A total of 94 clinical NSCLC samples and 86 adjacent tissues were enrolled in this study at the Second Hospital of Lanzhou University between 2017 and 2020. NSCLC samples were diagnosed by two independent pathologists. This investigation was performed according to the World Medical Association Declaration of Helsinki and approved by the Ethics Committee of the Second Hospital of Lanzhou University.

A549, H1299 and H1975 NSCLC cell lines were purchased from ATCC and then cultured in RPMI 1640 (Hyclone, USA), supplemented with 10% fetal bovine serum (Gibco, USA), antibiotics (100 U/mL, 1× penicillin/streptomycin, Gibco), and routinely maintained at 37°C in 5% CO₂. The GV115 (pGCSIL-GFP; Shanghai GeneChem Co., Ltd.) lentivirus was used for constructing COL8A1-knockdown vectors. Targeted sequences were as follows: shCOL8A1-1, 5’-GCCUAUGAGAUGCCUGCAUUUdTdT-3’; shCOL8A1-2, 5’-GCCACCACAAAUUCCACAAUAdTdT-3’; shCtrl, 5’-TTCTCCGAACGTGTCACGT-3’. The shCOL8A1 or shCtrl expression vectors, combined with Helper 1.0 and Helper 2.0 packaging vectors, were co-transfected into 293T cells using Lipofectamine™ 2000. The virus supernatants were collected by ultracentrifugation. After 72 hours transfection, the cells was used for subsequent experiments. For overexpression, the coding sequence of COL8A1 (NM_001850.5), were cloned into pCDNA3.1 vectors. The plasmids were transfected in to cells using Lipofectamine 3000 (Invitrogen). After 48 hours transfection, the cells were used for subsequent experiments.

siRNA was used to interfere iFIT1, iFIT3 or COL8A1 in A549, H1299 and H1975 cells. siRNAs against negative control, iFIT1 and iFIT3 were purchased from Huzhou Hippo Biotechnology Co., Ltd. and were transfected into A549, H1299 and H1975 cells by RNAiMAX (Invitrogen). 48 hours later, the cells were subjected to immunoblotting analysis of knockdown efficiency and functional experiments. Targeted sequences were as follows: siIFIT1-1, 5’-CUUCGGAGAAAGGCAUUAGAUdTdT-3’; siIFIT1-2, 5’-CGUCAAUGCAAUUAUCCAUUAdTdT-3’; siIFIT3-1, 5’-GCAAUAUGCUAUGGACUAUdTdT-3’; siIFIT3-2, 5’-GCAAGCAGCCAAAUGUUAUdTdT-3’; siCOL8A1-1, 5’- GCCUAUGAGAUGCCUGCAUUUdTdT-3’; siCOL8A1-2, 5’- GCCACCACAAAUUCCACAAUAAdTdT-3’; siCtrl, 5’-UUCUCCGAACGUGUCACGUdTdT-3’.

To quantify the expression of selected proteins, formalin-fixed paraffin-embedded tissue sections were analyzed by immunohistochemistry (IHC). Formalin-fixed, paraffin-embedded (FFPE) tissue samples were deparaffinized and rehydrated, then followed by heat-induced antigen retrieval. And then stained using primary anti-COL8A1 antibody (bs-7529R, Bioss antibodies), anti-iFIT1 (23247-1-AP, Proteintech), anti-iFIT3 (15201-1-AP, Proteintech) or anti-pEGFR (#3777S, CST). The intensity of expression was graded as follows: negative = score 0, weak = score 1, moderated = score 2 and strong = score 3. Low expression means refers to a score 0 or 1, and high expression means refers to a scoring score 2 or 3. Immuno-reactivity was assessed independently by two expert pathologists blind to all clinical data.

A Celigo Image Cytometer (Nexcelom) or CCK8 assay were used to determine the proliferation capacity of NSCLC cell lines, according to the manufacturer’s instructions. Celigo Image Cytometer (Nexcelom) was used in COL8A1 knockdown experiment. Briefly, H1299, A549 and H1975 cell lines (2.5 × 10³ cells/well) were seeded into 96 well plates, with three replicates of each treatment group, and then maintained at 37°C for 5 days. The number of cells with green fluorescence was calculated by Celigo cell count every 24 h after cell seeding.

CCK8 assay was used in the rest of cell viability assay. Briefly, a total of 1000 cells were seeded into 96-well plates. At indicated time, 10 μl of CCK8 regent was added into each well followed by incubation at 37°C for 3 hours. Then OD value at 450 nm was measured.

NSCLC cells (5 × 10³ cells/well/100 µl) subjected to corresponding treatments were seeded into 6-well plates and incubated for 10 days. At the end of experiments, cells were washed with PBS three times, fixed in 4% paraformaldehyde for 10 min, then stained with 1% crystal violet. After air-drying, the numbers of colonies > 2 mm diameter were quantified.

For the transwell assay (Corning Costar, USA), H1299, A549 and H1975 cells (density, 1.5 × 10⁵) were seeded on the noncoated membrane (for cell migration assay) or the Matrigel-coated membrane (for cell invasion assay) of upper chambers of 24-well units without serum, and 0.6 mL of medium supplemented with 10% FBS added to the lower chambers to serve as a chemoattractant. After culture for 18 h, cells that had not migrated to the lower surface of the chamber were completely removed using a cotton swab. Then, the chambers were fixed with methanol for 15 min and then stained with crystal violet at room temperature. The invasive cells were counted under a light microscope, with at least five random fields analyzed for each sample. The experiments were repeated three times.

Apoptosis was examined by flow cytometry. Briefly, cells subjected to different treatments were harvested, washed twice with cold PBS, and then incubated with Annexin V-FITC, or Annexin V-APC and propidium iodide (PI) staining solution (Shanghai YEASEN Biotech Co. Ltd) for 30 min at 37°C in dark conditions, following the manufacturer’s instructions. Annexin V-APC and propidium iodide (PI) staining was used in COL8A1 knockdown experiment, while Annexin V-FITC and propidium iodide (PI) staining was used in the rest of apoptosis assay. To induce cell apoptosis, the cells were treated with 2uM PAC-1 (Selleck, S2738). Then the cells were subjected to cell apoptosis assay. Cells were analyzed by flow cytometry (cytoFlex, Beckman Coulter, Inc.). The data were analyzed by Cytexpert (Version 2.4.0.28, Beckman Coulter, Inc.).

Cells were infected with the shCOL8A1 or shCtrl lentivirus. Cells were fixed with 70% cold ethanol at 4°C overnight, washed twice with ice-cold PBS, and incubated with 10 mg/ml RNase at 37°C. The cell cycle was monitored by propidium iodide (PI) staining of the nucleus. The fluorescence of DNA-bound PI in cells was measured by flow cytometry (FACS Calibur, Becton Dickinson).

In brief, total RNA was extracted using Trizol regent (Invitrogen) and subsequently digested with DNase enzyme (Invitrogen). qRT-PCR assays were conducted using a SYBR master mixture Kit (TaKaRa) on a StepOne Real-Time PCR system (Thermo Fisher). Primer sequences are presented in Supplementary Table 1 . Relative RNA expression levels were normalized to those of β-actin. The Ct2-ΔΔCt method was performed to examine the relative expression.

Whole cell lysates were extracted using RIPA reagent (Beyotime, China) supplemented with protease inhibitors (Roche cOmplete ULTRA tablets), and protein concentrations determined using a Bicinchoninic Acid Protein assay Kit (Thermo Fisher, USA). Total protein aliquots (20 μg) were separated by SDS-PAGE, as described previously. Primary antibodies against p-EGFR (#3777S, CST), EGFR (#2085, CST), p-AKT (#4060, CST), AKT (#4821, CST), IFIT-1 (23247-1-AP, Proteintech), IFIT-3 (15201-1-AP, Proteintech), and β-actin (#4970, CST) were applied in this study. Secondary antibodies used were IRDye 800CW Goat anti-rabbit 926-32211 (Lot No. C90723-19, LI-COR) and IRDye 800CW Goat anti-mouse 926-32210 (Lot No. C90408-08, LI-COR).

To evaluate the effects of COL8A1 expression on the growth of NSCLC cells in vivo, a nude mouse xenograft model was used. Female BALB/c nude mice (6–8 weeks old) were raised in a specific pathogen-free environment. Stable A549 cell lines expressing shCOL8A1 or control shRNA were sorted and maintained in growth medium supplemented with puromycin (2 µg/mL) for one week. NSCLC A549 cells transfected with shCtrl or shCOL8A1 (2 × 10⁶ cells/mouse) were injected subcutaneously into the right flanks of mice. After inoculation, tumor volume was evaluated by caliper measurements every 3 days and calculated according to the formula: volume = 1/2 × length × width². When tumor volumes in the shCtrl group reached 600 cm³, mice were killed and tumors harvested for analysis.

Results are presented as the mean ± standard deviation. Statistical differences between two groups were determined using a Student’s t-test. Statistical differences among ≥3 groups were determined using a one-way ANOVA followed by a Tukey’s post hoc test. Categorical data were analyzed using a Chi-Square test. Spearman’s correlation coefficients (rs) were calculated between COL8A1 staining score and IFIT1, IFIT3 or p-EGFR staining score. P < 0.05 was considered statistically significant.

To identify and characterize COL8A1 in NSCLC, we examined its expression levels in NSCLC and paired adjacent normal tissue samples. The results revealed that COL8A1 was highly expressed in NSCLC tissues. Moreover, of the 94 cases included in the IHC staining assay, COL8A1 was positively expressed in 74 NSCLC tissues (78.7%) ( Figure 1 and Table 1 ). To assess possible associations between COL8A1 expression and tumor characteristics, IHC scores for COL8A1 were analyzed in relation to clinicopathological parameters. As shown in Table 2 , high expression of COL8A1 was strongly associated with more advanced pathologic stage of NSCLC (II–III), compared to earlier pathologic stage (I). These results suggest that COL8A1 may be acted as an important factor in the development of NSCLC.

Our clinicopathological data clearly indicated that COL8A1 was associated with tumor pathological stage. To determine the role of COL8A1 in NSCLC, the cell lines, A549 and H1975, were transfected with lentivirus encoding COL8A1 shRNA (shCOL8A1-1 and shCOL8A1-2) and empty lentiviral vectors (shCtrl) to establish stable COL8A1-silenced cell lines. As shown in Figure 2A , COL8A1 was clearly suppressed in both A549 and H1975 cell lines. Knockdown of COL8A1 markedly suppressed the capacities of proliferation and colony formation in A549 and H1975 cells ( Figures 2B, C and Supplementary Figures 1A–C ). Cell cycle analysis indicated that A549, H1975 and H1299 cells expressing shCOL8A1 induced cell cycle arrest at G1 phase ( Figure 2D and Supplementary Figure 1D ). Further, we found that COL8A1 knockdown promoted apoptosis in A549, H1299 and H1975 NSCLC cell lines, indicating that silencing of COL8A1 could partly suppress cell growth by promoting apoptosis ( Figure 2E and Supplementary Figure 1E ).

As tumor progression and metastasis is a complex process involving cell motility, including cell invasion and migration, we next examined the effects of COL8A1 expression on NSCLC cell invasion using transwell assays. COL8A1-silenced A549 and H1975 cells exhibited significantly lower invasion ability compared with shCtrl cells ( Figure 2F , Supplementary Figure 1E ).

To further explore its roles acted as an oncogene, COL8A1 was overexpressed in A549, H1299 and H1975 cells, as confirmed by western blotting ( Figure 3A ). As shown in Figures 3B, C , COL8A1-overexpressing cells displayed markedly enhanced cell proliferation and colony formation capacities relative to control cells. To explore the apoptosis activity medicated by COL8A1, lung cancer cells were treated with an apoptosis inducer, PAC-1, and the results indicated that COL8A1 overexpression significantly suppressed apoptosis of A549, H1299 and H1975 cells ( Figure 3D ). These results demonstrate that COL8A1 overexpression promotes cell proliferation and colony formation, as well as suppressing apoptosis, of A549, H1299 and H1975 cells.

To further explore the mechanisms underlying COL8A1 involvement in NSCLC progression, we applied RNA-seq technology to determine whether COL8A1 knockdown influenced gene expression in NSCLC cells. RNA-seq results identified 3959 up-regulated and 5542 down-regulated genes in COL8A1 knockdown cells ( Figure 4A ), which were involved in apoptosis, p53, interferon alpha response signaling and interferon gamma responses, as well as the interferon alpha response pathway ( Figure 4B ). To confirm this finding, we detected the related gene expression in interferon alpha response signaling and interferon gamma response signaling by qPCR, the results demonstrated that the expression of genes related to interferon signaling were significantly changed after COL8A1 knockdown or COL8A1 overexpression ( Figures 4C, D ).

Activation of interferon signaling has vital roles in regulating NSCLC progression. In addition, IFIT1 and IFIT3 are critical for EGFR recycling and activation in other cancers (21). Hence, whether COL8A1 upregulation of IFIT1 and IFIT3, activates EGFR in NSCLC warrants investigation. Notably, COL8A1 knockdown in H1975 and H1299 cells robustly suppressed the expression of IFIT1, IFIT3, and phosphorylation of AKT and EGFR ( Figures 4E, F ). Conversely, COL8A1 overexpression remarkably increased the levels of phosphorylated of AKT and EGFR, as well as of IFIT1 and IFIT3, in H1975 and H1299 cells ( Figures 4E, F ). Moreover, according to correlation analysis using data from NSCLC tissues in TCGA database, IFIT1 and IFIT3 expression levels were positively correlated with those of COL8A1 ( Figures 4G, H ). Taken together, these results suggested that COL8A1 may activate EGFR by upregulating IFIT1 and IFIT3.

Next, we aimed to investigate the functional roles of IFIT1 and IFIT3 in NSCLC development. Western blotting showed that silencing of IFIT1 and IFIT3 effectively suppressed their expression and inhibited AKT and EGFR activity ( Figures 5A, B ). In addition, to explore the mechanism of COL8A1 upregulated IFIT1 and IFIT3-medicated EGFR activation, we overexpressed COL8A1 in lung cancer cells H1299 and H1975, and found that IFIT1, IFIT3 and pEGFR were upregulated by COL8A1 overexpression, whereas after co-transfected with COL8A1-overexpressing vectors and siIFIT1/siIFIT3, the expression of IFIT1, IFIT3 and pEGFR were suppressed, which indicated that COL8A1 promoted EGFR activation through upregulating IFIT1 and IFIT3 in lung cancer cells ( Supplementary Figure 2 ). Cell viability and colony formation assays suggested that knockdown of IFIT1 and IFIT3 significantly suppressed proliferation of H1299 and H1975 cells ( Figures 5C–H ). In addition, silencing of IFIT1 and IFIT3 inhibited NSCLC cell migration and invasion, and enhanced NSCLC cell apoptosis ( Figures 5I, J ).

To further investigate whether COL8A1-mediated tumor progression required IFIT1 and IFIT3 knockdown, we examined cell proliferation in COL8A1-overexpressing and IFIT1/IFIT3-silenced NSCLC cells. As shown in Figures 5K, L , IFIT1 or IFIT3 knockdown efficiently blocked COL8A1-enhanced NSCLC cell proliferation and colony formation capacity. Collectively, either IFIT1 or IFIT3 could restore the effects of COL8A1 on cell proliferation and migration in H1299 and H1975 cells.

To further explore the function of COL8A1 in vivo, we established a mouse xenograft model by subcutaneously injecting shCOL8A1-expressing cells into nude mice. COL8A1-silenced xenograft tumors grew significantly more slowly than those in the control group, with tumor volume and weight in COL8A1-silenced tumors lower than those expressing control shRNA, suggesting that knockdown of COL8A1 suppressed tumor growth capacity in vivo ( Figures 6A–C ). Western blot analysis of tumor tissues showed that COL8A1 knockdown reduced IFIT1 and IFIT3 expression, as well as EGFR phosphorylation ( Figure 6D ). Overall, our data demonstrate that COL8A1 is critical for NSCLC growth in vivo.

Finally, we assessed the correlations between COL8A1 and IFIT1/IFIT3 expression levels, and EGFR phosphorylation. To this end, a total of 50 NSCLC tissue samples were subjected to IHC staining analysis, which demonstrated that COL8A1 protein abundance was positively correlated with IFIT1/IFIT3 expression and EGFR phosphorylation ( Figure 7 and Table 3 ). A positive Spearman correlation co-efficient was also detected between IFIT1/3 expression and EGFR activity in human NSCLC samples ( Figure 7 and Table 3 ). Therefore, COL8A1 activation of EGFR through upregulation of IFIT1/IFIT3 may occur in human NSCLC.