RETRACTED: METTL14 Suppresses CRC Progression via Regulating N6-Methyladenosine-Dependent Primary miR-375 Processing

Introduction

Colorectal cancer (CRC) is the third most common cancer and the fourth most fatal malignancy in the world. Although surgery combined with system therapy has improved the prognosis of CRC patients in the past few decades, tumor recurrence and liver metastasis are still common in CRC patients and are closely correlated with poor survival., Therefore, a better understanding of the mechanism involved in CRC initiation and progression is essential.

At present, more than 100 types of RNA modifications have been reported,4, 5, 6 among which N6-methyladenosine (m6A) modification is the most prevalent internal modification in eukaryotic mRNAs.7, 8, 9 Dysregulation of this modification is closely associated with human diseases, including malignancies. In mammals, the formation of m6A modification is catalyzed by methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms tumor 1-associated protein (WTAP). The m6A modification can be erased by alkB homolog 5 (ALKBH5) and fat mass and obesity-associated protein (FTO).11, 12, 13, 14, 15

Recently, an increasing number of findings have revealed that m6A functions as a critical post-transcriptional modification that regulates mRNA and non-coding RNA biogenesis; for example, Lin et al. have found that METTL3 promoted translation initiation of many oncogenes in human lung cancer through m6A modification. Alarcón et al. reported that METTL3 depletion decreased the binding of DGCR8 to primary microRNAs (primiRNAs) and resulted in the reduction of mature miRNAs. Ma et al. also found that METTL14 interacted with DGCR8 and positively regulated the primary miR-126 (primiR-126) process in an m6A-dependent manner.

In our study, we explored the role of m6A modification in CRC and elucidated the mechanism by which m6A modification participates in the biological process of CRC. First, decreased METTL14 was found in CRC tissues and cell lines, and was closely correlated with overall survival (OS). Then, we demonstrated that METTL14 was associated with CRC progression in vitro and in vivo. Furthermore, we investigated the targets of METTL14 in the biological process of CRC. miR-375, functioning as a tumor suppressor in various cancers, was identified as a target of METTL14. METTL14 knockdown decreased miR-375 expression levels by modulating DGCR8 binding to primary miR-375 (primiR-375) in a m6A-dependent manner. Last, we further verified that METTL14 suppressed CRC progression via the miR-375/Yes-associated protein 1 (YAP1) and miR-375/SP1 pathways.

Results

METTL14 Was Obviously Downregulated in CRC Tissues and Cell Lines

We initially analyzed METTL14 expression in HCT116, HCT8, HT29, SW620, SW480, DLD-1, and FHC. Lower levels of METTL14 were detected in CRC cells compared with FHC (Figure 1A). Moreover, we also detected METTL14 expression in 112 pairs of CRC tissues and adjacent normal tissues (ANTs) using qRT-PCR. The results showed that lower levels of METTL14 were found in CRC tissues than in ANTs (Figure 1B).

Figure 1

METTL14 Is Downregulated in CRC Cell Lines and CRC Tissues and Is Negatively Associated with the Overall Survival Rate of CRC Patients

(A) qRT-PCR analysis of METTL14 mRNA expression in six CRC cell lines and FHC. (B) qRT-PCR analysis of METTL14 mRNA expression in 112 pairs of CRC tissues. GAPDH was used as an internal control. (C) IHC analysis of METTL14 protein expression in 112 pairs of CRC tissues. (D) Kaplan-Meier survival curves of OS in 112 CRC patients based on METTL14 IHC stains. Patients were divided into two subgroups according to the scores of median METTL14 IHC staining. **p < 0.01, ***p < 0.001.

METTL14 Was Closely and Negatively Associated with Clinicopathological Parameters

To further investigate whether the METTL14 protein is associated with the clinical progression of CRC, we performed immunohistochemistry (IHC) on 112 paired CRC tissue samples. The IHC analysis showed that the IHC scores of METTL14 in CRC tissues were markedly lower than those in matched ANTs (Figure 1C). Next, these CRC tissue samples were divided into two subgroups in accordance with the median METTL14 IHC staining score. Its expression was obviously correlated with TNM stage and differentiation (p < 0.05; Table S1). The results indicated that high expression of METTL14 is associated with malignant progression in CRC patients.

Kaplan-Meier survival analysis revealed that low METTL14 IHC staining was associated with poor OS in CRC patients (Figure 1D). Moreover, Cox regression analyses were performed to further assess the prognostic value of METTL14 in CRC. As shown in Table S2, univariate analysis revealed that lower METTL14 expression (hazard ratio [HR] = 1.535; 95% confidence interval [CI] = 1.121–2.313; p < 0.001) and a higher TNM stage (HR = 2.073; 95% CI = 1.339–3.209; p = 0.001) were clearly correlated with OS.

Multivariate survival analysis indicated that METTL14 expression was strongly associated with OS. Multivariate analysis also suggested that lower METTL14 expression (HR = 1.953; 95% CI = 1.492–2.397; p = 0.012) and a higher TNM stage (HR = 2.016; 95% CI = 1.152–3.526; p = 0.016) were dramatically associated with OS. Together, these results indicated that decreased METTL14 was correlated with CRC progression and could be a potential prognostic biomarker in CRC.

METTL14 Inhibits CRC Cell Proliferation

To determine whether m6A modification plays an important role in CRC tumorigenesis and progression, we knocked down METTL14, a key component of the m6A methyltransferase that exhibits a positive correlation with m6A levels. First, we inhibited METTL14 expression via transfection with shMETTL14 (shMETTL14-1, shMETTL14-2) in HCT116 and HCT8 cells (Figure 2A), and reduced m6A levels were found after METTL14 knockdown (Figure 2B). Next, we performed Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) to investigate whether METTL14 is involved in CRC growth. CCK-8 and EdU assays demonstrated that the proliferation of HCT116 and HCT8 cells significantly increased after the suppression of METTL14 (Figures 2C and 2D). Examination of the cell-cycle profile revealed a lower percentage of cells transfected with shMETTL14 in the G0/G1 phase and a higher percentage in the S phase (Figure 2E).

Figure 2

METTL14 Knockdown Promotes CRC Cell Proliferation

(A) METTL14 was detected by qRT-PCR after METTL14 knockdown in HCT116 and HCT8 cells. (B) The m6A contents of total RNAs were detected using the m6A RNA Methylation Assay Kit in METTL14-knockdown HCT116 and HCT8 cells. (C and D) The proliferative abilities of HCT116 and HCT8 cells after suppression of METTL14 were determined by CCK-8 (C) and EdU (D, original magnification ×200) assays. (E) Cell-cycle analysis was performed in HCT116 and HCT8 cells after METTL14 knockdown using flow cytometry. The results are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

To further determine the role of METTL14 in tumor growth, we also upregulated METTL14 in HCT116 and HCT8 cells using LV-METTL14 (Figure 3A), and increased m6A levels were detected after METTL14 overexpression (Figure 3B). Notably, METTL14 overexpression obviously impeded CRC cell proliferation in CCK-8 and EdU assays (Figures 3C and 3D). A cell-cycle assay demonstrated that the proportion of cells in the G0/G1 phase was significantly higher, and the proportion of cells in the S phase was markedly lower in METTL14-upregulating CRC cells (Figure 3E).

Figure 3

METTL14 Upregulation Inhibits CRC Cell Growth

(A) METTL14 expression levels were increased after transfection with METTL14 expression plasmid in HCT116 and HCT8 cells. (B) The m6A contents of total RNAs were detected using the m6A RNA Methylation Assay Kit in METTL14-overexpressing HCT116 and HCT8 cells. (C and D) The proliferation of HCT116 and HCT8 cells after METTL14 overexpression was evaluated using CCK-8 (C) and EdU (D) assays. (E) Cell-cycle analysis was performed in HCT116 and HCT8 cells after METTL14 overexpression using flow cytometry. The results are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

METTL14 Inhibits CRC Cell Migration and Invasion

Next, wound healing, Transwell migration, and invasion assays were performed to explore the role of METTL14 on CRC cell migration and invasion. Our data showed that the migration and invasion abilities of HCT116 and HCT8 cells were drastically elevated after METTL14 knockdown (Figures 4A and 4B), whereas METTL14 overexpression obviously impaired CRC cell migration and invasion (Figures 4C and 4D).

Figure 4

METTL14 Inhibits CRC Cell Migration and Invasion

(A) Wound healing (original magnification ×100) assays for HCT116 and HCT8 cells with METTL14 knockdown. (B) Transwell migration and Matrigel invasion (original magnification ×200) assays for HCT116 and HCT8 cells with METTL14 knockdown. (C) Wound healing (original magnification ×100) assays for HCT116 and HCT8 cells with METTL14 overexpression. (D) Transwell migration and Matrigel invasion (original magnification ×200) assays for HCT116 and HCT8 cells with METTL14 overexpression. The results are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

METTL14 Suppresses CRC Growth and Metastasis In Vivo

To further explore the role of METTL14 in vivo, we constructed stable METTL14-knockdown CRC cells using lentivirus vector containing shMETTL14-1 (LV-shMETTL14), as well as established stable METTL14-overexpressing CRC cells using LV-METTL14. Next, a subcutaneous xenograft model was performed, and the results showed that the growth of tumors from METTL14-depleted xenografts was obviously promoted, whereas METTL14 overexpression was markedly inhibited compared with the tumors from control-infected (control of LV-shMETTL14 [LV-shNC], LV-NC) HCT116 cells (Figures 5A and 5B). Moreover, IHC staining indicated that knockdown of METTL14 increased the expression of Ki67, whereas METTL14 overexpression decreased Ki67 expression levels in xenograft tissues (Figures 5C and 5D).

Figure 5

The Biological Function of METTL14 In Vivo

(A) Tumors collected from nude mice are exhibited. (B) Tumor volume curves of mice upon LV-shMETTL14 or LV-shNC (left), as well as LV-METTL14 or LV-NC (right) treatment, were analyzed. (C) Representative images of IHC staining of Ki67 expression in tumors from mice. (D) The IHC scores of Ki67 in xenograft tissues. (E) Representative lungs from mice (left), images of lung tissue sections stained using H&E (right). (F) The number of metastatic nodules in the lungs of mice (five sections evaluated per lung) from four groups (n = 6). The results are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Furthermore, we injected HCT116 cells through the tail vein to establish a lung metastasis tumor model in nude mice. HCT116 cells infected with LV-shMETT14 obviously promoted pulmonary metastasis, whereas those infected with LV-METTL14 markedly restrained pulmonary metastasis (Figures 5E and 5F). This difference was further determined by the examination of entire lungs through H&E staining of the mouse lung sections.

METTL14-Dependent m6A Methylation Modulates the Processing of miR-375 by DGCR8

An increasing number of studies have verified that either METTL3 or METTL14 is required for the engagement of primiRNAs by DGCR8 in various cancers.17, 18, 19

We herein identify whether DGCR8 governs the processes of primiRNAs that are dependent on METT14/m6A in CRC. Given that reduced m6A levels result in primiRNA process arrest and lead to decreased levels of corresponding miRNAs, the expression of METTL14/m6A-dependent miRNAs is positively correlated with METTL14. Using LinkedOmics (http://www.linkedomics.org/login.php), nine miRNAs, including miR-375 (r = 0.2324, p < 0.001), miR-30e (r = 0.1949, p < 0.001), miR-200a (r = 0.1418, p < 0.05), miR-625 (r = 0.1329, p < 0.05), miR-639 (r = 0.127, p < 0.05), miR-611 (r = 0.1262, p < 0.05), miR-941-1 (r = 0.1172, p < 0.05), miR-135a-2 (r = 0.1172, p < 0.05), and miR-107 (r = 0.1162, p < 0.05), were found to be positively correlated with METTL14 in CRC tissues (Figure 6A). We then measured these miRNAs in METTL14-deleted CRC cells and found that miR-375 was significantly downregulated in METTL14-deleted CRC cells (Figure 6B). We then examined the expression of miR-375, miR-200a, primiR-375, and primiR-200a in METTL14-knockdown or METTL14-overexpressing CRC cell lines. We found that mature miR-375 and miR-200a was downregulated in METTL14-knockdown CRC cells and upregulated in METTL14-overexpressing CRC cells (Figure 6C). Unprocessed primiRNA-375 was found to accumulate in METTL14-depleted CRC cells and accelerate in METTL14-overexpressing CRC cells, but METTL14 knockdown/overexpression had no effect on primiRNA-200a expression, unlike miR-375 (Figure 6D).

Figure 6

METTL14-Dependent m6A Methylation Modulates the Processing of miR-375 by DGCR8

(A) Search for miRNAs positively correlated with METTL14 through the LinkedOmics database. (B) The expression of miR-375 and miR-200a was determined by qRT-PCR in HCT116 and HCT8 cells after METTL14 knockdown. (C and D) miR-375, miR-200a (C), primiR375, and primiR-200a (D) were quantified using qRT-PCR upon METTL14 knockdown or upregulation in HCT116 and HCT8 cells. (E) Schematic representation of the reporters was employed to determine the role of the METTL14 on primiR-375 processing. Represented in red is the primiR-375 sequence and in green the control pri-miR-1-1. The top reporter contains a wild-type sequence of the primiR-375, and the potential sites of methylations are depicted as black dots. The reporter on the bottom contains a mutant version of primiR-375 in which the one putative adenine of the METTL14 motif was mutated. The bar graph represents the relative expression of mature miR-375 normalized to mature miR-1-1. (F) Immunoprecipitation of DGCR8-associated RNA from METTL14-overexpressing or control CRC cells followed by qRT-PCR to determine primiRNA binding to DGCR8. prilet-7e was used as a positive control, and primiR-30e was employed as a negative control. (G) Immunoprecipitation of m6A-modified RNA in METTL14-overexpressing or control CRC cells followed by qRT-PCR to evaluate the primiRNA m6A modification levels. The results are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

In order to verify a direct role of the m6A modification in the primiR-375 processing, SRAMP database (http://www.cuilab.cn/sramp) were performed to predict the m6A sites located in primiR-375 and found two RRACH m6A sequence motifs in primiR-375 (only one adenosine [A] present outside the pre-miRNA sequence) (Figure S1). We then modified a previously described reporter construct., In one reporter, a wild-type (WT) version of primiR-375 was performed, and in the other reporter, the A’s of the m6A motif present in the primiR-375 that were out of the premiR-375 region were mutated. Our findings showed that mutation of m6A motif in primiR-375 markedly decreased its processing to the mature form (Figure 6E).

We also immunoprecipitated DGCR8 from METTL14-overexpressing and control CRC cells and detected primiR-375 bound to DGCR8 using qRT-PCR, and found that the expression levels of primiR-375 bound to DGCR8 were significantly increased in METTL14-overexpressing CRC cells (Figure 6F). Furthermore, we immunoprecipitated m6A from RNAs in METTL14-overexpressing and control CRC cells, and found that METTL14 overexpression clearly increased the amount of primiR-375 modified by m6A (Figure 6G).

miR-375 Is a Downstream Target of METTL14

To explore the potential molecular mechanism of miR-375 in CRC progression as a downstream target of METTL14, we measured the expression of miR-375 in 112 pairs of CRC tissue samples and found that miR-375 was markedly decreased in CRC tissues in comparison with corresponding ANTs (Figure 7A), and its downregulation was associated with poor prognosis (Figure 7B). Additionally, a positive correlation between METTL14 and miR-375 was noted in CRC (r2 = 0.2542; Figure 7C).

Figure 7

miR-375 Is a Downstream Target of METTL14

(A) qRT-PCR analysis of miR-375 in CRC tissues and corresponding adjacent normal tissues (ANTs). (B) Kaplan-Meier survival curves of CRC patients with low and high miR-375 expression. (C) The relationship between miR-375 and METTL14 mRNA in CRC tissues. (D) qRT-PCR analysis of miR-375 in METTL14-overexpressing CRC cells after treating with miR-375 inhibitor (inhibitor), and U6 was used as an internal control. (E and F) The ability of proliferation in METTL14-overexpressing HCT116 and HCT8 cells after treating with inhibitor was assessed using CCK-8 (E) and EdU (F) assays. (G) Wound healing assays for METTL14-overexpressing HCT116 and HCT8 cells transfected with miR-375 inhibitor. (H) Transwell migration and invasion assays for METTL14-overexpressing HCT116 and HCT8 cells transfected with miR-375 inhibitor. (I) qRT-PCR analysis of miR-375 in METTL14 silencing HCT116 and HCT8 cells transfected with miR-375 mimic (mimic). (J and K) CCK-8 (J) and EdU (K) assays for METTL14 silencing HCT116 and HCT8 cells transfected with miR-375 mimic. (L) Wound healing assays for METTL14 silencing HCT116 and HCT8 cells transfected with miR-375 mimic. (M) Transwell migration and invasion assays for METTL14 silencing HCT116 and HCT8 cells transfected with miR-375 mimic. The results are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. NC, untransfected CRC cells.

Next, we downregulated miR-375 using miR-375 inhibitor (inhibitor) in stable METTL14-overexpressing HCT116 and HCT8 cells (Figure 7D), and found that inhibition of miR-375 could restore the decreased ability of proliferation (Figures 7E, 7F, and S2A), migration, and invasion (Figures 7G and S2B and Figures 7H and S2C, respectively) in HCT116 and HCT8 cells.

Lastly, we upregulated miR-375 in stable METTL14-deleting CRC cells (Figure 7I), and a series of restoration assays was performed in METTL14-deleting HCT116 and HCT8 cells. Our results showed that forced expression of miR-375 can reverse the effects of METTL14 knockdown on cell proliferation (Figures 7J, 7K, and S3A), migration, and invasion (Figures 7L and S3B and Figures 7M and S3C, respectively).

METTL14 Inhibits CRC Progression via the miR-375/YAP1 and miR-375/SP1 Pathways

It is well accepted that miR-375 is a crucial cancer-related miRNA, and many oncogenes have been verified as functional targets of miR-375, including YAP1, SP1, AEG1, PDK1, IGF1R, and JAK2. Here, we found that METTL14 overexpression significantly elevated the expression of miR-375 and reduced the expression of YAP1 and SP1 in HCT116 and HCT8 cells (Figures 8A and 8B). Moreover, we also detected the expression of YAP1 and SP1 in xenograft tissues using IHC staining and showed that METTL14 overexpression decreased the expression of YAP1 and SP1, whereas METTL14 knockdown increased YAP1 and SP1 expression levels in xenograft tissues (Figure 8C). Therefore, we speculated that METTL14 might exert its biological function through the miR-375/YAP1 and miR-375/SP1 pathways. To verify this assumption, a direct interaction of miR-375 and YAP1 or SP1 was further substantiated via cloning the predicted WT and mutated (Mut) 3′ UTR binding site in a dual-luciferase reporter plasmid, and decreased luciferase activity after miR-375 mimics cotransfection in 293T and HCT116 cells was observed in the YAP1 and SP1 WT sequences, but the mutated binding site did not alter the luciferase activity (Figure 8D).

Figure 8

METTL14 Regulates CRC Progression through the miR-375/YAP1 and miR-375/SP1 Pathways

(A) qRT-PCR analysis of miR-375 in METTL14-overexpressing HCT116 and HCT8 cells. (B) The expression of YAP1 and SP1 in METTL14-overexpressing HCT116 and HCT8 cells was detected using western blot. (C) The expression of YAP1 and SP1 in xenograft tissues was detected using IHC. (D) miR-375 and its putative binding sequence in the wild-type (WT) and mutant (Mut) 3′ UTRs of SP1 or YAP1. Relative luciferase activity of the indicated 3′ UTR vector in 293T and HCT116 cells cotransfected with miR-375. (E) Western blot was performed to detect the expression of METTL14, YAP1, and SP1 in HCT116 and HCT8 cells after transfecting or not. GAPDH was used as an internal control. (F and G) CCK-8 (F) and EdU (G) assays for METTL14-overexpressing HCT116 and HCT8 cells transfected with YAP1 expression plasmid. (H) Wound healing for METTL14-overexpressing HCT116 and HCT8 cells transfected with SP1 expression plasmid. (I) Transwell migration and invasion assays for METTL14-overexpressing HCT116 and HCT8 cells transfected with SP1 expression plasmid. The results are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. NC, untransfected CRC cells.

Furthermore, we elevated YAP1 and SP1 expression using YAP1 and SP1 expression plasmids in METTL14-overexpressing CRC cells (Figure 8E), and a series of restoration assays was performed in METTL14-overexpressing HCT116 and HCT8 cells. As shown in Figures 8F, 8G, and S4A, YAP1 increased the capability of proliferation, and SP1 elevated the ability of migration and invasion in stable METTL14-overexpressing HCT116 and HCT8 cells (Figures 8H and S4B and Figures 8I and S4C, respectively). Together, these results indicate that METTL14 inhibits CRC progression via the miR-375/YAP1 and miR-375/SP1 pathways.

Discussion

Various chemical modifications of proteins and DNA have been well verified, and their pathological and biological functions have been extensively studied.22, 23, 24, 25 m6A modification, one type of RNA epigenesis, has been increasingly studied in human disease, including cancer.,

With the advance of m6A sequence technology, m6A modification has been identified in the mRNAs of more than 7,000 genes in mammalian organisms., Recently, an increasing number of studies have revealed that m6A modification in mRNAs or non-coding RNAs plays pivotal roles in stem cell self-renewal and tumorigenesis; in cell growth in cancer; and in the fate and functions of RNA, including mRNA stability, splicing, location, and translation, RNA-protein interactions, and primiRNA processes.,,27, 28, 29, 30, 31 Li et al. verified that FTO, a key RNA demethylase, was highly expressed and functioned as an oncogene via regulation of ASB2 and RARA in certain subtypes of acute myeloid leukemia (AML). METTL14, a key component of the m6A methyltransferase complex, has been reported to be dysregulated and play critical roles in the tumorigenesis of glioblastoma, AML, and breast cancer.

At present, the studies on the role of m6A modification in CRC remain elusive. Bai et al. reported that YTH m6A RNA binding protein 1 (YTHDF1) is a core factor in RNA methylation modification and plays a vital oncogenic role in CRC.

In our study, we first revealed that the major m6A “writer,” METTL14, was significantly downregulated in CRC and was obviously correlated with TNM stage. Low expression of METTL14 was dramatically associated with poor OS. Additionally, we also detected that METTL14 knockdown promoted and METTL14 overexpression inhibited CRC cell proliferation, migration, and invasion in vitro, which was further verified by in vivo experiments. Based on the above findings, we concluded that METTL14 functions as a tumor suppressor in CRC.

Ma et al. reported that METTL14, a main factor involved in RNA m6A modification, interacted with DGCR8 and positively modulated primiR-126 processes in an m6A-dependent manner. Herein, we verified that METTL14 depletion decreased the binding of DGCR8 to primiR-375 and resulted in the global reduction of miR-375 and concomitant accumulation of unprocessed primiR-375 in CRC cells. Additionally, we also found that METTL14 affects the expression of miR-200a, but not primiR-200a; therefore, we speculate that METTL14 does not regulate the expression of miR-200a in a METTL14-dependent m6A modification. miR-375, a well-known cancer-related miRNA, is frequently downregulated and acts as a tumor suppressor in multiple types of cancers., Nishikawa et al. reported that YAP1, the key target for the Hippo pathway, showed a reverse correlation with miR-375 and growth-inhibitory activities in lung cancer with neuroendocrine (NE-lung cancer). Wang et al. reported that miR-375 was downregulated in cervical cancer cells and suppressed cell migration and invasion via targeting the transcription factor SP1. In our studies, we found that miR-375 was a downstream target of METTL14 and could reverse the inhibitory effect caused by METTL14 overexpression in CRC. Furthermore, we noticed that METTL14 suppressed CRC cell growth through the miR-375/YAP1 pathway while restraining CRC cell migration and invasion via the miR-375/SP1 pathway.

Conclusions

In summary, our findings have identified METTL14 as a tumor suppressor in CRC. METTL14-mediated m6A modification regulates the process of primiR-375 with respect to its functional effect on CRC progression. Furthermore, we demonstrated that METTL14 suppressed CRC cell growth, migration, and invasion via the miR-375/YAP1 and miR-375/SP1 pathways. Our work revealed a critical function for METTL14 and provided insight into crucial roles of METTL14/m6A methylation in CRC.

Materials and Methods

Patient Samples

A total of 112 fresh CRC tissues and their corresponding ANTs were obtained from patients with CRC who received radical surgery at Nanjing First Hospital, Nanjing Medical University between January 2001 and December 2007. None of the patients achieved system treatment before surgery. All of these samples were immediately snap frozen in liquid nitrogen and stored at −80°C until RNA extraction. Informed consent was obtained from each of these patients, and this study was approved by the Institutional Review Board of Nanjing First Hospital, Nanjing Medical University. The clinicopathological characteristics of these patients are listed in Table S1.

Cell Culture and Treatment

Human CRC cell lines HCT116, HCT8, HT29, SW620, SW480, and DLD-1, and corresponding normal colonic epithelial cell (FHC) were purchased from ATCC. These cells were cultured in DMEM (GIBCO, USA) with 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% fetal bovine serum (FBS; GIBCO, USA) at 37°C supplied with a 5% CO2.

Cell Transfection and Stable Cell Line Construction

CRC cells were transfected with short hairpin RNAs (shRNAs) (shMETTL14-1, shMETTL14-2), miR-375 inhibitors, and mimics or their negative control (shNC, mimic-NC, inhibitor-NC), as well as YAP1 and SP1 overexpression plasmid or blank vector using Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s protocol.

To obtain cell lines stably expressing METTL14, METTL14 cDNA was amplified and subcloned into the lentiviral vector (Genecreat, Wuhan, China). Recombinant lentiviruses containing the METTL14 gene (LV-METTL14) were obtained from Genecreat (Wuhan, China). Two CRC cell lines (HCT116 and HCT8) were infected, and stable cells were selected by treatment with puromycin (1.5 μg/mL) for 2 weeks.

The lentivirus vector containing shMETTL14-1 (LV-shMETTL14) was amplified and cloned by Genecreat (Wuhan, China). HCT116 and HCT8 cells were infected with concentrated virus; then the cells were selected by treatment with puromycin (2 μg/mL) for 2 weeks. All shRNA, mimics, and inhibitor sequences are listed in Table S3.

EdU Assays

A total of 1.5 × 105 transfected cells were seeded into a 24-well plate overnight. The next day, 50 μM 5-ethynyl-2′-deoxyuridine (EdU; Cell Light EdU DNA Imaging Kit, RiboBio, China) was added, and the cells were grown for an additional 2 h. The cells were stained in accordance with the following instructions: cells were fixed with 4% paraformaldehyde at room temperature for 30 min, and then glycine was added (2 mg/mL) for 5 min and 0.5% Trion X-100 for 10 min. After washing twice using PBS, the cells were stained with Apollo fluorescent dye and Hoechst 33342. The images were captured using inverted microscopy (Nikon, Japan), and the percentage of EdU-positive cells was detected from five random fields in three wells.

CCK-8, Wound Healing, Transwell Migration, and Invasion Assays

CCK-8, wound healing, Transwell migration, and invasion assays were performed as previously described.,

Cell-Cycle Distribution Analysis

The trypsinized cells (1 × 106) were fixed with 75% ethanol at 4°C for 24 h. The fixed cells were incubated with RNase A (for 30 min at 37°C) after being washed with PBS, and 5 μL of propidium iodide (PI) (KeyGen BioTECH, China) was added to the cell suspension. The mixture was incubated at room temperature for 30 min in the dark. The suspended cells were analyzed for cell cycle using the FACSCalibur Flow Cytometer (BD Biosciences, CA, USA). The percentages of cells in the G0/G1, S, and G2/M phases were counted and compared.

Western Blot Analysis

The total proteins were extracted from cells with radioimmunoprecipitation assay (RIPA) lysis buffer mixed with PMSF, protein inhibitors, and phosphatase inhibitors (KeyGEN BioTECH, China). Equal amounts of proteins were separated with 10% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% BSA for 1.5 h and then incubated with primary antibodies: rabbit polyclonal anti-METTL14 (1:1,000, ab46154; Abcam, UK), anti-YAP1 (1:2,000; 103584-1-AP; Proteintech, China), anti-SP1 (1:1,000, 21962-1-AP; Proteintech, China), and anti-GAPDH (1:10,000, 10494-1-AP; Proteintech, China). Proteins were then detected by an enhanced chemiluminescence system (ECL) reagent (KeyGEN BioTECH, China) after incubation with secondary antibodies for 1 h at room temperature.

RNA Isolation and qRT-PCR

TRIzol reagent (Invitrogen, USA) was used to extract total RNA in accordance with the manufacturer’s instructions. The expression of mature miRNAs was detected by a Hairpin-it microRNA and U6 snRNA normalization RT-PCR Quantitation Kit (Genepharma, China), and the primers for miRNAs were obtained from Genepharma (China). For METTL14 mRNA, cDNA was synthesized using a PrimeScript reagent kit with gDNA Eraser (Takara, Dalian, China), and qRT-PCR was performed using SYBR Premix Ex Taq kit (Takara, Dalian, China). The expression of primiR-375, primiR-200a, pri-let-7e, and primiR-30e was detected using a TaqMan primiRNA assay (HS03303515_pri, Hs03303376_pri, Hs03295173_pri, and Hs03303424_pri). The relative expression of miRNA or mRNA was analyzed using the 2−ΔΔCT method. All results were normalized to GAPDH or U6. The primers for METTL14 mRNA and GAPDH are listed in Table S4.

Luciferase Reporter Assay

For miR-375 target gene luciferase reporter assays, target sequences containing predicted miR-375 binding sites were respectively synthesized and inserted into the pmirGLO luciferase vector (GeneCreat, China), and then co-transfected with miR-375 mimics (mimic) or its control (mimic-NC) into 293T and HCT116 cell lines using Lipofectamine 2000. Dual-luciferase reporter assay system (Promega, Madison, WI, USA) was used to detect the relative luciferase activity and normalized to Renilla luciferase activity.

RNA m6A Quantification

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA). The m6A RNA Methylation Assay Kit (ab185912; Abcam, UK) was used to detect the m6A content in the total RNA. In brief, 2 μL of the negative control, 2 μL of the diluted positive control, and 200 ng of sample RNA were added to a 96-well plate. Then, 50 μL of diluted capture antibody, detection antibody, and diluted enhancer solution were added into each well. Signaling was detected after adding 100 μL diluted developer solution and stop solution to each well within 2–10 min using a microplate reader at 450 nm. A simple calculation of the percentage of m6A in total RNA can be carried out using the following formula: m6A% = ([sample OD − NC OD] ÷ S)/([PC OD − NC OD] ÷ P) × 100%, where S is the amount of sample RNA in nanograms, and P is the amount of positive control in nanograms.

RNA Immunoprecipitation

An RNA immunoprecipitation (RIP) assay was performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) in accordance with the manufacturer’s protocol. Cells were lysed in RIP lysis buffer, and then 100 μL whole-cell extract was incubated with magnetic beads conjugated with anti-DGCR8 (ab90579; Abcam, UK) or immunoglobulin G (IgG) for 6 h at 4°C. After that, the beads were incubated with Proteinase K with shaking to remove protein. Finally, the coprecipitated RNAs were extracted and subjected to qRT-PCR using primers for primiRNAs and normalized to input.

For the m6A RNA binding assays, the Magna MeRIP m6A Kit (Millipore, Billerica, MA, USA) was used. In brief, RNAs were chemically fragmented to ∼100 nt, and fragmented RNA was then incubated with magnetic beads conjugated with m6A antibody (Millipore, Billerica, MA, USA) for immunoprecipitation. The enrichment of m6A-containing mRNA was then analyzed through qRT-PCR and normalized to input.

Immunohistochemistry

The CRC tissues, ANTs, and xenograft CRC tissues were collected, paraffin embedded, and cut into 4-μm-thick sections. Sections were incubated with rabbit anti-METTL14, anti-Ki67, anti-YAP1, and anti-SP1 at 4°C overnight, followed by secondary antibodies. The sections were visualized under an inverted microscope (Nikon, Japan) at ×200 magnification. The intensity of staining was scored by two independent pathologists in the following four categories: no staining = 0, weak staining = 1, moderate staining = 2, and strong staining = 3. The stain-positive sections were categorized into four grades: 0 (0%), 1 (1%–33%), 2 (34%–66%), and 3 (67%–100%). The final IHC score was calculated by multiplying the percentage of positive cells with the intensity score.

Ectopic Reporter Constructs

Ectopic reporter constructs were employed to analyze the primiR-375 process as previously described., We used the primiRNA reporter construct developed by Auyeung et al. and replaced the miRNA control pri-miR-1-1 with its altered version in which the adenosines (A’s) of the potential m6A motifs were mutated. Then we placed the query miRNA, either WT pri-miR-375 or a mutant version in which the A’s of the putative m6A motifs were mutated to thymidines (Ts), upstream of the pri-miR-1-1 control. Then these two constructs were used to transfect HEK293T cells using Lipofectamine 2000. The RNA was extracted 48 h later, and qRT-PCR was performed to evaluate the production of mature miR-375 and mature miR-1-1.

In Vivo Experiments

All animal experiments were approved by the Animal Care Committee of Nanjing First Hospital, Nanjing Medial University (acceptance no. SYXK 20160006). Six-week-old BALB/c nude mice were purchased from the College of Veterinary Medicine, Yang Zhou University. For the xenografted tumor model, 1 × 107 HCT116 cells in 0.2 mL PBS were subcutaneously injected into BALB/c nude mice, which were randomly divided into four groups (six mice per group). The volume of the tumors was calculated with the following equation: V = 0.5 × (length × width2). For metastasis experiments, 2 × 106 cells in 0.2 mL PBS were injected into the tail vein of nude mice, which were randomly divided into four groups (six mice per group). After 40 days of injection, the mice were sacrificed, and their lungs were removed and stained by H&E staining.

Statistical Analysis

All experiments were performed in triplicate. The data are expressed as the mean ± SD and analyzed by SPSS 22.0 software. Student’s t test and one-way ANOVA were used to estimate the differences between groups. Mann-Whitney U test or χ2 test was performed to analyze the relationship between METTL14 expression and clinicopathological features. The Kaplan-Meier method was applied to assess OS. The survival curves were compared with the log-rank test. Follow-up time was censored if the patient was lost to follow-up. A Cox proportional hazards model was used to perform multivariate analysis and calculate the 95% CI. A p value <0.05 was considered to be statistically significant.

Author Contributions

S.W. conceived the study. X.C. and M.X. designed the experiments. X. Xu, K.Z., X.L., L.S., and B.P. performed the in vitro experiments. X. Xu and K.Z. performed the in vivo experiments. B.H., Y.P., and H.S. provided assistance for data acquisition, data analysis, and manuscript editing. X.C. wrote the manuscript. The revision of this manuscript was guided by X. Xia.

Conflicts of Interest

The authors declare no competing interests.