A total of 24 paired samples of tumorous and non-tumorous tissues were collected from surgery at Tumor Hospital of Shaanxi Province with the consent of patients. The histological analysis of each sample was confirmed by pathologists in a double-blind manner. All the procedures were approved by the Ethics Committee of the Tumor Hospital of Shaanxi Province Hospital.

Human NSCLC cell lines NCI157, A549, H1299, H460, H1703, H1975, and BEAS control cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI-1640 medium with 10% fetal bovine serum (Gibco, United States). All the NSCLC cell lines were authenticated by short tandem repeat (STR) analysis and were tested for Mycoplasma contamination.

Total RNA of NSCLC cells was extracted with TRIzol reagent (Invitrogen, United States). The relative fold expression was calculated using the comparative threshold cycle (2^−ΔΔCt). The primer is presented as follows: β-actin: Forward (5′-3′): CCC​ACT​CCT​CCA​CCT​TTG​AC, Reverse (5′-3′): CAT​ACC​AGG​AAA​TGA​GCT​TGA​CAA; IGF2BP2: Forward (5′-3′): GTT​GGT​GCC​ATC​ATC​GGA​AAG​G, Reverse (5′-3′): TGG​ATG​GTG​ACA​GGC​TTC​TCT​G; MALTA1: Forward (5′-3′): GAA​TTG​CGT​CAT​TTA​AAG​CCT​AGT​T, Reverse (5′-3′): GTT​TCA​TCC​TAC​CAC​TCC​CAA​TTA​AT.

Total protein of NSCLC cells was extracted with RIPA reagent (Beyotime Biotechnology, China). An equal amount of total protein lysate (30 μg) was separated by 7.5–12% SDS-PAGE and transferred onto a PVDF membrane, followed by incubation with primary antibody overnight at 4°C. Then the bands were incubated with secondary antibody for 1 h at room temperature (RT). The bands were detected using a Bio-Rad ChemiDoc XRS system. The primary antibodies were presented as follows: anti-IGF2BP2 (1:1,000, abcam, ab124930), anti-ATG12 (1:1,000, abcam, ab109491), and anti–β-actin (1:2,000, abcam, ab8226).

Cell proliferation was analyzed using CCK8 assay and colony formation assay. CCK8 assay was performed using a Cell Counting Kit-8 (CCK-8, Biotool, China). Cells were seeded in 96-well plates at a density of 3.5 × 10³ cells/well. The CCK8 reagent was added to each well at different time points. After incubation for 4 h at 37°C, the absorbance at 450 nm was measured. For colony formation assay, cells were seeded in 6-well plates and cultured for 14 days. Then the cells were fixed and stained with crystal violet. The number of colonies was countered for five representative fields.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University. Mice (male and 6 weeks old) were subcutaneously injected with NSCLC cells (1.0*10⁶ cells/200 μl). The mice were terminated after 4 weeks of induction, and the tumor volume and tumor weight were measured.

IGF2BP2 antibody was used to pull down MALTA1. The IGF2BP2 antibody was then recovered with protein A/G beads, and RNA level of MALTA1 in the precipitates was measured by qRT-PCR. For m6A RIP, m6A antibody (MABE1006) (Millipore Sigma, Burlington, MA) was used to pull down m6A modified MALTA1, and RNA level of MALTA1 in the precipitates was measured by qRT-PCR.

Statistical analyses were performed using the GraphPad Prism program. The t-test was used to compare the mean of a continuous variable between two groups. OS and disease-free survival (DFS) curves were calculated using the Kaplan–Meier method and were analyzed with the log-rank test. p values < 0.05 were considered as significant.

We first checked the TCGA database to investigate the role of IGF2BP2 in NSCLC patients. As compared with normal tissues, the IGF2BP2 mRNA level was upregulated in primary NSCLC tissues (Figure 1A). Moreover, a higher IGF2BP2 mRNA level was positively correlated with poor overall survival (OS) and disease-free survival (DFS) (Figures 1B,C). The upregulation of IGF2BP2 mRNA was further validated in fresh NSCLC samples and the paired adjacent non-tumor sites (Figure 1D, n = 24, p < 0.01). Consistently, the upregulation of IGF2BP2 was presented in NSCLC cell lines compared with the BEAS cells (Figures 1E,F). These results highlight the clinical significance of IGF2BP2 in NSCLC.

To gain an insight into IGF2BP2 in NSCLC progression, we ectopically expressed IGF2BP2 in A549 cells (Figures 2A,B) and generated IGF2BP2 knockdown in H1975 cells (Figures 2C,D). We found that IGF2BP2 promoted cell proliferation, as determined using CCK8 and colony formation assays (Figures 2E,F). However, knockdown of IGF2BP2 showed an opposite effect on NSCLC cell proliferation (Figures 2G,H). Then we established the subcutaneous tumor bearing nude mice model to better understand the oncogenic role of IGF2BP2 in NSCLC. The results confirmed that IGF2BP2 promoted NCCLC proliferation, as indicated by larger tumor volume and heavier tumor weight (Figures 2I–K). Conversely, IGF2BP2 knockdown repressed tumor growth in nude mice (Figures 2L–N). All the results indicate the oncogenic role of IGF2BP2 in NSCLC.

It is widely recognized that lncRNA MALAT1 is a key regulator in NSCLC initiation, progression, and metastasis (Tang et al., 2018; Song J. et al., 2020; Li M. et al., 2021). Because IGF2BP2 usually functions as a key regulator of IncRNAs, we speculate whether lncRNA MALAT1 can be regulated by IGF2BP2. As expected, IGF2BP2 overexpression upregulated the MALAT1 level, whereas MALAT1 was significantly downregulated by IGF2BP2 knockdown (Figures 3A,B). We further investigate the mechanisms by which IGF2PB2 regulates MALAT1 expression. Since a central role of IGF2BP2 in carcinogenesis is to regulate RNA stability, we examined the stability of MALAT1 by using actinomycin D (2 μg/ml). The results showed that the MALAT1 decay was slowed down via upregulating of IGF2BP2 (Figure 3C). Conversely, knockdown of IGF2BP2 accelerates MALAT1 decay compared with control cells (Figure 3D). In addition, RIP-PCR assay further validated the interaction between IGF2BP2 and MALAT1 (Figure 3E). As for N6-methyladenosine (m6A), it is the most abundant internal modification on RNAs (Jin et al., 2019; Xue et al., 2021). We detected whether IGF2BP2 promotes MALAT1 stability via m6A modification. RIP assays with m6A antibody identified enrichment of MALAT1 via upregulating of IGF2BP2 (Figure 3F). All these data suggested that IGF2BP2 regulates MALTA1 stability in NSCLC.

It is known that ATG12 is a key downstream regulator of MALAT1, and ATG12 is required for NSCLC progression (He et al., 2020). Therefore, we tested whether IGF2BP2 can regulate ATG12 expression via MALAT1 in NSCLC. Western blot assay revealed that IGF2BP2 overexpression upregulated the ATG12 level, whereas ATG12 was significantly downregulated by IGF2BP2 knockdown (Figures 4A,B). We also confirmed that MALAT1 overexpression upregulated the ATG2 level, whereas ATG12 was significantly downregulated by MALAT1 knockdown (Figures 4C,D). To further validate whether IGF2BP2 can regulate NSCLC proliferation via ATG12, we knocked down ATG12 expression in IGF2BP2 overexpressing cells and ectopically expressed ATG2 in IGF2BP2 knockdown cells (Figures 4E,F). CCK8 assay indicated that knockdown ATG12 expression can repress cell proliferation in IGF2BP2 overexpressing cells, while ectopically expressed ATG12 promotes cell proliferation in IGF2BP2 knockdown cells (Figures 4G,I). Colony formation assay showed the same trend as the CCK8 experiment (Figures 4H,J). All the data suggested that IGF2BP2 promotes NSCLC proliferation via the lncRNA MALAT1/ATG12 axis.