Study On The Function Of N6-methyladenosine RNA Binding Protein 1(YTHDF1) And N6-methyladenosine RNA Binding Protein 2(YTHDF2) On Gastric Cancer Cells

Gastric cancer,one of the most common malignant tumors in the world,has the characteristics of difficult to early diagnosis and poor prognosis,thus seriously endangering human health.Some studies have shown that epigenetics plays an important role in the development of gastric cancer.N6-methyladenosine modification(m~6A)is widely conserved among in eukaryotic species with a regulation of post-transport phase,and its main involved proteins include methylation of enzymes called“writers”,demethylation of transferase called“erasers”and m~6A methylation binding proteins called“readers”.Objective:To study the relationship between m~6A methylation banding protein YTHDF1 and YTHDF2 in gastric cancer,and to observe the function of gastric cancer cells by knocking down YTHDF1 or YTHDF2 in gastric cancer cells,and providing evidence in vitro on the development of gastric cancer.To establish the relationship between gastric cancer and m~6A methylation modification.Methods:The Cancer Genome Atlas(TCGA)database was downloaded from UCSC Cancer browser and to search for the differential expressions of YTHDF1 and YTHDF2 mRNA in gastric cancer tissues.Short hairpin RNA(ShRNA)targeting YTHDF1 or YTHDF2 were designed and cloned into lentivirus expression vector pLKO.1.Furthermore,MGC-803 and HGC-27 gastric cancer cells were transfected with p LKO.1-shRNA to knockdown the expression of YTHDF1 or YTHDF2,which was confirmed by the detection of mRNA and protein expression using Real-Time quantitative PCR and Western blotting,respectively.Then cell function tests were done to detect qualified cells,cell proliferation was detected by CCK-8 assay,cell migration and cell invasion were observed by Transwell assay,and cell cycle and apoptosis were examined by flow cytometry.The total RNA was extracted from the cells which successfully knocking down the protein level,and the differential gene was screened after the next generation sequencing by bioinformatics.Results:According to the TCGA database,the expression of YTHDF1or YTHDF2 mRNA in gastric cancer were significantly higher than that in the normal tissues.Gastric cancer cells stably expressing YTHDF1-shRNA/YTHDF2-shRNA were successfully established.Furthermore,the proliferation capacity of YTHDF1-shRNA-expressing MGC-803 cells and HGC-27 cells were significantly inhibited compared with the controls.Similarly,YTHDF1 could repress MGC-803 cells and HGC-27 cells migration and invasion.360 differentially expressed genes were identified by the next sequencing of MGC-803 cells with stable expression of YTHDF1-shRNA,with 197 genes raised and 163 genes reduced.The proliferation capacity of YTHDF2-shRNA-expressing MGC-803cells was significantly inhibited compared with the controls and the percentage of YTHDF2-shRNA-expressing MGC-803 cells in G1 phase increased and in S phase decreased compared with the controls.Meanwhile,cell apoptosis ratio of YTHDF2-shRNA-expressing MGC-803 was significantly higher compared with the control groups.Conclusion:Knockdown of YTHDF1 in MGC-803 cells and HGC-27cells may inhibit cell proliferation,repress cells migration and invasion.360differentially expressed genes were identified by the next sequencing of MGC-803 cells with stable knock-down the expression of YTHDF1 mRNA,with 197 genes raised and 163 genes reduced.Knockdown of YTHDF2 in MGC-803 cells may inhibit cell proliferation and promote apoptosis.