GOLPH3促进前列腺癌雄激素非依赖性转换作用机制的研究

Androgen dependent prostate cancer (ADPC) converted into androgen independent prostate cancer (AIPC), extremely high mortality rate of AIPC has become a major health hazard for older men. The studies on the mechanism of AIPC pathogenesis is a hot research field, mainly about androgen receptor (AR) gene amplification, androgen receptor mutation, apoptosis gene abnormalities and so on. But the above theory can not reasonably explain the pathogenesis of AIPC. AIPC still need further study. GOLPH3 is a bona fide oncogene with potent transforming activity, which is the first oncogene localized to the trans-Golgi Network. Our previous in vitro and in vivo studies first confirmed that GOLPH3 promote prostate cancer converted from ADPC into AIPC, but the exact mechanism is nuclear. By prostate cancer tissue samples miRNA microarray analysis, bioinformatics and combined with pre-experimental results, we found that three miRNAs may be complementary combinated in AIPC in the GOLPH3 3'UTR, regulating its expression and function. We also found that AR may be the major downstream target protein of GOLPH3. On this basis, this project intends to research the mechanism of the conversion process from two aspects, one is upstream miRNAs and the other is downstream signaling regulatory pathway. First, we study the target miRNAs expression and its relationship with GOLPH3(qRT-PCR and in situ hybridization and Luciferase report system) and determine the exact miRNA which regulate the expression of GOLPH3 from the cells and the overall level (target miRNA overexpression and interfere experiments ). Then, we intend to screen GOLPH3 and AR interacting protein by gene chip, proteomics and yeast two-hybrid system. The target protein will be verificated by GST-pull down, Co-IP and Laser scanning confocal co-localization analysis, and the pathway of GOLPH3 play the conversion role will be clear. We expect to fine a new signal pathway with GOLPH3 and AR series. The succseeful completion of this project will provide an effective target for blocking the prostate cancer convert from ADPC to AIPC.

前列腺癌从雄激素依赖向雄激素非依赖转换的发生机制尚不明确。我们前期体内体外研究证实，新发现的首个定位于反面高尔基网的癌基因GOLPH3促进前列腺癌的激素非依赖性转换。本项目在前期研究及预实验基础上，拟从调控GOLPH3上游miRNA和下游信号调控通路两个方面系统研究GOLPH3促进前列腺癌激素非依赖转换的作用机制：首先，普查前列腺癌中目标miRNA表达情况及其与GOLPH3的关系（qRT-PCR、原位杂交和Luciferase报告系统），从细胞和整体水平（目标miRNA过表达及干扰实验）明确调控GOLPH3的miRNA；然后，通过基因芯片结合蛋白质组、酵母双杂交筛选GOLPH3和AR的相互作用蛋白，运用GST-pull down、Co-IP和激光共聚焦共定位分析等技术对目标蛋白进行验证，探寻GOLPH3通过AR发挥促转换作用的信号调控通路，为阻断前列腺癌激素非依赖性转换的治疗提供有效靶点。

前列腺癌从雄激素依赖向雄激素非依赖转换的发生机制尚不明确。我们前期体内体外研究证实，新发现的首个定位于反面高尔基网的癌基因GOLPH3促进前列腺癌的激素非依赖性转换。本项目在前期研究及预实验基础上，从调控GOLPH3上游miRNA和下游信号调控通路两个方面系统研究GOLPH3促进前列腺癌激素非依赖转换的作用机制：首先，普查前列腺癌中目标miRNA 表达情况及其与GOLPH3的关系，从细胞和整体水平明确，miR-186结合GOLPH3的3’UTR从而降低GOLPH3蛋白的表达，通过降低p21和p27蛋白的表达、提高CyclinD1蛋白的表达和Rb蛋白的磷酸化水平，说明miR-186通过阻滞细胞分裂周期G1/S期，从而降低细胞有丝分裂。利用真核生物表达谱芯片筛选出与GOLPH3功能相关基因TGF-β2、PLAT、SPP1、FLNA等，结合文献挖掘及RT-PCR、IHC的验证，提出GOLPH3可能通过与TGF-β信号通路、Hedgehog信号通路相关的通路，调控前列腺癌细胞与细胞外基质的相互作用，从而促进激素依赖性前列腺癌的演进。本项目已发表研究性论文17篇，其研究成果为后续研究奠定了坚实的基础，为激素非依赖性前列腺癌发病机制和靶向治疗研究提供了新的线索。