Downregulation of miR-132 by promoter methylation contributes to pancreatic cancer development

Shuyu Zhang 0 y 0 Jun Hao 0 y 0 Fang Xie 0 Xiangui Hu 0 Cong Liu 0 Jian Tong 0 Jundong Zhou 0 Jinchang Wu 0 Chenghao Shao 0 0 Tissue samples Twenty pancreatic cancer tissue samples and matched normal adjacent pan- creatic tissue samples were obtained postoperatively in 2009 from the Depart- ment of General Surgery, Changhai Hospital, Second Military Medical University ( Shanghai, China ). All patients gave signed, informed consent for their tissues to be used for scientific research. Ethical approval for the study was obtained from the Department of General Surgery, Changhai Hospital, Second Military Medical University ( Shanghai, China ). All diagnoses were based on pathological and/or cytological evidence. The histological features of the specimens were evaluated by a senior pathologist according to the WHO classification criteria. Tissues were obtained before chemotherapy and radia- tion therapy and were immediately frozen and stored at 80 C prior to the microarray, real-time polymerase chain reaction (PCR) and methylation analyses The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: - MicroRNAs (miRNAs), which regulate gene expression by partial complementarity to the 3# untranslated region of their target genes, have been implicated in cancer initiation and progression. However, the molecular mechanism underlying the regulation of miRNA expression during pancreatic tumorigenesis has not been extensively reported. In this study, we first compared the miRNA expression in human pancreatic cancers and adjacent normal tissues by miRNA array and identified 12 differentially expressed miRNAs. miR-132, which is downregulated in tumors, was further studied in greater detail. Decreased expression of miR-132 was confirmed in 16 of 20 pancreatic carcinomas (P < 0.0001), compared with their respective benign tissues by TaqMan miRNA assays. miR-132 expression was remarkably influenced by promoter methylation in PANC1 and SW1990 cells. Promoter hypermethylation was observed in tumor samples but not in the normal counterparts, and the expression of miR-132 negatively correlated with its methylation status (P 5 0.013). miR-132 was transcribed by RNA polymerase II, and Sp1 played a major role in miR-132 transcription. The expression of Sp1 correlated with that of miR-132 in tissues. Moreover, cancerous tissues showed significantly lower Sp1-binding affinity to the miR132 promoter, relative to non-tumor samples. Proliferation and colony formation of pancreatic cancer cells were suppressed in cells transfected with miR-132 mimics and enhanced in cells transfected with miR-132 inhibitor by negatively regulating the Akt-signaling pathway. Our present findings illustrate the mechanism driving miR-132 downregulation and the important role of miR-132 in pancreatic cancer development. Introduction Pancreatic cancer is an aggressive malignancy with one of the worst mortality. It is the sixth leading cause of death from malignant disease in China and the fourth leading cause of cancer related death in the USA (1,2). The majority of patients have developed an aggressive form of the disease by the time of diagnosis, limiting the potential Abbreviations: CREB, cyclic adenosine 3#,5#-monophosphate response element-binding; ChIP, chromatin immunoprecipitation; ERK, external signalregulated kinase; mRNA, messenger RNA; miRNA, microRNA; OD, optical density; PCR, polymerase chain reaction. yThese authors contributed equally to this work. for therapeutic intervention (3). At this stage, several genetic and epigenetic changes have taken place, resulting in the silencing of tumor suppressors, overexpression of oncogenes and ultimately tumor progression (4). In recent years, there have been important advances in understanding the molecular biology of pancreatic cancer and genetic analyses have shown that the basis of this dismal disease is extremely complex and heterogenous (5). MicroRNAs (miRNAs) are small non-coding regulatory RNAs that suppress gene expression through partial complementary elements in the 3# untranslated regions of their target messenger RNAs (mRNAs) (6). Most animal miRNAs are evolutionarily conserved and are often found in clusters (7). Primary miRNAs with the stem-loop structure are transcribed by RNA polymerases (RNA Pols) and are processed both in the nucleus and cytoplasm by Drosha and Dicer to generate mature miRNAs (8,9). The mature miRNA is assembled into a miRNA-induced silencing complex, which then directs its binding to the cognate sequence in the 3# untranslated region of the target mRNAs. miRNAs are implicated in a wide variety of biological processes including cell proliferation, apoptosis, metabolism, cell differentiation and tumor initiation and promotion (10), and their identification may provide new insights in the elucidation of cancer progression (11). Over the past several years, advanced technologies have been used to quantify miRNAs in pancreatic tumor tissues and several deregulated miRNAs have recently been revealed including miR-21, miR-146, miR-221 and miR-196a (12). These findings suggest the involvement of miRNAs in the pathogenesis of pancreatic cancer. The deregulation of miRNAs might be attributed to disorders in transcription and subsequent procession. Therefore, transcriptional regulation of miRNAs may represent an underexplored etiology of pancreatic cancer. However, few studies have demonstrated the mechanism of miRNA transcription during the development of pancreatic cancer. Furthermore, the regulatory role of only a few miRNAs has been elucidated in pancreatic cancer initiation and progression. More extensive investigations of miRNA function are warranted. In this study, we first analyzed miRNA expression between normal and cancerous pancreatic tissues and identified 12 preferentially expressed miRNAs. We further investigated the underlying mechanism of miR-132 transcription and the consequence of its deregulation. Cis-modulating elements and trans-factors were identified in the miR-132 promoter. Downregulation of miR-132 in tumors was caused by promoter hypermethylation and decreased Sp1-binding affinity to its promoter. Moreover, miR-132 attenuates proliferation and colony formation of pancreatic cancer cells via the Akt-signaling pathway. Materials and methods miRNA microarray and real-time PCR analyses Total RNA was extracted from cells or tissues with Trizol reagent (Invitrogen, Carlsbad, CA). Microarray-based miRNA expression profiling was performed using the miRCURY LNA human microRNA Array (Exiqon, Vedbaek, Denmark). The microarrays contained 1200 assay probes corresponding to all of the annotated human and non-human primate miRNA sequences (miRBase, version 12, 2008; the Wellcome Trust Sanger Institute, Cambridgeshire, UK). Total RNA labeling and hybridization were performed using standard conditions according to manufacturer instructions. The expression levels of matur (...truncated)