ATP5B在细胞膜异位表达对人前列腺癌细胞转移能力的影响及机制探究

发布时间：2018-06-25 22:57

本文选题：前列腺癌 + 细胞膜ATP5B　；参考：《吉林大学》2017年硕士论文

【摘要】：前列腺癌(Prostate cancer,PCa)是男性泌尿生殖系统常见的恶性肿瘤之一,发生转移后治疗难度加大,且预后不佳。因此,阐明PCa转移机制、识别高转移潜能前列腺癌的潜在诊疗靶点尤为重要。在本课题组前期工作中,我们选用前列腺正常上皮细胞,前列腺癌细胞PC-3和前列腺癌高转移细胞PC-3M通过噬菌体七肽库进行差减筛选,得到了只与PC-3M特异性结合的短肽B04,并且证实B04能够有效抑制PC-3M细胞的增殖、转移。随后,通过蛋白质组学等方法检测到了短肽B04的受体蛋白为PC-3M细胞膜上的ATP5B,并验证了ATP5B在PC-3M细胞的细胞膜异位表达。ATP5B通常表达于线粒体内膜,在某些细胞中可以异位表达于细胞膜,其功能并未明确。异位表达于PC-3M膜表面的ATP5B的功能有待鉴定,因此在本实验中,我们旨在发现和探究ATP5B在细胞膜异位表达对人前列腺癌细胞侵袭转移能力的影响及其机制。实验结果:1.ATP5B在前列腺癌及癌旁组织的表达及定位通过免疫组化染色法检测98例前列腺癌组织芯片中ATP5B蛋白的表达情况,前列腺组织芯片包括58例前列腺癌组织、37例前列腺癌旁组织和3例正常前列腺组织。并对ATP5B在细胞膜异位表达情况进行分析,结果显示:(1)ATP5B在前列腺癌组织中可出现肿瘤细胞膜的异位表达;且细胞膜异位表达阳性率在浸润扩散的前列腺癌组织中明显增高(p0.05)。(2)ATP5B在细胞膜异位表达阳性率在Gleason≥8的前列腺癌组织中明显增高(p0.05)。2.ATP5B在细胞膜异位表达对前列腺癌细胞侵袭转移能力的影响⑴细胞膜ATP5B过表达与抑制表达的模型建立分别用胆固醇和白皮杉醇处理PC-3M细胞,采用Western blot、实时定量PCR、ATP水平检测等技术从蛋白、基因和功能水平探究胆固醇和白皮杉醇对于PC-3M细胞膜ATP5B的表达是否有影响。结果显示:(1)蛋白水平:胆固醇处理后PC-3M的线粒体蛋白ATP5B降低,膜蛋白ATP5B增加,总蛋白ATP5B无明显变化;白皮杉醇处理后PC-3M的线粒体蛋白ATP5B无明显变化,膜蛋白ATP5B降低,总蛋白ATP5B降低,(2)基因水平,胆固醇处理PC-3M后,ATP5B m RNA相对表达量无明显变化;白皮杉醇处理PC-3M后,ATP5B m RNA相对表达量降低。(3)功能水平,胆固醇促进PC-3M细胞外ATP生成,白皮杉醇抑制细胞外ATP生成。以上结果提示,胆固醇促使ATP5B由线粒体异位至细胞膜,增加细胞膜ATP5B的表达;而白皮杉醇可以抑制细胞膜ATP5B的表达。⑵ATP5B在细胞膜异位表达促进前列腺癌侵袭转移的体内外研究采用细胞划痕实验、Transwell小室迁移/侵袭实验来检测改变ATP5B在细胞膜异位表达量后,PC-3M的侵袭转移功能在体外的改变。结果显示:胆固醇处理PC-3M后,ATP5B在细胞膜异位表达量增高,其体外迁移、侵袭均提高(p0.05);细胞膜ATP5B抑制剂白皮杉醇处理PC-3M后,其体外迁移、侵袭均明显下降(p0.05)。随后采用人前列腺癌鸡胚绒毛尿囊膜转移模型检测抑制细胞膜ATP5B表达前后PC-3M半体内转移能力变化。结果显示:抑制PC-3M细胞膜ATP5B,其转移能力明显下降(p0.05)。以上结果提示ATP5B在细胞膜异位表达可促进前列腺癌侵袭转移。⑶ATP5B在细胞膜的异位表达促进前列腺癌侵袭转移机制初步探索采用Westren Blot、PCR检测改变ATP5B在细胞膜异位表达量后,c-Myc、VEGFA的蛋白以及m RNA的变化。结果显示:ATP5B在PC-3M细胞膜异位表达量的改变会引起c-Myc、VEGFA蛋白及转录水平的变化,并且改变ATP5B在细胞膜的异位表达量后其体外诱导HUVEC细胞管型形成的能力也随之发生变化。推测:ATP5B在细胞膜异位表达可能通过激活c-Myc原癌基因并上调VEGFA来促进前列腺癌的侵袭转移。以上实验结果说明了ATP5B在高级别前列腺癌和浸润扩散的前列腺癌组织的细胞膜上高表达;可分别通过胆固醇和白皮杉醇处理PC-3M细胞来构建细胞膜ATP5B过表达和抑制表达模型;ATP5B在细胞膜异位表达促进前列腺癌细胞体内外侵袭转移,其机制可能是通过激活C-Myc原癌基因并上调VEGFA来促进前列腺癌的侵袭转移。以上结果也进一步说明ATP5B在细胞膜异位表达可促进前列腺癌细胞的侵袭转移,有望成为前列腺癌转移的检测指标或治疗靶点。
[Abstract]:Prostate cancer (PCa) is one of the common malignant tumors in the male genitourinary system. The treatment is difficult and the prognosis is not good after the metastasis. Therefore, it is very important to clarify the mechanism of PCa transfer and identify the potential diagnostic target of high metastatic potential prostate cancer. In our early work, we chose normal epithelium of the prostate. Cells, prostate cancer cell PC-3 and high metastatic prostate cancer cell PC-3M were screened by phage seven peptide library, and a short peptide B04 was obtained only with PC-3M specific binding, and it was proved that B04 could effectively inhibit the proliferation and metastasis of PC-3M cells. Then, the receptor protein of short peptide B04 was detected by proteomics as PC-3M. ATP5B on the cell membrane, and verifies that the ectopic expression of ATP5B in the cell membrane of PC-3M cells is usually expressed in the mitochondrial membrane, which can be expressed ectopic to the cell membrane in some cells. The function of the.ATP5B is not clear. The function of ATP5B expressed on the surface of the PC-3M membrane needs to be identified. In this experiment, we aim to discover and explore ATP5B The effect of ectopic expression of cell membrane on the invasion and metastasis of human prostate cancer cells and its mechanism. Experimental results: the expression and localization of 1.ATP5B in prostate cancer and para cancer tissue were detected by immunohistochemical staining in 98 cases of prostate cancer tissue microarray, and 58 cases of prostate cancer tissue were included in the anterior gland tissue chip. The heterotopic expression of ATP5B in the cell membrane was analyzed in 37 para cancer tissues and 3 normal prostate tissues. The results showed that (1) the ectopic expression of the tumor cell membrane could be found in the prostate cancer tissue (1) and the positive rate of ectopic expression of the cell membrane was significantly increased in the infiltrating and diffusing prostate cancer tissues (P0.05). (2) ATP5B was ATP5B in the prostate cancer tissue. The positive rate of ectopic expression of cell membrane increased significantly in the prostate cancer tissues of Gleason > 8 (P0.05). The effect of ectopic expression of.2.ATP5B on the invasion and metastasis of prostate cancer cells (1): the ATP5B overexpression of cell membrane and the model of inhibiting expression of the cell membrane, PC-3M cells were treated with cholesterol and paclitaxel respectively, and Western blot was used in real time. Quantitative PCR, ATP level detection and other techniques explored whether cholesterol and paclitaxel had an effect on the expression of ATP5B in the PC-3M cell membrane from protein, gene and functional levels. The results showed: (1) protein level: the mitochondrial protein ATP5B decreased in PC-3M after cholesterol treatment, the membrane protein ATP5B increased, and the total protein ATP5B had no obvious changes; after the treatment of paclitaxel The mitochondrial protein ATP5B of PC-3M had no obvious changes, the membrane protein ATP5B decreased, the total protein ATP5B decreased, and (2) the relative expression of ATP5B m RNA was not obviously changed after PC-3M treatment of PC-3M, and the relative expression of ATP5B m RNA decreased after the treatment of PC-3M. (3) the function level, the cholesterol promoted the formation of extracellular matrix, and the inhibition of taxol inhibition. These results suggest that cholesterol promotes ATP5B from mitochondrial to cell membrane and increases the expression of ATP5B in cell membrane, while paclitaxel can inhibit the expression of ATP5B in cell membrane. (2) ATP5B in cell membrane ectopic expression to promote the invasion and metastasis of prostate cancer in vivo and in vitro study by cell scratch test, Transwell cell migration / invasiveness experiment was used to detect the changes in the invasion and transfer function of PC-3M after ATP5B's ectopic expression of cell membrane. The results showed that after cholesterol treatment PC-3M, the ectopic expression of ATP5B increased in cell membrane, and its invasion in vitro increased (P0.05). After the treatment of PC-3M, the ATP5B inhibitor of the cell membrane was migrated and attacked in vitro. Significantly decreased (P0.05). Then the transfer ability of PC-3M in the cell membrane was detected by the chick chorioallantoic membrane transfer model of human prostate cancer. The results showed that the metastasis ability of PC-3M cell membrane was significantly decreased (P0.05). The results suggested that the ectopic expression of ATP5B in the cell membrane could promote the invasion of prostate cancer. Metastasis. Heterotopic expression of ATP5B in cell membrane promotes the mechanism of invasion and metastasis of prostate cancer by using Westren Blot. PCR changes the changes in c-Myc, VEGFA protein and m RNA changes after ATP5B in cell membrane ectopic expression. The results show that ATP5B in PC-3M cell membrane ectopic expression can cause c-Myc, protein and transcriptional water The ability to induce the formation of HUVEC cells in vitro after ATP5B's ectopic expression in the cell membrane also changes. It is speculated that the ectopic expression of ATP5B in the cell membrane may promote the invasion and migration of the prostate cancer by activating the c-Myc proto oncogene and up regulation of VEGFA. These results indicate that ATP5B is at a high level. The cell membrane of prostate cancer and infiltrating prostate cancer tissue is highly expressed, and PC-3M cells can be treated with cholesterol and paclitaxel to construct the ATP5B overexpression and inhibition expression model of the cell membrane, and the ectopic expression of ATP5B in the cell membrane promotes invasion and metastasis of the prostate cancer cells in vivo and in vivo. The mechanism may be by activating the primary cancer of C-Myc. The gene and up regulation of VEGFA can promote the invasion and metastasis of prostate cancer. The above results also suggest that ATP5B can promote the invasion and metastasis of prostate cancer cells by ectopic expression of the cell membrane. It is expected to be a detection target or therapeutic target for the metastasis of prostate cancer.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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