YY1/miR-186/ZHX1 Circuitry Controls Invasion And Proliferation In Human Pancreatic Cancer

Objective :zinc finger and homeobox 1,known as a transcription repressor,has been reported being associated with the progression of cancers,such as gastric cancer,cholangiocarcinoma,liver cancer.However,the role of ZHX1 in pancreatic cancer remains unknown and gene regulation of ZHX1 is rarely studied.In this study,we aim to investigate the regulation mechanism of ZHX1 and functional roles of ZHX1 in pancreatic cancer.Methods:(1)Immunofluorescence analysis was used to investigate the location of ZHX1 in pancreatic cell.Immunohistochemistry was used to access the expression of ZHX1 in 53 pairs of pancreatic tumor tissues and adjacent non-tumor pancreatic tissues.We also analyzed the relationship between ZHX1 expression and clinicopathological characteristics.(2)In vitro experiments,RT-PCR and Western blot were performed to detect expression of ZHX1 in pancreatic cell lines SW1990,CFPAC-1,Patu8988,PL45,and Panc-1,and to examine the transfection efficiency of transfection after overexpression or knockdown by si RNA or plasmid.(3)Cell invasion assays,wound healing assay,CCK-8 assay and plate clone forming assay were used to investigate invasion,migration and proliferation of pancreatic cancer cells.(4)Through bioinformatics analysis,it was found that mi R-186-5p specifically binds to 3'-UTR of ZHX1 m RNA;transcription factor YY1 can bind to the promoter region of ZHX1 and the promoter region of mi R-186;(5)Chromatin immunoprecipitation(Ch IP)analysis and Luciferase reporter assay in pancreatic cancer cells is to confirm effect and sites of YY1 on ZHX1 promoter region and mi R-186 promoter region;RT-PCR and Western blot is performed to confirm effect and sites of mi R-186-5p on ZHX1 m RNA.(6)Western blot was conducted to analyse the level of phosphorylation of AKT after upregulation or downregulation of ZHX1 in pancreatic cancer.The AKT allosteric inhibitor MK2206 validated the role of p-AKT phosphorylation in ZHX1 regulation of pancreatic cancer invasion,migration and proliferation.Results:(1)Immunostaining results showed the ZHX1 was mainly located in the nucleus and slightly located in cytoplasm.ZHX1 was positive in 26.4%(14/53)of pancreatic cancer tissues and 77.3%(41/53)of adjacent non-tumor pancreatic tissues.Difference between the positive rate of ZHX1 in the pancreatic cancer tissues group and non-tumor tissues group is statistically significant(?2=27.549,P<0.05).In addition,ZHX1 was associated with tumor size,tumor differentiation and clinical stage(P<0.05,respectively).(2)In pancreatic cancer cells,downregulation of ZHX1 inhibited cell invasion,migration,and proliferation,whereas the opposite results were seen in the up-regulated group.(3)YY1/mi R-186 could regulate the expression of ZHX1 in pancreatic cancer cells,and YY1 could bind to the promoter of ZHX1 and mi R-186.(4)ZHX1 inhibited cell invasion,migration and proliferation in pancreatic cancer via inhibiting AKT phosphorylation.Conclusion:The expression of ZHX1 in pancreatic cancer tissues was at a low level,and low expression of ZHX1 was correlated with tumor size,tumor differentiation and clinical stage.Our results revealed a regulatory network in which YY1 exerts repression on ZHX1 at transcription level or promotion on mi R-186 transcription which repressing ZHX1 at posttranscriptional level.ZHX1 acts as a cancer suppressor in pancreatic cancer through inhibiting AKT phosphorylation.