IGF2BP3 Promotes Lung Cancer Progression Through FTO Dependent m6A Modication by Stabilizing N-myc

Background: N6-methyladenosine modication has been involved in various biological processes. However, its role in non-small cell lung cancer has not been well studied. Here, we show that IGF2BP3, as an transcription factor, plays a critical oncogenic role in non-small-cell lung cancer carcinogenesis through activating FTO expression and inducing aberrant m6A modication. Methods: To evaluate the role of IGF2BP3 in non-small-cell lung cancer, we performed cell proliferation and cell cycle assays in three lung cancer cell lines. Lung cancer mouse model is used to examine the effects of IGF2BP3/FTO/N-myc on lung proliferation potentials in vivo. We analyzed the correlation between IGF2BP3 and FTO, IGF2BP3 and N-myc protein in colon cancer patients by Pearson correlation. To nally explore the relationship of IGF2BP3/FTO/N-myc, we used western blots, proliferation and cell cycle assays to conrm that IGF2BP3 may regulate lung cancer progression through FTO dependent m6A modication by stabilizing N-myc. Results: We rst identied that IGF2BP3 overexpressed in non-small-cell lung cancer tissue and cells. Then, we showed that FTO was the dysregulated factor responsible for the abnormal N6-methyladenosine modication in non-small-cell lung cancer. The loss-of-function assay demonstrated that IGF2BP3 enhances FTO-mediated cell proliferation and promotes cell apoptosis, through regulating expression of target gene N-myc by reducing m6A level in mRNA transcript. Conclusion: Our study demonstrates the functional importance of IGF2BP3 and N6-methyladenosine methylation modication in the tumor progression of non-small-cell lung cancer, and provides profound insights into lung carcinogenesis and drug response. the Modication

m6A is modi ed by the m6A methyltransferases METTL3 and METTL14, erased by a-ketoglutaratedependent dioxygenase ALKB homolog 5 (ALKBH5) or fat-mass and obesity-associated protein (FTO), and read by YTH N6-Methyladenosine RNA Binding Proteins (YTHDFs) or IGF2BPs [14,15,16]. The dysfunction of the m6A process was observed in various types of tumorigenesis. In gastric cancer, METTL3-mediated m6A modi cation is critical for epithelial-mesenchymal transition and metastasis [10]. Suppression of m6A reader YTHDF2 promotes hematopoietic stem cell expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal [17]. Despite these recent discoveries between m6A modi cation with malignant cancer development and treatment, the status of m6A modi cation and the underlying regulatory mechanism in lung cancer remains little known.
N-myc, a member of the Myc family of basic-helix-loop-helix-zipper (bHLHZ) transcription factors, is an oncogene associated with a range of cellular processes, including growth,proliferation, apoptosisand differentiation [18]. N-Myc induce malignance by binding to speci c DNA sequences and modulating target gene transcription, leading to cell proliferation [19]. Accumulating evidence demonstrates that Nmyc exerts diverse biological functions in cancer development [20,21,22], however, the impact of N-myc in NSCLC development and progression has yet to be investigated.
Our previous study has indicated that TRIM11 acts as an oncogene in lung cancer through promoting cell growth, migration and invasion [23]. In the current study, we investigated the role of m6A modi cation and IGF2BP3 in lung cancer and addressed the underlying mechanisms by which m6A participates in the biology of lung cancer. Initially, by data mining of the TCGA database, we found that the expression of IGF2BP3 increased in lung cancer compared to corresponding normal tissues and higher IGF2BP3 predicted a poor prognosis in lung cancer patients. Then by loss and gain-of-function assays, we found that IGF2BP3 facilitated cell proliferation and accelerated cell cycle by regulating downstream gene FTO.
Finally, m6A-seq combined with RNA-seq analysis revealed that IGF2BP3 may regulate N-myc stability via modulating the 3'UTR and 5'UTR region of its transcript.

Bioinformatics analysis
The gene expression data were obtained from the TCGA dataset (LIHC) for lung adenocarcinoma, including 526 cases of tumor tissues and 59 cases of normal lung tissues.For RNA sequencing, each sample was cleaned up on aRNeasy Mini Column (Qiagen, Limburg, Netherlands), treated with DNase, and analyzed for quality on an Agilent 2100 Bioanalyzer. Samples were on an IlluminaHiSeq 4000 for 2 × 150-bp paired-end sequencing. The sequenced reads were aligned to the human genome GRCh38 by HISAT2 [44]. FeatureCounts [45]

Western blot
Proteins in tissues or cells were extracted by RIPA buffer (containing protease and phosphatase inhibitor; Solarbio, R0010) and quanti ed by BCA Quantitation Kit (Thermo, PICPI23223). About 25 μg of protein was isolated by 10% SDS-PAGE (Shanghai JRDUN Biotechnology) and then transferred to a polyvinylidene uoride (PVDF) membrane (Millipore, HATF00010) by electro-blotting. The cells were blocked for 1 h in 5% skim milk (BD Biosciences, BYL40422), and incubated for 2h at room temperature.
The cells were washed 5 times with PBST and incubated with secondary antibody (1:1000, Beyotime) at 37°C for 1 h in the dark. The chemiluminescence detection reagent (Millipore, WBKLS0100) was added and incubated for 5 min in the dark and then exposed on the ECL (Tanon, Tanon-5200). Relative protein levels were calculated using Image J software.

Cell proliferation
Cells in the logarithmic growth phase were trypsinized and counted under a microscope to prepare a cell suspension of 3 x 10 4 cells/ml. Control and transfected NSCLC cells were planted into 96-well culture plates and cultured at 37°C overnight. Cell Counting Kit-8 was added to the cells at 0, 24, 48, and 72 h.
After incubating for 2h for 37°C in a 5% CO 2 incubator, the absorbance at 450 nm wavelength was measured with a microplate reader.

Cell cycle
Cells in logarithmic growth phase were trypsinized and inserted into a six-well plate at a density of 300,000 cells/well. NSCLC cells were seeded into 6-well plates and transfected with siRNA or plasmid. 48 h after transfection, cells were harvested and xed with pre-chilled 70% ethanol at -20°C overnight. After centrifugation, the supernatant was discarded and the cells were washed twice with pre-chilled PBSthe next day. Cells were then stained with 500 μL PI/RNase staining buffer in the dark for 15 min. Finally, samples were analyzed using a uorescence activated cell sorting ow cytometer.

Immuno uorescence
The pre-treated lung cancer cells were seeded in eight-well chamber slides. Cells were incubated with the corresponding antibodies for 1 h at room temperature and incubated with uorescent secondary antibody IgG for half an hour. After DAPI staining and slides mounting, images were captured using a laserscanning confocal microscope.

Dual Luciferase Assay
Lung cancer cells were transfected with the combinations of N-myc 3'UTR, IGF2BP3 siRNA or control siRNA, and phRL (Renilla luciferase) TK plasmid with Lipofectamine 2000 (Invitrogen, USA). After 12-24 h, the luciferase activity was measured using the dual luciferase reporter gene assay system (Promega, USA). Luciferase activity was measured by a luminometer.

RNA Immunoprecipitation
RNA immunoprecipitation (RIP) analysis was performed using the Magna RIP kit (Millipore). Cells were lysed in RIP lysis buffer and the RNA-protein complexes were immuno-precipitated by using anti-IGF2BP3, anti-YTHDF2 antibodies, and normal rabbit IgG. The co-precipitated RNA was puri ed using phenol: chloroform: isoamyl alcohol and analyzed by real-time PCR. Prior to immunoprecipitation, the input RNA was subjected to control ampli cation.
RNA stability assay NSCLC cells were treated with Actinomycin D for 0h, 3h and 6h. After total RNA was extracted from treated cells, Real-time PCR was performed to obtain the relative levels of N-myc mRNA. In short, the degradation rate of mRNA (Kdecay) is calculated by the following formula: ln (C / C0) =-Kdecayt. t is the time of transcription suppression and C is the mRNA level at time t. C0 is the mRNA level at 0 hours in the equation, that is, the mRNA level before the decay begins. Therefore, the half-life (t1 / 2) of the mRNA can be calculated by the following equation:

In vivo xenograft model formation
Mouse experiments were performed in concordance with the NIH Guidelines. To investigate thefunction of IGF2BP3 on tumor growth in vivo, the male BALB/c nude mice at 5-week-old were used in our study.

PC9
(1 x 10 6 ) cells were injected subcutaneously into these mice to form a lung cancer xenograft model. Then the mice were randomly divided into four groups when the tumor diameter reached 4mm, and mice were injected with Control shRNA, IGF2BP3shRNA, FTO, and FTO with IGF2BP3shRNA for 20 days by multi-point intratumoral injection. Tumor volume (mm 3 ) is estimated by the following formula: tumor volume (mm 3 ) = shorter diameter 2 × longer diameter/2.

Statistical analysis
All the statistical analyses in our study were carried out using SPSS 20.0 software and GraphPad Prism 7. Results were presented as means ± SEM. Survival analysis was evaluated by the Kaplan-Meier survival curve and Log-rank tests. Statistical signi cance was assessed by unpaired two-tailed Student's test. p < 0.05 was considered to be statistically signi cant.

Results
Upregulated-IGF2BP3 is clinical relevant in NSCLC First, we discovered that the expression of IGF2BP3 was signi cantly increased in lung cancer samples in the TCGA database, compared with normal tissues ( Figure 1A). And the survival rate of the patients with high IGF2BP3 expression was signi cantly lower than that with low expression ( Figure 1B). Moreover, we detected IGF2BP3 expression in 35 paired lung cancer and adjacent tissues by using qPCR technology in cohort 1, and found that the expression of IGF2BP3 was signi cantly higher in lung carcinoma than the adjacent tissue ( Figure 1C). Besides, we detected IGF2BP3 protein level in cohort 2 by using IHC technology and the survival analysis revealed that patients with high IGF2BP3 expression had a poor prognosis ( Figure 1D). In addition, we found that high IGF2BP3 expression was positively corelated with lung cancer poor differentiation and lymph node metastasis (Table 1). Moreover, IGF2BP3 was signi cantly elevated in multiple lung cancer cell lines A549, H1975, H358, H1299, and PC9, which compared with bronchial epithelial HBE cells (Supplementary Figure 1A-1B). All those data indicated that IGF2BP3 may play a crucial role in lung cancer progression, and its high expression may be associated with poor prognosis of lung cancer. IGF2BP3 positively regulate FTO expression To evaluate the speci c mechanism of IGF2BP3 involved in lung cancer. We next detected the expression of demethylase (FTO, ALKBH5) and methyltransferase (METTL 3 and METTL 14) in lung cancer cells transfected with IGF2BP3 siRNA or control siRNA. It was found that FTO expression signi cantly decreased in IGF2BP3 downregulated cells ( Figure 2F). These data suggested that IGF2BP3-regulated cell proliferation depends on FTO in lung cancer. To address the role of IGF2BP3 in tumorigenic potential in vivo, we injected PC9 cells into nude mice to constructed a subcutaneous xenograft mouse model. The results showed that knockdown of IGF2BP3 dramatically reduced tumor weight ( Figure 4A-4B) and tumor volume ( Figure 4C), and increased the survival rate in nude mice ( Figure 4D). However, overexpression of FTO in IGF2BP3 siRNA transfected cells signi cantly rescued IGF2BP3 down-regulation induced decrease in tumor volume and the survival rate ( Figure 4A-4D). In support of the pro-tumor role of IGF2BP3, Ki67 staining revealed that the downregulation of IGF2BP3 decreased tumor cell proliferation in vivo, which could be relieved by FTO overexpression ( Figure 4E). Furthermore, we found that downregulation of IGF2BP3 decreased FTO expression in vivo both in mRNA and protein level ( Figure 4F). These data strongly indicated that IGF2BP3 modulate lung cancer tumor growth by regulating the expression of FTO. Transcriptome-wide m6A-seq and RNA-seq assays to identify potential targets of IGF2BP3 in NSCLC To identify the potential mRNA targets of IGF2BP3 whose m6A levels were increased upon IGF2BP3 downregulation. We conducted transcriptome-wide m6A-sequencing (m6A-seq) (Supplementary Table 2) and RNA-seq (Supplementary Table 3) assay in PC9 cell transfected with IGF2BP3 or control siRNA, separately. The results showed that the downregulation of IGF2BP3 notably increased the total m6A level ( Figure 5A). Consisted of the previous researches, the most common m6A motif 'GGAC' was signi cantly enriched in the m6A peaks ( Figure 5B). Furthermore, most of the FTO-binding sites were enriched in CDS region and 3'UTR ( Figure 5C). We next compared the genes with altered-m6a modi cations and mRNA expression between IGF2BP3 siRNA and control siRNA group. The analysis of m6A-seq and RNA-seq revealed a signi cantly increased m6A methylation and reduced mRNA level in the transcription level of N-myc after knockdown of IGF2BP3 in PC9 cells ( Figure 5D-5E). The real-time PCR further con rmed that the expression of N-myc was signi cantly downregulated in IGF2BP3 knockdown cells ( Figure 5F). In addition, the MeRIP-qPCR analyses showed the m6A levels of N-myc was dramatically improved in IGF2BP3 downregulated cells ( Figure 5G). To further address the effect of m6A modi cation on N-myc expression, we constructed N-myc luciferase reporter plasmid, which contained the 3'UTR or 5'UTR of Nmyc in the m6A sites. As expected, compared with control siRNA, IGF2BP3 siRNA transfection signi cantly increased the luciferase activity ( Figure 5H). To con rm the effect of m6A increase on the stability of N-myc, we conducted RNA stability assay and the results showed that knowndown of IGF2BP3 reduced the half-life of N-myc mRNA in PC9 cells ( Figure 5I). We further investigated the role FTO in the regulation of N-myc. The results demonstrated that, compared with control group, FTO downregulation notably decreased N-myc expression ( Figure 5J), the luciferase activity of 5'/3' UTR of Nmyc ( Figure 5K-5L) and reduced the half-life of N-myc mRNA ( Figure 5M), in accordance with our previous results. As reported, YTHDF2 is an m6A reader responsible for mRNA decay [24]. Downregulation of YTHDF2 signi cantly improved the mRNA level of N-myc ( Figure 5N, Supplementary Figure 2G). RIP assay indicated that YTHDF2 might directly bind to the 3'UTR and 5'UTR and of N-myc ( Figure 5O-5P).
Furthermore, the half-life of N-myc mRNA was notably decreased in NSCLC cells transfected with YTHDF2 siRNA ( Figure 5Q). Altogether, our data demonstrated that IGF2BP3/FTO-mediated m6A modi cation increased N-myc expression through YTHDF2-dependent mRNA stability. N-myc is functionally important target gene of IGF2BP3 in NSCLC N-myc is a pivotal oncogene in carcinogenesis [25,26]. Previous studies have demonstrated that N-myc is remarkably increased in various malignant tumors, as well as in lung cancer [27,28]. Here we nd that N-myc is signi cantly upregulated in lung cancer tissue, and the expression of N-myc was positively correlated with IGF2BP3 in our cohort ( Figure  6A-6B). N-myc expression was signi cantly decreased in PC9 and H1975 cells transfected with IGF2BP3 siRNA compared with the control group both in mRNA and protein level ( Figure 6C-6D). Ectopic expression of IGF2BP3 improved N-myc mRNA and protein levels ( Figure 6E). To further investigate the function of N-myc in IGF2BP3 mediated carcinogenesis in lung cancer. We co-transfected N-myc plasmid with IGF2BP3 siRNA in PC9 and H1975 cells (Supplementary Figure 2H, 2I). The results showed that Nmyc overexpression promoted cell proliferation and rescued IGF2BP3 downregulation induced decrease in cell proliferation and cell cycle arrest in PC9 and H1975 cells ( Figure 7A-7B, 7D-7E, 7G-7H).
Downregulation of N-myc declined lung cancer cell proliferation and blocked IGF2BP3-induced increase in lung cancer cell proliferation and cell cycle acceleration ( Figure 7C, 7F, 7L, Supplementary Figure 2J). Taken together, these data indicated that N-myc involved in IGF2BP3 mediated pulmonic carcinogenesis.

Discussion
Upregulated-IGF2BP3 is clinical relevant in NSCLC First, we discovered that the expression of IGF2BP3 was signi cantly increased in lung cancer samples in the TCGA database, compared with normal tissues ( Figure 1A). And the survival rate of the patients with high IGF2BP3 expression was signi cantly lower than that with low expression ( Figure 1B). Moreover, we detected IGF2BP3 expression in 35 paired lung cancer and adjacent tissues by using qPCR technology in cohort 1, and found that the expression of IGF2BP3 was signi cantly higher in lung carcinoma than the adjacent tissue ( Figure 1C). Besides, we detected IGF2BP3 protein level in cohort 2 by using IHC technology and the survival analysis revealed that patients with high IGF2BP3 expression had a poor prognosis ( Figure 1D). In addition, we found that high IGF2BP3 expression was positively corelated with lung cancer poor differentiation and lymph node metastasis (Table 1). Moreover, IGF2BP3 was signi cantly elevated in multiple lung cancer cell lines A549, H1975, H358, H1299, and PC9, which compared with bronchial epithelial HBE cells (Supplementary Figure 1A-1B). All those data indicated that IGF2BP3 may play a crucial role in lung cancer progression, and its high expression may be associated with poor prognosis of lung cancer.

IGF2BP3 positively regulate FTO expression
To evaluate the speci c mechanism of IGF2BP3 involved in lung cancer. We next detected the expression of demethylase (FTO, ALKBH5) and methyltransferase (METTL 3 and METTL 14) in lung cancer cells transfected with IGF2BP3 siRNA or control siRNA. It was found that FTO expression signi cantly decreased in IGF2BP3 downregulated cells (Figure 2A, Supplementary Figure 2A-2B). We further detect the expression of FTO in lung cancer tissues. The results showed that FTO signi cantly increased in lung cancer, and was positively correlated with the expression of IGF2BP3 ( Figure 2B-2C). Down-regulation of IGF2BP3 expression in lung cancer cell lines PC9 and H1975 resulted in decreased expression of FTO in mRNA and protein level ( Figure 2D-2E). Meanwhile, overexpression of IGF2BP3 notably increased FTO expression ( Figure 2F, Supplementary Figure 2C). It demonstrated that FTO may be downstream of IGF2BP3.

IGF2BP3-induced cell survival depends on FTO in NSCLC cell
We next hypothesized that FTO mediated the biological function of IGF2BP3 in lung carcinoma, we transfected IGF2BP3 siRNA alone and in combination with FTO overexpression plasmids into PC9 and H1975 cell lines (Supplementary Figure 2D,2E). It was found that down-regulation of IGF2BP3 inhibited cell proliferation, arrested cell cycle and decreased the expression of FTO ( Figure 3A-3B, 3D-3E, 3G-3H). FTO overexpression signi cantly rescued IGF2BP3 down-regulation induced decrease in cell proliferation and cell cycle arrest ( Figure 3A-3B, 3D-3E, 3G-3H). To further con rm the results, we transfected IGF2BP3 overexpression plasmid in H358 cell line, it was found that upregulation of IGF2BP3 promoted cell proliferation, cell cycle and increased the expression of FTO ( Figure 3C, 3F, 3I). While knockdown of FTO signi cantly blocked IGF2BP3-induced increase in lung cancer cell proliferation and cell cycle acceleration ( Figure 3C, 3F, 3I, Supplementary Figure 2F). These data suggested that IGF2BP3-regulated cell proliferation depends on FTO in lung cancer.
To address the role of IGF2BP3 in tumorigenic potential in vivo, we injected PC9 cells into nude mice to constructed a subcutaneous xenograft mouse model. The results showed that knockdown of IGF2BP3 dramatically reduced tumor weight ( Figure 4A-4B) and tumor volume ( Figure 4C), and increased the survival rate in nude mice ( Figure 4D). However, overexpression of FTO in IGF2BP3 siRNA transfected cells signi cantly rescued IGF2BP3 down-regulation induced decrease in tumor volume and the survival rate ( Figure 4A-4D). In support of the pro-tumor role of IGF2BP3, Ki67 staining revealed that the downregulation of IGF2BP3 decreased tumor cell proliferation in vivo, which could be relieved by FTO overexpression ( Figure 4E). Furthermore, we found that downregulation of IGF2BP3 decreased FTO expression in vivo both in mRNA and protein level ( Figure 4F). These data strongly indicated that IGF2BP3 modulate lung cancer tumor growth by regulating the expression of FTO.
Transcriptome-wide m6A-seq and RNA-seq assays to identify potential targets of IGF2BP3 in NSCLC To identify the potential mRNA targets of IGF2BP3 whose m6A levels were increased upon IGF2BP3 downregulation. We conducted transcriptome-wide m6A-sequencing (m6A-seq) (Supplementary Table 2) and RNA-seq (Supplementary Table 3) assay in PC9 cell transfected with IGF2BP3 or control siRNA, separately. The results showed that the downregulation of IGF2BP3 notably increased the total m6A level ( Figure 5A). Consisted of the previous researches, the most common m6A motif 'GGAC' was signi cantly enriched in the m6A peaks ( Figure 5B). Furthermore, most of the FTO-binding sites were enriched in CDS region and 3'UTR ( Figure 5C). We next compared the genes with altered-m6a modi cations and mRNA expression between IGF2BP3 siRNA and control siRNA group. The analysis of m6A-seq and RNA-seq revealed a signi cantly increased m6A methylation and reduced mRNA level in the transcription level of N-myc after knockdown of IGF2BP3 in PC9 cells ( Figure 5D-5E). The real-time PCR further con rmed that the expression of N-myc was signi cantly downregulated in IGF2BP3 knockdown cells ( Figure 5F). In addition, the MeRIP-qPCR analyses showed the m6A levels of N-myc was dramatically improved in IGF2BP3 downregulated cells ( Figure 5G). To further address the effect of m6A modi cation on N-myc expression, we constructed N-myc luciferase reporter plasmid, which contained the 3'UTR or 5'UTR of Nmyc in the m6A sites. As expected, compared with control siRNA, IGF2BP3 siRNA transfection signi cantly increased the luciferase activity ( Figure 5H). To con rm the effect of m6A increase on the stability of N-myc, we conducted RNA stability assay and the results showed that knowndown of IGF2BP3 reduced the half-life of N-myc mRNA in PC9 cells ( Figure 5I). We further investigated the role FTO in the regulation of N-myc. The results demonstrated that, compared with control group, FTO downregulation notably decreased N-myc expression ( Figure 5J), the luciferase activity of 5'/3' UTR of Nmyc ( Figure 5K-5L) and reduced the half-life of N-myc mRNA ( Figure 5M), in accordance with our previous results. As reported, YTHDF2 is an m6A reader responsible for mRNA decay [24]. Downregulation of YTHDF2 signi cantly improved the mRNA level of N-myc ( Figure 5N, Supplementary Figure 2G). RIP assay indicated that YTHDF2 might directly bind to the 3'UTR and 5'UTR and of N-myc ( Figure 5O-5P). Furthermore, the half-life of N-myc mRNA was notably decreased in NSCLC cells transfected with YTHDF2 siRNA ( Figure 5Q). Altogether, our data demonstrated that IGF2BP3/FTO-mediated m6A modi cation increased N-myc expression through YTHDF2-dependent mRNA stability.
N-myc is functionally important target gene of IGF2BP3 in NSCLC N-myc is a pivotal oncogene in carcinogenesis [25,26]. Previous studies have demonstrated that N-myc is remarkably increased in various malignant tumors, as well as in lung cancer [27,28]. Here we nd that Nmyc is signi cantly upregulated in lung cancer tissue, and the expression of N-myc was positively correlated with IGF2BP3 in our cohort ( Figure 6A-6B). N-myc expression was signi cantly decreased in PC9 and H1975 cells transfected with IGF2BP3 siRNA compared with the control group both in mRNA and protein level ( Figure 6C-6D). Ectopic expression of IGF2BP3 improved N-myc mRNA and protein levels ( Figure 6E). To further investigate the function of N-myc in IGF2BP3 mediated carcinogenesis in lung cancer. We co-transfected N-myc plasmid with IGF2BP3 siRNA in PC9 and H1975 cells (Supplementary Figure 2H, 2I). The results showed that N-myc overexpression promoted cell proliferation and rescued IGF2BP3 downregulation induced decrease in cell proliferation and cell cycle arrest in PC9 and H1975 cells ( Figure 7A-7B, 7D-7E, 7G-7H). Downregulation of N-myc declined lung cancer cell proliferation and blocked IGF2BP3-induced increase in lung cancer cell proliferation and cell cycle acceleration ( Figure 7C, 7F, 7L, Supplementary Figure 2J). Taken together, these data indicated that Nmyc involved in IGF2BP3 mediated pulmonic carcinogenesis.

Conclusion
Taken together, here we provide compelling in vitro and in vivo evidence demonstrating that the IGF2PB3 plays a critical oncogenic role in cell proliferation and pulmonary carcinogenesis mediated by FTO, through regulating m6A level in mRNA transcripts of its target gene N-myc. Our study highligts the functional importance of the m6A modi cation maghinery in lung cancer. Given the importance of Differences between groups were done by the Chi-square test.      H358 cells with different treatments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Vector+shNC or shNC+Vector; #p < 0.05, ###p < 0.001 vs. Vector+shIGF2BP3 or shNC+oeIGF2BP3.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.