m1A-regulated DIAPH3 promotes the invasiveness of colorectal cancer via stabilization of KRT19.

Background: In recent years, the emphasis has shifted to understanding the role of N1-methyladenosine (m1A) in tumor progression as little is known about its regulatory effect on mRNA and its role in the metastasis of colorectal cancer (CRC).

Methods: We performed methylated RNA immunoprecipitation sequencing of tumor tissues and tumor-adjacent normal tissues from three patients with CRC to determine the m1A profile of mRNA in CRC. The expression of diaphanous-related formin 3 (DIAPH3) and its correlation with clinicopathological characteristics of CRC were evaluated using immunohistochemistry and online datasets. The role of DIAPH3 in the migration and invasion of CRC cells was evaluated using wound healing assay, Transwell assay and xenograft metastatic model. The downstream targets of DIAPH3 were screened using mass spectrometry. By co-transfecting DIAPH3 siRNA and a keratin 19 (KRT19) ectopic plasmid into CRC cells, the role of DIAPH3-KRT19 signaling axis was confirmed.

Results: The mRNA level of DIAPH3 and its m1A modifications increased simultaneously in the CRC tissues. In addition, high DIAPH3 expression in CRC tissues is significantly associated with metastasis and progression to an advanced stage. After the knockdown of DIAPH3, the migration and invasion capabilities of CRC cells suffered a notable decline, which could be rescued by overexpressing KRT19. In addition, the proteasome inhibitor MG132 could block the degradation of KRT19 induced by DIAPH3 silencing.

Conclusions: Our study reveals that DIAPH3 mRNA was modified in CRC cells by m1A methylation. Silencing DIAPH3 suppresses the migration and invasion of CRC cells, potentially through the proteasome-dependent degradation of downstream KRT19.