Identification of RegIV as a novel GLI1 target gene in human pancreatic cancer.

Feng Wang,Ling Xu,Aiwu Ke,Lijuan Yang,Yinshi Huang,Xingpeng Wang,Shanshan He,Wenhui Mo,Chuanyong Guo,Guoyong Hu,Xuanfu Xu,Vladimir N Uversky

doi:10.1371/journal.pone.0018434

Abstract

Background and AimsGLI1 is the key transcriptional factor in the Hedgehog signaling pathway in pancreatic cancer. RegIV is associated with regeneration, and cell growth, survival, adhesion and resistance to apoptosis. We aimed to study RegIV expression in pancreatic cancer and its relationship to GLI1.MethodsGLI1 and RegIV expression were evaluated in tumor tissue and adjacent normal tissues of pancreatic cancer patients and 5 pancreatic cancer cell lines by qRT-PCR, Western blot, and immunohistochemistry (IHC), and the correlation between them. The GLI1-shRNA lentiviral vector was constructed and transfected into PANC-1, and lentiviral vector containing the GLI1 expression sequence was constructed and transfected into BxPC-3. GLI1 and RegIV expression were evaluated by qRT-PCR and Western blot. Finally we demonstrated RegIV to be the target of GLI1 by chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assays (EMSA).ResultsThe results of IHC and qRT-PCR showed that RegIV and GLI1 expression was higher in pancreatic cancer tissues versus adjacent normal tissues (p<0.001). RegIV expression correlated with GLI1 expression in these tissues (R = 0.795, p<0.0001). These results were verified for protein (R = 0.939, p = 0.018) and mRNA expression (R = 0.959, p = 0.011) in 5 pancreatic cancer cell lines. RegIV mRNA and protein expression was decreased (94.7±0.3%, 84.1±0.5%; respectively) when GLI1 was knocked down (82.1±3.2%, 76.7±2.2%; respectively) by the RNAi technique. GLI1 overexpression in mRNA and protein level (924.5±5.3%, 362.1±3.5%; respectively) induced RegIV overexpression (729.1±4.3%, 339.0±3.7%; respectively). Moreover, CHIP and EMSA assays showed GLI1 protein bound to RegIV promotor regions (GATCATCCA) in pancreatic cancer cells.ConclusionGLI1 promotes RegIV transcription by binding to the RegIV gene promoter in pancreatic cancer.

Highlights

Pancreatic cancer (PC) is the fourth most common cancerrelated cause of mortality in the Western world [1,2,3] and has a dismal prognosis despite considerable progress in management
GLI1 and RegIV expression in pancreatic cancer tissues To study GLI1 and RegIV expression in PC, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and IHC were used in 12 paired biopsy tissues
GLI1 expression was higher in 9 cases (9/12) compared with adjacent normal pancreatic tissues (p = 0.011; Figure 2); RegIV expression was higher in 9 cases (9/12) (p = 0.011; Figure 2)

Summary

Introduction

Pancreatic cancer (PC) is the fourth most common cancerrelated cause of mortality in the Western world [1,2,3] and has a dismal prognosis despite considerable progress in management. Research over the last two decades has shown that PC is a genetic disease fundamentally, caused by inherited germline and acquired somatic mutations in cancer-associated genes. Multiple alterations in genes that are important in PC progression have been identified, for example K-ras, INK4A, p53, and SMAD4/DPC4 [4,5]. PC is characterized by nearuniversal mutations in K-ras and frequent deregulation of crucial embryonic signalling pathways, including the Hedgehog (HH) signaling pathway [6,7]. GLI1 is the key transcriptional factor in the Hedgehog signaling pathway in pancreatic cancer. We aimed to study RegIV expression in pancreatic cancer and its relationship to GLI1

Objectives

Methods

Results

Conclusion