ALKBH5 Inhibits Epithelial-mesenchymal Transition In Pancreatic Cancer Cells Via Wnt/?-catenin Signalling

Objective: To detect expression of ALKBH5(alk B homolog 5)in human pancreatic cancer cell lines and investigate its effects on migration,invasion and epithelial-mesenchymal transition(EMT)of pancreatic cancer cells,and to explore its possible molecular mechanisms and clinical significance.Methods: 1.The levels of ALKBH5 gene m RNA expression in normal pancreatic and pancreatic carcinoma were explored in ONCOMINE database to explore its clinical significance.ALKBH5 m RNA and protein expression levels in three human pancreatic cancer cell lines were detected by Real-time PCR and Western blot.2.ALKBH5 sh RNA interference plasmid p LKO.1-sh-ALKBH5(sh-ALKBH5)was constructed according to ALKBH5 sh RNA sequences and its interference effect was identified in m RNA and protein levels.ALKBH5 CDS sequences were cloned into the eukaryotic expression vector p3 x FLAG-CMV-24 to construct ALKBH5 overexpression plasmid 3x Flag-ALKBH5,and its overexpression effect was identified in m RNA and protein levels.3.sh-ALKBH5 interference plasmid was transfected into pancreatic cancer cell lines Pa Tu8988 and SW1990.3x Flag-ALKBH5 was transfected into SW1990 and Bx PC3 cells.Transwell migration assay and Transwell invasion assay were used to detect the effects of ALKBH5 on the biological behavior of migration and invasion in pancreatic cancer cells.4.sh-ALKBH5 interference plasmid was transfected into pancreatic cancer cell lines Pa Tu8988 and SW1990.3x Flag-ALKBH5 was transfected into SW1990 and Bx PC3 cells.EMT markers,metastasis-associated protein MMP2,MMP9 and related protein levels of Wnt/?-catenin signaling pathway were detected by Western blot.The ?-catenin/TCF-4 transcription activities were detected by Dual-Luciferase reporter assay.Results: 1.The levels of ALKBH5 gene expression in pancreatic cancer patients were explored in TCGA database and ALKBH5 high group shows longer-term overall survival.The relative protein and m RNA expression of ALKBH5 were differed in different pancreatic cancer cells,and were high in Pa Tu8988 and Bx PC3,low in SW1990.2.PCR identification and sequencing results showed that sh-ALKBH5 and 3x Flag-ALKBH5 plasmids were constructed successfully.In this study,the constructed plasmid sh-ALKBH5 can effectively inhibit the expression of ALKBH5 in pancreatic Pa Tu8988 and SW1990 cells.The constructed plasmid 3x Flag-ALKBH5 could efficiently overexpress ALKBH5 in SW1990 and Bx PC3 cells.3.Compared with the control group,the cell migration and invasion abilities were increased in Pa Tu8988 and SW1990 cells after down-regulating ALKBH5.On the contrary,the migration and invasion abilities were decreased in SW1990 and Bx PC3 cells after up-regulating ALKBH5.4.Knocking down of ALKBH5,the protein levels of EMT mesenchymal markers,metastasis-associated protein MMP2,MMP9 were increased in Pa Tu8988 and SW1990 cells,and the protein levels of ?-catenin and p-GSK-3? and ?-catenin/TCF-4 transcription activity in Wnt/?-catenin signaling pathway were significantly increased.On the contrary,up-regulating ALKBH5,the protein levels of EMT mesenchymal markers,metastasis-associated protein MMP2,MMP9 were decreased in SW1990 and Bx PC3 cells,and the protein levels of ?-catenin and p-GSK-3? and ?-catenin/TCF-4 transcription activity in Wnt/?-catenin signaling pathway were significantly reduced.Conclusion: 1.ALKBH5 may contribute to the migration and invasion of pancreatic cancer cells by regulating the Wnt/?-catenin signaling pathway,however,the specific mechanism needs further study.2.ALKBH5 plays a role of tumor-suppressive gene in pancreatic cancer migration,invasion and EMT in vitro.