The m6A demethylase FTO promotes esophageal cancer progression via the YTHDF1/AKT3 signaling network

doi:10.21203/rs.3.rs-1731913/v1

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The m6A demethylase FTO promotes esophageal cancer progression via the YTHDF1/AKT3 signaling network

https://doi.org/10.21203/rs.3.rs-1731913/v1

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Background

As the most abundant modification in eukaryotic messenger RNAs (mRNAs), the N⁶-methyladenosine (m⁶A) plays vital roles in many biological processes.

Results

Here we applied the methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) to screen the m⁶A targets in esophageal cancer patients. We found that the m⁶A demethylase FTO was significantly up regulated in esophageal cancer cell lines and patient tissues. In vivo and in vitro assays demonstrated that FTO was involved in the proliferation, migration, invasion, and apoptosis of esophageal cancer cells. Moreover, we found that the m⁶A methyltransferase METTL14 negatively regulates FTO function on esophageal cancer progression. By using transcriptome-wide m⁶A-seq and RNA-seq assays, we identified that AKT3 is a downstream target of FTO, which acts in concert to regulate the tumorigenesis and metastasis of esophageal cancer.

Conclusions

All these findings revealed insights into the m⁶A-regulated tumorigenesis in esophageal cancer, which is applicable for designing new therapeutic strategy.

esophageal cancer

N6-methyladenosine (m6A)

FTO

AKT3

YTHDF1

The mRNA m⁶A modification was identified in the 1970s [1, 2] and is the most abundant in eukaryotic mRNAs with unique distribution patterns [3]. In mammalian cells, the m⁶A modification is catalyzed by a methyltransferase complex (“writers”) consisting of the proteins methyltransferase-like 3 (METTL3), METTL14, Wilms tumor 1 associated protein (WTAP), VIRMA (KIAA1429), and RBM15 [4, 5]. Notably, the first RNA demethylase, fat mass and obesity-associated protein (FTO), was identified to function as an m⁶A “eraser” to remove m⁶A modification from RNA, revealing that RNA modification is reversible [6]. Afterwards, the alkylation repair homolog protein 5 (ALKBH5) was proven to be another “eraser” of m⁶A modification [7], indicating a dynamic nature of m⁶A methylation. Since then, numerous studies have focused on the dynamics of m⁶A modification [8–12]. Moreover, m⁶A-binding proteins with a YTH domain, including the cytoplasmic proteins YTHDF1, YTHDF2, and YTHDF3 and the nuclear protein YTHDC1, have been identified as the “readers” of m⁶A and modulate mRNA stability and translation [9, 10, 13, 14].

Recently, accumulated studies have focused on the biological functions of m⁶A modification in mRNA [15]. It has been reported that m⁶A modification is involved in various biological processes, including the heat-shock response [13], DNA damage response [16, 17], mRNA clearance [8], neuronal functions [18], cortical neurogenesis [19], progenitor cell specification [20], and T cell homeostasis [21]. Moreover, m⁶A modification has been found to be associated with the tumorigenesis and progression of various cancers [22].

The m⁶A demethylase FTO was found to play critical roles in regulating fat mass, adipogenesis, and body weight [23–25]. In addition, large-scale epidemiological studies have demonstrated the association of the FTO SNP risk genotype with the development of cancers such as breast, kidney, prostate, and pancreatic cancers, as well as leukemia, lymphoma and myeloma [26–28]. Some studies have shown that FTO plays a carcinogenic role in esophageal cancer and other solid tumors [29–31]. A previous study showed that FTO plays an oncogenic role in cell transformation and leukemogenesis [32]. However, the definitive role of FTO in cancer remains limited.

In this study, we systematically investigated the role of m⁶A modification in the tumorigenesis of esophageal cancer, which remains one of the most common forms of cancer worldwide [33, 34]. We found a significantly increased level of FTO in esophageal cancer cells. The following functional studies revealed an oncogenic role of FTO in esophageal cancer tumorigenesis. Moreover, we found that the m⁶A methyltransferase METTL14 is also involved in FTO-regulated m⁶A modification, which acts in concert with FTO to regulate AKT3 methylation in the tumorigenesis and metastasis of esophageal cancer.

FTO expression is upregulated in esophageal cancer

We first investigated the m⁶A levels in the mRNAs of esophageal cancer cells. By using MeRIP-PCR, we identified that the m⁶A levels of mRNAs isolated from five esophageal cancer cell lines were statistically (p < 0.05, t test) less abundant than those of one normal control cell line (Fig. 1A). The m⁶A/A level of mRNA is decreased to ~ 10% in esophageal cancer cells compared with ~ 40% in normal cells (Fig. 1A). These results indicate that m⁶A in mRNAs should be demethylated in esophageal cancer cells. We thus detected the expression of the N⁶-methyladenosine RNA demethylase FTO in the tissues of different esophageal cancer patients. Compared to normal tissues, tissues from esophageal cancer patients had an upregulated level of FTO expression (Fig. 1B). Moreover, esophageal cancer patients with higher FTO expression had shorter overall survival (Fig. 1C), which suggests that FTO expression might serve as a prognostic marker for esophageal cancer patients. The subsequent western blotting analysis also showed significantly higher expression of FTO in esophageal cancer cells, especially in KYSE150 cells, than in normal esophageal cells (Fig. 1D). In addition, the esophageal cancer patients had a higher expression of FTO proteins, as analyzed by western blotting (Fig. 1E). The expression of FTO was also significantly higher in esophageal cancer patient tissues than in paracancerous tissues (PT) (Fig. 1F). Collectively, these results clearly demonstrated that FTO is highly expressed in esophageal cancer patients, which correlates with a lower survival rate of esophageal cancer patients.

FTO is involved in the proliferation, migration, invasion, and apoptosis of esophageal cancer cells

To investigate the potential roles of FTO in EC cells, we performed a series of functional assays to characterize the effect of FTO in EC cells. We first down regulated the expression of FTO by transfection of FTO sh-RNAs in KYSE150 cells (Fig. 2A). The following CCK-8 assays showed that down regulation of FTO significantly inhibited the proliferation of KYSE150 cells (Fig. 2B). Moreover, FTO knockdown in KYSE150 cells decreased the cell migration and invasion capability compared to that of the control cells (Fig. 2C). Previous studies have shown that E-cadherin is a biomarker for cell migration [35, 36]; thus, we detected the expression of E-cadherin in FTO knockdown cells. The results showed that down regulation of FTO correlates with the higher expression of E-cadherin (E-cad) in KYSE150 cells (Fig. 2D), indicating that FTO might be associated with E-cadherin-regulated cell migration in esophageal cancer cells.

Afterwards, we overexpressed FTO in HEECs by transfection of FTO-overexpressing lentivirus (Fig. 2E). The cell proliferation rate was significantly increased upon overexpression of FTO in HEECs (Fig. 2F). Moreover, the cell migration and cell invasion capabilities were also increased in FTO-overexpressing HEEC cells (Fig. 2G). In addition, the colony formation assays showed that compared to the control cells, FTO overexpression significantly promoted cell proliferation in KYSE150 cells, whereas FTO knockdown largely impeded cell proliferation (Fig. 2H). Moreover, compared to the control cells, FTO overexpression in HEEC cells drastically decreased the apoptotic cells from 25–10%, as analyzed by flow cytometry (Fig. 2I), indicating a decreased cell apoptosis rate with FTO overexpression. All these results indicated that the oncogenic role of FTO is involved in cell proliferation, migration, invasion, and cell apoptosis in esophageal cancer cells.

FTO negatively correlates with METTL14 in esophageal cancer cells

Our results showed that a higher FTO level correlates with a poor prognosis in ES patients (Fig. 1C). To further characterize whether m⁶A methylation is indeed associated with ES prognosis, we introduced the ratio of FTO/METTL14 to represent the methylation rate in the cells. Given that METTL14 is a well-characterized m⁶A methyltransferase [37, 38], a higher FTO/METTL14 ratio represents a lower level of m⁶A methylation in cells. Analysis of ES patients with higher FTO/METTL14 ratios showed a rather poor prognosis rate compared to that of patients with lower FTO/METTL14 ratios (Fig. 3A). Further analysis of the differentially expressed genes by RNA-seq showed that FTO overexpression and METTL14 overexpression resulted in a total of 157 shared differentially expressed genes (Fig. 3B). These results indicated that FTO might be functionally associated with METTL14. To further examine the correlation between FTO and METTL14, we compared the proliferation ability of KYSE150 cells with the reversal changes of FTO or METTL14. As expected, down regulation of FTO or up regulation of METTL14 reduced the proliferation ability, which could be restored via down regulation of METTL14 (Fig. 3C). Moreover, the decreased migration or invasion of KYSE150 cells via FTO knockdown could also be restored by METTL14 overexpression (Fig. 3D). In contrast, METTL14 knockdown has the opposite effect of an elevated capability of migration or invasion in KYSE150 cells. All these results suggest that FTO and METTL14 negatively correlate with each other in functioning as biomarkers in esophageal cancer patients.

Akt3 is regulated by FTO-mediated m⁶A modification in esophageal cancer cells

To investigate the potential role of FTO in tumor progression, we detected the m⁶A contents of the total mRNA with the EpiQuik™ m⁶A RNA Methylation Quantification Kit (Colorimetric) in FTO-overexpressing HEEC and FTO-silenced KYSE150 cells. As expected, FTO overexpression significantly decreased the m⁶A content in HEECs, whereas FTO silencing dramatically increased the m⁶A content in KYSE150 cells (Figs. 4A-4B). As analyzed by the RMBase database, the genes with m⁶A modification have a consensus motif of U/AGGAC (Fig. 4C), which is the common feature among the genes with m⁶A methylation [39, 40].

We take the intersection of the down-regulated peak after overexpression of FTO and the up-regulated peak after interference with FTO, and then enrich the KEGG function of the intersected genes. The results show that cell cycle and other pathways are significantly enriched in the genes with different peaks. Compared to the control cells, 128 genes showing a 1.5-fold m⁶A change in the expression level were identified in esophageal cancer cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that a handful of genes were associated with the different metabolic pathways in various cancer cells. The overall level analysis of the m⁶A modification after FTO knockout indicated that FTO was related to the methylation of m⁶A (Fig. 4D). Among these genes, we selected the top seven most differentially expressed members and detected their expression in KYSE150 cells by real-time PCR. The results showed that METTL1, CAMKK1, PKN2, and TGFBR1 were upregulated in FTO-silenced KYSE150 cells, whereas ERBB2, AKT3 and PAK1P1 were downregulated (Fig. 4E). Among the three downregulated genes, Akt3, which is a serine/throne kinase from the Akt family [41, 42], is involved in the biogenesis of many different types of cancers [43]. The correlation prediction of FTO and AKT3 by the GEPIA database gave an R-value of 0.55, which strongly indicated the physical interaction between Fto and Akt3 genes, the correlation of genes suggests that the two proteins may have some physical interaction (Fig. 4F). We thus detected the m⁶A abundance on Akt3 mRNA transcripts in KYSE30 and KYSE150 cells by m⁶A-seq, and the results showed that m⁶A methylation was enriched in the exon and 3’UTR regions of Akt3 with a clustered distribution (Fig. 4G).

To further investigate the role of Akt3 in FTO-regulated m⁶A methylation, we detected the m⁶A content in KYSE150 cells by MeRIP-PCR. The results showed that FTO knockdown retained the m⁶A methylation in Akt3, as shown by an elevated m⁶A content in KYSE150 cells (Figs. 4H and 4I). To examine the role of m⁶A methylation on the Akt3 3’UTR, containing firefly' luciferase reporters were generated, followed by the wild-type Akt3 3’UTR, mutant1 or mutant2 3’UTR. The 3’UTR-reporter luciferase assay showed that compared to the control cells, the mutant1 3’UTR slightly but not significantly reduced Akt3 expression, whereas the mutant2 3’UTR had a minor effect on Akt3 expression (Fig. 4J). Dot plot analysis of the overall level of m⁶A modification in esophageal cancer cells before and after knockdown of the Akt3 m⁶A modification site. The results indicated that m⁶A methylation of the 3’UTR might not be involved in the m⁶A modification-regulated Akt3 expression.

AKT3 is involved in m⁶A-regulated esophageal cancer tumorigenesis and metastasis

We then characterized the roles of Akt3 in esophageal cancer cell functions by several in vitro experiments. Notably, overexpression of FTO in HEEC cells largely increased the expression of both Akt3 mRNA and protein (Fig. 5A), whereas FTO knockdown in KYSE150 cells decreased the expression of Akt3 mRNA and protein (Fig. 5B). To further investigate whether FTO affects the stability of Akt3 mRNA, we tested AKT3 mRNA levels in KYSE150 cells with FTO knockdown after treatment with actinomycete D, which is ametabolic inhibitor [35, 44]. The results showed that the mRNA level dramatically decreased overtime with FTO knockdown (Fig. 5C), indicating that FTO might increase the stability of Akt3 mRNA, which results in a higher level of AKT3 protein expression.

Next, we downregulated AKT3 expression in KYSE150 cells and tested its effect on KYSE150 cellular functions. Transfection of either of the three AKT3 siRNAs in KYSE150 cells significantly decreased the expression of AKT3 at both the mRNA and protein levels (Fig. 5D). In addition, overexpression of AKT3 also increased the expression of Vimentin (Fig. 5E), indicating that AKT3 promotes tumor cell migration. Accompanied by the decrease in AKT3 with si-AKT3 transfection in KYSE150 cells, the cell proliferation ratio also decreased over time (Fig. 5F). Moreover, the cell invasion and migration capabilities were also decreased with AKT3 knockdown in KYSE150 cells (Fig. 5G). All these results indicated that AKT3 is involved in the tumorigenesis of esophageal cancer progression.

FTO and AKT3 act in concert to regulate esophageal cancer cell tumorigenesis and metastasis

To further investigate the correlation between FTO and AKT3 in esophageal cancer tumorigenesis, we overexpressed AKT3 in KYSE150 cells accompanied by FTO knockdown. As shown in Fig. 6A, compared to the transfection of AKT3-overexpressing vector (AKT3-OE), which increased AKT3 mRNA expression ~ 43.5-fold, AKT3 expression in FTO-silenced KYSE150 cells transfected with AKT3-overexpressing vector (sh-FTO + AKT3-OE) increased ~ 15.3-fold, which largely compromised the upregulation of AKT3 mRNA. The results also coincide with the notion that FTO-regulated m⁶A demethylation promotes AKT3 mRNA stability (Fig. 5C). As a result, the AKT3 protein levels were also upregulated in the AKT3-OE and sh-FTO + AKT3-OE KYSE150 cells (Fig. 6B) but were slightly lower in the sh-FTO + AKT3-OE cells. To test whether AKT3 could restore the effect of FTO knockdown, we tested the proliferation ratio of KYSE150 cells using CCK-8 assays. The results showed that FTO knockdown decreased the proliferation ratio, which could be restored by AKT3 overexpression in sh-FTO + AKT3-OE cells (Fig. 6C). Similarly, wound healing, migration, invasion and colony formation assays showed that the effects of FTO knockdown in KYSE150 cells could also be restored by AKT3 overexpression (Figs. 6D-6G). These results also indicated that FTO-regulated m⁶A demethylation of AKT3 is associated with the tumorigenesis and metastasis of esophageal cancer cells.

YTHDF1 maintains AKT3 mRNA stability in an m⁶A-dependent manner

Previous studies have identified two major families of m⁶A “readers” that might play a specific role in controlling the fate of methylated mRNAs, such as the YTH family and the IGF2BP family [5, 45, 46]. To identify the specific m⁶A readers of AKT3 and determine the m⁶A-dependent mechanism of AKT3 regulation, we performed FLAG RNA pull-down assays in KYSE150 cells to screen AKT3-related m⁶A readers. Notably, YTHDC1 and YTHDF1, but not other members in the YTH family, specifically bind to the AKT3 full-length transcripts in KYSE150 cells (Fig. 7A). Biotin-based pull-down assays also confirmed the direct interactions of AKT3 mRNA with both YTHDC1 and YTHDF1 (Figs. 7B-7C), indicating a potential positive regulatory mechanism. Further detection of the expression levels in the tumor tissues compared to the control groups revealed that only YTHDF1 had an upregulated expression level in esophageal cancer cells (Fig. 7D). In contrast, YTHDC1 showed no significant changes in the expression level compared to the control groups (Fig. 7E).

To further test the role of YTHDF1 in AKT3 stability, we inhibited or increased the expression of YTHDF1 in KYSE150 cells. As a result, AKT3 mRNA and protein expression were decreased upon the overexpression of YTHDF1 in KYSE150 cells (Fig. 7F). Moreover, AKT3 mRNA and protein expression were upregulated after siRNA inhibition of YTHDF1 in KYSE150 cells (Fig. 7G). Taken together, our results suggested that the methylated AKT3 transcripts might be directly recognized by YTHDF1, which maintained the stability of the AKT3 transcripts.

FTO and AKT3 promote esophageal cancer progression in vivo

To test the potential role of FTO and AKT3 in esophageal cancer biogenesis in vivo, we injected sh-FTO or AKT3-OE KYSE150 cells subcutaneously into nude mice. Then, the mice were killed when the tumor volumes were approximately 1000 mm³ for each group. Compared to the control groups, transfection of sh-FTO KYSE150 cells significantly decreased the tumor weight, whereas transfection of AKT3-OE KYSE150 cells increased the tumor weight (Fig. 8A). To further determine the impacts of m⁶A methylation on in vivo metastasis, sh-FTO, AKT3-OE or sh-FTO & AKT3-OE KYSE150 cells were injected into nude mice by tail vein injection to analyze lung colonization. As shown in Figure. 8B, the number of lung tumors derived from FTO knockdown KYSE150 cells showed no significant changes; however, AKT3-OE or sh-FTO&AKT3-OE significantly promoted the number of lung tumors compared with control cells, suggesting that AKT3 overexpression promoted tumor metastasis in vivo. All these results suggested that FTO and AKT3 are involved in esophageal cancer progression in vivo.

Increasing evidence indicates that mRNA m⁶A modification participates in a number of biological functions and in the progression of cancer cells [32, 35, 47]. In this study, we demonstrated that mRNA m⁶A modification can regulate the progression of esophageal cancer. In brief, the expression level of FTO in esophageal cancer cells was negatively correlated with the m⁶A levels in mRNA. Changes in FTO and METTL14 levels largely affect the in vitro proliferation, migration, and invasion of cancer cells. Further investigations identified AKT3 as one of the targets of FTO. In particular, AKT3 is a phosphatidylinositol-3-kinase, and the protein kinase B family is a key element of the PI3K/AKT signaling pathway. The AKT pathway was found to regulate many hallmarks of cancer and the metastatic cascade in breast cancer [48–50]. In addition, much effort has been made to develop targeted therapies for AKT signaling in breast cancer [51–53]. Thus, the PI3K/AKT pathway is a promising target for cancer therapy owing to the high frequency of its dysregulation in human breast cancer [54]. Here, we showed that AKT3 is involved in the progression of esophageal cancer, which provides the basis for further targeting the AKT3 pathway for clinical therapy of esophageal cancer.

Knowledge of the roles of mRNA modification in controlling cancer progression remains limited. As the first characterized m⁶A demethylase, FTO has been reported to regulate the tumorigenesis of different types of cancers. FTO was found to enhance leukemic oncogene-mediated cell transformation and leukemogenesis by reducing the m⁶A levels of its targets [32]. In addition, pharmaceutical inhibition of FTO by a chemical inhibitor Inhibit tumor progression and significantly prolong the life of glioblastoma stem cell transplanted mice [47]. On the other hand, METTL14, which is the methyltransferase of mRNA m⁶A, exhibits several functions in cancer cells, such as regulating leukemogenesis and proliferation of hematopoietic stem/progenitor cells (HSPCs) [38]. Targeting METTL14, especially combined with differentiation inducers, may be an effective new therapeutic strategy for the treatment of AML. In addition, METTL14 and METTL3 form a stable heterodimer core complex and play a role in cell m⁶A deposition, which can inhibit the metastatic potential by regulating primary microRNA 126 treatment in an m⁶A dependent manner [55]. In this study, we provided a link between the regulation of AKT3 mRNA m⁶A methylation by FTO and METTL14. We found that downregulation of FTO or overexpression of METTL14 have a similar effect on multiple aspects of esophageal cancer progression, including migration, invasion, proliferation, and tumorigenesis, which also suggests that FTO function could be restored by METTL14 in esophageal cancer. Our results describe the roles of m⁶A and FTO in cancer progression and provide a basis for the development of therapeutic strategies against esophageal cancer metastasis. Notably, the METTL14/FTO/AKT3 signaling network might have other normal functions in addition to influencing tumorigenesis in esophageal cancer. More investigations are needed to clarify the fine regulatory circuit of these players in cellular functions.

The m⁶A modification modulates all stages in the life cycle of RNA, such as RNA processing, nuclear export, and translation modulation [14, 56, 57]. For example, m⁶A modification can promote the de alkenylation of RNA through the first characterized m6A "reader" protein YTHDF2, thereby triggering mRNA degradation [9]. Here, we showed that the stability of AKT3 mRNA transcripts is enhanced by the reader protein YTHDF1. Previous studies have shown that YTHDF1 is linked to the progression of various cancers, including non-small-cell lung cancer [58], colorectal carcinoma [59], and ovarian cancer [60]. In our study, we found that knockdown of YTHDF1 decreased AKT3 at both the mRNA and protein levels (Fig. 7F), whereas YTHDF1 overexpression significantly enhanced AKT3 mRNA and protein levels. These data support that AKT3 is the direct target of YTHDF1 in esophageal cancer. The results indicated that YTHDF1 might regulate the transcription and translation of AKT3. However, the detailed mechanism by which YTHDF1 regulates AKT3 expression needs further investigation.

In conclusion, we provide a large amount of in vitro and in vivo evidence that m⁶A modification can regulate the progression of esophageal cancer by promoting the growth, survival and invasion of cancer cells. Importantly, we uncovered that FTO operates a regulatory network of m⁶A modification that involves METTL14, YTHDF1 and AKT3 signaling, providing the first insight into the mechanism of FTO-mediated esophageal cancer progression.

Samples, cell lines, and plasmids

The 28 human esophageal cancer samples were obtained from abdominal surgery at the First Affiliated Hospital of University of Science and Technology of China, and patients provided informed consent. The pathological condition was determined by an experienced surgical specialist. The comprehensive clinical and pathological information of esophageal cancer patients is shown in SI Appendix, Table S1. The esophageal cancer cell lines KYSE140, KYSE180, KYSE450, KYSE30,and KYSE150 and the human normal esophageal epithelial cell line HEEC were obtained from the Chinese Academy of Cell Resource Center (Shanghai, China) [61].

Lentiviruses for overexpressing and silencing the expression of FTO, METTL14 and AKT3 were constructed from Hanbio Biotechnology Co., Ltd. Plasmids for the expression of Flag-tagged wild-type (YTHDF1-WT, YTHDF2-WT, YTHDF3-WT, YTHDC1-WT, YTHDC2-WT) were constructed withthep3xFLAG-Myc-CMV vector. Detailed information regarding the primers used for plasmid construction is depicted in SI Appendix, Table S2. For shRNA plasmids used in lentivirus-mediated interference, complementary sense and antisense oligo nucleotides encoding shRNAs targeting YTHDF1 were synthesized, annealed and cloned into the pHBLV-U6-MCS-EF1-mcherry-T2A-PURO vector. The related sequences of shRNAs are shown in Table S2 of the SI Appendix.

Gene expression and survival analysis in esophageal cancer datasets

Kaplan–Meier plotter (http://kmplot.com/analysis/) was used to assess the prognostic value of FTO and METTL14 expression in patients with esophageal cancer. The mRNA expression of FTO, YTHDC1, and YTHDF1 in cancer tissues and matched adjacent normal tissues of esophageal cancer patients was obtained from TCGA (The Cancer Genome Atlas) database. GEPIA2 (http://gepia.cancer-pku.cn/) was used to assess the correlation analysis of FTO and AKT3.

m⁶A content analysis

The EpiQuik TM m⁶A RNA Methylation Quantification Kit (Epigentek) was used to analyze the content of m⁶A in total RNA.

Dot blot assays

mRNA was obtained according to the PolyA Ttract® mRNA Isolation System (Promega) instruction manual. mRNA (50 ng/100 ng) was diluted to 2 µl with DEPC water and placed in a PCR instrument for thermal denaturation at 65 ℃ for 10 minutes. The denatured RNA was evenly dotted onto a positively charged nylon membrane (Beyotime). UV irradiation was placed under the operating platform for 15 minutes, and RNA was fixed to the membrane. The fixed nylon film was washed in 1 x PBST 3 times for 5 minutes each time. Ten milliliters of 5% skim milk sealant was prepared, and the membrane was placed in the sealant and sealed at room temperature for 2 hours. Rabbit m⁶A primary antibody (active motif) was formulated at a ratio of 1:1000. The nylon membrane was dipped in the primary antibody and incubated overnight at 4 ℃. The nylon film incubated overnight was washed with PBST 3 times for 5 minutes each time. Rat anti-rabbit secondary antibody was prepared according to 1:2000. The nylon membrane was immersed in the secondary antibody and incubated at room temperature for 2 hours. The nylon film was washed 4 times with TBST for 5 minutes each time. The ECL color solution was prepared, and the nylon film was immersed in the color solution for 10 seconds. The nylon film was removed and observed under developer. Then, the nylon film was stained with 0.2% methylene blue dye (pH 5.2, corrected by 0.3 M sodium acetate for pH) for 0.5 hours. Photographs were taken, and the sample load volume of each sample was compared.

The m⁶ART–PCR

According to described protocol, m⁶A-RT–PCR was conducted [62]. To obtain the m⁶A pull-down portion, 2 µg RNA was used for immunoprecipitation with m⁶A antibody in 500 µl IP buffer. m⁶A RNA was immunoprecipitated with Dynabeads ® Protein A and then eluted twice with elution buffer. m⁶A IP RNA was recovered by ethanol precipitation. Then, 2 ng of the total RNA and m⁶A IP RNA were used as templates in qRT–PCR.

In vitro cell assays

Total of 5 × 10³ cells per well were seeded onto a 96-well plate and checked every 24 hours (0, 24, 48, 72 and 96 hours), and cell proliferation was measured using CCK-8.

The cell migration test was performed in a 24-well plate with an 8 µm pore size Transwell chamber (Corning). A total of 200 µl of a suspension containing 5 ×10⁴ cells made of RPMI 1640 without FBS was inoculated into the upper part of the chamber. Then,600 µl of RPMI 1640 medium containing 20% FBS was added to the lower part of the chamber. After incubation for 30 hours at 37 ℃ and 5% CO₂, the transwell chamber was removed, the culture medium in the well was discarded, the cells were washed twice with PBS, fixed with methanol for 5 minutes, stained with 0.1% crystal violet for 30 minutes, the upper layer of cells was wiped off with a cotton swab, and the cells were washed with PBS 3 times. Five fields of view were randomly taken under the microscope to observe the cells and count them.

Cell invasion assays were performed in a 24-well plate with 8 µm pore size chamber inserts (Corning). A total of 8 ×10⁴ cells were seeded in the upper portion of the invasion chamber with 200 µl of RPMI 1640 without FBS. The lower portion of the chamber contained 600 µl of medium supplemented with 20% FBS and glutamine. After incubation for 36 hours at 37 ℃ and 5% CO₂, the noninvading cells were removed from the upper surface of the membrane. Cells that moved to the bottom surface of the chamber were stained with 0.1% crystal violet for 30 minutes. The cells were then imaged and counted in four separate areas with an inverted microscope.

RNA pull-down assays

RNA was taken from the Megascript® T7 Transcription Kit (Ambion) through the in vitro Transcription Kit, and then the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific) was used to conduct the RNA pull-down experiment. In short, a Pierce RNA 3' End Desthiobiotinylation Kit (Thermo Scientific) was used to biotin the RNA. Then, 50 pmol of biotinylated RNA, 50 µl of streptavidin magnetic beads and 200 µg of cell lysates were incubated at a suitable temperature for a certain period of time, and the supernatant was collected after repeated washing for real-time PCR and Western blotting analysis. Detailed information regarding the primers used for real-time PCR analysis is depicted in SI Appendix, Table S2.

RIP assays

A Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) was used for RIP according to the instructions [63]. Briefly, approximately 2–4 × 10⁷ KYSE150 cells were lysed before centrifugation, incubated with magnetic beads and coated with antibodies for 4 hours or overnight at 4 ℃. Then, the complexes were washed and incubated with proteinase K. Then, the samples were centrifuged and placed on a magnetic separator, and the supernatants were used to extract RNA with an RNA extraction kit (Bioline). Purified RNA was used for analysis. Detailed information regarding the primers used for PCR analysis is depicted in SI Appendix, Table S2.

m⁶A-seq and data analysis

Total RNA was isolated from KYSE150 and KYSE30 FTO knockdown (sh-FTO) cells using TRIzol reagent (Tiangen). RNA fragmentation, m⁶A-seq, and library preparation were performed according to the manufacturer’s instructions [64]. An RNA Library Prep Kit (NEB, USA) was used for library preparation. The m⁶A-seq data were analyzed according to the manufacturer’s protocols.

Vector and m⁶A mutation assays

The potential m⁶A sites of full-length AKT3 transcripts were predicted using an online tool, SRAMP (http://www.cuilab.cn/sramp/). The m⁶A motif-depleted 3’UTR regions were cloned into pGL3 for luciferase reporter gene analysis. The specific sequences are shown in SI Appendix, Table S2.

RNA stability

To measure the RNA stability of FTO knockdown in KYSE150 cells, actinomycete D (6 μg/ml)-treated control cells and down regulated FTO cells were used to block RNA transcription at 0, 2, 4, 6, and 8 hours. AKT3 mRNA residue was detected by quantitative PCR, and the stability of mRNA was calculated.

Luciferase reporter assays

FTO knockdownKYSE150 cells were transfected with pGL3, pGL3-WT-3’UTR, pGL3-Mut1-3’UTR, or pGL3-Mut2-3’UTR in a 6-well plate. After transfection for 8 hours, each cell line was reseeded into a 96-well plate. After 24 hours of incubation, both firefly and Renilla luciferase activities were measured 24 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega) [65]. Detailed information regarding the primers used for plasmid construction is depicted in SI Appendix, Table S2.

In vivo xenograft model

For the subcutaneous transplanted model, sh-control, sh-FTO, NC-OE and AKT3-OE KYSE150 cells (6 × 10⁶ per mouse, n = 3 for each group) were diluted in 100 μl of PBS + 100 μl Matrigel (BD) and subcutaneously injected into immuno deficient male mice to investigate tumor growth. When the tumor volume in each group reached ~100 mm³, the mice were anesthetized with a small flow of carbon dioxide to make them unconscious. Then, the mice were killed completely by increasing the flow, and by pulling the leg of the mouse to see if there was muscle tension, the mice were judged as dead. Then, the tumors were removed and weighed for use in immunohistochemistry assays and further studies. The equation V=0.5×D×d² was used to calculate the tumor volume (V: volume, D: longitudinal diameter, d: latitudinal diameter). For the in vivo lung metastasis model, mice were injected with WT (wild-type), sh-FTO, AKT3-OE and sh-FTO+AKT3-OE KYSE150 cells (1 × 10⁶ per mouse, n = 3 for each group). Six weeks after injection, mice were killed, and metastatic lung tumors were analyzed.

Immunohistochemistry assays

Tissue arrays were constructed using 28 pairs of esophageal cancer and paracancerous tissues as well as animal experimental specimens. Paraffin embedding, sectioning, and immunohistochemistry (IHC) were programmed to detect FTO expression in esophageal cancer and paracancerous tissues and Vimentin, E-cadherin, and MMP2 expression in animal specimens. Pictures were taken usinga LEICA DM 4000B microscope.

Statistical analysis

Microsoft Excel software and GraphPad Prism were used to assess the differences between experimental groups. Statistical significance was analyzed by a two-tailed Student’s t test and one-way ANOVA. p values less than 0.05 were considered to be statistically significant: *, p value < 0.05; **, p value < 0.01; ***, p value < 0.001.

m⁶A: N⁶-methyladenosine;

FTO: Fatmass and obesity-associated protein;

AKT3: AKT serine/threonine kinase 3;

METTL14: Methyltransferase-like 14;

WTAP: Wilms tumor 1 associated protein;

ALKBH5: alkylation repair homolog protein 5;

YTHDF1: YTH N⁶-methyladenosine RNA binding protein 1;

YTHDF2: YTH N⁶-methyladenosine RNA binding protein 2;

IGF2BP1: insulin-like growth factor 2 mRNA binding protein 1;

IGF2BP2: insulin-like growth factor 2 mRNA binding protein 2;

E-cad: E-cadherin;

VIM: Vimentin;

MMP2: Matrix metallopeptidase 2;

ActD: Actinomycetes D;

MeRIP-seq: Methylated RNA immunoprecipitation;

RNA-seq: Transcriptomic RNA sequencing;

IHC:Immunohistochemical;

RIP:RNA immunoprecipitation;

RT–qPCR: Quantitative real-time PCR;

SDS–PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis;

3′UTR: Three prime untranslated region;

CDS: coding sequence;

WT: wide-type;

PVDF: polyvinylidene fluoride;

TCGA: The Cancer Genome Atlas;

USTC: University of Science and Technology of China

Ethics approval and consent to participate

The animal study proposal was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Science and Technology of China(2021-N(A)-139). All of the mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China.

Consent for publication

Not applicable.

Availability of data and materials

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Anhui Provincial Natural Science Foundation of China (No. 2008085MH299). The Chen xiao-ping Foundation for the Development of Science and Technology of Hubei province (CXPIJHL2000005-07-34). The fundamental Research Funds for the Central Universities (WK9110000086). The Postdoctoral Research Funding of Anhui Province in 2019 (2019B371). The Youth Technical Backbone Fund of West Branch of the First Affiliated Hospital of USTC granted to CBZ and LSK, respectively.

Authors' contributions

RW and CBZ carried out the structure of the experiments and revised the manuscript; FFZ, YHL and CBZ drafted the initial manuscript; ZYL and LSK conceived of the study, participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Prof. YongLiang Jiang on proofreading the manuscript and the staff who participated in this study.

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The m6A demethylase FTO promotes esophageal cancer progression via the YTHDF1/AKT3 signaling network

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Results

FTO expression is upregulated in esophageal cancer

FTO is involved in the proliferation, migration, invasion, and apoptosis of esophageal cancer cells

FTO negatively correlates with METTL14 in esophageal cancer cells

Akt3 is regulated by FTO-mediated m6A modification in esophageal cancer cells

AKT3 is involved in m6A-regulated esophageal cancer tumorigenesis and metastasis

FTO and AKT3 act in concert to regulate esophageal cancer cell tumorigenesis and metastasis

YTHDF1 maintains AKT3 mRNA stability in an m6A-dependent manner

FTO and AKT3 promote esophageal cancer progression in vivo

Discussion

Materials And Methods

Samples, cell lines, and plasmids

Gene expression and survival analysis in esophageal cancer datasets

m6A content analysis

Dot blot assays

The m6ART–PCR

In vitro cell assays

RNA pull-down assays

RIP assays

m6A-seq and data analysis

Vector and m6A mutation assays

RNA stability

Luciferase reporter assays

In vivo xenograft model

Immunohistochemistry assays

Statistical analysis

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

Akt3 is regulated by FTO-mediated m⁶A modification in esophageal cancer cells

AKT3 is involved in m⁶A-regulated esophageal cancer tumorigenesis and metastasis

YTHDF1 maintains AKT3 mRNA stability in an m⁶A-dependent manner

m⁶A content analysis

The m⁶ART–PCR

m⁶A-seq and data analysis

Vector and m⁶A mutation assays