Study On The Pim1 Affect Ribosome Biogenesis In The Prostate Cancer

Objective:To identify the role of Pim1 in impacting ribosome biogenesis in prostate cancer and primary touched on its molecular pathways.Methods:By building and extracting high concentration of Pim1 shRNA from the E.coli DH5α which transfected with Pim1 shRNA, then useing Pim1 shRNA that obtain from the above process transfection PC3 cells, the transfected PC3 cell culture with the medium which contains puro. To obtain stable low expression Pim1 cell lines by monoclonal cell culture. Real-time qPCR and westernblot technology were utilized to detect Pim1 mRNA expression and protein level. Using sucrose gradient centrifugation separation technology to separate the ribosome 80 S, 60 S large subunit and 40 S small subunit of PC3 cell. Total layers containing the ribosome 80 S, 60 S large subunit and 40 S small were collected and the protein of each gradients were extracted. The distribution of Pim1 protein and the association between Pim1 and ribosome were indentified by Westernblot; Using sucrose gradient centrifugation technology to separate the ribosome 80 S, 60 S large subunit and 40 S small subunit of Pim1 low expression PC3 cell, PC3 cell and vector, respectively. The level of ribosome 80 S, 60 S large subunit and 40 S small subunit were detected by a UV detector for continuous absorbance measurements at 254 nm. Level of the ribosomal proteins of the ribosome subunit which is affected by Pim1 were indentified by RT-qPCR. All the data were calculated by using SPSS18.0 software.Results:1. Pim1 shRNA was transfected into PC3 through Lipofectamine 2000, the transfected cells were culture with puro then, finally, Pim1 stable low expressed cells were obtain by monoclonal cell culture, after the RT-qPCR and westernblot indentify the mRNA and protein level of Pim1;2. Sucrose gradient centrifugation and Westernblot detection found that the distribution of Pim1 was more closely related to ribosome 80 S and 40 S small subunit,and has nothing to do with the distribution of 60 S ribosome large subunits, thedifference has statistical significance;3. While Pim1 low expression in PC3 cell, the sucrose gradient centrifugation and UV detector found that the level of ribosome 40 S small subunit, 80 S and polysome reduced; While Pim1 low expression in PC3 cell, RT-qPCR detection found that the level of ribosomal protein RPS6 and RPS21 decreased.Conclusions:1. Pim1 was more closely related to ribosome 80 S and 40 S small subunit, and has nothing to do with the distribution of 60 S ribosome large subunits, the difference has statistical significance, it indicates that Pim1 could interact with ribosome 80S、40S small subunit and ploysome;2. While Pim1 low expression in PC3 cell, the level of ribosome 40 S small subunit,80 S and polysome reduced and RT-qPCR detection found that the level of ribosomal protein RPS6 and RPS21 decreased. It suggests that Pim1 causing the ribosome stress and affecting the development of prostate cancer. By affecting ribosome 80 S and 40 S small subunit biosynthesis through regulating the level of the expression of RPS6 and RPS21.