miR-4770 promotes colorectal cancer progression by targeting PRKAA2 via m6A modification

Abstract

BACKGROUND

Colorectal cancer (CRC) is a malignant tumor with high incidence, and carries a high risk of morbidity and mortality. Although microRNAs and N6-methyladenosine (m6A) modification have been shown to be involved in the progression of CRC, the specific role of miR-4770 and its m6A-regulated mechanism in CRC is still unclear.

AIM

To investigate whether METTL3-mediated m6A modification regulates miR-4770 expression, and to clarify the functional significance of miR-4770/PRKAA2 axis in the malignant progression of CRC cells.

METHODS

CRC tissue samples and their matched normal counterparts were acquired from ten patients. For in vitro analyses, we used FHC (normal colon epithelial) cells, along with HCT116 and SW480 CRC cells. The amounts of miR-4770, METTL3, PRKAA2, and pri-miR-4770 transcripts were measured via qRT-PCR. Cell viability, migration, and invasion were assessed by MTT and Transwell chamber assays. Western blot analysis was performed to assess PRKAA2 protein abundance and the expression status of important components within the AMPK/mTOR signaling axis. The degree of m6 A modification of pri-miR-4770 and its binding to DGCR8 protein were analyzed by MeRIP and RIP techniques. Dual luciferase reporter assay was used to verify whether PRKAA2 is a direct downstream target of miR-4770. Finally, rescue experiments were carried out by simultaneously inhibiting miR-4770 and knocking down PRKAA2.

RESULTS

In CRC tissues and cell lines, the expression levels of miR-4770 and METTL3 were increased, while the expression of PRKAA2 was decreased. There was a positive correlation between the expression of METTL3 and miR-4770. Knockdown of METTL3 inhibited the expression of miR-4770, reduced the m6A modification level of pri-miR-4770, and reduced the enrichment of pri-miR-4770 and DGCR8 proteins. Conversely, overexpression of METTL3 produced the opposite effect. Functional experiments revealed that inhibiting miR-4770 lowered CRC cell viability, motility, and invasiveness, while forced expression of miR-4770 intensified these aggressive traits. PRKAA2 was identified as a direct target of miR-4770, which negatively modulates its expression at the post-transcriptional level. Downregulating PRKAA2 partly reversed the decreases in CRC cell viability, migration, and invasion that resulted from miR-4770 silencing. Additional experiments in SW480 cells verified that miR-4770 modulates PRKAA2 expression and the activity of the AMPK/mTOR signaling pathway.

CONCLUSION

According to this study, METTL3 likely promotes the processing of miR-4770 in an m6A-dependent manner. Meanwhile, miR-4770 enhances CRC cell viability, migration, and invasion, an effect that is at least partly mediated by direct targeting of PRKAA2 and subsequent regulation of the AMPK/mTOR pathway. The METTL3/miR-4770/PRKAA2 molecular axis could be a potential molecular target for future CRC research on mechanisms and treatment.

Keywords: Colorectal cancer; miR-4770; N6-methyladenosine methylation; METTL3; PRKAA2; Malignant progression

Core Tip: This study investigated the role of miR-4770 in colorectal cancer (CRC) and its regulation by N6-methyladenosine (m6A) modification. The results showed that miR-4770 and METTL3 were upregulated in CRC tissues and cells, whereas PRKAA2 was downregulated. METTL3 may promote the maturation of pri-miR-4770 through m6A modification, thereby increasing miR-4770 expression. Functionally, miR-4770 enhanced CRC cell viability, migration, and invasion by directly targeting PRKAA2 and modulating the AMPK/mTOR signaling pathway. These findings suggest that the METTL3/miR-4770/PRKAA2 axis may provide a potential mechanistic basis for CRC progression.