前列腺外周带基质细胞高表达LMO2促进前列腺癌发展的分子机制研究

Prostate cancer (PCa) occurs predominantly in the peripheral zone, and prostate stromal cells derived from the peripheral zone influence the development and progression of PCa.Therefore the study on the molecular mechanism is essential. In our preliminary studies, we showed that LMO2 was highly expressed in peripheral zone stroma cells compared with transitional zone stromal cells , and its expression level was closely associated with PCa cell proliferation, invasion and tumorgenesis, and LMO2 can negatively regulate miR-15a/16 expression, which could be one potential mechanism of PCa predilection in the peripheral zone but rarely occurs in the transitional zone. However, its mechanism has not yet been fully understood. In this research plan, we will use the co-culture system of prostate cells and stromal cells to further investigate the molecular mechanism of stromal cells activation, cytokine secretion, proliferation and invasion of PCa cells stimulated by LMO2. We will further study the mechanism of regulation between LMO2 with miR-15a/16. We will define the upstream signal pathways that modulate expression of miR-15a/16 and identify the downstream targets of miR-15a/16. The function and mechanism of miR-15a/16 as well as its upstream and downstream components in prostate tumorigenesis will be studied. Data generated from these studies will advance our understanding of the molecular mechanism of prostate cancer predilection in the peripheral zone, and provide a new theoretical and experimental basis for repression and intervention on the development, progression and metastasis of PCa.

前列腺癌好发于前列腺外周带，外周带基质细胞影响着前列腺癌的发生发展。我们前期研究发现：前列腺外周带基质细胞表达LMO2高于移行带基质细胞，其表达量与前列腺癌细胞增殖、侵袭能力密切相关，且负向调控miR-15a/16的表达，这可能是前列腺癌好发于外周带的重要原因，但其机制尚不清楚。本研究拟采用前列腺上皮细胞-基质细胞微环境模型，研究LMO2促进基质细胞活化、细胞因子分泌、前列腺癌细胞增殖及侵袭转移的分子机制，深入研究LMO2与miR-15a/16的相互调控关系，鉴定miR-15a/16的下游靶基因，探索miR-15a/16的上游调控信号通路和下游靶基因在前列腺基质微环境中的功能机制。研究结果将阐释LMO2在前列腺基质微环境中对前列腺癌发生发展的分子机制，从而揭示前列腺癌好发于外周带的机制，为干预前列腺癌发生发展提供新的理论与实验依据。

前列腺癌好发于前列腺外周带，外周带基质细胞影响着前列腺癌的发生发展。我们研究发现前列腺外周带和移行带基质细胞存在差异基因，其中包括编码和非编码基因的差异。通过免疫组织化学、免疫印迹和实时定量PCR证实LMO2基因在前列腺不同带基质细胞以及不同带前列腺组织标本中有差异表达，同样我们运用实时定量PCR发现miR-15a/16在前列腺外周带的表达量较前列腺移行带降低。前列腺基质细胞过表达LMO2基因后，基质细胞可以活化为活性基质细胞。过表达LMO2基因的基质细胞与前列腺上皮细胞共培养，体内外实验发现前列腺上皮细胞增殖、浸润转移能力增强。沉默前列腺外周带基质细胞的LMO2基因，外周带基质细胞对前列腺上皮细胞增殖及迁移的作用随之减弱。研究发现过表达LMO2基因的基质细胞上清液中FGF9、FGF2的含量增高，通过激活前列腺上皮细胞的STAT信号通路，从而导致BPH-1、PC3细胞增殖、迁移。启动子荧光素酶报告基因等实验证明了外周带基质细胞内LMO2基因对miR-15a/16的转录调控。通过生物信息学预测发现FGF9、FGF2是miR-15a/16的靶基因。将携带miR-15a/16的载体转染至基质细胞中，通过实时定量PCR和Western Blotting分别检测FGF9、FGF2的mRNA和蛋白水平的变化，发现miR-15a/16可以调控FGF2、FGF9的表达。构建人FGF9、FGF2 3'UTR荧光素酶报告基因和删除对应于miR-15a/16“种子序列”的突变报告基因，通过双荧光素酶报告基因验证了miR-15a/16和FGF9、FGF2之间的靶向调控关系。在过表达LMO2基因的基质细胞与前列腺上皮细胞的共培养体系中，使用特异性抗体封闭细胞因子FGF9、FGF2的表达，检测基质细胞对前列腺上皮细胞作用的改变，结果发现与前列腺上皮细胞生长能力、克隆形成能力以及侵袭能力相关的STAT信号通路被阻断了。本项目研究结果阐释LMO2基因在前列腺基质微环境中对前列腺癌发生发展的分子机制，前列腺外周带基质细胞对前列腺上皮细胞的促进作用是通过LMO2/miR-15a/16/FGF9、FGF2通路介导的，揭示了前列腺癌好发于外周带的分子机制，为干预前列腺癌发生发展提供了新的理论与实验依据。