Functional Studies Of NES1 In Prostate Cancer

AimTo investigate the role of NES1 gene in prostate cancer.Materials and MethodsThe expression of NES1 protein in 40 cases prostate cancer tissues were detected by immunohistochemistry,and were compared with that of adjacent normal tissues,as well as that of 20 cases benign prostatic hyperplasia tissues.The relationship between the NES1 protein expression in prostate cancer tissue and several clinical evaluation methods,including Gleason score and pathological grade,serum prostate-specific antigen,were analysed.Besides,the correlation of the tumor tissue NES1 protein expression and the occurrence of bone metastases were evaluated.Furthermore,the real-time PCR and Western blot were used to detect the expression of NES1 mRNA and protein in different prostate cancer cell lines.The results were compared with the expression of NES1 in the normal prostate epithelial cell line.The recombinant lentiviral vector?pLVX-NES1-IRES-ZsGreen1?was constructed and transferred into LNCaP and PC-3 prostate cancer cell lines,with the characteristics of low NES1 expressing,in order to up-regulate the NES1 expression.The flow cytometry was used to select and obtain the high-purity,high NES1 stable expressing cell lines,LNCaP-NES1 and PC-3-NES1.Experiments of cell proliferation,apoptosis,migration and invasion,as well as the proliferation in vivo,were done with these cell lines,to study the function of NES1 gene in the androgen-dependent and independent prostate cancers.Small animal ¹⁸F-FLT PET/CT imaging and ¹⁸F-FDG PET/CT imaging were used to evaluate the tumor proliferation and glucose metabolism between the NES1 over-expressing and control groups of nude mices.Finally,the NES1,Ki-67,bcl-2,bax protein expressions of the xenografts were detected by immunohistochemistry with the purpose to primarily explore the mechanism of NES1 gene in prostate cancer.Results?1?NES1 protein expression was low in prostate cancer tissues,normal in adjacent normal tissues and high in benign prostatic hyperplasia tissues.The NES1 expressions in prostate cancer cell lines?PC-3,DU-145 and LNCaP?were significantly lower than that in normal prostate epithelial cell line?RWPE-1?.?2?No significant correlation had been found between the prostate tumor tissue NES1protein expression and the Gleason grading,the occurrence of bone metastases,the serum PSA,respectively.?3?Stable NES1 overexpression of LNCaP-NES1 and PC-3-NES1 cell lines were validated by realtime PCR and western blot.?4?No significant inhibition in cell proliferation,apoptosis and migration could be seen in NES1 overexpressed androgen-dependent prostate cancer cell line LNCaP,except for cell invasion,with a mild suppressive effect.NES1 gene in androgen-independent prostate cancer cell line PC-3 could inhibit the tumor cell proliferation,promote early apoptosis,and inhibit the migration and invasion.?5?Xenograft proliferation inhibition effect in NES1 overexpressed PC-3 nude mice was shown by general experiment and small animal ¹⁸F-FLT PET/CT imaging.Small animal ¹⁸F-FDG PET/CT imaging showed the inhibition of xenograft glucose metabolism in NES1 overexpressed PC-3 nude mice.Down-regulation of Ki-67,bcl-2protein expressions and bcl-2/bax ratio,slight up-regulation of bax protein expression in NES1 overexpressed group were detected in xenograft immunohistochemistry.ConclusionsIn androgen-independent prostate cancer,NES1 genes might play a role in tumor suppression by inhibiting cell proliferation,migration and invasion,and by promoting early cell apoptosis.