Abstract
BackgroundProstate cancer (PC), a common malignant tumor, is the second-leading cause of cancer death among American men. Its successful treatment greatly relies on the early diagnose. Engrailed-2 (EN2) has been confirmed being existed with a high level in the urine of PC patients. In this study, to explore the application of EN2 in PC, we detected the immunohistochemical staining difference and EN2 expression level between benign prostatic hyperplasia (BPH) and PC.MethodsWe developed a monoclonal antibody against the helix 3 in EN2 and confirmed its specificity with Western blotting (WB) and immunofluorescence detecting the subcellular localization of endogenous and exogenous EN2 in three PC cell lines (LNCap, PC3, and DU145). We conducted immunohistochemical staining using this homemade antibody, and RT-PCR to detect the expression of EN2 in 25 PC and 25 BPH cases, and analyzed the correlation of EN2 expression and PC clinical staging.ResultsThe results of WB and immunofluorescence showed our homemade EN2 monoclonal antibody could specifically bind endogenous and exogenous EN2 protein in three different PC cell lines. Endogenous EN2 was generally expressed in the cytoplasm and exogenous EN2 mostly existed in the nucleus of these cell lines. Immunohistochemical staining in PC had extremely stronger signals than that in BPH, suggesting a higher EN2 expression level in PC, which was confirmed by RT-PCR. Interestingly, the stained areas in BPH tissues were mainly in nucleus and cytoplasm, while in PC tissues were mainly on cytomembrane. Moreover, the expression level of EN2 was positively correlated with the PC clinical staging.ConclusionUsing our homemade EN2 antibody, we have found different staining patterns and expression level of EN2 in BPH and PC,which may be helpful to predict prostatic disease progression.
Highlights
Prostate cancer (PC), a common malignant tumor, is the second-leading cause of cancer death among American men
The band indicated by the red arrow in the lane of “EN2-Red fluorescent protein (RFP)+” was the EN2-RFP fusion protein expressed by transfected 293 T cells which did not appear in the lane of “EN2-RFP-”
The endogenous and exogenous EN2 identified by Western blotting (WB) using our homemade EN2 monoclonal antibody was shown in Fig. 1d, where two bands appeared in “EN2-RFP+” line while only one band appeared in “EN2-RFP-” line
Summary
Ethics statement This study involving human participants was approved by the ethics committee of The First Affiliated Hospital of Zhengzhou University. Total proteins were extracted with Cell Culture Lysis 1 × Reagent (#53711–5399, Promega Inc., USA) according to the product instruction and incubated on ice for 10 min. Immunofluorescence The recombinant pcDNA3.1-EN2-Red fluorescent protein (RFP) was transfected into 293 T or prostate cancer cell lines PC-3, DU-145 or LNCap, using PEI (polyethylenimine linear, #23966, Polysciences Inc., USA). The culture medium was aspirated, and cells were fixed by incubating with 4% paraformaldehyde (#P1110,Solarbio Inc., China) for 15 min at room temperature. After washing twice in PBS buffer, freshly prepared 0.2% Triton X-100 (#T8200, Solarbio Inc., China) was added and incubated for 10 min at room temperature. The Goat anti-mouse IgG/FITC (#SF131, Solarbio Inc., China) was added and incubated for 35 min at 37 °C in the dark. In BPH tissues, the relative transcription level of each target was obtained by subtracting the transcription level of an internal reference (GAPDH) from the level of each target
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