Clinical sample

This study included 84 lung cancer patients who underwent surgical treatment at our hospital from June 2020 to June 2022 as research subjects. Based on the occurrence of metastasis, these 84 lung cancer patients were divided into the non-metastasis group (n = 58) and the metastasis group (n = 26). There were no significant differences in clinical baseline data, including gender, age, BMI, etc., between the two groups. Tumor tissues and matched normal lung tissues were collected from all patients and analyzed using qRT-PCR. All tumor and matched normal tissues were confirmed by histopathological examination. This study was approved by our hospital’s ethics committee and adheres to the principles of the Helsinki Declaration. Informed consent forms were signed by all participants before their involvement. The baseline of the LC patients is displayed in Supplementary Table 1.

RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

RNA isolation from tissues or cells was performed using Trizol (Invitrogen, Carlsbad, CA, USA). The purity and concentration of the extracted RNAs were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA). A total of 500 ng of RNA was used to synthesize cDNA with the PrimeScript reverse transcriptase reagent kit (Takara, Shiga, Japan). Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) was carried out on the Light 7500 Real-Time PCR System (Applied Biosystems, USA) using SYBR Premix Ex TaqII (Takara) to determine the indicated RNA levels. The thermal cycling conditions were set as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 1 min. Primer sequences are provided in Table1. GAPDH served as a normalized reference. The relative RNA levels were analyzed using the 2-ΔΔCt method. All primers were designed and synthesized by Sangon (Shanghai, China). Primers were exhibited in Table 1.

Table 1 qRT-PCR primer sequencesCell culture

The human lung cancer cell lines A549, H358, H1299, Calu-3, and normal human lung epithelial cells BEAS-2B (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM/RPMI-1640/Ham’s F-12 K medium containing 10% FBS (GIBCO) and 1% penicillin-streptomycin at 37 °C in a 5% CO2 humidified incubator. The human NK cell line NK92 cells (ATCC) were cultured in MEMa supplemented with 12.5% FBS, 2 mM L-glutamine, and 12.5% horse serum (Gibco). For NK92 cell activation, cells were stimulated with 100 U/mL IL-2 (Gibco, PHC0023) for 24 h. For co-culture experiments, the activated NK-92 cells were co-cultured with A549 or H1299 cells at a ratio of 10:1 for 4 h.

Cell transfection

The lentiviral vectors pSIH1-H1-copGFP (8,619,936, BioVector Science Lab Inc., Beijing, China) and pLV-EGFP-N (VL3211, Inovogen Tech Co., Ltd., Beijing, China) were utilized to construct lentiviral short hairpin RNAs (shRNAs) targeting LINC00665 (sh-LINC00665) and lentiviral vectors for overexpressing HHLA2 (oe-HHLA2). These constructs were separately used to infect A549/H1299 lung cancer cells of the corresponding groups. Transfections were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The sequences of the shRNAs used in this study are provided in Table 2.

Table 2 Sequences of shRNAs used in this studyCCK-8

Cells were seeded at a density of 5000 cells per well in 96-well plates (Solarbio, Beijing, China). Subsequent to transfection based on experimental requirements, cells were cultured and subjected to detection using the Cell Counting Kit-8 assay every 24 h. The optical density (OD450) values were measured and analyzed using a microplate reader (Bio-RAD, USA).

Flow Cytometric Analysis (FACS)

After 24 h of transfection, A549/H1299 cells from each treatment group were collected, and staining for apoptosis was conducted using the Annexin V-FITC Apoptosis Detection Kit (BD Bioscience, Oxford, UK) following the manufacturer’s guidelines. Cell apoptosis rates were assessed using flow cytometry. In addition, following a protocol referenced from previous research [19], NK92 cells were co-cultured with A549/H1299 cells at a 1:1 ratio for 5–6 days. Flow cytometry was employed to evaluate NK92 cell proliferation (Cell-Trace or Ki67), degranulation (CD107a), and IFN-γ production. In brief, cell surface and intracellular protein staining were performed using antibodies against IFNγ, Ki67 (proliferation), and CD107a (degranulation). The assessment of NK cell phenotype and function was conducted following fixation and permeabilization with the eBioscience kit according to the manufacturer’s instructions. NK cells were labeled with CellTrace proliferation dye (ThermoFisher Scientific) before co-culture with A549/H1299. Fixable live/dead cell dye (ThermoFisher Scientific) was used to determine viable cells. All cells were acquired using the LSRFortessa flow cytometer and analyzed with FlowJo 10.7.

Transwell assays

In the transwell experiment with A549/H1299 cells, we utilized transwell chambers (Corning, USA) coated with Matrigel. A total of 1 × 10^4 cells were initially cultured in the upper transwell chamber with only DMEM, while the lower chamber was filled with 10% FBS (fetal bovine serum) and DMEM. After 24 h, the medium and noninvasive cells were removed from the upper chamber, and the invasive cells were subsequently counted under an optical microscope (Olympus, Japan). Additionally, NK cell migration was assessed in transwell assays designed to permit only active migration of NK cells, which were placed on the upper insert, towards the bottom where A549/H1299 cells were cultured. The number of NK cells was determined by relative CD56 percentages using flow cytometry.

Cytotoxicity assay

After stimulating NK92 cells with 100 U/mL IL-2 (Gibco, PHC0023) for 24 h, activated NK-92 cells were co-cultured with A549/H1299 cells at a ratio of 10:1 for 4 h. The CytoTox96 Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI, USA) was then used to measure the lactate dehydrogenase (LDH) levels in A549/H1299 cells. Briefly, cells were plated at a density of 2.3 × 10^5 cells/mL in a 96-well plate. Fifty microliters of CytoTox96 Reagent was added to each well and incubated for 30 min at room temperature. This was followed by the addition of Stop Solution (50 µL). The absorbance at 490 nm (A490) was determined using a microplate reader (Bio-Rad). Percent cytotoxicity was calculated as follows: Specific lysis (%) = [optical density (OD) experimental group – OD target cell natural release control] / (OD target cell maximum release control – OD target cell natural release control) × 100. The maximum release was measured after treatment with the lysis agent provided by the manufacturer.

ELISA

The levels of IFN-gamma and TNF-alpha in the cell culture medium were measured using ELISA kits (BMS228 and BMS223-4, Invitrogen). In brief, the culture medium was collected and centrifuged at 1400 rpm for 1 min. The ELISA assay was conducted following the manufacturer’s instructions, and the absorbance at 450 nm (A450) was determined using a microplate reader (Bio-Rad).

RNA-FISH subcellular localization

RNA-FISH was employed to investigate the cellular localization of LINC00665. DNA oligo probes for LINC00665 (labeled with FAM) were obtained from GenePharma (Shanghai, China). A549 cells (1 × 10^5) were seeded in a 24-well plate. After 24 h, the culture medium was removed, and cells were washed with PBS three times. Cells were fixed using paraformaldehyde and prehybridized with PBS containing 0.5% Triton X-100. Subsequently, the cells were hybridized with LINC00665 probes in hybridization buffer overnight at 42 °C. DAPI (Beyotime) was used for nuclear counterstaining. Finally, observation and imaging were performed under a Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany).

Nucleus-cytoplasm separation experiment

According to the manufacturer’s instructions, the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) was used to extract nuclear and cytoplasmic fractions separately. The expression of LINC00665 in the nuclear and cytoplasmic extracts was then detected by RT-qPCR.

RNA immunoprecipitation (RIP)

Anti-TCF7 antibody (PAB27159, Wuhan AmyJet Scientific Inc.) was used for RIP analysis of E2F1, with IgG as a control. RIP experiments were conducted using the Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore, Merck KGaA, Germany) following the manufacturer’s guidelines. The purified RNA was then subjected to qRT-PCR to detect co-precipitated LINC00665 and HHLA2 mRNA.

RNA fish and immunefluorescence

Briefly, H1299 or A549 cells incubated with anti-α-TCF7 or HHLA2 antibody (1:200, CST) and then incubated with Alexa Fluor 555-conjugated secondary antibody (1:10000, A32727, Thermo Fisher). The sections were dehydrated with ethanol and rehydrated in 50% formamide and then hybridized with a 20 nM 5′-digoxigenin-labeled LINC00665 probe (Ribobio) and anti-digoxigenin-FITC. DAPI was used for counterstaining the nuclei, and images were observed with laser scanning confocal microscopy (LSM710, Zeiss).

Western blot

Protein was extracted using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific), and the concentration was determined with a bicinchoninic acid kit (Beyotime). Thirty micrograms (30 µg) of protein were separated by 10% SDS-PAGE, then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking in 5% skim milk for 2 h, the membrane was incubated overnight at 4 °C with primary antibodies against TCF7 (1:1000, ab159646), HHLA2 (1:500, ab214327), and β-actin (1:5000, ab6276) (all antibodies were obtained from Abcam). Following TBST washing, an HRP-conjugated secondary antibody (ab205718, 1:2000) was added and incubated at room temperature for 1 h, followed by visualization using ECL reagent (EMD Millipore, USA). Image J software (Media Cybernetics, USA) was used for grayscale quantification of bands in the western blot images, with β-actin serving as an internal reference. Each experiment was repeated three times. Original uncropped blot images was displayed in Supplementary File 1.

Animal study

Female NOD-SCID mice (4 to 6 weeks old, n = 6 per group) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China). The transfected A549 cells were subcutaneously injected into the flank of the mice. Tumor size was monitored every 7 days and calculated using the formula: Volume = 1/2 × length × width^2. IL-2 activated NK92 cells were injected through the tail vein on day 3 and 7 post-inoculation. On day 28, the xenograft tumors were harvested and weighed. All animal experiments were conducted in accordance with established protocols and approved by the Animal Protection and Use Committee of our institution, adhering to the “Guidelines for the Care and Use of Laboratory Animals.

Immunohistochemistry (IHC)

The deparaffinized sections underwent antigen retrieval. After blocking with 1% BSA, the slides were incubated with the Ki67 antibody (ab15580), followed by incubation with an HRP-conjugated secondary antibody. The signal was detected using the DAB color development kit (Beyotime).

Statistical analysis

Data statistical analysis and plotting were conducted using SPSS 21.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 8.01 software (GraphPad Software Inc., San Diego, CA, USA). Measurement data with a normal distribution, confirmed by the Shapiro-Wilk test, are presented as mean ± standard deviation. Pairwise/independent sample t-tests were used for comparisons between two groups. One-way ANOVA analysis was employed for comparisons among multiple groups, followed by Tukey’s multiple comparisons test for post hoc analysis. A significance level of P < 0.05 was considered statistically significant.