Experimental Study On The Function And Mechanism Of MAZ Gene In Prostate Cancer

Background:Prostate cancer (PCa) remains an important disease and leads to a large numberof male deaths each year worldwide. PCa in America is the most commonlydiagnosed malignancy and the second leading cause of male cancer mortality afterlung cancer. In China the incidence of PCa is lower, but with the improvement ofliving standard and the growth of aging population, the changing of diet structure andthe improvement of the techniques of medical testing in recent years, the incidenceof PCa is increasing with each passing year. The quality of life and life expectancy ofmen over50in China are being affected by PCa, which becomes a more and moreimportant issue in urological field.Unlike other tumors, the most significant feature of PCa is that the biologicalbehavior of PCa is highly heterogeneous and the prognosis of it cannot be accuratelypredicted. Some PCa patients can rapidly progress in one or two years, others cannever to be found and no life-threatening. Another notable feature of PCa is androgendependence. During its early stages, the progression of PCa relies on androgen.Because of this, except for prostatectomy and radiotherapy in localized prostatecancer, the main treatment for advanced prostate cancer is androgen ablation bysurgery or chemical castration. In the early period of androgen ablation therapy, it canmake a more satisfactory effect. However, after12~18months of anti-androgentherapy, androgen-dependent (AD) PCa inevitably progresses to an androgen-independent (AI) stage with an eventual fatal prognosis. At present, we consider thatthe development and progression of PCa is a very complex process for it involvesmany factors and many steps, not only involves genetic factors, but also involves age,race, dietary habits and environmental factors. To date, the mechanism for thedevelopment and progression of PCa remains largely unknown.MAZ(Myc-associated Zinc-finger protein)gene is located on chromosome16p11.2. This gene is transcribed as an mRNA of2.7kilobases(kb) that encodes477amino acids, a60-kDa MAZ protein. MAZ gene expression was proved in humanheart, brain, lung, liver, skeletal muscle, pancreas and prostate. According to thedocuments results, it has been demonstrated that deregulated expression of MAZ geneclosely related to the development and progression of cancer. MAZ plays animportant role in regulating transcription of some genes from promoter, includingC-MYC, RAG-2, serotonin1α receptor, ENT-1, CLC-K1, PNMT, and PPAR-γ1. In addition, MAZ has been shown that it also plays an important role in regulatingtranscription of some genes from terminator, including eNOS, Sp4, and telomerase, aswell. Therefore, MAZ appears to be a transcription factor with a dual role in theinitiation and termination of transcription.To our knowledge, the expression of MAZ in different stages of PCa and its rolein PCa tumorigenesis and progression as well as in androgen transformation have notbeen evaluated so far. Therefore, it is of great essential to study the role of MAZ inPCa cell lines to elucidate the mechanism of MAZ in PCa tumorigenesis andprogression. Then we can further explore the molecular mechanism of PCadevelopment and progression. We hope that our study can find out a new target in thetreatment of PCa and a new prognosis factor for PCa detection.Objective:To study the MAZ expression in different stages of PCa; to study the role andmechanism of MAZ in PCa tumorigenesis and progression as well as in androgentransformation; to study the correlative role between MAZ and androgen receptor; tofurther explore the role of MAZ in the molecular mechanism of PCa development andprogression; try to find out a new target in the treatment of PCa and a new prognosisfactor for PCa detection.Methods:1. qRT-PCR was used to assess the MAZ differential expression levels in a set of15pairs of androgen dependent PCa and adjacent normal tissues.Immunohistochemistry was used to assess the MAZ differential expression levelsin paraffin sections of40androgen dependent PCa,10androgen independent PCa,10benign prostatic hyperplasia,8PIN.2. qRT-PCR was used to assess the MAZ expression levels in5human PCa celllines and2human normal prostatic cell lines. One PCa cell line with the highestMAZ expression was transfected with MAZ siRNA. Then the biologic behaviorwas evaluated by the experiments of cell proliferation and apoptosis, cell cycle,colony formation, migration and invasion.3. qRT-PCR was used to assess the MAZ and AR expression levels in a set of15pairs of androgen independent PCa and adjacent normal tissues to explore theinterrelationship between MAZ and AR. LNCaP-AD was separate transfected with AR siRNA and MAZ siRNA, then qRT-PCR and Western blotting were usedto assess the MAZ and AR expression levels.Results:1. qRT-PCR was used to assess the MAZ expression levels in a set of15pairs ofandrogen dependent PCa and adjacent normal tissues. We found that MAZ washighly expressed in the tumor tissue (p<0.05). IHC assessed that MAZ expressionlevels in PIN samples were higher than BPH samples. Androgen dependent PCawere divided into3groups, which are Gleason Scroe <7,=7and>7. The MAZexpression evaluated by IHC was compared in pairwise comparison, but nostatistically significant difference. However, the test showed statisticallysignificant difference between androgen dependent PCa group and androgenindependent PCa group (p=0.0406). MAZ was highly expressed in the androgendependent PCa tissue.2. qRT-PCR was used to assess the MAZ expression levels in5human PCa celllines and2human normal prostatic cell lines. We found that MAZ was higherexpressed in the tumor tissue LNCaP-AD and C4-2cell lines which also hadhigher expression levels of AR, but PC3cell line which has a lowest ARexpression had the lowest MAZ expression. After knockdown of MAZ, themigration and invasion capacity of LNCaP-AD cell line significantly declined, thecell proliferation notably inhibited, the colony formation declined during thepassage process and the rate of apoptosis significantly rose. The tests showedstatistically significant difference between MAZ715siRNA and NC,MOCK(p<0.05). In cell cycle experiment, LNCaP-AD cells which transfected by MAZ715siRNA were blocked in G0/G1phase and the number of cells in S phase wasdecreased (p<0.05).3. qRT-PCR was used to assess the MAZ and AR expression levels in a set of15pairs of androgen independent PCa and adjacent normal tissues. The MAZexpression was positively related with AR expression. Knockdown of MAZsignificantly increased AR, knockdown of AR significantly decreased MAZ.Conclusion:MAZ may play an important role in PCa tumorigenesis and progression. MAZprobably affect PCa progression through regulating a series of oncogenes and proto oncogenes. We confirmed that MAZ and AR are interrelated. However, theexpression of MAZ will decline when PCa progress to androgen independent. Furtherstudies are required to clarify the molecular mechanisms underlying reciprocalregulation of MAZ and AR. Finally, we conclude that overexpression of MAZ has animportant role in PCa pathogenesis, and MAZ could be a potential therapeutic target.