Because of the apparent absence of the LRH1 receptor in these cells, we used AsPC-1 and Capan-1 to establish stable cell lines for further experiments. == Fig. == Pancreatic cancer (PC) is the fourth-leading cause of cancer mortality in the United States, with an estimated 43,920 new cases and 37,390 deaths in 2012.[12] PC has the lowest 5-year survival rate of all malignancies due to aggressive progression and multiple-drug resistance.[12] Previous studies have revealed that signaling pathways involved in the development of the embryonic pancreas also play major roles in the malignant transformation of the adult pancreas.[9,13] We hypothesize that liver receptor homolog 1 (LRH1) (also called Nuclear Receptor Subfamily 5 Group A Member 2 [NR5A2]) is a driving factor that is shared between pancreatic embryogenesis and oncogenesis.[7] LRH1 is a promoter binding factor and an alpha-fetoprotein transcriptional factor. LRH1 is a key downstream target of the pancreatic and duodenal homeobox 1 regulatory cascade that regulates pancreatic development, differentiation, and function.[1] Importantly, accumulating evidence indicates participation of LRH1 in the pathogenesis of multiple tumors, such as gastric, [15] breast, ML 161 [14] colon, [11] and PC.[4,10] For example, LRH1 affects ML 161 tumor response to anti-estrogen drugs by regulating the expression of estrogen receptor in breast cancer cells.[14] In the intestine, LRH1 and -catenin/Tcf4 signaling induces G1/S-specific cyclin D1/E1-mediated cell proliferation and self-renewal.[5] LRH1 contributes to intestinal tumor formation by ML 161 affecting both cell cycle control and the inflammatory process.[11] Notably, a recent genome-wide association study identified single nucleotide polymorphisms of theLRH1gene associated with PC risk.[10] LRH1 mRNA level was higher in PC cell lines compared to a normal pancreatic ductal epithelium derived cell line, and is associated with increased protein levels in pancreatic tumors.[4] LRH1 expression upregulated mRNA levels of cyclin D1/E1 in PC cell lines.[4] Accordingly, selective blocking of LRH1 by siRNA decreased mRNA levels of cyclin D1/E1.[4] However, the mechanism by which LRH1 promotes PC development remains unclear. To explore the oncogenic property of LRH1, we investigated how LRH1 contributes to PC growth using cell-based assays, xenografts in nude mice, and tissue specimens of PC. == 2. MATIERIALS AND METHODS == == 2.1 Cell ML 161 culture == The human PC cell lines AsPC-1, BxPC-3, Capan-1, HPAF-II, Hs 766T, MIA PaCa-2, PL45 and Panc-1, liver hepatocellular carcinoma cell line HepG2 (ATCC), and non-neoplastic human pancreatic duct epithelial cell line HPDE (Applied Biological Materials Inc.) were employed. All the cell lines have been authenticated by short tandem repeat (STR) profiling to reduce the frequency of cell misidentification.[8] Cells were cultured at 37 C in a humidified atmosphere containing 5% CO2in corresponding medium supplemented with 10% FBS and antibiotics (penicillin and streptomycin). Cells were passaged when they reached 80% confluence. == 2.2 Lentiviral infected stable PC cell lines with overexpression of LRH1 == We established two stable Capan-1 and AsPC-1 cell lines with constitutive expression of LRH1 using the lentiviral expression system Rabbit Polyclonal to PRKCG (GeneCopoeia, #EX-Z2607-Lv105). After 48 h of transfection with packaging plasmids and pLentiviral plasmids of target genes in 293T cells, the supernatants containing lentiviral particles were filtered by 0.45 m nitrocellulose membrane and mixed with polybrene at 8 g/ml. Twenty-four hours after lentiviral infection, cells were incubated with the corresponding media for 24 h, then selected with ML 161 8 g/mL (for Capan-1) or 3 g/mL (for AsPC-1) puromycin overtime to eliminate un-infected cells. The LRH1 protein level in stable PC cell lines was confirmed by Western blot. The selected cell lines were routinely cultured in corresponding puromycin media until two days before the experiments, the cells were cultured in media without puromycin. == 2.3 Western Blot Analysis == Cell lysates were treated with ConA-sepharose beads overnight followed by centrifugation to remove cadherin-bound -catenin. Total Cell lysates and non-membrane bound cell lysates were separated by SDS PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed using primary antibodies against LRH1 (Abcam, ab18293), cyclin D1 (Santa Cruz, sc-8396), cyclin E1 (Santa Cruz, sc-247), calpain1 (Santa Cruz, sc-7531), -catenin (Cell signaling Technology, #9562), estrogen receptor alpha (ER) (Santa Cruz, sc-7207). Protein bands were visualized by IRDye 680RD Infrared Dye and IRDye 800CW Infrared Dye and exposed on Odyssey image system (LI-COR). == 2.4 MTT Assay == PC cells (vectors vs. LRH1 overexpressing Capan-1 and AsPC-1) (1.7104cells per well) were seeded in 24 well.
