An Alternatively Spliced Variant of METTL3 Mediates Tumor Suppression in Hepatocellular Carcinoma

Abstract

Many post-transcriptional mRNA processing steps play crucial roles in tumorigenesis and the progression of cancers, such as N6-methyladenosine (m⁶A) modification and alternative splicing. Upregulation of methyltransferase-like 3 (METTL3), the catalytic core of the m⁶A methyltransferase complex, increases m⁶A levels and results in significant effects on the progression of hepatocellular carcinoma (HCC). However, alternative splicing of METTL3 has not been fully investigated, and the functions of its splice variants remain unclear. Here, we analyzed both our and online transcriptomic data, obtaining 13 splice variants of METTL3 in addition to canonical full-length METTL3-A in HCC cell lines and tissues. Validated by RT-qPCR and Western blotting, we found that METTL3-D, one of the splice variants expressing a truncated METTL3 protein, exhibits higher levels than METTL3-A in normal human livers but lower levels than METTL3-A in HCC tumor tissues and cell lines. Further functional assays demonstrated that METTL3-D expression decreased cellular m⁶A modification, inhibited the proliferation, migration, and invasion of HCC cells, and was negatively associated with the malignancy of patient tumors, exhibiting functions opposite to those of full-length METTL3-A. This study demonstrates that the METTL3-D splice variant is a tumor suppressor that could potentially be used as a target for HCC therapy.

Keywords: METTL3; RNA m6A; hepatocellular carcinoma; splice variants; tumor suppressor.

Figure 1

Splice variants of METTL3 in human tissues and HCC cell lines. (A) Analyses of alternative splicing of METTL3 using online and our transcriptomic data. Upper, Sashimi plot of METTL3 using the aligned RNA-seq reads from HepG2 cells. Middle, comparison of the four METTL3 splice variant transcripts. The data were downloaded from Ensembl. Lower, comparison of the METTL3 splice variant protein domains. (B) mRNA levels of METTL3-A, B, C, and D in human tissues. Analyses were performed using data from GTExPortal. (C) mRNA levels of METTL3-A, -B, -C, and -D in human normal liver, HCC tumor tissues, and HepG2 cells. The data are expressed as means ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001 (t test).

Figure 2

METTL3-D is downregulated in HCC and correlates with tumor stage. (A) mRNA levels of METTL3-A, -B, -C, and -D in normal and HCC tissues from the GEPIA2 database. (B) RT–qPCR analyses of the mRNA levels of METTL3-A, -B, -C, and -D in NATs and tumor tissues from our patients. The clinical characteristics of all the patients are listed in Table S1. (C) mRNA levels of METTL3-A and METTL3-D are correlated with tumor stage. The mRNA levels were analyzed by RT–qPCR, and patients were grouped as early-stage HCC (Stage I) and late-stage HCC (Stage III). (D) The protein levels of METTL3-A were upregulated in HCC tumor tissues, while METTL3-D was downregulated. Right, Western blot analysis of Flag-tagged METTL3 variants on the pcDNA3 vector in HepG2 cells using the anti-FLAG antibody. Left, Western blot analysis of the endogenous METTL3 variants in HCC patient NATs and tumor tissues using the anti-METTL3 antibody. (E) Information concerning patients in panel (D). (F) The protein levels of METTL3-A and METTL3-D in patients were correlated with their tumor stages. The data are expressed as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (t test). Black and red asterisks have the same meaning.

Figure 3

METTL3-D mediates RNA m⁶A modification in HCC cells. (A, B) OE of the METTL3 splice variants (-B, -C, and -D) decreases the m⁶A levels of total RNAs in HepG2 cells, while OE of METTL3-A increases the m⁶A levels. (A) Dot blot assay. (B) Colorimetric m⁶A quantification assay. The signal intensities of dot blots were quantitated by ImageJ. (C) Elevation of the m⁶A levels in HCC tumor tissues. m⁶A of mRNAs was evaluated using the colorimetric m⁶A quantification assay. (D) Significant correlation between the m⁶A levels and tumor stages among HCC patients. The m⁶A levels of total RNA from each patient were analyzed by the dot blot assay and quantitated by ImageJ. (E) HCC patients with higher levels of METTL3-D had longer overall survival times (TCGA data). (F) HNSC patients with higher levels of METTL3-D had longer overall survival times (TCGA data). The data are expressed as means ± SD. ** p < 0.01, **** p < 0.0001 (t test).

Figure 4

An illustration of the relationship between METTL3-D expression and expected medication response (TCGA data).

Figure 5

METTL3-D inhibits the proliferation, migration, and invasion of HCC cells. (A) Expression of METTL3-D decreases the proliferation rate of HCCLM3 cells. Left, CCK-8 assay analysis; right, cell counting assay analysis. (B) The MTT assay showed that METTL3-D expression decreases the proliferation rate of HCCLM3 (left) and HepG2 (right) cells. (C) Expression of METTL3-D inhibits the migration of MHCC97L cells. Representative images are shown at 0 h and 48 h after transfections. (D) Expression of METTL3-D inhibits the invasion ability of MHCC97L cells. The data are expressed as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (t test).

Figure 6

The METTL3-D splice variant has an opposite function to that of the downstream genes of the canonical full-length METTL3-A. (A) RT–qPCR analyses of the mRNA levels of downstream target genes in METTL3-A- or METTL3-D-overexpressing HepG2 cells. SOCS2 and BATF2 were previously reported as mRNA stability-decreased target genes of METTL3, while EGFR and TAZ were previously reported as mRNA translation-affected target genes [31,32,33]. (B) mRNA levels of SOCS2 and BATF2 in two HCC patients. Compared with patient #58, patient #23 had higher levels of METTL3-D and higher levels of SOCS2 and BATF2. (C) Correlation coefficient between METTL3-D and BATF2 expression in HCC patients. The original data were downloaded from GEPIA2. The data in the bar graphs are expressed as means ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001 (t test).