TFDP3的表达调控及其对前列腺癌PC3细胞凋亡的影响

发布时间：2018-09-11 21:44

【摘要】：前列腺癌(prostate cancer，PCa)在男性肿瘤中具有很高的发病率，在美国已成为男性癌症死因中仅次于肺癌的第二大杀手[1]。传统的临床前列腺癌治疗方法包括雄激素阻断治疗、手术治疗和药物去势治疗等。但是随着时间的推移，几乎全部患者的前列腺癌类型都将由激素依赖性转变为非依赖性，也就是激素非依赖性前列腺癌(androgen independent prostate cancer，AIPC)，使得原本激素依赖性的癌细胞增殖加速而不受低雄激素水平抑制，最终促进了前列腺癌的进展或转移。到目前为止，对AIPC还尚未发现有效的治疗办法。但随着现代分子生物学的发展，基因治疗将有望成为癌症治疗的新的有效手段。因此阐明前列腺癌的分子发病机制及转录调控机理具有重要的科学和临床意义。 TFDP3是一个新的E2F1的直接调控因子。本课题组前期应用组织芯片和原位杂交方法进行了正常组织和癌组织的TFDP3表达分析。实验证明，该基因并非所谓的睾丸/肿瘤特异的癌基因，而是广泛表达于多种组织细胞。同时，它的确在多种癌组织中表达增强，尤其是相对于正常情况下不表达TFDP3的肝和前列腺组织中。研究表明TFDP3不仅可以抑制E2F1诱导的细胞增殖活性，而且可以抑制E2F1诱导的细胞凋亡的作用；可以诱导前列腺癌细胞的自噬作用；提高激素依赖的前列腺癌细胞LNCap的生存能力。TFDP3诱导的自噬功能和抗凋亡效应极有可能是前列腺癌由激素依赖性向非依赖性转变过程中的关键的自我保护机制之一。本研究通过进一步对TFDP3启动子区转录结合位点的分析，发现TFDP3可能与E2F1转录因子结合。在此基础上，本课题首先我们构建了包含TFDP3基因启动子的荧光报告载体，从启动子水平证实了转录因子E2F1对TFDP3的调控作用，其次通过western-blot初步探索TFDP3在E2F1刺激下的蛋白水平的变化，并且利用流式细胞术在功能水平上证实了TFDP3与E2F1的相互作用对前列腺癌细胞凋亡所造成的影响。最后，通过免疫组化分析前列腺癌组织和正常组织中TFDP3和E2F1的表达水平，证明TFDP3和E2F1在前列腺癌组织中的表达水平相较于正常组织的表达水平均增高；同时，随着前列腺癌等级的增高，二者的表达水平也增强，并且增高的趋势相一致。主要研究结果如下： 1.首先，通过NCBI基因组数据库查找到TFDP3基因ATG上游约1064bp序列，并针对此序列设计引物。以人的前列腺癌PC3细胞基因组DNA为模板，采用PCR技术扩增TFDP3启动子片段并克隆到pGL3-Basic荧光素酶报告基因上游多克隆位点处，在此基础上构建包含TFDP3基因启动子和荧光素酶报告基因的重组载体pGL3-TFDP3-promoter。最后，将构建的重组载体pGL3-TFDP3-promoter转染PC3细胞后，检测荧光素酶的表达活性，以验证克隆的片断存在TFDP3的启动子序列或转录调控序列。进一步与E2F1表达载体共转染PC3细胞后，TFDP3启动子诱导的荧光素酶活性较单独转染pGL3-TFDP3-promoter显著升高(1.14±0.06vs0.61±0.05, P0.05)，证明E2F1可诱导TFDP3转录。 2．通过转染E2F1表达载体pCMV-E2F1-HA于PC3细胞中，提取蛋白后，经Western blot检测，得到以下实验结果：相比未转染组，转染E2F1表达载体组的E2F1和TFDP3的表达均有所增加，IOD（TFDP3）/IOD(β-actin)约增加2.7倍（0.089±0.02vs0.24±0.03，P0.05）。可见E2F1对TFDP3的蛋白表达具有上调作用。 3.通过流式细胞仪检测TFDP3与E2F1相互作用对前列腺癌细胞凋亡的影响可以发现：转染E2F1表达载体后细胞凋亡率显著上升(2.66%±0.001vs7.1%±0.003，P0.05)，而与pcDNA3.1-TFDP3共转染后细胞凋亡率明显下降（7.1%±0.003vs4.92%±0.002，P 0.05）。因此，，进一步验证了TFDP3可以在一定程度上抑制由E2F1诱导的前列腺癌细胞凋亡。 4.通过免疫组化检测前列腺癌组织标本以及正常前列腺组织标本中TFDP3和E2F1的表达情况，结果显示：相较正常前列腺组织，前列腺癌组织中TFDP3和E2F1的表达水平均增强，同时随着前列腺癌等级的增高，TFDP3和E2F1的表达也随之增长，并且二者的增长趋势相一致。综上所述，本研究通过构建TFDP3启动子区荧光报告载体pGL3-TFDP3-promoter，并利用双荧光素酶检测系统及Western blot初步确定E2F1对TFDP3基因启动子区具有转录水平和表达水平的调控作用，证明了E2F1参与该基因表达调控。此外通过免疫组化检测正常前列腺组织和前列腺癌组织中TFDP3和E2F1表达水平，并结合流式细胞术检测证实TFDP3可以结合E2F1进而有效抑制前列腺癌细胞的凋亡，从而在保障前列腺癌细胞生存中发挥了重要作用。这一现象提示TFDP3可能通过抑制激素缺失条件下前列腺细胞的凋亡，从而促进前列腺癌细胞的生存。
[Abstract]:Prostate cancer (PCa) has a high incidence in male tumors, and has become the second leading cause of cancer death in the United States after lung cancer [1].Traditional clinical treatments for prostate cancer include androgen blockade therapy, surgical treatment and drug castration. Hormone-independent prostate cancer (AIPC) can accelerate the proliferation of hormone-dependent cancer cells without being inhibited by low androgen levels, and ultimately promote the progression or metastasis of prostate cancer. However, with the development of modern molecular biology, gene therapy is expected to become a new and effective method for cancer treatment. Therefore, it is of great scientific and clinical significance to elucidate the molecular pathogenesis and transcriptional regulation mechanism of prostate cancer.
TFDP3 is a new direct regulator of E2F1. Our team used tissue microarray and in situ hybridization to analyze the expression of TFDP3 in normal and cancer tissues. It has been proved that TFDP3 is not a so-called testicular/tumor specific oncogene, but is widely expressed in a variety of tissues and cells. Increased expression in tissues, especially in liver and prostate tissues where TFDP3 is not normally expressed, has been shown to inhibit not only E2F1-induced cell proliferation but also E2F1-induced apoptosis, induce autophagy of prostate cancer cells, and enhance hormone-dependent prostate cancer. The autophagy and anti-apoptosis effects induced by TFDP3 may be one of the key self-protection mechanisms in the transition of prostate cancer from hormone dependence to non-dependence.
In this study, we further analyzed the transcriptional binding sites of TFDP3 promoter region and found that TFDP3 may bind to E2F1 transcription factors. On this basis, we first constructed a fluorescent reporter vector containing TFDP3 gene promoter, and confirmed the regulatory effect of transcription factor E2F1 on TFDP3 at promoter level. Secondly, Western-blo was used to investigate the role of TFDP3 transcription factor E2F1 in TFDP3 transcription. T preliminarily explored the changes of TFDP3 protein level stimulated by E2F1, and confirmed the effect of TFDP3-E2F1 interaction on apoptosis of prostate cancer cells by flow cytometry. Finally, the expression levels of TFDP3 and E2F1 in prostate cancer tissue and normal tissue were analyzed by immunohistochemistry. The expression level in prostate cancer tissues was higher than that in normal tissues. At the same time, with the increase of prostate cancer grade, the expression level of both increased, and the trend was consistent.
1. First, a sequence of about 1064bp upstream of ATG of TFDP3 gene was found by NCBI genomic database, and primers were designed for this sequence. Using human prostate cancer PC3 cell genomic DNA as template, TFDP3 promoter fragments were amplified by PCR and cloned to the upstream polyclonal site of pGL3-Basic luciferase reporter gene. The recombinant vector pGL3-TFDP3-promoter containing TFDP3 gene promoter and luciferase reporter gene. Finally, the constructed recombinant vector pGL3-TFDP3-promoter was transfected into PC3 cells and the luciferase expression activity was detected to verify the existence of TFDP3 promoter sequence or transcriptional regulatory sequence in the cloned fragment.
After co-transfection with E2F1 expression vector, the luciferase activity induced by TFDP3 promoter was significantly higher than that induced by pGL3-TFDP3-promoter alone (1.14 + 0.06vs0.61 + 0.05, P 0.05). It was demonstrated that E2F1 could induce TFDP3 transcription.
2. After transfection of E2F1 expression vector pCMV-E2F1-HA into PC3 cells, the protein was extracted and detected by Western blot. The results showed that the expression of E2F1 and TFDP3 was increased in the E2F1 expression vector group, and the expression of IOD (TFDP3) / IOD (beta-actin) was increased by 2.7 times (0.089 + 0.02vs0.24 + 0.03, P 0.05) compared with the untransfected group. The protein expression was up-regulated.
3. The effect of TFDP3-E2F1 interaction on apoptosis of prostate cancer cells was detected by flow cytometry. It was found that the apoptosis rate of prostate cancer cells increased significantly after transfection of E2F1 expression vector (2.66%+0.001 vs 7.1%+0.003, P 0.05), but decreased significantly after co-transfection with pcDNA3.1-TFDP3 (7.1%+0.003 vs 4.92%+0.002, P 0.05). The results showed that TFDP3 could inhibit the apoptosis of prostate cancer cells induced by E2F1 to some extent.
4. The expression of TFDP3 and E2F1 in prostate cancer tissue and normal prostate tissue were detected by immunohistochemistry. The results showed that compared with normal prostate tissue, the expression of TFDP3 and E2F1 in prostate cancer tissue were increased. At the same time, the expression of TFDP3 and E2F1 increased with the increase of prostate cancer grade. The growth trend of the two is the same.
To sum up, we constructed a fluorescent reporter vector pGL3-TFDP3-promoter for TFDP3 promoter region, and preliminarily determined that E2F1 could regulate the transcriptional and expression levels of TFDP3 promoter region by using dual luciferase detection system and Western blot, which proved that E2F1 was involved in the regulation of TFDP3 gene expression. The expression levels of TFDP3 and E2F1 in normal prostate tissue and prostate cancer tissue were measured. Flow cytometry showed that TFDP3 could bind to E2F1 and inhibit the apoptosis of prostate cancer cells effectively, thus playing an important role in protecting the survival of prostate cancer cells. The apoptosis of prostate cells promotes the survival of prostate cancer cells.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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