Androgen Receptor(AR) Up-regulation Of RNA Binding Protein QKI In Castration Resistant Prostate Cancer

?Background:?Prostate cancer is the third leading cause of incidence rate for male urogenital system malignant tumor in C hina.The prostate cancer is insidious without typical symptoms,so it is always in the late stage that we can do nothing.We don't know how to treat the difficult disease.The early diagnosis of patients mainly depends on PSA lever in serum and Digital Rectal Exam is also a main auxiliary mean.Prostate cancer endocrine therapy effect is remarkable,but after 12 to 18 months of androgen deprivation therapy,the patient inevitably entered the stage of castration resistant prostate cancer.Castration resistant prostate cancer with poor prognosis and quality of life in patients with serious decline,has very serious consequences for patients.Therefore,clear castration resistant prostate cancer development mechanism,may provide new methods for the clinical treatment of prostate cancer.Androgen receptor plays a very important role in the process of castration resistant prostate cancer development.At present,the treatment target for the CRPC are concentrated in the AR upstream,and from the perspective of the mechanism of C RPC,just upstream of the inhibition of AR pathway,does not stop AR excessive activation of downstream genes.So,starting from the AR signaling pathways downstream of or can explore sensitization CRPC cell potential therapeutic targets.QKI(quaking)belongs to the STAR(signal transduction and activation of RNA)family an RNA-binding protein,the participation in the process of development of the nervous system myelin development.Different mutant mice qki gene can behave before birth death from cardiovascular muscle system developmental disorders,or in 10 days after birth for quick tremor and tonic convulsion attacks as adults.Except for the formation of the myelin sheath in the nervous system play an important function,most of QKI protein is also involved in the cell biological processes,such as angiogenesis,apoptosis,cell adhesion,cell growth and morphogenesis and organogenesis,and so on.At present,the study found QKI may regulate as many as 1430 downstream target genes,of which 24% of the genes involved in cell proliferation,metastasis,highly suggest possible QKI involed in tumor proliferation and metastasis.Whether there is a regulation relationship between AR and QKI,if there is a regulation relationship,then has what effect to the development of prostate cancer has yet to see the related literature reports.Therefore,clear relationship between the regulation and its influence to the prostate cancer will help to further understand the evolution of prostate cancer,also helps to provide a new treatment strategies.?Objective:?1.To clear different prostate cancer cells and clinical tumor tissue samples of AR and QKI molecules and protein expression level,clear the potential regulation relationship does exist.2.To clear expression of endogenous AR prostate cancer cells in the case of ADT intervention,the expression of AR and QKI changes in prostate cancer cells,as well as the change of the AR in CRPC cell mutants.3.To clear the influence of AR expression of QKI silence,as well as to the different function of cell cycle and apoptosis of prostate cancer cell line.4.To clear silence QKI influence about the existence of AR expression,as well as the influence on cell cycle and the function,preliminary discussion may influence the possible mechanism.5.To clear whether silenced QKI can influence the drug sensitivity of Casodex in CRPC.?Methods and results:?1.In our clinical tumor tissue samples by extracting protein and different protein in prostate cancer cells and molecules,respectively through the q RT-PCR and western blotting to observe different prostate cancer cells of AR and QKI mRN A and protein expression.We found that in AR high expression of tumor tissue samples and department of prostate cancer cells,the expression of QKI also increased obvious ly.This suggests that AR may be able to regulate the expression of QKI.Through our forecast QKI promoter region of transcription factors,found that there may be AR potential binding sites.We analyze QKI promoter region binding sites through dual luciferase reporter gene system,found there are AR binding sites,and can positively regulate the activity of QKI.Therefore,the AR can regulate the expression of QKI positively.2?Carbon adsorption serum is used to culture prostate cancer cells to rule out other serum hormone and cytokine activation of AR.We use carbon adsorption serum and physiological concentration of serum DHT to verify the androgen regulation of AR activation.Western blotting and qRT-PCR are used to detect expression of AR and QKI mRNA and protein in different prostate cancer cells.The q RT-PCR is used to detect AR mutants and the change of the expression of target genes under the condition of ADT.We found that expression of AR and QKI are decreased in prostate cancer cells LNCaP and C4-2 cells under the condition of ADT.After joining DHT,the expression of AR and QKI have a certain degree of recovery.AR can regulate the expression of QKI were further verified.The expression of AR mutant show contrast change in C4-2 cells.In castration resistant prostate cancer cells,AR mutants can be in the absence of androgen exist,since the activation,which is beneficial to cell survival.3?Small interference RNA and transfection whit liposome 2000 are used to silence the expression of AR.Western blotting and q RT-PCR are used to detect expression of AR and QKI mRNA and protein in different prostate cancer cells.Flow cytometry is used to detect silenced AR's influence on the function of prostate cancer cell cycle and apoptosis.We found that the expression of AR and QKI is decreased,cell cycle is arrested and apoptosis is increased.LNCaP and C4-2 cell performance is different in the cell cycle arrest.The former is mainly the G1/S phase of the block.The latter for the G2/M block.UBE2 c is the main regulatory molecules of G2/M,which is significantly higher in C4-2 cells.This may be the reason of the G2/M block in C4-2 cells.4?Small interference RNA and transfection whit liposome 2000 are used to silence the expression of QKI.WE use the qRT-PCR and western blotting to detect cells AR,QKI,PSA and HSP90 mRNA and protein expression.Flow cytometry is used to detect silenced QKI's influence on the function of prostate cancer cell cycle and apoptosis.We found that the expression of AR and QKI is decreased,cell cycle is arrested and apoptosis is increased.LNCaP and C4-2 cell performance is different in the cell cycle arrest.The former is mainly the G1/S phase of the block,the latter for the G2/M block.We also tested the endoplasmic reticulum apoptosis protein caspase – 12,G1/S phase protein P27,G2/M phase protein P21.The results show that apoptosis protein and cycle protein were significantly increased.5?MTT is used to detect the function of cell proliferation.We found that silenced QKI can influence the drug sensitivity of Casodex in CRPC.?Conclusion:?1.The expression of QKI was positively correlated with AR levels in both cell lines and clinical samples.2.AR up-regulated the expression of QKI in prostate cancer cell lines,such as LNCaP and C4-2.3.Silenced AR and QKI arrest the LNCaP cell in the G1/S phase and the C4-2 cells in G2/M phase.Silenced AR and QKI can increase the cell apoptosis.4.Silencing QKI can enhanced the sensitivity of casodex in CRPC.