Deregulation of the MiR-193b-KRAS Axis Contributes to Impaired Cell Growth in Pancreatic Cancer

April Deregulation of the MiR-193b-KRAS Axis Contributes to Impaired Cell Growth in Pancreatic Cancer Xianglan Jin 0 1 2 Yang Sun 0 1 2 Haiyan Yang 0 1 2 Ji Li 0 1 2 Shuangni Yu 0 1 2 Xiaoyan Chang 0 1 2 Zhaohui Lu 0 1 2 Jie Chen 0 1 2 0 Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tsinghua University , Beijing , People's Republic of China 1 Funding: This work was supported by grants from the National Natural Science Foundation of China (Nos. 81172334 and 30471970) (URL: http://www. nsfc.gov.cn/), and the National Key Technology R&D program (No. 2006BAI02A14) (URL:http://www.most. gov.cn/index.htm). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Academic Editor: Martin Fernandez-Zapico, Schulze Center for Novel Therapeutics, Mayo Clinic , UNITED STATES Modulation of KRAS activity by upstream signals has revealed a promising new approach for pancreatic cancer therapy; however, it is not clear whether microRNA-associated KRAS axis is involved in the carcinogenesis of pancreatic cancer. Here, we identified miR-193b as a tumor-suppressive miRNA in pancreatic ductal adenocarcinoma (PDAC). Expression analyses revealed that miR-193b was downregulated in (10/11) PDAC specimens and cell lines. Moreover, we found that miR-193b functioned as a cell-cycle brake in PDAC cells by inducing G1-phase arrest and reducing the fraction of cells in S phase, thereby leading to dampened cell proliferation. miR-193b also modulated the malignant transformation phenotype of PDAC cells by suppressing anchorage-independent growth. Mechanistically, KRAS was verified as a direct effector of miR-193b, through which the AKT and ERK pathways were modulated and cell growth of PDAC cells was suppressed. Taken together, our findings indicate that miR-193b-mediated deregulation of the KRAS axis is involved in pancreatic carcinogenesis, and suggest that miR-193b could be a potentially effective target for PDAC therapy. - Competing Interests: The authors have declared that no competing interests exist. MicroRNAs (miRNAs) are a class of very small, non-coding RNAs that are evolutionarily conserved in many organisms. miRNAs suppress the expression of protein-coding genes in metazoans by binding to the 30 untranslated region (30-UTR) or even the coding region of their corresponding mRNA [13]. In this process, specific miRNAs pair-bond with target genes, leading to translational repression or/and mRNA destabilization [4]. Recent bioinformatic analyses have indicated that a great number of mRNAs are conserved target transcripts of miRNAs in mammals [5, 6]. miRNAs regulate a variety of cellular and developmental processes, including cell proliferation, survival, differentiation, animal development and disease [79]. Importantly, miRNAs display aberrant expression patterns in tumors and have emerged as important regulators of tumorigenesis and cancer progression, acting as tumor-suppressor genes or oncogenes [10, 11]. Pancreatic cancer is a malignancy with an extremely poor prognosis, with patients exhibiting dismal five-year relative survival rates of 6% [12]. This poor outcome is partly attributable to the inadequacy of currently available therapies and the fact that the cancer is usually diagnosed at late stages when these limited therapeutic options are no longer effective. Molecular alterations associated with pancreatic tumorigenesis and progression have been extensively investigated. Among the most frequent molecular alterations is KRAS, an oncogene that, when activated, causes cell growth and survival. Although KRAS mutations act as a key event in pancreatic carcinogenesis [13], targeting upstream signals that modulate KRAS activity may be a promising future approach for treating pancreatic cancer [14]. In silico screens using TargetScan (http://www.targetscan.org) have revealed that KRAS is targeted by the miRNA, miR-193b. In addition, Calin et al. found a high correlation between miRNA gene loci and cancer-associated genetic alterations [15]. Notable in this context, the miR-193b gene is located at 16p13, a region within chromosome 16 that exhibits genetic imbalance in pancreatic adenocarcinoma [16][17]. Moreover, aberrant expression of miR-193b has been detected in several human tumors [1820] and miR-193b is associated with Mitogenactivated Protein Kinase (MAPK) signaling in pancreatic cancer [21]. However, the expression and function of miR-193b is not well characterized in pancreatic diseases. In this study, we found that the expression of miR-193b was downregulated in pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, compared to adjacent benign pancreatic tissue. In order to investigate the role of miR-193b in these disorders, we performed an in vitro gain-of-function analysis by transfecting cell lines with miR-193b mimics. miR-193b function in these cells was assessed by examining cell viability, proliferation, apoptosis and colony-formation ability, and the underlying molecular mechanism was probed by testing KRAS as a target of miR-193b. This study was approved by the Peking Union Medical College Hospital Institutional Review Board. Written informed consent was obtained from all the patients. Cell lines and pancreatic tissue samples The pancreatic cancer cell lines, MIA PaCa-2, PANC-1, AsPC-1 and BxPC-3, and hTERT-HPNE (Human Pancreatic Nestin Expressing) human pancreatic duct epithelial cells were from American Type Culture Collection (ATCC). The type of hTERT-HPNE cells is intermediary cells formed during acinar-to-ductal metaplasia (according to ATCC information). All cell lines were cultured in complete growth medium containing Dulbecco's Modified Eagle's Medium (DMEM) and 10% fetal bovine serum (FBS) at 37C in a humidified 5% CO2 atmosphere. Cells were transfected with miRIDIAN hsa-miR-193b mimic or Negative Control #2 (Dharmacon, Thermo Fisher Scientific) at a final concentration of 25 nM using the transfection reagent DharmaFECT 4 (Dharmacon), according to the manufacturers instructions. Cell density was measured using a Countess Automated Cell Counter (Invitrogen). Pancreatic tissue samples were collected from the Department of Pathology at Peking Union Medical College Hospital. Fresh samples were obtained from patients undergoing pancreatic resection and were immediately snap-frozen and stored in liquid nitrogen. In addition to histological confirmation of PDAC in paraffin-embedded sections immediately after surgery, the cancer status of PDAC and matched adjacent pancreas was reconfirmed in frozen sections of each specimen prior to RNA extraction. Patient characteristics, including sex, age, pathological diagnosis, differentiation and lymph node involvement, are presented in S1 Table. RNA quantitation and in situ hybridization Total RNA was extracted from frozen tissue samples (...truncated)