-Aldrich, St. Louis, MO) was used as the primary antibody, and tissue samples were incubated overnight at 4 ℃ with the antibody. Negative control was established using phosphate-buffered saline (PBS) instead of the primary antibody. Following this, the samples were incubated at 37 ℃ for 30 min with a biotin-labeled secondary antibody (MaiXin, Fuzhou, China), and DAB staining was performed.

Five randomly selected fields of view were assessed for each tissue slice, with a count of 100 cells per field under an optical microscope (Nikon, Japan). ZMIZ2 expression levels were categorized into five stages based on the percentage of stained cells: 0 (no staining), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (> 76%). Additionally, based on the intensity of cell staining, ZMIZ2 expression was further classified into three levels: 0 (no staining), 1 (light yellow particles), and 2 (deep yellow or yellowish-brown particles). Each tissue slice received both percentage and coloring scores, which were multiplied to obtain the final score. Because the staining scores in most normal lung bronchial and alveolus epithelia were less than 4, a score ≥ 4 was considered a positive expression, whereas a score < 4 was considered a negative expression.

Non-small-cell lung cancer (NSCLC) cell lines and culture

The human immortalized bronchial epithelial cell line (HBE, no. AC338600) and lung adenocarcinoma cell line (SPC-A-1, no. XY-XB-1057) were obtained from ATCC Cell Library (Manassas, VA). The LK2 cell line was received from Doctor Hiroshi Kijima (Department of Pathology and Bioscience, Hirosaki University Graduate School of Medicine, Japan). NCI-A549 (no. TCHu150), NCI-H1299 (no. SCSP-589), Calu-1 (no. TCHu192), NCI-H661 (no. SCSP-5071), and NCI-H460 (no. SCSP-584) cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were subjected to short tandem repeat (STR) analysis and mycoplasma testing. Calu-1 cells were cultured in Myco5A medium, and the remaining six lung cancer cell lines were cultured in RPMI-1640 medium. All culture media contained 10% calf serum (Invitrogen, Carlsbad, CA) and 100 U/mL penicillin (Sigma-Aldrich). The cell lines were cultured in a 5% CO2 incubator.

ZMIZ2 overexpression plasmid, CRISPR-CAS9-sgRNA lentivirus, and cell transfection

Myc-tagged pCMV6 empty vector and pCMV6-ZMIZ2 plasmids were purchased from OriGene (no. PS-100001 and no. RC-216992, respectively; Rockville, MD). The pcDNA3.1 empty vector (no. 52535), pcDNA3.1-FLAG-SIRT1 (no. 1791), pcDNA3.1-FLAG-SIRT1-H363Y (no. 1792), GFP- β-catenin (no. 71367), pGL3b 8xGTIIC luciferase (no. 34615), and Super 8 × Topflash (no. 12456) plasmids were purchased from Addgene (Cambridge, MA). The pRL-TK vector (no. E2241) was purchased from Promega (Madison, Wisconsin). HA-TCF4 (NM_001083962), HA-TEAD (NM_021961), pEGFP-N1 empty vector, pEGFP-N1-YAP (NM_001130145), and Myc-ZMIZ2 (NM_001300959) mutant plasmids (ZMIZ2-ΔNLS, ZMIZ2-ΔMIZ, and ZMIZ2-ΔPro-rich) were constructed by Baihao Company (Shenyang, China). Control siRNA (sc-37007), siRNA-ZMIZ2 (sc-89373), and siRNA-SIRT1 (sc-40986) were purchased from Santa Cruz Technology Inc. (CA). Lentivirus-ZMIZ2 and sgRNA-ZMIZ2 lentivirus plasmids were purchased from GeneChem (Shanghai, China). Cells were transiently transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. In case of stable transfection, purinomycin (Sigma-Aldrich) was employed during clone screening.

Protein extraction and western blotting

Assays were performed as described previously [6]. ZMIZ2 (sc-163547, IB/1:200, IP/1:50), GFP-Tag (sc-9996, IB/1:500, IP/1:50), TCF4 (sc-166699, IB/1:200), and GAPDH (sc-293335, IB/1:1000) were purchased from Santa Cruz Biotechnology. SIRT1 (no. 8469, IB/1:1000, IP/1:50), Myc Tag (no. 2276, IB/1:1000, IP/1:50), FLAG-Tag (no. 14793, IB/1:1000), HA Tag (no. 3724, IB/1:1000), C-myc (no. 9402, IB/1:1000), MMP7 (no. 3801, IB/1:1000), CyclinD1 (no. 2922, IB/1:1000), CTGF (no. 86641, IB/1:1000), CYR61 (no. 14479, IB/1:1000), MST (no. 3682 IB/1:1000), p-MST (no. 49332, IB/1:1000), p-LATS1 (no. 8654, IB/1:1000) LATS1 (no. 3477, IB/1:1000), YAP (no. 14074, IB/1:1000, IP/1:50), action (no. 3700, IB/11000), MOB1 (no. 13730, IB/1:1000), SAV1 (no. 13001, IB/1:1000), β-catenin (no. 8480, IB/1:1000, IP/1:50), acetyl-β-catenin (Lys49, no. 9534, IB/1:1000), acetyl lysine antibody (no. 9441, IB/1:1000), LaminB1 (no. 13435, IB/1:1000), and Tublin (no. 2148, IB/1:1000) were all purchased from Cell Signalling Technology (Danvers, MA). After incubation with peroxidase-coupled anti-mouse IgG and anti-rabbit IgG (Santa Cruz Biotechnology) antibodies at 37 °C for 1 h, bound proteins were viewed using ECL (Thermo Fisher Scientific, Waltham, MA) and detected using Bio-Rad Systems (California, USA). ImageJ software (version 18.0) was used to measure grayscale values on strips [20], and the relative expression level was determined by normalizing to GAPDH. Human recombination protein Wnt3a (no. 5036-WN, R&D Systems, France) was dissolved in PBS containing 0.2% BSA to achieve a concentration of 10 μg/mL and was utilized in the assays at a final concentration of 50 ng/mL. The experiments were repeated three times, and the mean was subsequently calculated.

RNA extraction and real-time quantitative polymerase chain reaction (RT–qPCR)

Total RNA was extracted from cells using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), and RT–qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) with 20 μL sample in a 7900HT Fast real-time quantitative PCR system (Applied Biosystems, Foster City, CA). The PCR conditions included an initial step at 50 ℃ for 2 min, followed by 95 ℃ for 10 min, and then a cycling step at 95 ℃ for 40 s and 60 ℃ for 60 s. GAPDH was used as an internal reference, with the relative expression level of the gene represented as ΔCt (Ct value of the gene—Ct value of the internal reference). The changes in the relative expression of genes were calculated as 2–ΔΔCt using the Ct method [21], and all experiments were conducted in triplicate. Table 1 presents the primer sequences used in RT–qPCR analysis.

Table 1 Primers for real-time reverse transcriptase polymerase chain reactionDual-luciferase reporter assay

YAP/TEAD transcriptional activity was measured using a luciferase assay based on the pGL3b_8xGTIIC-luciferase plasmid purchased from Addgene (plasmid no. 34615); β-catenin/TCF4 transcriptional activity was measured using Super 8xTopflash plasmid purchased from Addgene (plasmid no. 12456). Cells were transfected to express the indicated proteins, and Renilla luciferase was used as a control for signal normalization. Dual luciferase assays were performed according to the manufacturer’s protocol (Progema, WI). Three independent transfections were performed for each experiment. Data were normalized to the empty vector control and presented as mean ± SD.

Nuclear-cytoplasmic protein separation

The cells were trypsinized and washed with cold PBS, and the cell pellets were resuspended in an ice-cold lysis buffer supplemented with protease inhibitors (210 mM mannitol, 70 mM sucrose, 5 mM Tris, pH 7.5, and 1 mM EDTA). After 15 min of incubation on ice, the cells were homogenized. The nuclei were isolated via centrifugation (12,000 rpm, 10 min, 4 °C), and the nuclear pellets were washed with PBS and resuspended on ice for 5 min in RIPA buffer (Santa Cruz Biotechnology, CA). Following centrifugation and sedimentation, the supernatant (nuclear fraction) was collected and transferred to a new 1.5-mL tube. The lysate was quantified using the Bradford assay, and an equivalent amount of total protein was subjected to western blotting.

Mass spectrometry analysis, immunoprecipitation, and Kyoto encyclopaedia of genes and genomes (KEGG)

For the immunoprecipitation assay, whole-cell extracts were prepared after transfection and incubated overnight with indicated antibodies and Protein A/G beads (no. sc-2003, Santa Cruz Technology). Beads were then washed three times with lysis buffer, and immunoprecipitates were eluted for detection using mass spectrometry. The results obtained from mass spectrometry were integrated and statistically analyzed, subsequently incorporating KEGG signaling pathway enrichment analysis through OmicShare Tools (omicshare.com/tool/). The BioGird online protein–protein interaction database (thebiogrid.org) was applied to predict the underlying interacted proteins with ZMIZ2.

Cellular immunofluorescence staining

Cells were fixed using 4% paraformaldehyde and sealed with 3% BSA for 1 h. ZMIZ2 rabbit polyclonal antibody (HPA040716, 1:50; Sigma Aldrich), β-catenin (1:100; Cell Signalling Technology), and YAP (no. 14074, 1:100) antibodies were incubated with the cells overnight at 4 ℃. Subsequently, the cells were incubated with a goat anti-rabbit/mouse secondary antibody (1:5000; Cell Signalling Technology). Nuclei were stained with DAPI and observed via confocal scanning using a Radiance 2000 laser confocal microscope (Carl Zeiss, Thornwood, NY).

Colony formation, matrix adhesive invasion, and MTT assays

Colony formation assay: Following a 48-h period post overexpression or interference in the tumor cell transfection experiment, the cells were seeded into a 6-cm cell culture dish (1000 cells/dish) and incubated for 12 days. Subsequently, the dish was washed with PBS and stained with haematoxylin. Colonies with > 50 cells were then counted.

MTT assay: Approximately 3000 transfected-NCI-H1299 and Calu-1 cells were cultured in a 96-well plate with 10% serum for 24 h. Subsequently, 20 μL of a 5 mg/ml MTT (thiazolyl blue) solution was added to each well, followed by incubation at 37 ℃ for 4 h. After removing the solution, MTT crystals were dissolved in 150 mL of dimethyl sulfoxide (DMSO), and absorbance was measured at 490 nm using a spectrophotometer (Bio-Rad, CA).

Cell matrix adhesive invasion assay: The matrix adhesive (BD Biosciences, CA) was diluted in Dulbecco’s modified Eagle medium (DMEM) medium (1:3 ratio) in a 24-well plate, excluding any additives, and seeded in an 8-μm pore size upper chamber (Costa, Shanghai, China). After 48 h of transfection, the tumor cells (5 × 105 cells) were placed in the upper chamber and incubated for 16 h. A culture medium containing 10% calf serum was placed in the lower chamber. After 24 h of cultivation post-seeding, the cells were fixed in ice-cold methanol for 15 min and stained with haematoxylin. Ten randomly selected fields of view were used to count the number of cells invading the subventricular space using a Nikon optical microscope (Japan). The experiment was repeated three times, and the average value was calculated.

Animal experiments

Animal experiments were performed in accordance with the ethical regulations governing animal studies at the China Medical University (no. CMU20231358). The maximal tumor size permitted by the ethics committee/ was 2000 mm3, and the maximal tumor size in our study was not exceeded. Female BALB/c nude mice (n = 25), aged 4 weeks and weighing 16–20 g, were purchased from the Charles River Company (Beijing, China). Both the feed and drinking water were sterilized in a semibarrier system maintained at constant temperature and humidity. The cell concentration in each group (0.2 mL) was adjusted to 5 × 106 pieces/mL and subcutaneously injected into the dorsal regions of the nude mice. Following continuous observation for 5 weeks postinjection day, the mice were euthanized, and the weight and volume of the subcutaneously transplanted tumors were recorded. Tumor volume was calculated using the equation: length × width2 /2.

Ethics statement

The study design was approved by the Institutional Review Board of China Medical University (no. LS [2019] 003). All participants signed the informed consent form, and the study was conducted in accordance with the principles of Declaration of Helsinki. The animals used in this study were treated according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 8023, revised 1978).

Statistical analysis

All data were statistically analyzed using SPSS version 27.0 (Chicago, IL). The chi-square test was employed to examine the association between ZMIZ2 expression and clinical pathological factors. Kaplan–Meier analysis was used to assess the relationship between ZMIZ2 and the overall prognosis of patients with lung cancer. The differences between groups were analyzed using a t-test or two-way analysis of variance (ANOVA). A significance level of P < 0.05 was considered statistically significant.

Role of the funding source

The funders (National Natural Science Foundation of China) had no role in the study design, sample collection and examination, animal experiments, data analysis and interpretation, or writing of the manuscript. The corresponding authors had full access to all the data and final responsibility for the decision to submit for publication.