Methylation recognition protein YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) regulates the proliferation, migration and invasion of osteosarcoma by regulating m6A level of CCR4-NOT transcription complex subunit 7 (CNOT7).

N6-methyladenosine (m6A) is one of the most significant modifications in human mRNAs. Emerging evidence indicates that m6A participates in the initiation and development of malignant tumors. Nevertheless, the biological roles and mechanism of m6A in osteosarcoma (OS) remain unclear. The purpose of this study was to investigate the role and mechanism of the methylation recognition protein-YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in OS. The YTHDF1 expression in OS was detected by qRT-PCR and Western blot assay. M6A quantification was utilized to measure the methylation level of OS. Cell counting kit-8 (CCK8), 5-Ethynyl-2'-deoxyuridine (EdU) assay and transwell experiments were conducted to confirm the biological effects of YTHDF1 on OS cells. The bioinformatics websites and in vitro assays were conducted to analyze the downstream targets of YTHDF1 was upregulated in OS tissues at mRNA and protein level. The results showed that the expression level of YTHDF1 might be closely associated with the poor prognosis for OS patients. Inhibition of YTHDF1 could suppress the proliferation, migration and invasion of the OS cells. Moreover, we found that CCR4-NOT transcription complex subunit 7 (CNOT7) might be the potential target of YTHDF1, which was upregulated in OS tissues. YTHDF1 could recognize the m6A sites of CONT7 and promote its expression in an m6A manner. Moreover, methyltransferase-like 3 (METTL3) could promote the m6A level of CONT7. YTHDF1 was upregulated in OS and could promote cell proliferation, migration and invasion. The METTL3-CONT7-YTHDF1 regulatory axis might be the potential target for the prognosis and therapy of OS.

Effect of CNOT7 Gene Knockdown on the Immune Microenvironment of HepG2 Cells by Reduced TGF-β1 Secretion / 中国医科大学学报

Objective To study the effect of human CCR4-NOT transcription complex subunit 7 (CNOT7) gene knockdown on the immune microenvironment of HepG2 cells and explore its significance. Methods We designed a cell transfection protocol and performed the experiment with three groups:CNOT7-targeted knockdown group, control group, and CNOT7 overexpression group. The transfection efficiency was assessed using inverted fluorescence microscopy, and the expression level of CNOT7, transforming growth factor-β1 (TGF-β1), and nuclear factor-kappa B (NF-κB) p65 proteins was determined by Western blotting. The concentration of TGF-β1 secreted in the cell culture supernatant was measured by ELISA. The sensitivity of tumor cells to the killing function of natural killer (NK) cells was detected by flow cytometry. Results Compared with the control group, the expression level of TGF-β1 and NF-κB p65 proteins was significantly decreased in the CNOT7-targeted knockdown group, and the TGF-β1 concentration in the culture supernatant was also significantly reduced. However, in the CNOT7 overexpression group, the expression level of the two proteins and TGF-β1 concentration were significantly increased. NK cells were co-cultured with tumor cells, and the apoptosis rate of HepG2 cells transfected with CNOT7-specific shRNA was significantly increased. However, in the CNOT7 overexpression group, the apoptosis rate was significantly decreased. Conclusion CNOT7 forms the immune microenvironment of hepatocellular carcinoma. Targeted knockdown of CNOT7 can reduce TGF-β1 secretion and enhance the killing function of NK cells toward HepG2 cells.