miR-373调控乳腺癌侵袭转移的功能机制及临床意义研究

发布时间：2018-04-26 00:10

本文选题：miR-373 + 乳腺癌　；参考：《暨南大学》2017年博士论文

【摘要】：第一部分 miR-373介导的TRPS1下调促使乳腺癌侵袭转移的分子机制及临床意义研究背景:乳腺癌是最常见的女性恶性肿瘤,是癌症导致女性死亡的最主要原因。尽管经过长期深入研究,早期诊断和治疗乳腺癌有了长足的进步,但是转移率和术后复发率仍然很高。因此,阐明乳腺癌转移复发的机制,发现新的预后标记物,具有重要的科学意义和临床价值。乳腺癌的侵袭转移是一个多步骤、多因素参与的复杂过程,许多癌基因、抑癌基因、转移相关基因以及一些非编码RNA参与了这个过程的调控。非编码RNA miR-373的异常表达与乳腺癌的侵袭转移密切相关,但miR-373在乳腺癌侵袭转移的分子机制目前仍不清楚。我们在前期工作中发现TRPS1可能是miR-373的一个新的候选靶标基因。目前,TRPS1主要集中在分化和发育方面的研究,TRPS1在乳腺癌中的生物学功能尚不明确,通过什么样的作用机制影响肿瘤的发生发展还不清楚。本论文在前期研究的基础上,旨在探讨miR-373调控候选靶标基因TPRS1在乳腺癌侵袭转移中的功能、分子机制和临床意义,为干预乳腺癌远端转移提供新的靶点线索。研究方法:1.分别沉默和过表达miR-373,Western Blotting检测TRPS1的表达。2.生物信息学预测结合双荧光报告基因、点突变等实验验证miR-373直接抑制TRPS1的表达。3.Western Blotting检测不同转移潜能的肿瘤细胞中TRPS1的表达。4.Transwell和Western Blotting检测干扰和过表达TRPS1对肿瘤细胞的迁移侵袭能力以及EMT分子表型的变化。5.NOD-SCID小鼠尾静脉注射TRPS1稳定沉默表达细胞株检测TRPS1对肿瘤转移能力的影响。6.构建野生型和突变型的TRPS1载体,Western Blotting和Transwell检测miR-373high的乳腺癌细胞中的TRPS1的表达、迁移侵袭能力的变化。7.同时沉默TRPS1和抑制miR-373的组合处理,Western Blotting和Transwell检测TRPS1的表达、迁移侵袭能力的变化。8.SILAC定量蛋白质组学技术筛选受TRPS1调控的下游分子FOXA1。9.分别干扰和过表达TRPS1,q PCR和Western Blotting检测FOXA1的表达。10.生物信息学预测结合双荧光报告基因、点突变、Ch IP-q PCR等实验验证TRPS1转录激活FOXA1的表达。11.同时沉默TRPS1和过表达FOXA1的组合处理,Transwell和Western Blotting检测乳腺癌细胞的迁移侵袭能力以及EMT分子表型的变化。12.在临床样品数据分析中确认TRPS1与FOXA1之间的病理学相关性。深入分析TRPS1水平的高低与病理学指标和生存期之间的关系。研究结果:1.TRPS1是miR-373的直接靶标基因,miR-373通过结合TRPS1的3’UTR区下调TRPS1的表达。2.TRPS1在高转移潜能的肿瘤细胞中下调,TRPS1的下调促进了乳腺癌EMT和侵袭转移。3.在动物水平证实稳定沉默TRPS1促进乳腺癌的远处转移。4.TRPS1是miR-373促使乳腺癌EMT和侵袭转移的主要靶标基因。5.TRPS1作为转录激活因子,直接结合FOXA1的启动子区促进FOXA1的转录。6.TRPS1通过上调EMT负调控因子FOXA1的表达,从而抑制乳腺癌的侵袭转移和EMT。7.临床样品中TRPS1与FOXA1的表达存在正相关关系。8.TRPS1低表达的乳腺癌病人预后较差,提示了TRPS1低表达可以作为乳腺癌预后差的一个指标。结论:本论文首次发现了TRPS1作为转录激活因子,激活EMT负调控因子FOXA1的转录。阐明miR-373介导了TRPS1在乳腺癌中的低表达,下调FOXA1的表达,最终促进乳腺癌的EMT和侵袭转移的分子机制。第二部分miR-373-TXNIP-HIF1α-TWIST-miR-373信号反馈环在乳腺癌侵袭转移的作用及机理研究研究背景:我们前期工作证实了,miR-373通过结合抑癌基因TXNIP的3’UTR区下调TXNIP的表达,从而促进乳腺癌EMT和迁移侵袭。本论文是在前期研究的基础上,进一步探讨miR-373如何通过TXNIP促使乳腺癌侵袭转移的功能机制,以及miR-373下调的TXNIP促使乳腺癌侵袭转移的分子机制和临床意义。研究方法:1.NOD-SCID小鼠尾静脉分别注射miR-373、miR-373/TXNIP-3’UTR和miR-373/TXNIP稳定表达细胞株,检测肿瘤的肺转移能力。2.分别过表达miR-373或沉默TXNIP,同时加入1μM浓度的氧化剂H2O2处理12h,Transwell检测乳腺癌细胞的迁移侵袭能力的变化。3.分别抑制miR-373或过表达TXNIP,同时加入2m M浓度的抗氧化剂NAC处理24h,Transwell检测乳腺癌细胞的迁移侵袭能力的变化。4.分别沉默和过表达HIF1α,Western Blotting检测TWIST的表达。5.同时过表达TXNIP和miR-373的组合处理,Western Blotting和IF检测HIF1α和TWIST的表达、核定位行为的变化。6.分别沉默和过表达HIF1α或TWIST,q PCR检测miR-373的表达。7.同时沉默TWIST和过表达HIF1α的组合处理,q PCR检测miR-373的表达。8.生物信息学预测结合双荧光报告基因、点突变、Ch IP-q PCR等实验验证TWIST转录激活miR-373的表达。9.在临床样品数据分析miR-373-TXNIP-HIF1α-TWIST-miR-373信号反馈环的临床意义。研究结果:1.在前期研究成果的基础上,我们进一步在动物水平证实miR-373通过靶向抑制TXNIP促进乳腺癌的远处转移。2.miR-373介导的TXNIP的沉默,引起细胞内的ROS水平下调,促进了乳腺癌的侵袭转移。3.由miR-373过表达和TXNIP沉默介导的ROS水平下调,促进了HIF1α及其下游TWIST的移位入核和表达上调。4.HIF1α及其下游TWIST存在对miR-373的正反馈调节,TWIST通过结合miR-373的启动子区促进miR-373的转录。5.阐明miR-373-TXNIP-HIF1α-TWIST-miR-373信号反馈环介导了乳腺癌EMT和侵袭转移。这一调控机制与乳腺癌病人的预后差显著相关,提示了此信号反馈环的激活可作为乳腺癌病人预后差的一项诊断指标。结论:本论文在前期研究的基础上,阐明了由miR-373激活的miR-373-TXNIP-HIF1α-TWIST-miR-373信号反馈环促使乳腺癌EMT和侵袭转移的分子机制,证实了该信号反馈环的激活可作为乳腺癌的预后标志物。
[Abstract]:Part 1: the molecular mechanism and clinical significance of miR-373 mediated TRPS1 downregulation in breast cancer invasion and metastasis: breast cancer is the most common female malignant tumor, and is the most important cause of cancer leading to female death. Although long - term in-depth study, the early diagnosis and treatment of breast cancer have made considerable progress, but the transfer rate The recurrence rate is still very high. Therefore, it is of great scientific significance and clinical value to elucidate the mechanism of metastatic recurrence of breast cancer and discover new prognostic markers. The invasion and metastasis of breast cancer is a multistep, complex process involving multiple factors, many oncogenes, tumor suppressor genes, metastasis related genes and some non coded RNA. The abnormal expression of non coding RNA miR-373 is closely related to the invasion and metastasis of breast cancer, but the molecular mechanism of miR-373 in the invasion and metastasis of breast cancer is still unclear. We found that TRPS1 may be a new candidate target gene for miR-373 in earlier work. At present, TRPS1 is mainly concentrated in the differentiation and development side. The biological function of TRPS1 in breast cancer is not clear. What kind of mechanism of action affects the development of the tumor is not clear. On the basis of previous studies, this paper aims to explore the function, molecular mechanism and clinical significance of the miR-373 regulation candidate target gene TPRS1 in the invasion and metastasis of breast cancer, in order to intervene in mammary gland. Distal metastasis of cancer provides new target clues. Study methods: 1. silence and overexpression of miR-373, Western Blotting detection of TRPS1 expression,.2. bioinformatics prediction combined with double fluorescent reporter gene, point mutation and other experiments verify that miR-373 directly inhibits TRPS1 expression by.3.Western Blotting to detect TRPS1 in tumor cells with different metastatic potential. Expression of.4.Transwell and Western Blotting detection of interference and overexpression of TRPS1 to tumor cell migration and invasion and changes in the phenotype of EMT molecule.5.NOD-SCID mouse tail vein injection TRPS1 stable silent expression cell lines detection of TRPS1 on tumor metastasis ability,.6. construction of wild type and mutant TRPS1 vector, Western Blotti Ng and Transwell detection of the expression of TRPS1 in breast cancer cells of miR-373high, changes in migration and invasion ability.7. simultaneously silence TRPS1 and combination of inhibition of miR-373, Western Blotting and Transwell detection TRPS1 expression, migration and invasion ability change.8.SILAC quantitative proteomics technology screening downstream molecules regulated by downstream molecules 1.9. interference and overexpression of TRPS1, Q PCR and Western Blotting detection of FOXA1,.10. bioinformatics prediction combined with double fluorescent reporter gene, point mutation, Ch IP-q PCR, etc. Changes in cell migration and invasion and changes in EMT molecular phenotype.12. confirm the pathological correlation between TRPS1 and FOXA1 in clinical sample data analysis. The relationship between the level of TRPS1 and the pathological indexes and life period is analyzed. The results of the study: 1.TRPS1 is the direct target gene of miR-373, miR-373 by combining TRPS1's 3 'UTR. The expression of TRPS1 is down regulated in the tumor cells with high metastatic potential. The down regulation of TRPS1 promotes the EMT and invasion and metastasis of the breast cancer EMT and the metastasis.3. in the animal level, which is stable and silent TRPS1 to promote the distant metastasis of breast cancer..4.TRPS1 is the main target gene of miR-373 to promote breast cancer EMT and invasion and metastasis.5.TRPS1 as a transcription activator. Directly combining the promoter region of FOXA1 to promote the transcriptional.6.TRPS1 of FOXA1 by up regulation of the expression of EMT negative regulator FOXA1, thus inhibiting the invasion and metastasis of breast cancer and the expression of TRPS1 and FOXA1 in the EMT.7. clinical samples, the prognosis of breast cancer patients with low expression of positive correlation.8.TRPS1 is poor, suggesting that the low expression of TRPS1 can be used as mammary gland. Conclusion: TRPS1 is first discovered as a transcription activator and activates the transcription of EMT negative regulator FOXA1. MiR-373 mediates the low expression of TRPS1 in breast cancer, down regulation of FOXA1 expression, and ultimately promotes the molecular mechanism of EMT and invasion and metastasis of breast cancer. Second part miR-373-TXNIP-HIF1 alpha -TWIS The research background of the role and mechanism of T-miR-373 signal feedback loop in the invasion and metastasis of breast cancer: our previous work has confirmed that miR-373 reduces the expression of TXNIP by combining the 3 'UTR region of the tumor suppressor gene TXNIP, thus promoting the EMT and migration of breast cancer. This paper is based on the earlier research, and further explores how miR-373 passes through TX. The molecular mechanism and clinical significance of NIP to promote invasion and metastasis of breast cancer and the molecular mechanism and clinical significance of miR-373 down-regulation of TXNIP to promote invasion and metastasis of breast cancer. Research methods: miR-373, miR-373/TXNIP-3 'UTR and miR-373/TXNIP in 1.NOD-SCID mice were injected respectively with miR-373, miR-373/TXNIP-3' UTR and miR-373/TXNIP, and the lung metastasis of the tumor was detected by.2. over expression of miR. -373 or TXNIP, while adding 1 micron M concentration of oxidant H2O2 to 12h, Transwell to detect the migration and invasion of breast cancer cells,.3. inhibits miR-373 or overexpression TXNIP respectively. The expression of HIF1 alpha and Western Blotting in the detection of TWIST expression.5. and the combination of TXNIP and miR-373. Western Blotting and IF detect the expression of HIF1 alpha and TWIST. CR detection of miR-373 expression.8. bioinformatics prediction combined with double fluorescent reporter gene, point mutation, Ch IP-q PCR and other experiments to verify the clinical significance of TWIST transcriptional activation miR-373 expression.9. in the clinical sample data analysis miR-373-TXNIP-HIF1 alpha -TWIST-miR-373 signal feedback loop. Study fruit: 1. on the basis of previous research results, we entered One step at animal level shows that miR-373 promotes the distant metastasis of.2.miR-373 mediated TXNIP silencing through target inhibition of TXNIP, causing the ROS level down regulation in cells, promoting the invasion and metastasis of breast cancer,.3., miR-373 overexpression and TXNIP silencing mediated ROS downregulation, promoting HIF1 A and its downstream TWIST translocation and translocation. The expression of.4.HIF1 alpha and its downstream TWIST are positive feedback regulation of miR-373, and TWIST promotes miR-373 transcriptional.5. by combining the promoter region of miR-373 to clarify that miR-373-TXNIP-HIF1 a -TWIST-miR-373 signal feedback loop mediates breast cancer EMT and invasion and metastasis. This regulation mechanism is significantly related to the poor prognosis of breast cancer patients. The activation of this signal feedback loop can be used as a diagnostic indicator of poor prognosis in breast cancer patients. Conclusion: on the basis of previous studies, this paper elucidates the molecular mechanism of miR-373 activated miR-373-TXNIP-HIF1 alpha -TWIST-miR-373 signal feedback loop for breast cancer EMT and invasion and metastasis, and confirms that the activation of the signal feedback loop can be used as a result. Prognostic markers for breast cancer.

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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