Molecular Mechanism Of NOB1 Promoting The Distant Metastasis Of Prostate Cancer

ObjectiveProstate cancer(PCa),an aggressively invasive tumor,is one of the most prevalent malignancies in men and the second cause of male cancer death.It is a clinically heterogeneous-multifocal disease and its incidence is rising.The mechanisms influencing the progression and prognosis of prostate cancer involve a multi-step process.Novel effective diagnostic and prognostic biomarkers for PCa are needed to prevent overtreatment of indolent tumors,and to ensure early detection of aggressive PCa that requires timely treatments.Nin one binding protein(NOB1),encoded by NOB1 gene,is a subunit of the 26 S proteasome and plays a key role in protein degradation pathway.NOB1 protein composed of a PIN domain and a C terminal zinc ribbon domain,which has been reported to function as a transcription regulator.It is reported that NOB1 may play a role in cell cycle progression,drug resistance and oncogenesis.A recent study shows that knockdown of NOB1 expression inhibits cellular proliferation and migration in human gliomas,suggesting that NOB1 promotes glioma cell growth and migration and could be a candidate for molecular targeting.Those findings indicate that NOB1 may be involved in various tumors and may serve as an oncogenic factor.However,the expression of NOB1 and its significance in PCa has been uncertain.In this study,we investigated the NOB1 expression to evaluate its prognostic significance in PCa patients with long-term follow-up data and its association with clinicopathological features.Given the virtues of lentiviral vector system,we early constructed a kind of lentiviral vector via shRNA targeted on down-regulating NOB1 gene.Through study,we find that the expression level of NOB1 could impact cell biology behavior on hormone resistance prostate cancer,and a solid foundation has been laid for NOB1 targeted molecular treatment of prostate cancer in the future.Methods and ResultsExpression of NOB1 in prostate cancer tissues and cell linesTo study the function of NOB1 gene in prostate cancer,the prostate cancer tissue and adjacent normal tissue were stained with anti-NOB1 antibody.Prostate cancer tissues were strongly stained with NOB1 antibody(lower panel),compared to the adjacent normal tissue.The expression levels of NOB1 were detected with qPCR in seven pairs of prostate tissues,which were all significantly up-regulated in the cancer tissues(PCa)compared to the adjacent tissues.Then we analyzed NOB1 expressions in prostate cancer cell lines.NOB1 expression levels in DU145,PC-3,and 22RV1 were higher than that in the androgen-sensitive prostate cancer cell LNCap that shows less aggressiveness than other three prostate cancer cell lines.These results implied that NOB1 might be related to the tumorigenicity of prostate cancer.Knockdown efficiency of NOB1 in high aggressive prostate cancer cellsIn early study,we constructed a kind of lentiviral vector via shRNA targeted on down-regulating NOB1 gene.PC-3 and DU145 cell with a relatively high level of NOB1 expressions were therefore chosen for the further investigation.Both PC-3 and DU145 cells were transfected with Lv-shNOB1 to knockdown NOB1 gene expression.The efficient transfection of lentivirus after 72 h were visualized by GFP expression.The knockdown effects on the protein and mRNA levels were detected with western blot and qPCR anlaysis in PC-3 and DU145 cells.Result shows that shRNA lentiviral vector could significantly lower the expression of NOB1 in PC-3 and DU145 cells.Influence of NOB1 knockdown on proliferation,cell cycle progression and migration capacity of prostate cancer cells PC-3 and DU145.In our mainly experimental part,we lower the expression of NOB1 via shRNA lentiviral vector in order to the influence of NOB1 gene on cell biology of hormone resistant prostate tumor.PC-3 and DU145 cells were transfected with Lv-shNOB1 and subjected to MTT analysis for cell proliferation.Compared to the Lv-shcon transfected cells,Lv-shNOB1 significantly suppressed cell proliferation of both cells,indicating a important role of NOB1 in the regulation of prostate cancer cell growth.To assess the function of NOB1 in cell colony formation,Lv-shNOB1 transfected PC-3 and DU145 cells were seeded onto 6-well plates.After 15 days' cultivation,colonies were fixed and stained with Giemsa.Compared to control,NOB1 silence reduced colony sizes,and caused nearly 6 fold and 3 fold reductions in colony formations of PC-3 and DU145 cells,respectively.Then,we future apply flow cytometry assay to analyze the role of NOB1 on cell cycle progression.Compared to the control or Lv-shcon transfected groups,the percentage of Lv-shNOB1 induced G0/G1 phase arrest in PC-3 and DU145 cells.In addition,from our result,we could see there were slight decreases in both cell lines on the S phase.This result demonstrated that knockdown of NOB1 might affect cell cycle progression,which might lead to growth inhibition.Thereby,we speculated that NOB1 gene may facilitate the growth of prostate cancer cell.In the end,the transwell migration assay was applied to investigate whether NOB1 had an impact on the mobility of prostate cancer cells.Result shows that NOB1 knockdown obviously inhibited the migration capacity of PC-3 and DU145 cells.ConclusionsIn conclusion,to explore the function of NOB1 in human prostate malignancy,we analyzed the expression of NOB1 in prostate cancer and found that NOB1 was elevated in prostate cancer tissues compared to the adjacent normal tissues.Knockdown of NOB1 by lentivirus-shRNA inhibited the proliferation and colony-formation ability of PC-3 and DU145 prostate cancer cells.Cell cycle analysis showed that silencing of NOB1 caused G0/G1 phase arrest and a slight decrease in S phase.Furthermore,knockdown of NOB1 significantly suppressed the mobility of PC-3 and DU145 prostate cancer cells.Collectively,these findings suggested that NOB1 might be involved in tumorigenecity of prostate cancer,and could be a potential molecular target for high aggressive prostate cancer gene therapy.