前列腺外周带基质细胞过表达LMO2基因对前列腺上皮细胞的生物学影响

发布时间：2018-09-12 10:48

【摘要】：目的：根据前列腺带性结构差异学说，研究前列腺外周带基质细胞（Peripheral Zones Stromal Cells，PZs)与移行带基质细胞(Transitional ZonesStromal Cells，TZs)编码基因及非编码基因表达差异，研究差异基因LMO2在PZs中过表达的意义，进一步探讨LMO2基因在基质微环境中对前列腺细胞增殖、迁移的生物学影响，并解释产生上述现象可能的机制。方法：1.原代培养正常前列腺PZs、TZs，并通过免疫组织细胞化学鉴定，应用Affymetrix基因表达谱芯片及miRNA芯片筛选前列腺不同带基质细胞之间差异的编码及非编码基因。RT-PCR、Western-blotting、免疫荧光、免疫组织化学验证差异基因LMO2在PZs、TZs中的表达。2.构建LMO2的过表达慢病毒载体，感染前列腺基质细胞WPMY-1后，获得稳定表达LMO2的前列腺基质细胞株（WPMY-1-LMO2）；构建LMO2特异性shRNA质粒，转染PZs，获得shRNA-LMO2前列腺基质细胞（PZsshRNA-LMO2）。荧光显微镜、RT-PCR、Western-blot检测LMO2的表达及沉默效率。3.利用Transwell系统构建上皮-基质共培养模型，观察前列腺上皮细胞增殖及迁移能力的特性变化。蛋白因子芯片及RT-PCR检测细胞因子FGF-9、IL-11的表达。CCK-8、EdU实验及细胞划痕实验观察FGF-9及IL-11对BPH-1、PC3细胞增殖及细胞迁移的影响。Western-blot检测上皮-基质共培养模型中BPH-1及PC3细胞相关蛋白分子的变化。4.裸鼠前列腺原位移植瘤模型及皮下移植瘤模型，观察成瘤时间、肿瘤体积等，验证前列腺基质细胞WPMY-1-LMO2对PC3细胞增殖及侵袭能力的影响，动物活体成像技术观察肿瘤的体内转移，免疫组化方法检测淋巴结转移及移植瘤增殖能力。结果：1.成功原代培养PZs、TZs，基因芯片筛选512种基因在前列腺不同带基质细胞有差异表达。miRNA芯片筛选发现在PZs、TZs中存在27种差异的miRNA。RT-PCR、Western-blot、免疫组化、免疫荧光技术证实LMO2在PZs中过表达。2.成功构建了稳定表达LMO2基因的慢病毒细胞株WPMY-1-LMO2；构建的shRNA-LMO2质粒能有效干扰外周带基质细胞中LMO2基因，获得PZsshRNA-LMO2细胞株。3.体外建立上皮-基质共培养模型，，前列腺上皮细胞BPH-1及PC3与WPMY-1-LMO2共培养及用WPMY-1-LMO2的条件培养基孵育后，BPH-1及PC3增殖及迁移能力与对照组比较明显增强（P0.05），STAT3及AKT磷酸化水平升高，CCND1表达增加。沉默PZs中的LMO2基因，PZsshRNA-LMO2对前列腺细胞增殖、迁移能力减弱。蛋白因子芯片发现FGF-9及IL-11在WPMY-1-LMO2上清液中表达升高， RT-PCR结果发现FGF-9mRNA在WPMY-1-LMO2较对照组细胞平均上调4.1倍，IL-11mRNA在WPMY-1-LMO2较对照组细胞平均上调5.5倍（P0.05）。4. WPMY-1-LMO2基质细胞可以促进肿瘤生长、转移较对照组效果显著（P0.05）。结论：前列腺外周带和移行带基质细胞中存在差异基因表达，筛选LMO2基因在前列腺外周带基质细胞中过表达；前列腺基质细胞LMO2蛋白过表达，明显增加前列腺上皮细胞增殖和迁移等能力，并增加肿瘤的生长、侵袭和远处转移；前列腺基质细胞表达LMO2后，诱导FGF-9、IL-11等细胞因子高表达，并通过旁分泌效应刺激前列腺上皮细胞生长、迁移；进一步证明前列腺基质-上皮相互作用在前列腺癌发生进展中的重要性，特别是基质的关键作用。以前列腺基质为研究切入点，为研究前列腺癌发生具有带性差异的分子机制、前列腺基质作为靶向治疗和预后判断的研究奠定坚实的实验基础。
[Abstract]:Objective: According to the theory of zonal structural difference of prostate, to study the expression of coding and non-coding genes in peripheral zone stromal cells (PZs) and transitional zone stromal cells (TZs) of prostate, and to study the significance of overexpression of LMO2 in PZs. The biological effects of genes on the proliferation and migration of prostate cells in the matrix microenvironment and the possible mechanisms underlying these phenomena are explained.
Methods: 1. Primary culture of normal prostate PZs, TZs, and through immunohistochemical identification, Affymetrix gene expression profiling chip and microRNA chip screening prostate stromal cells with different coding and non-coding genes. RT-PCR, Western-blotting, immunofluorescence, immunohistochemical verification of the differential gene LMO2 in PZs, TZ. LMO2-specific shRNA plasmid was constructed and transfected into PZs to obtain shRNA-LMO2 prostate stromal cells (PZs shRNA-LMO2). Fluorescence microscopy, RT-PCR and Western-blot were used to detect LMO2 expression. Expression and Silencing Efficiency. 3. Establishment of an epithelial-matrix co-culture model using Transwell system to observe the proliferation and migration of prostatic epithelial cells. Expression of cytokines FGF-9 and IL-11 was detected by protein chip and RT-PCR. Proliferation and cell proliferation of BPH-1 and PC3 cells were observed by CCK-8, EdU and cell scratch assays. Western-blot was used to detect the changes of BPH-1 and PC3 cell-related protein molecules in the co-culture model of epithelium and matrix. 4. Orthotopic prostate transplantation tumor model and subcutaneous prostate transplantation tumor model in nude mice were used to observe the tumor formation time, tumor volume, etc. To verify the effect of prostatic stromal cells WPMY-1-LMO2 on the proliferation and invasion of PC3 cells, and animal survival. The tumor metastasis in vivo was observed by body imaging, and the lymph node metastasis and the proliferation of transplanted tumor were detected by immunohistochemistry.
Results: 1. Primary culture of PZs, TZs, gene chip screening 512 genes differentially expressed in prostate stromal cells with different zones. Microarray screening found 27 different microRNAs. RT-PCR, Western-blot, immunohistochemistry, immunofluorescence technology confirmed that LMO2 was overexpressed in PZs. 2. The stable expression of LMO2 gene was successfully constructed. A lentiviral cell line WPMY-1-LMO2 was constructed, and the plasmid shRNA-LMO2 could effectively interfere with the LMO2 gene in the peripheral zone stromal cells. 3. Establishment of an epithelial-matrix co-culture model in vitro, co-culture of BPH-1 and PC3 with WPMY-1-LMO2 and incubation with WPMY-1-LMO2, BPH-1 and PC3 proliferated. Compared with the control group, the migration ability and STAT3 and AKT phosphorylation level increased, CCND1 expression increased. LMO2 gene in PZs was silenced, and the proliferation and migration ability of prostate cells were weakened by PZsshRNA-LMO2. The expression of FGF-9 and IL-11 in the supernatant of WPMY-1-LMO2 were increased by protein chip, and the expression of FGF-9 mRNA in WPMY-1-LMO2 was detected by RT-PCR. The expression of IL-11 mRNA in WPMY-1-LMO2 was 5.5 times higher than that in control group (P 0.05). 4. WPMY-1-LMO2 stromal cells could promote tumor growth and metastasis significantly (P 0.05).
Conclusion: Differential gene expression exists in the stromal cells of the peripheral zone and transitional zone of prostate, LMO2 gene overexpression is screened in the stromal cells of the peripheral zone of prostate, LMO2 protein overexpression in prostatic stromal cells significantly increases the proliferation and migration of prostatic epithelial cells, and increases tumor growth, invasion and distant metastasis. Glandular stromal cells expressing LMO2 induce the overexpression of cytokines such as fibroblast growth factor-9 and interleukin-11, and stimulate the growth and migration of prostatic epithelial cells by paracrine effect. This further proves the importance of prostatic stromal-epithelial interaction in the development of prostate cancer, especially the key role of stroma. In order to study the molecular mechanism of banding differences in prostate cancer, prostatic stroma as a targeted therapy and prognostic evaluation lays a solid experimental foundation.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

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