Previous studies have reported that this expression of TFF3 is relatively high in gastrointestinal epithelial cells and that it may induce tumorigenesis17C20, which supports the potential oncogenic role of TFF3 overexpression in the prostate. TFF3 IHC score in the tumor tissues was significantly higher than that in the normal tissues (4.702 vs. 0.311, gene fusion occurs in 40C50% of primary PCa tissues7, and over 90% of PCa specimens show overexpression of PCA38. However, their clinical utility is still controversial9. The allelic loss of 8p12C21 is usually reported to commonly occur in PCa ( 90%) and high-grade prostatic intraepithelial neoplasia ( 60%)10. The loss of em NKX3C1 /em , located at 8p12C21, is also reported to be associated with cancer progression and the poor prognosis of PCa11,12. Recent next generation sequencing analyses of PCa reveal recurrent somatic mutations, such as em SPOP /em , em MED12 /em , and em FOXA1 /em 13C16. However, since PCa has variable biological backgrounds, more reliable biomarkers are required to understand its tumorigenesis mechanism and realize precision medicine for PCa. Trefoil factor family 3 (TFF3) belongs to the trefoil factor family, which includes two other members (TFF1 and TFF2). TFF3 is a secreted peptide that is predominantly expressed in the mucous epithelia of the gastrointestinal tract17. TFF3 and other TFF members are known to be involved in the protection of the gastrointestinal tract against mucosal injury and subsequent repair18. In addition to mucosal restitution, TFFs are known to be involved in the migration/invasion of tumor cells, antiapoptosis signaling and the prevention of anoikis in epithelial cells18C20, which are the key features of cancer progression. Alterations in TFF3 expression are observed in diverse cancers, such as breast21,22, gastric23, pancreatic24, colorectal25, and prostate cancers26. Specifically, TFF3 is usually reported to be commonly overexpressed in PCa. Garraway et al. and Faith et al. consistently reported that TFF3 was significantly overexpressed in PCa tissues compared with normal prostate tissues (42% vs. 10 and 47% vs. 18.8%, respectively), suggesting that TFF3 is a useful biomarker for PCa26,27. Regarding its oncogenic roles, Perera et al. reported that this overexpression of TFF3 significantly increased cell proliferation, anchorage-independent growth, 3-dimensional colony formation, wound healing, cell migration, and radio-resistance28. However, the clinicopathologic features and oncogenic mechanisms associated with TFF3 overexpression in PCa are not clear26,27, suggesting the necessity MAP3K11 of further investigation into its biological roles and underlying mechanisms. In this study, we aimed to elucidate the roles of TFF3 overexpression in prostate tumorigenesis by knocking down the overexpressed TFF3. We also explored the molecular mechanisms behind its tumorigenic roles and decided that TFF3 is usually involved in prostate carcinogenesis via blocking the mitochondria-mediated apoptosis pathway. Materials and methods Cell lines The LNCap.FGC (hereinafter called LNCap), PC-3, and WPMY-1 (prostate stromal cell line) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). They were maintained in RPMI 1640 with 10% FBS (LNCap and PC-3) or DMEM with 5% FBS (WPMY-1). Transfection of TFF3 siRNAs Three different TFF3-specific siRNAs (siTFF3-1, siTFF3-2, and siTFF3-3) were purchased from Invitrogen (Carlsbad, CA). Their sequences are available in Supplementary Table?S1. To estimate the sequence-specific effectiveness of the TFF3-specific siRNAs, we also used a negative control siRNA (siCtrl) (Invitrogen) that has no significant homology with any known sequences in the human genome. JTV-519 free base Since siTFF3-3 JTV-519 free base showed the best performance of the three constructs (data not shown), we used siTFF3-3 for all JTV-519 free base the downstream experiments (hereinafter called siTFF3). siTFF3 was transfected into the cells at a final concentration of 50?nM using Lipofectamine? 2000 transfection reagent (Invitrogen). The cells were seeded in growth medium at a density of 40C50% one day before transfection. The cells were harvested at different time points for the following tests. TFF3-specific qRT-PCR Total RNA was isolated from the cells with TRIzol reagent (Invitrogen). Total RNA (5?g) was reverse transcribed using oligo dT primers and SuperScript? III reverse transcriptase (Invitrogen). Quantitative real-time reverse transcription-PCR (qRT-PCR) was performed with the ViiA? 7 Real-Time PCR System using THUNDERBIRD? SYBR? qPCR Mix (Toyobo, Osaka, Japan) and the TFF3-specific primer set: TFF3-F, 5-CCC TGC AGG AAG CAG AAT-3 and TFF3-R, 5-GGG AGC AAA GGG ACA GAA A-3. GAPDH was used for normalization. Western blot analysis Seventy-two hours following transfection with TFF3-siRNA, the cells were harvested and lysed in RIPA cell lysis buffer with EDTA and a protease inhibitor (GenDEPOT, Houston, TX). The resulting supernatant was collected as total cellular protein and electrophoresed on 12C15% SDS-polyacrylamide gels. The separated proteins were transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Billerica, MA) using a BioRad Mini-PROTEAN? Tetra Cell (BioRad, Hercules, CA). The membranes were blocked in 5% skim milk and then incubated overnight at 4? with antibodies, including a rabbit polyclonal anti-TFF3 antibody (Santa Cruz, Dallas, TX), a mouse monoclonal anti-TFF3 antibody (Santa Cruz), a rabbit monoclonal anti-cleaved caspase-3 antibody (Cell Signaling Technology, Danvers, MA), a rabbit polyclonal anti-caspase-9 antibody (Cell Signaling Technology), a rabbit polyclonal anti-PARP-1 antibody (Santa Cruz), a mouse monoclonal anti-BCL2 antibody (Santa Cruz), a rabbit monoclonal anti-BAX antibody (Abcam, Cambridge, UK), a mouse monoclonal anti-COX IV antibody (Abcam), and a mouse monoclonal anti–actin antibody.