[The Regulatory Effect of RNA m 6 A Methylation Modification on KDM4B Gene Expression in t (8;21) AML Cells by MeRIP-qPCR].

OBJECTIVE: To confirm the direct regulatory effect of WTAP -mediated RNA m6 A modification on the KDM4B gene in t (8;21) acute myeloid leukemia (AML) cells through MeRIP combined with reverse transcription real-time quantitative PCR (RT-qPCR) technology.

METHODS: The lentivirus-mediated shRNA target WTAP or KDM4B gene was used to transfect the t (8;21) AML cell lines: Kasumi-1 and SKNO-1, and cells transfected with randomly shuffled shRNA as the control. Using the Ultrapure RNA Extraction Kit (DNase I) to extract RNA. The Magna MeRIPTM m6 A Kit was used to enrich methylated modified fragments, and detect the m6 A methylated RNA regions by RT-qPCR, and the protein and mRNA expression levels of WTAP and KDM4B in cells were detected by Western blot and reverse transcription real-time quantitative PCR (RT-qPCR). Colony formation assays were used to detect the colony ability of cells in vitro .

RESULTS: Silencing the expression of WTAP in Kasumi-1 cells, the enrichment of m6 A methylation modification was significantly decreased in the 3'UTR of KDM4B mRNA( P < 0.01), and the protein( P < 0.001) and mRNA (Kasumi-1: P < 0.001; SKNO-1: P < 0.01) expression levels of KDM4B were also significantly inhibited in Kasumi-1 and SKNO-1 cells upon WTAP knockdown (all P < 0.01), accompanied by a significant decrease in the colony-forming ability of both cell lines (both P < 0.01).

CONCLUSION: In t(8;21) AML cell lines, WTAP could regulate the expression of KDM4B by regulating the m6 A modification of the 3'UTR of KDM4B mRNA, and silencing the expression of KDM4B could inhibit the cellular proliferation in vitro .