YTHDF2 Orchestrates Proliferation/Epithelial Mesenchymal Transition Dichotomy In Pancreatic Cancer Cells

Objective: To investigate the expression profile of YTHDF2 in pancreatic cancer tissues and 3 different pancreatic cell lines and the effect of YTHDF2 on the proliferation,migration and invasion of pancreatic cancer cell.To find the preliminary mechanism of the effect of EMT,which provide new strategy for the diagnosis and treatment of pancreatic cancer.Methods:(1)We first analyzed the YTHDF2 mRNA and protein expression in normal pancreas and pancreatic cancer by public database;(2)Western blot and qPCR was performed to detect the relative mRNA and protein expression of YTHDF2 in pancreatic cancer cell lines(PaTu8988,SW1990,BxPC3).YTHDF2 short hairpin RNA(shRNA)sequence was designed and inserted into pLKO.1-TRC vector,then packaging lentivirus to infect SW1990 and Bx PC3 cells.Stable infected cells were obtained by puromycin screening.ShRNA silencing efficiency was tested by Western blot and qPCR;(3)Colony formation assay and CCK8 were applied to assess the growth ability of cells with different treatments,proliferation-related protein CyclinD1 and upstream protein were detected by Western blot;(4)Transwell migration assay and wound healing assay were performed to evaluate the migration abilities,Transwell invasion assay was performed to evaluate the invasion abilities,the expression of cell invasion-related protein MMP2 and MMP9 at both mRNA and protein levels were detected by qPCR and Western blot;(5)The expression of EMT-related protein E-cadherin,Vimentin,Snail and YAP were detected by Western blot,and illuminated the mechanism of epithelial-mesenchymal transiton effected by YTHDF2.Results:(1)Public database showed that YTHDF2 mRNA and protein levels were lower in normal pancreatic tissues than that in pancreatic cancer tissues,YTHDF2 expression increased successively in stage I,stage II,stage III and stage IV groups,and the stage I group presented the lowest and stage IV the highest YTHDF2 expression levels,YTHDF2 expression in Pathologic T1 and T2 was lower than that in Pathologic T3 and T4;(2)The expression of YTHDF2 was higher in SW1990 and Bx PC3 cells,The specific shRNA against YTHDF2 plasmid(sh-YTHDF2)was successfully constructed and infected intoSW1990 and Bx PC3 cells,Western blot and qPCR displayed that the expression of YTHDF2 was efficiently inhibited at both protein and mRNA levels;(3)YTHDF2down-regulation caused the ability of cell colony formation and proliferation were significantly inhibited in SW1990 and Bx PC3 cells,YTHDF2 knockdown resulted in down-regulation of p-Akt,p-GSK3β and CyclinD1;(4)It revealed that YTHDF2 depletion promoted migration and invation,led to up-regulation of MMP2 and MMP9 at both mRNA and protein levels;(5)YTHDF2 knockdown induced down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal markers Vimentin,Snail,promoted EMT probably via up-regulation of YAP but not TGF-β/Smad signaling in pancreatic cancer cells.Conclusions:(1)YTHDF2 is up-regulated in pancreatic cancer;(2)YTHDF2 orchestrates epithelial-mesenchymal transition /proliferation dichotomy in pancreatic cancer cells;(3)YTHDF2 depletion promoted EMT probably via up-regulation of YAP but not TGF-β/Smad signaling in pancreatic cancer cells.