Shengshu Li1, Xiaoxin Lu2, Dongyang Zheng3, Weizong Chen4, Yuzhu Li5, Fang Li6. 1. Department of Pulmonary and Critical Care Medicine, The 8th Medical Center of Chinese, PLA General Hospital, Beijing, 100091, China. 2. Department of Oncology, Hainan Hospital of PLA General Hospital, Haitang District, Sanya, 572013, China. 3. Department of Pulmonary and Critical Care Medicine, Hainan Hospital of PLA General Hospital, Haitang District, Sanya, 572013, China. 4. Xinglong Red Cross Hospital, Wanning, 571533, China. 5. Department of Pulmonary and Critical Care Medicine, Hainan Hospital of PLA General Hospital, Haitang District, Sanya, 572013, China. lidocors@163.com. 6. Department of Oncology, Hainan Hospital of PLA General Hospital, Haitang District, Sanya, 572013, China. e9125333songfang@163.com.
Abstract
OBJECTIVE: Lung cancer (LC) remains a threatening health issue worldwide. Methyltransferase-like protein 3 (METTL3) is imperative in carcinogenesis via m6A modification of microRNAs (miRNAs). This study estimated the effect of METTL3 in LC by regulating m6A methylation-mediated pri-miR-663 processing. METHODS: miR-663 expression in 4 LC cell lines and normal HBE cells was determined using RT-qPCR. A549 and PC9 LC cells selected for in vitro studies were transfected with miR-663 mimics or inhibitor. Cell viability, migration, invasion, proliferation, and apoptosis were detected by CCK-8, Transwell, EdU, and flow cytometry assays. The downstream target genes and binding sites of miR-663 were predicted via Starbase database and validated by dual-luciferase assay. LC cells were delivered with oe-METTL3/sh-METTL3. Crosslinking between METTL3 and DGCR8 was verified by co-immunoprecipitation. Levels of m6A, miR-663, and pri-miR-663 were measured by m6A dot blot assay and RT-qPCR. m6A modification of pri-miR-663 was verified by Me-RIP assay. Finally, the effects of METTL3 in vivo were ascertained by tumor xenograft in nude mice. RESULTS: miR-663 was upregulated in LC cells, and miR-663 overexpression promoted cell proliferation, migration, invasion, and inhibited apoptosis, but miR-663 knockdown exerted the opposite effects. miR-663 repressed SOCS6 expression. SOCS6 overexpression annulled the promotion of miR-663 on LC cell growth. METTL3 bound to DGCR8, and METTL3 silencing elevated the levels of pri-miR-663 and m6A methylation-modified pri-miR-663, and suppressed miR-663 maturation and miR-663 expression. METTL3 facilitated tumor growth in mice through the miR-663/SOCS6 axis. CONCLUSION: METTL3 promotes LC progression by accelerating m6A methylation-mediated pri-miR-663 processing and repressing SOCS6.
OBJECTIVE: Lung cancer (LC) remains a threatening health issue worldwide. Methyltransferase-like protein 3 (METTL3) is imperative in carcinogenesis via m6A modification of microRNAs (miRNAs). This study estimated the effect of METTL3 in LC by regulating m6A methylation-mediated pri-miR-663 processing. METHODS: miR-663 expression in 4 LC cell lines and normal HBE cells was determined using RT-qPCR. A549 and PC9 LC cells selected for in vitro studies were transfected with miR-663 mimics or inhibitor. Cell viability, migration, invasion, proliferation, and apoptosis were detected by CCK-8, Transwell, EdU, and flow cytometry assays. The downstream target genes and binding sites of miR-663 were predicted via Starbase database and validated by dual-luciferase assay. LC cells were delivered with oe-METTL3/sh-METTL3. Crosslinking between METTL3 and DGCR8 was verified by co-immunoprecipitation. Levels of m6A, miR-663, and pri-miR-663 were measured by m6A dot blot assay and RT-qPCR. m6A modification of pri-miR-663 was verified by Me-RIP assay. Finally, the effects of METTL3 in vivo were ascertained by tumor xenograft in nude mice. RESULTS: miR-663 was upregulated in LC cells, and miR-663 overexpression promoted cell proliferation, migration, invasion, and inhibited apoptosis, but miR-663 knockdown exerted the opposite effects. miR-663 repressed SOCS6 expression. SOCS6 overexpression annulled the promotion of miR-663 on LC cell growth. METTL3 bound to DGCR8, and METTL3 silencing elevated the levels of pri-miR-663 and m6A methylation-modified pri-miR-663, and suppressed miR-663 maturation and miR-663 expression. METTL3 facilitated tumor growth in mice through the miR-663/SOCS6 axis. CONCLUSION: METTL3 promotes LC progression by accelerating m6A methylation-mediated pri-miR-663 processing and repressing SOCS6.
Authors: Isaia Barbieri; Konstantinos Tzelepis; Luca Pandolfini; Junwei Shi; Gonzalo Millán-Zambrano; Samuel C Robson; Demetrios Aspris; Valentina Migliori; Andrew J Bannister; Namshik Han; Etienne De Braekeleer; Hannes Ponstingl; Alan Hendrick; Christopher R Vakoc; George S Vassiliou; Tony Kouzarides Journal: Nature Date: 2017-11-27 Impact factor: 49.962