Analyzing The Effects of ProL1 Upregulation On PC3 Global Gene Expression. Analyzing The Effects of ProL1 Upregulation On PC3 Global Gene Expression

Purpose: To examine the effects of proL1 upregulation on global gene expression in PC3 cells using RNA sequencing (RNA-Seq) Methods: PC3 cells were transformed to overexpress proL1 using lentiviral vectors. The proL1 overexpressing variant (PC3 proL1+) and their parental cell line were plated in 6-well plates in triplicate (3 wells for each cell line). After 3 days, their RNA was isolated using the Qiagen RNeasy Mini kit and their global gene expression patterns were analyzed using RNA-Seq Results: We identified 1698 differentially expressed genes between PC3 proL1+ and its parental cell line. Of these differentially expressed genes (DEGs), 209 were in common with LNCaP when proL1 was overexpressed. To identify biological functions that may be regulated through overexpression of proL1 in LNCaP cells, the list of DEGs was submitted to the DAVID, GOC, and KEGG databases, which identified 1250, 1366, and 480 unique genes within each database, respectively, which were then used for ontological analysis. According to these databases, proL1 overexpression regulated gene expression in ontological groups involved "anatomical structure development/morphogenesis" (more specifically "prostate gland morphogenesis"), extracellular matrix organization," "signaling," and "signal transduction. DEGs were also overrepresented in ontological groups potentially involved in vascularization and blood flow regulation. Analysis of the list of DEGs using the KEGG database identified the ontologic group "pathways in cancer" as having the greatest number of DEGs. DEGs were also identified with significant overrepresentation in specific biochemical pathways, such as the P13K-Akt, VEGF, and MAPK signaling pathways as well as steroid metabolic pathways. However, there were differences in the regulation of specific genes related to the androgen response between LNCaP and PC3. Unlike PC3, where overexpression of ProL1 caused a significant increase in expression of the androgen receptor gene (AR) and a decrease in expression of the estrogen receptor (ESR1), in LNCaP neither of these genes were changed in expression. In contrast, proL1 overexpression in LNCaP reduced expression of the progesterone receptor gene (PGR) but not in PC3 cells. Conclusion: Global analysis of the changes in gene expression caused by proL1 overexpression in PC3 and LNCaP cells supports that it has a role both in the modulation of genetic pathways involved in both overcoming hypoxia and the development of androgen-insensitivity. Opiorphins have previously been shown to be directly involved in regulation of blood-flow to tissues through their modulation of smooth muscle tone, and therefore their upregulation in tumors may directly contribute to overcoming the hypoxic barrier that develops in the growing tumor. In addition, overexpression of ProL1 in both PC3 and LNCaP cells modulated expression of genes involved in angiogenesis and morphogensis; the activation of these pathways would likely be involved in vascularization of the tumor and thereby also contribute to overcoming hypoxia. Overexpression of proL1 also affected genes involved in steroid metabolism and response pathways in both LNCaP and PC3, which could potentially contribute to the modulation of androgen sensitivity. In PC3 proL1+ cells we observed a significant upregulation of the Androgen Receptor (AR). Increased activity of the AR could subsequently lead to the activation of secondary messengers involved in modulating the activity of other signaling pathways such as the PI3K-Akt signaling and MAPK pathways, which had significant over-representation of DEGs in PC3 proL1+ cells. The PI3K-Akt signaling pathway is considered one of the most commonly dysregulated pathways in prostate cancer with approximately 40% of early stage, and 70-100% of advanced staged cancer exhibiting dysregulation of this pathway. Overall design: Total RNA of PC3 cells overexpressing proL1 and their parental cell line

研究目的：本研究借助RNA测序（RNA-Seq）技术，探究proL1上调对PC3细胞全局基因表达的影响。
实验方法：利用慢病毒载体将PC3细胞转染以过表达proL1。将过表达proL1的PC3细胞株（PC3 proL1+）及其亲本细胞株以每株3个复孔的密度接种于6孔板中。培养3天后，使用凯杰（Qiagen）RNeasy迷你试剂盒提取总RNA，并通过RNA测序（RNA-Seq）分析全局基因表达谱。
实验结果：本研究在PC3 proL1+细胞株与其亲本细胞株之间共鉴定出1698个差异表达基因（DEGs）。其中，209个差异表达基因与过表达proL1的LNCaP细胞中共有的差异基因重合。为鉴定LNCaP细胞中proL1过表达所调控的生物学功能，我们将该差异基因列表提交至DAVID、GOC及京都基因与基因组百科全书（KEGG）数据库，各数据库分别筛选出1250、1366及480个独特基因用于本体分析。基于上述数据库的分析结果显示，proL1过表达所调控的基因主要富集于以下本体类群：“解剖结构发育/形态发生”（具体为“前列腺腺形态发生”）、“细胞外基质组织”、“信号传导”及“信号转导”。差异表达基因同时在与血管生成及血流调控相关的本体类群中显著富集。通过KEGG数据库对差异基因列表进行分析发现，“癌症通路”是富集差异基因最多的本体类群。此外，差异基因在PI3K-Akt、血管内皮生长因子（VEGF）、丝裂原活化蛋白激酶（MAPK）信号通路及类固醇代谢通路等特定生化通路中亦存在显著富集现象。不过，LNCaP与PC3细胞中与雄激素应答相关的特定基因调控模式存在差异：在PC3细胞中，proL1过表达可显著上调雄激素受体（AR）基因的表达，并下调雌激素受体（ESR1）基因的表达；而在LNCaP细胞中，这两个基因的表达均未发生显著变化。与之相反，LNCaP细胞中proL1过表达可下调孕酮受体（PGR）基因的表达，该现象在PC3细胞中并未出现。
研究结论：对PC3及LNCaP细胞中proL1过表达所引发的基因表达变化进行全局分析后证实，proL1在调控与缺氧耐受及雄激素不敏感发生相关的遗传通路中发挥作用。此前已有研究表明，阿片样肽（opiorphins）可通过调节平滑肌张力直接参与组织血流调控，因此肿瘤中proL1的上调可能直接促进肿瘤生长过程中缺氧屏障的突破。此外，PC3与LNCaP细胞中proL1过表达均可调控血管生成及形态发生相关基因的表达，此类通路的激活可能参与肿瘤血管生成，进而同样有助于缺氧耐受。ProL1过表达还可影响LNCaP与PC3细胞中类固醇代谢及应答通路相关基因的表达，这可能对雄激素敏感性的调控产生潜在影响。在PC3 proL1+细胞中，我们观察到雄激素受体（AR）的显著上调。AR活性的增强可进一步激活参与调控其他信号通路（如PI3K-Akt及MAPK通路）活性的第二信使，而上述通路在PC3 proL1+细胞中均存在差异基因的显著富集。PI3K-Akt信号通路被认为是前列腺癌中最常见的失调通路之一，约40%的早期前列腺癌及70%-100%的晚期前列腺癌患者均存在该通路的失调。
实验整体设计：对过表达proL1的PC3细胞及其亲本细胞株的总RNA进行测序分析。