【作者】 胡芬；

【导师】 朱天慧；

【作者基本信息】 南开大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 第一部分：δEF1在乳腺癌细胞增殖中的功能研究乳腺癌是女性最常见的多发恶性肿瘤之一，无限增殖是几乎所有肿瘤细胞的特征之一，乳腺癌也毫不例外，乳腺癌细胞的异常增殖以及随后发生的转移过程所涉及的生物学过程及其原理尚不很清楚，这严重影响了乳腺癌的预防和治疗进展。 δEF1(δ-crystallinenhancer-binding factor1)隶属于一类锌指结构的转录因子超家族，具有广泛的转录抑制功能。该转录抑制功能主要依靠其两端的锌指结构簇与被调控基因的上游启动子中E2-box的结合而实现。虽然已知δEF1是癌细胞增殖和分化的一个重要的调节因子，但是关于它在调控细胞周期进程中的潜在功能还不是很清楚。我们用CCK-8试剂盒和Brdu参入流式细胞仪检测的方法研究δEF1在乳腺癌细胞周期中的作用。CCK-8实验结果表明，在MDA-MB-231细胞中过表达δEF1蛋白，可以明显促进细胞数目的增多；敲除掉内源的δEF1，则反之。BrdU处理并用流式细胞仪检测，结果证实在MDA-MB-231细胞中，δEF1的过表达可以非常明显使细胞周期处于S期的细胞的数目增加；用RNA干扰技术敲除掉δEF1，则可以得到相反的效果，这就说明δEF1可以促进乳腺癌细胞G1-S期的转换。在初步证实了δEF1可以促进乳腺癌细胞从G1向S期的转化的情况下，我们进一步对其分子机制进行了研究：在MDA-MB-231细胞中过表达δEF1，无论在转录水平还是翻译水平，均可看到CDK2和CDK4表达量的增加，p21表达量得减少。如果敲除掉内源性的δEF1，则CDK2和CDK4的表达量明显减少，p21的表达量明显被上调。这就证实在MDA-MB-231细胞中δEF1可以下调p21的表达，同时可以上调CDK2和CDK4的表达。为了进一步分析δEF1在转录水平是怎样调控p21的表达，首先，我们构建了一系列不同截短长度的人p21启动子的荧光素酶报告载体，并用网上的在线工具TRANSFAC和TESS，对人p21启动子进行了分析，结果发现在人p21启动子545/540处存在一个E2-box(CAGGTG)。荧光素酶实验和CHIP实验均证实δEF1通过结合到p21启动子的E2-box上，从而抑制p21的转录。我们又在雌激素受体ER阴性的MDA-MB-231，ER阳性的MCF-7、ZR-75-1、T47-D四种人乳腺癌细胞中用实时定量PCR和western blot方法检测到δEF1和p21表达呈明显的负相关。进一步在22例人乳腺癌组织临床标本中，实时定量PCR也检测δEF1和p21表达呈负相关。这都支持δEF1通过调控p21的表达从而调控乳腺癌细胞周期进程。综上所述，在乳腺癌细胞中，δEF1通过下调p21的表达，同时上调CDK2和CDK4的表达双重机制促进MDA-MB-231细胞的增殖。δEF1对细胞周期调控方面的研究，必将为我们如何防治乳腺癌提供一些新的思路。第二部分：δEF1在乳腺癌细胞骨转移中的功能研究在乳腺癌转移的很多阶段中，上皮细胞向间充质细胞的转化EMT是癌细胞扩散机制的基础，δEF1是含有锌指结构域的转录因子超家族的成员，在乳腺癌EMT和骨重建中起着重要的作用。我们发现δEF1在具有高迁移倾向的MDA-MB-231细胞中表达量较高，在具有低迁移倾向的MCF-7、T47-D和ZR-75-1细胞中表达量较低，并且在MDA-MB-231中过表达δEF1可以促进乳腺癌细胞的迁移和侵袭，而敲除掉内源性的δEF1的表达，则乳腺癌细胞的迁移和侵袭会受到抑制。骨是乳腺癌发生转移的主要靶器官之一，乳腺癌骨转移的发生往往伴随着成骨细胞和破骨细胞功能的紊乱。因此我们研究了MDA-MB-231细胞中δEF1表达的变化对乳腺癌诱导的破骨细胞成熟，成骨细胞成熟和钙沉积的影响。结果表明，过表达δEF1的MDA-MB-231细胞条件培养基不仅具有较高的诱导RAW264.7细胞TRACP活性的能力，进而促进破骨细胞的分化；而且可以抑制BMP-2诱导的C2C12细胞ALP活性的升高，以及抑制BMP-2诱导的C2C12细胞中osterix和骨钙素的表达增加，从而抑制成骨细胞的成熟；还可以减少MC3T3-E1细胞被茜素红染色的细胞数从而抑制成骨细胞的钙沉积。这就证实用过表达δEF1的MDA-MB-231中收集的条件培养基可以明显的诱导破骨细胞的成熟，并且同时抑制成骨细胞的分化和矿化；另一方面，敲除δEF1表达的MDA-MB-231细胞的条件培养基则表现出相反的作用。为了探索δEF1在乳腺癌骨转移中的具体分子机制，我们检测发现在MDA-MB-231细胞中，δEF1从转录和翻译两个水平都可以显著的上调基质金属蛋白酶-1MMP-1的表达。用转录因子数据库TESS分析MMP-1的启动子，发现在70/60的位置存在了一个AP-1位点(CATGAGTCAG)，荧光素酶实验结果显示，TPA可以上调MMP-1启动子的活性，而姜黄素(Curcumin)可以抑制MMP-1启动子的活性。而TPA，Curcumin和δEF1对人MMP-1启动子突变后的AP-1元件完全没有任何作用响应。ChIP实验证实过表达δEF1可以明显的使内源性的MMP-1启动子募集到更多的AP-1成员c-Jun/c-Fos。荧光素酶实验和CHIP实验结果都证实δEF1通过MMP-1启动子上的AP-1应答元件激活其启动子的活性。AP-1的组成元件已被证明是MAPK信号通路的下游分子，Western blot实验证实δEF1可以显著激活MDA-MB-231细胞中JNK信号通路，并且荧光素酶实验表明特异性JNK信号通路抑制剂sp600125大大削弱δEF1对MMP-1表达的诱导。可见δEF1诱导MMP-1表达这个过程是通过MAPK信号通路进行。总而言之，这些结果表明δEF1通过调节肿瘤微环境中生长因子来促进乳腺癌的骨转移过程中发挥着重要的作用。更多还原

【Abstract】 PartⅠ：δEF1promotes breast cancer cell proliferation throughdown-regulating p21expressionBreast cancer is the most common cancer in women. Abnormalproliferation of breast cancer cells and subsequent transfer process is amulti-gene participation through the completion of complex multi-stepprocess, its principle is not very clear. δEF1(δ-crystallin enhancer factor1) is a member of the zinc finger homeodomain transcription factorfamily. Studies revealed that δEF1is a widely expressed transcriptionalrepressor, working through its zinc finger clusters binding to consensusE2-box-like sequences. Although δEF1is implied as a regulatory factorat the crossroad between proliferation and differentiation incarcinogenesis, its potential effect in the regulation of cell cycleprogression has not been well elucidated. First, we assessed the effect of δEF1in regulating cell cycleprogression of breast cancer using cell proliferation assay. CCK-8asssyindicated that in δEF1-overexpressing cells, cell number increasedsignificantly, in comparison to the empty vector transfected cells. Incontrast, depletion of δEF1in MDA-MB-231cells exhibited a decreasedproliferative rate, compared to the control cells, indicating a promotingeffect of δEF1on proliferation of MDA-MB-231cells. Furthermore, flowcytometric measurements after BrdU incorporation confirmed an increasein the number of cells in the S phase after δEF1overexpression inMDA-MB-231cells. That knockdown of δEF1using RNA interferencewould result in a decrease of cell number in the S phase. Those resultshighlighting a potent role of δEF1to promote G1-S transition of breastcancer cells.Second,After known that δEF1can promote G1-S transition ofbreast cancer cells, we further studied the molecular mechanisms: Overexpressing δEF1in MDA-MB-231cells, we found thatCDK2,CDK4expression levels increased and the p21expressionreduced both at the transcriptional level and protein level; whereas δEF1knockdown exhibited an opposite effect.Further, having found that δEF1inhibited p21expression inMDA-MB-231cells, we were curious about whether δEF1is a bona fiderepressor of p21transcription. In this present report, a search using thetranscription factor database TRANSFAC and TESS identified an E2-box(CAGGTG) that is located at position545/540of human p21promoter.Luciferase and CHIP assays confirmed that δEF1inhibited thetranscription of p21through engaging the E2box on the p21promoter.To test whether δEF1mediates the transcriptional repression of p21in human tumor cells, we found that the expressions of δEF1and p21isinverse correlation in MDA-MB-231,MCF-7, T47D and ZR-75-1fourhuman breast cancer cell lines. We are further found a strong inverse relationship between the expressions of δEF1and p21in human breastcancer tissue samples. p21is one of critical regulatory molecules in cellcycle regulation. δEF1may play an irreplaceable role in breast cancercell cycle regulation trough regulate p21expression.Moreover, we demonstrated that δEF1down-regulated p21andconcurrently up-regulated the expressions of CDK2and CDK4duringthis process. δEF1inhibited p21transcription by recruiting to the E2-boxelement on the p21promoter. Depletion of endogenous δEF1inMDA-MB-231cells was sufficient to allow an inherent release of p21expression, thus resulting in the cell cycle arrest. In addition, thestimulatory effect of δEF1on cell proliferation through p21regulationwas supported by an inverse correlation of δEF1and p21expressionsobserved in both breast cancer cell lines and clinical tumor specimens.Taken together, these observations suggest a dual effect of δEF1inpromoting breast cancer cell proliferation, by differentially regulating the cell cycle regulatory proteins. This lay a good foundation for us to furtherresearch and development a novel target molecule for repression ofsystemic breast cancer progression.Part Ⅱ：δEF1promotes osteolytic metastasis of MDA-MB-231breast cancer cells by regulating MMP-1expressionBreast cancer metastasis is supposed to involve several stages inwhich epithelial-mesenchymal transition (EMT) is regarded as themechanistic basis for the behavior of cancer cells. Our recent studieshave implicated that δEF1is required for governing both breast cancerEMT and bone remodeling.We found that δEF1was highly expressed in MDA-MB-231, anosteolytic breast cancer cell line, but it was weakly expressed in theosteoblastic cell line, ZR-75-1. Its expression was also hardly detected inMCF-7and T47-D, two cell lines with low-metastatic potential. The results of cell migration and Matrigel invasion assays indicated thatMDA-MB-231cells overexpressing δEF1presented a distinct increase ofmigrating and invasive ability in MDA-MB-23cells, whereas depletionof δEF1revealed a decreased mobility and invasive ability inMDA-MB-231cells. the results confirmed that high expression of δEF1in MDA-MB-231cells might promote invasion and migration in vitro,thus triggering osteolytic bone metastasis of breast cancer.Bone metastase of breast cancer cells is closely related to theactivity of osteoblasts and osteoclasts. Therefore, we went on to studyhow changes in δEF1expression in breast cancer cells could affect theextent of breast cancer cell-induced osteoclast maturation, osteoblastmaturation and calcium. RAW264.7cells cultured in conditioned mediafrom δEF1overexpressing MDA-MB-231cells had significantly higherTRACP activities; and C2C12cells had a lower activity of ALP whentreated with conditioned medium from δEF1overexpressing MDA-MB-231cells;In addition, quantitative RT-PCR results showedthat the conditioned media from δEF1overexpressing MDA-MB-231cells exhibited an inhibition on BMP-2-induced expressions of osterixand osteocalcin, which are two othermarker genes of osteoblastdifferentiation; Alizarin red S staining assay show that retained dye ofMC3T3-E1cells treated with the conditioned medium from δEF1overexpressing MDA-MB-231was much less. In the present study, wefurther demonstrated that conditioned medium derived fromδEF1-overexpressing MDA-MB-231cells significantly inducesosteoclast maturation and concurrently represses osteoblastdifferentiation and mineralization. On the contrary, conditioned mediumderived from δEF1-interfered MDA-MB-231cells exhibits an oppositeeffect, thus confirming the effect of δEF to mediate osteolytic metastasisof breast cancer.To understand the molecular changes, we examined the MDA-MB-231-secreted factors in medium, we found that, δEF1remarkably up-regulates matrix metalloproteinase-1(MMP-1) expressionin MDA-MB-231cells. A search using the transcription factor databaseTESS identified an AP-1site (CATGAGTCAG) that is located atposition70/60of the MMP-1promoter. Luciferase assay resultsindicated that TPA up-regulated the promoter activity of MMP-1,whereas Curcumin repressed its activity in MDA-MB-231cells. Theresults of the luciferase assay also demonstrated that the mutation of theAP-1element on human MMP-1promoter totally depleted its response toTPA, Curcumin, and δEF1. CHIP assays show that overexpression ofδEF1in MDA-MB-231cells significantly increases the recruitment ofthe AP-1components, c-Jun/c-Fos, to the endogenous MMP-1promoter.Importantly, the results of luciferase and CHIP assays indicated thatδEF1activates MMP-1promoter activity through the AP-1responseelement. The AP-1components have been demonstrated to functiondownstream of the MAPK pathway.Western blot result show thatoverexpression of δEF1in MDA-MB-231cells significantly activatedphosphorylation of JNK signaling. The results of quantitative RT-PCRand luciferase assay showed that the blockade of the JNK signaling byspecific JNK inhibitor sp600125dramatically weakened δEF1-inducedexpression of MMP-1. δEF1up-regulates MMP-1expression could bemediated via the MAPK signaling pathway. In conclusion, theseobservations suggest a potent role of δEF1to promote breast cancermetastasis to bone by regulating secretion of growth factors in the tumormicroenvironment.更多还原