Background

Colorectal carcinoma (CRC) is the third most common cancer and second most common cause of cancer-related deaths worldwide [1]. Because of tumor metastasis and other complications, the mortality rate of CRC remains high. Therefore, a timely clarification of the molecular mechanism and effective therapeutic targets of CRC is warranted.

Ribonucleic acid (RNA) N6-methyladnosine (m6A) is the most common and abundant RNA modification in eukaryotes [2, 3]. M6A modification is mainly mediated by m6A methyltransferases, demethylases, and reader proteins. M6A methyltransferases mainly comprise methyltransferase-like 3 (METTL3) [4], methyltransferase-like 14 (METTL14) [5], Wilms tumor 1–associated protein (WTAP) [6], RNA binding motif protein 15 [7], and vir-like m6A methyltransferase associated [8]. M6A demethylases mainly comprise fat-mass and obesity-associated protein (FTO) [9] and alkylation repair homolog protein 5 (ALKBH5) [10]. Reader proteins mainly comprise YT521-B homology domain–containing family protein 1/2/3 (YTHDF1/2/3), YT521-B homology domain-–containing 1/2, insulin-like growth factor 2 mRNA binding proteins 1/2/3, and heterogeneous nuclear ribonucleoprotein family [11,12,13]. Several studies have verified that m6A modification plays a key role in cancer progression [9, 14]. METTL3 and METTL14 can regulate the progression of multiple types of cancer, including bladder cancer [15, 21]. FTO plays a central role in oral squamous cell carcinoma [22], hepatocellular carcinoma [5a), and a qPCR analysis indicated a substantial increase in CRB3 level after METTL3 knockdown (Fig. 5b and c). The results obtained from The Cancer Genome Atlas (TCGA) database revealed a substantial downregulation of CRB3 level in the CRC group (Additional file 2: Fig. S2b). A survival analysis indicated that the patients with CRC with high CRB3 level exhibited higher overall survival and disease-free survival relative to those without high CRB3 level (Additional file 2: Fig. S2c). Although METTL3 knockdown also promoted bridging integrator 1 (BIN1) expression, a substantial increase in BIN1 expression was observed in the CRC group (Additional file 2: Fig. S2a and 2d). The survival analysis did not indicate a correlation between BIN1 level and survival in patients with CRC (Additional file 2: Fig. S2e). In addition, the m6A level of CRB3 was substantially reduced after METTL3 knockdown (Fig. 5d). Luciferase reporters were revealed to determine the effect of m6A modification on CRB3 expression. For the variant form of CRB3, the adenosine bases in m6A consensus sequences (GGAC) were replaced by cytosine; thus, m6A modification was abolished. The results indicated that the transcriptional level of wild-type CRB3 significantly increased after METTL3 knockdown but not its level of variation (Fig. 5e). An RNA stability assay revealed that METTL3 knockdown substantially inhibited the degradation of CRB3 mRNA (Fig. 5f). Moreover, data from the TCGA database indicated that METTL3 mRNA expression in CRC tissues was negatively associated with CRB3 levels (Fig. 5g). Studies have reported that YTHDF2 can target mRNAs by recognizing m6A motif in CRC [5, 12]; thus, we explored the effect of YTHDF2 on CRB3. The results indicated that the expression of YTHDF2 was also significantly higher in adenoma and CRC tissues relative to normal tissues (Additional file 3: Fig. S3), and YTHDF2 knockdown substantially increased the level of CRB3 (Fig. 5h and i). RIP assays also verified the direct interaction between the YTHDF2 and CRB3 mRNA, and this direct interaction was impaired after METTL3 inhibition in SW620 and HCT116 cells (Fig. 5j and k). The results indicated that METTL3 regulated the expression of CRB3 in an m6A-YTHDF2-dependent manner.

Fig. 5
figure 5

Methyltransferase-like 3 regulated the expression of crumbs3 in an N6-methyladnosine -dependent manner. a Overlap** differentially N6-methyladnosine (m6A)-methylated genes between m6A methylation level and quantity level as filtered through a Venn diagram; b Crumbs3 (CRB3) mRNA level as measured after methyltransferase-like 3 (METTL3) knockdown; c CRB3 protein level as measured after METTL3 knockdown; d The m6A level of CRB3 as measured after METTL3 knockdown; e Luciferase reporters performed to determine the effect of m6A modification on CRB3 expression; f CRB3 mRNA expression as detected with or without treatment of actinomycin D at indicated time points; g The correlation between METTL3 and CRB3 expression in The Cancer Genome Atlas database for CRC; h CRB3 mRNA level as measured after YTH domain–containing family protein 2 (YTHDF2) knockdown; i CRB3 protein level as measured after YTHDF2 knockdown; j-k The direct interaction was verified between the YTHDF2 and CRB3 mRNA. Data are presented as means ± standard deviations (SDs). *P < 0.05, **P < 0.01, ***P < 0.001

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CRB3 inhibited CRC proliferation and invasion

Our results also indicated that CRB3 level was substantially lower in the adenoma and CRC groups than in the normal group, which was consistent with the data from TCGA database (Fig. 6a and b, Additional file 2: Fig. S2b). To further investigate the function of CRB3 in CRC, CRB3 knockdown was performed in HCT116 and SW620 cells. CRB3 knockdown significantly promoted the proliferation, migration and invasion of HCT116 and SW620 cells (Fig. 6c-e). CRB3 knockdown also significantly increased the migration speed of HCT116 and SW620 cells (Fig. 6f and g). These results verified that CRB3 regulated CRC progression.

Fig. 6
figure 6

Crumbs3 inhibited colorectal carcinoma proliferation and invasion. Crumbs3 (CRB3) expression in both adenoma and colorectal carcinoma (CRC) as assayed through (a) qPCR and (b) Immunofluorescence; c Proliferation of HCT116 and SW620 cells as measured after CRB3 knockdown; Transwell assays performed with CRB3 knockdown in (d) HCT116 cells and (e) SW620 cells; Wound healing assay performed with CRB3 knockdown in (f) HCT116 cells and (g) SW620 cells. Data are presented as means ± standard deviations (SD). *P < 0.05, **P < 0.01, ***P < 0.001

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METTL3 regulated CRC proliferation and invasion by CRB3-hippo pathway

Studies have demonstrated that CRB3 can regulate the Hippo pathway [30, 31]. In the present study, we discovered that CRB3 knockdown reduced MST1, LATS1, MOB1, and YAP phosphorylation levels, and it reduced the SAV1 levels in HCT116 and SW620 cells (Fig. 7a and b). Conversely, METTL3 knockdown substantially increased MST1, LATS1, MOB1, and YAP phosphorylation levels in HCT116 and SW620 cells (Fig. 7c and d). Studies have demonstrated that YAP enters the nucleus and acts as an oncogene; thus, we detected the level of YAP in nuclei. The results indicated that CRB3 knockdown markedly increased YAP protein levels in the nuclei of HCT116 and SW620 cells (Fig. 7e and f); however, METTL3 knockdown substantially reduced the YAP protein levels in the nuclei of HCT116 and SW620 cells (Fig. 7g and h).

Fig. 7
figure 7

Methyltransferase-like 3 and crumbs3 could both regulate Hippo pathway. Hippo pathway as detected after (a, b) Crumbs3 (CRB3) knockdown or (c, d) Methyltransferase-like 3 (METTL3) knockdown in HCT116 and SW620 cells; Yes1-associated transcriptional regulator (YAP) protein level as detected after (e, f) CRB3 knockdown or (g, h) METTL3 knockdown in nuclei of HCT116 and SW620 cells. Data are presented as means ± standard deviations (SD). *P < 0.05, **P < 0.01, ***P < 0.001

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To test whether CRB3 functions downstream of METTL3, we knocked down CRB3 in the METTL3 knockdown background. The results revealed that the effects of METTL3 knockdown on cell proliferation, migration, and invasion were rescued by the CRB3 knockdown (Fig. 8a-d). In addition, CRB3 knockdown reduced the MST1, LATS1, MOB1, and YAP phosphorylation levels caused by METTL3 knockdown, indicating that CRB3 knockdown repressed activation of hippo pathway that was caused by METTL3 knockdown (Fig. 8e and f).

Fig. 8
figure 8

The effect of methyltransferase-like 3 on colorectal carcinoma progression was rescued by crumbs3. The proliferation of HCT116 (a) and SW620 cells (b) was rescued after crumbs3 (CRB3) knockdown; Migration and invasion was rescued after CRB3 knockdown in HCT116 (c) and SW620 cells (d); The activation of hippo pathway was reversed after CRB3 knockdown in HCT116 (e) and SW620 cells (f). Data are presented as means ± standard deviations (SD). *P < 0.05, **P < 0.01, ***P < 0.001

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Discussion

The incidence of CRC, which is a malignant tumor, has increased. The 5-year survival rate of patients with CRC is 65% [32], but it is extremely low in advanced-stage CRC. Therefore, techniques that aid the implementation of treatment strategies for CRC are urgently required because they can improve patient survival [27]. Studies have reported that m6A modification plays a key role in CRC [4, 5, 12, 33]. However, the changes to m6A in both adenoma and CRC are still unknown. In addition, the relationship between m6A level and the survival of patients with CRC requires further clarification. In the present study, we discovered that m6A levels are significantly increased in both adenoma and CRC tissues, indicating that m6A modification may be involved in the adenoma-to-CRC transition. A further examination indicated that the patients with CRC with high m6A level exhibited shorter overall survival.

M6A modification is mainly mediated by m6A methyltransferases, demethylases and reader proteins, and it regulates pre-mRNA splicing, miRNA processing, translation, and mRNA decay [34]. In the present study, we discovered that the protein level of METTL3 and m6A methylase activity were both significantly increased in CRC, indicating that METTL3 could be involved in the CRC progression. Furthermore, patients with CRC with high METTL3 level exhibited shorter overall survival, suggesting that METTL3 can also serve as a prognostic marker of CRC. These results are consistent with those reported by other studies [4, 35]. Subsequently, we verified the function of METTL3 in CRC. METTL3 knockdown inhibited the proliferation of HCT116 and SW620 cells, and it also substantially inhibited the migration and invasion of HCT116 and SW620 cells. These results indicated that METTL3 acts as an oncogene that promotes the progression of CRC. Studies have also reported on the key role of METTL3 in various cancers. METTL3 can regulate MALAT1 stabilization through m6A modification, and it activates NF-κB activity to promote the malignant progression of glioma [36]. METTL3 increases miR-1246 levels through m6A modification, thereby promoting non-small-cell lung cancer progression [37]. Moreover, METTL3 regulates the m6A modification of SPHK2 to promote the progression of gastric cancer [38]. These findings verified the role of METTL3 in cancers such as CRC. Therefore, METTL3 can be a new treatment target for cancers.

Through an m6A epitranscriptomic microarray analysis, we revealed that CRB3 might be the downstream target of METTL3. METTL3 knockdown substantially reduced the m6A level of CRB3 and inhibited the degradation of CRB3 mRNA, finally to increase CRB3 expression. Studies have reported that the m6A consensus sequences are GGAC [5]. Our study revealed that METTL3 knockdown increased the transcriptional level of CRB3. When the adenosine bases of GGAC in CRB3 were replaced by cytosine, the transcriptional level of CRB3 did not change. In addition, YTHDF2 could also regulate the CRC progression in an m6A-dependent manner [5, 12, 39]. In the present study, we discovered that YTHDF2 knockdown substantially increased the level of CRB3. The direct interaction between the YTHDF2 and CRB3 mRNA was also verified, and this direct interaction was impaired after METTL3 inhibition. These results indicated that METTL3 regulated the expression of CRB3 in an m6A-YTHDF2-dependent manner. CRB3 is a protein of cell polarity, and it is associated with contact inhibition [40]. Studies have demonstrated that CRB3 plays a central role in cancers such as CRC [30, 41, 42]. In the present study, we discovered that CRB3 levels in both adenoma and CRC were substantially lower than in normal tissues, and we also revealed that patients with CRC with high CRB3 level exhibited higher overall survival and disease-free survival. CRB3 knockdown significantly promoted the proliferation, migration, and invasion of HCT116 and SW620 cells. These results indicated that CRB3 regulates CRC progression.

The depletion of CRB3 can inhibit the Hippo pathway and lead to increased nuclear localization of YAP [30, 31, 43]. The Hippo pathway plays a crucial role in regulating CRC progression [44,45,46,47]. In the present study, we also observed that CRB3 knockdown inhibited the Hippo pathway and increased the nuclear localization of YAP, suggesting that CRB3 regulates CRC progression through the Hippo pathway. Conversely, METTL3 knockdown activated the Hippo pathway and reduced the nuclear localization of YAP. Finally, our results revealed that the effects of METTL3 knockdown on cell proliferation, migration, and invasion were rescued by the CRB3 knockdown. CRB3 knockdown reversed the activation of hippo pathway caused by METTL3 knockdown. Therefore, our study indicated that METTL3 facilitated CRC progression by regulating the m6A-CRB3-Hippo pathway, which is a novel mechanism for regulating CRC. Even though we demonstrated the regulatory mechanism of METTL3 in CRC, further studies are required. First, although a study reported that the selective first-in-class catalytic inhibitor of METTL3 (i.e., STM2457) can be used in treatment strategies for acute myeloid leukemia [48], the inhibitor of METTL3 has not yet been identified for the treatment of CRC. Therefore, further studies are required to identify the inhibitor of METTL3. Second, we discovered substantially elevated m6A levels in both adenoma and CRC; METTL3 level was substantially elevated in only CRC, not in adenoma. This suggests that other enzymes may also be involved in the m6A modification in adenoma, and further clarification was required.

Conclusion

In summary, we demonstrated that m6A and METTL3 levels are significantly increased in CRC. Patients with CRC with high m6A or METTL3 levels exhibit shorter overall survival, and METTL3 promoted CRC progression. Mechanistically, METTL3 regulates the progression of CRC by regulating the m6A–CRB3–Hippo pathway. These findings provide a new perspective for the implementation of targeted therapy for CRC.