Role Of RNA M~6A Demethylase ALKBH5 In Regulation Of Invasion And Metastasis In Pancreatic Cancer

Object:To provide experimental basis for further study of the function and potential mechanism of m6A demethylase ALKBH5 involved in regulation of pancreatic ductal adenocarcinoma(PDAC).Methods:1.IHC were used to detect the expression of m6A methyltransferase METTL3/METTL14/WTAP,demethylase ALKBH5/FTO and binding protein YTHDF1/YTHDF2/YTHDF3 in PDAC,correlation with clinicopathological features and prognosis were analyzed.2.Analysis on migration and invasion capability of ALKBH5 stable overexpression and knockdown pancreatic cancer MIA PaCa-2 and PANC-1 cells through transwell and wound healing.3.ALKBH5 stable overexpression MIA PaCa-2 cells were injected into nude mice through tail vein,metastasis status was detected by in vivo imaging system.4.RNA immunoprecipitation sequencing(RIP-seq),methylated RNA immunoprecipitation sequencing(MeRIP-seq)and wertern blot(WB)were used to explore the potential mechanism involved in ALKBH5-mediated regulation of pancreatic cancer.Results:1.Expression of m6A regulators in PD AC(1)In three m6A methyltransferases,WTAP was upregulated in cancer cells when compared with normal ductal epithelial cells,METTL3/METTL14/WTAP was downregulated in stroma cells when compared with cancer cells.Expression of METTL3 in cancer cells and stroma cells is related to gender,METLL14 in stroma cells is related to bile duct invasion,WTAP in cancer cells is related to neurological invasion.Expression of METTL3/METTL14/WTAP has no association with PDAC survival time.(2)In two m6A demethylase,ALKBH5/FTO was downregulated in cancer cells when compared with normal ductal epithelial cells,expression of ALKBH5/FTO had no difference in stroma cells when compared with cancer cells.Expression of ALKBH5/FTO has no relation to clinicopathological features either in cancer cells nor in stroma cells.Expression of ALKBH5 in tumor cells and FTO in stroma cells is associated with PDAC survival time.High expression level indicates better prognosis.(3)In three m6A binding proteins,YTHDF3 was upregulated in cancer cells when compared with normal ductal epithelial cells,YTHDF1/YTHDF2/YTHDF3 was downregulated in stroma cells when compared with cancer cells.Expression of YTHDF3 in stroma cells is related to gender.Expression of YTHDF1 in stroma cells is associated with PDAC survival time.High expression level indicates better prognosis.2.ALKBH5 inhibits invasion and metastasis of pancreatic cancer cells MIA PaCa-2 and PANC-1 in vitro.3.ALKBH5 inhibits metastasis of MIA PaCa-2 in vivo.4.Sequencing analysis indicates that functions of differential expression genes after ALKBH5 overexpression are related to cell adhesion,cell junction and cadherin binding.These differential expression genes including metastasis-associated genes N-Cadherin,Vimentin,EXT1,MAP3K4,EIF3C,EIF3D,EIF3L,etc.Their expression changes were verified in ALKBH5 stable overexpression and knockdown MIA PaCa-2.5.ALKBH5 inhibts expression of EMT markers N-Cadherin and Vimentin expression in MIA PaCa-2.6.ALKBH5 has no obvious enrichment of N-Cadherin and Vimentin,but EXT1,MAP3K4,EIF3C,EIF3D,EIF3L can be enriched by ALKBH5,7.ALKBH5 elavates m6A level of EXT 1.Conclusions:1.ALKBH5 was downregulated in PD AC and associated with poor prognosis.2.ALKBH5 inhibits invasion and metastasis of pancreatic cancer in vitro and in vivo.3.Inhibition of pancreatic cancer invasion and metastasis is related to regulation of EMT through m6A-mediated EXT,MAP3K4,EIF3C,EIF3D and EIF3L.