The Role Of NOB1Gene On Cell Biology Research Of High Aggressive Human Prostate Cancer

ObjectiveProstate cancer is a common malignant tumor of the medium-elderly men and secondmost frequently diagnosed cancer in male world-wide. More than300,000men arediagnosed with prostate cancer each year in Europe, and there are240,000new cases and33,000deaths of prostate cancer in the United States in the year of2011. In USA, Pca isthe most common malignancy and the second leading cause of deaths from cancer. In ourcountry, the morbidity and mortality of prostate cancer is lower than that of developedcountries. But in recent years, there is a significant increasing trend in the incidence ofprostate cancer. Recently, some research reported that prostate cancer in shanghai withhigh incidence of7.7/100,000ranked the top of male urinary tract tumors in China.Therefore, prostate cancer has become a common disease that draw people ’s attention, alsobeen one hot topics in the field of urology research.Recently, the therapy of androgen deprivation is always powerful standard for prostatemalignancy. Whether surgical castration or drug castration can also reach the effect thattumor cells are killed abundantly. A Study detected that, after a median of18-24months,the men who underwent androgen deprivation would progress to hormone refractory. Forhormone-resistant prostate cancer(HRPC), recent chemotherapy is just alleviativetreatment, there is no cure for advanced prostate cancer.NOB1(Nin one binding-1), a kind of zinc protein, has been first discovered inSaccharomyces cerevisiae as an essential gene encoding the Nin one binding protein by ayeast two-hybrid assay. It plays an important role in the assembly and maturation oflysosomes and the formation of ribosomal RNA. Human NOB1, including nine exons andeight introns, locates in the chromosomes16q22.1. It contains an open reading frame witha molecular weight of46kDa, which is composed of33~1271bp and encodes412aminoacid residues. NOB1contains two important domains, the PIN (PilT amino terminus)domain and Zinc ribbon motif (amino acids208-296), through which NOB1may mediateribosome biogenesis and protein synthesis by PIN domain, while regulate transcription byzinc ribbon motif, respectively. Expression of NOB1protein in human various organs arevisible, but there are different expression quantity in different organs. For example,expression of NOB1was mainly detected in adult human lung, liver and spleen while thatwas less in human kidney, prostate, ovary and colon. In recent years, some studiessuggested that NOB1may participate in development process of many malignant tumors,reduced expression of NOB1may inhibit the proliferation of colorectal cancer,hepatocellular carcinoma, breast cancer, colon cancer, ovarian cancer cells, papillarythyroid carcinoma, human gliomas, non-small cell lung cancer. However, the role of NOB1 in prostate cancer, especially hormone-resistant prostate cancer, has no clear known. Giventhe virtues of lentiviral vector system, we early constructed a kind of lentiviral vector viashRNA targeted on down-regulating NOB1gene. Through study, we find that theexpression level of NOB1could impact cell biology behavior on hormone resistanceprostate cancer, and a solid foundation has been laid for NOB1targeted moleculartreatment of prostate cancer in the future.Methods and ResultsExpression of NOB1in prostate cancer tissues and cell linesTo study the function of NOB1gene in prostate cancer, the prostate cancer tissue andadjacent normal tissue were stained with anti-NOB1antibody. Prostate cancer tissues werestrongly stained with NOB1antibody (lower panel), compared to the adjacent normaltissue. The expression levels of NOB1were detected with qPCR in seven pairs of prostatetissues, which were all significantly up-regulated in the cancer tissues (PCa) compared tothe adjacent tissues. Then we analyzed NOB1expressions in prostate cancer cell lines.NOB1expression levels in DU145, PC-3, and22RV1were higher than that in theandrogen-sensitive prostate cancer cell LNCap that shows less aggressiveness than otherthree prostate cancer cell lines. These results implied that NOB1might be related to thetumorigenicity of prostate cancer.Knockdown efficiency of NOB1in high aggressive prostate cancer cellsIn early study, we constructed a kind of lentiviral vector via shRNA targeted ondown-regulating NOB1gene. PC-3and DU145cell with a relatively high level of NOB1expressions were therefore chosen for the further investigation. Both PC-3and DU145cells were transfected with Lv-shNOB1to knockdown NOB1gene expression. Theefficient transfection of lentivirus after72h were visualized by GFP expression. Theknockdown effects on the protein and mRNA levels were detected with western blot andqPCR anlaysis in PC-3and DU145cells. Result shows that shRNA lentiviral vector couldsignificantly lower the expression of NOB1in PC-3and DU145cells.Influence of NOB1knockdown on proliferation, colony formation, cell cycleprogression and migration capacity of prostate cancer cells PC-3and DU145.In our mainly experimental part, we lower the expression of NOB1via shRNAlentiviral vector in order to the influence of NOB1gene on cell biology of hormoneresistant prostate tumor. PC-3and DU145cells were transfected with Lv-shNOB1andsubjected to MTT analysis for cell proliferation. Compared to the Lv-shcon transfectedcells, Lv-shNOB1significantly suppressed cell proliferation of both cells, indicating aimportant role of NOB1in the regulation of prostate cancer cell growth.To assess the function of NOB1in cell colony formation, Lv-shNOB1transfected PC-3and DU145cells were seeded onto6-well plates. After15days’ cultivation, colonieswere fixed and stained with Giemsa. Compared to control, NOB1silence reduced colonysizes, and caused nearly6fold and3fold reductions in colony formations of PC-3andDU145cells, respectively.Then, we future apply flow cytometry assay to analyze the role of NOB1on cell cycleprogression. Compared to the control or Lv-shcon transfected groups, the percentage ofLv-shNOB1induced G0/G1phase arrest in PC-3and DU145cells. In addition, from ourresult, we could see there were slight decreases in both cell lines on the S phase. This resultdemonstrated that knockdown of NOB1might affect cell cycle progression, which mightlead to growth inhibition. Thereby, we speculated that NOB1gene may facilitate thegrowth of prostate cancer cell.In the end, the transwell migration assay was applied to investigate whether NOB1had animpact on the mobility of prostate cancer cells. Result shows that NOB1knockdownobviously inhibited the migration capacity of PC-3and DU145cells.ConclusionsIn conclusion, to explore the function of NOB1in human prostate malignancy, weanalyzed the expression of NOB1in prostate cancer and found that NOB1was elevated inprostate cancer tissues compared to the adjacent normal tissues. Knockdown of NOB1bylentivirus-shRNA inhibited the proliferation and colony-formation ability of PC-3andDU145prostate cancer cells. Cell cycle analysis showed that silencing of NOB1causedG0/G1phase arrest and a slight decrease in S phase. Furthermore, knockdown of NOB1significantly suppressed the mobility of PC-3and DU145prostate cancer cells.Collectively, these findings suggested that NOB1might be involved in tumorigenecity ofprostate cancer, and could be a potential molecular target for high aggressive prostatecancer gene therapy.