丙戊酸钠通过上调miR-34a的表达抑制SMAD4在前列腺癌中的表达

发布时间：2018-03-07 17:23

本文选题：丙戊酸钠　切入点：SMAD4　出处：《山东大学》2016年博士论文　论文类型：学位论文

【摘要】：研究背景前列腺癌作为一种男性恶性肿瘤,在欧美等国非常常见。近年来随着我国经济生活水平的提高,饮食及文化相关的发展,社会的老龄化及查体的普及,前列腺癌在我国的发病率呈逐年上升的趋势。前列腺癌患者发病时一般较晚,许多患者发现时已发生转移,其最常见的转移为骨转移。而研究发现前列腺癌患者的高病死率与其高转移率密切相关,因此,人们越来越关注针对前列腺癌患者转移相关的治疗。上皮间质转化(EMT)是指原位肿瘤细胞,在受到物理或化学因素的作用下,从而失去了细胞极性、细胞间黏附等上皮细胞的特性,并获得间质细胞的特性时,转化成为能够向细胞外基质侵袭并向远处转移的细胞。上皮间质转化(EMT)已被证实在前列腺癌转移过程中发挥着重要作用,其调控机制由多个信号通路综合参与引导,包括GF-β、Notch、Wnt以及ERK/MAP K等通路。其中TGF-β/SMAD4通路对肿瘤特别是晚期时的EMT发展密切相关。SMADs是哺乳动物TGF-β超家族信号传导的下游信号蛋白,它们可将GF-β信号由细胞膜转导进入细胞核,并与DNA及一些转录因子相互作用。SMAD4即在TGF-β/SMAD4信号通路中起到关键的“渡船”的作用。因此当抑制SMAD4的表达时,可以抑制TGF-β通路信号传递,阻止上皮间质转化(EMT)在肿瘤细胞中发生。近年来发现的miRNA是一组非编码小RNA,其作用涉及众多生理、病理过程,主要表现为抑制mRNA的翻译或诱导其降解,从而改变靶蛋白的表达水平。关于miRNA的研究成为当前的热点,大量研究证明miRNA在不同的肿瘤中均有不同的表达,且发挥着重要的作用：例如部分miRNA对肿瘤细胞中的EMT起促进作用,而部分miRNA对肿瘤细胞中的EMT起抑制作用。因此将来治疗肿瘤通过研究针对miRNA靶向治疗药物会有很大的发展前景。目前大量的研究表明,丙戊酸钠(VPA)可抑制前列腺癌(PCa)细胞转移并且相应的下调SMAD4蛋白表达水平。然而VPA对前列腺癌细胞中SMAD4表达产生调控的过程中,是否存在miRNA的参与,这其中涉及的具体机制目前尚不清楚。如果通过实验找到影响SMAD4表达的核心miRNA,就可以针对该miRNA的作用机制研究相关的药物治疗。这种针对性更强的治疗可能会效果更好且可以减少并发症,对前列腺癌的治疗将有着重要的意义。目的前期研究已证实丙戊酸钠(VPA)可抑制前列腺癌(PCa)细胞转移,并且能通过下调SMAD4表达水平阻断EMT的发生。通过本实验,我们研究丙戊酸钠(VPA)通过何种途径下调SMAD4蛋白表达水平,寻找是否存在相关的miRNA与其调控密切相关,找到这种miRNA并探索能否通过该miRNA的调控,改变SMAD4的表达水平,从而确定其是否在SMAD4的表达过程中起到关键作用,为前列腺癌的治疗寻找新的治疗靶点。方法我们通过使用www.targetscan.com查找可以与SMAD4互补结合miRNAs,然后通过在PubMed上搜索及进行文献复习确定与VPA相关的miRNA,将同时与SMAD4及VPA相关的miRNA作为我们的研究对象。实验设计首先培养两种前列腺癌细胞株(PC3、LNCaP),在应用VPA处理后,检测SMAD4的蛋白和mRNA水平有无变化,变化有无统计学差异。再次用VPA处理前列腺癌细胞株,观察是否对作为研究对象的miRNA表达水平产生影响。选择应用VPA处理后的变化有统计学差异的miRNA进行下一步实验。通过质粒转染技术,分别植入转染相应miRNA前体及miRNA抑制剂后,检测其对前列腺癌细胞中SMAD4表达的影响。选出对SMAD4表达产生有统计学意义影响的miRNA作为最终目标。最后将通过相应的miRNA抑制剂将选出的目标miRNA的表达进行抑制,观察其能否消除VPA对SMAD4的影响,以评价其是否在VPA抑制SMAD4的表达过程中起到关键的作用。结果通过使用www.targetscan.com查找可以与SMAD4互补结合miRNAs,然后通过在PubMed上搜索及文献复习确定与VPA相关的miRNA,我们将同时与SMAD4及VPA相关的5种miRNA作为我们的研究对象进行实验,分别是miR-20a, miR-34a, miR-124a,miR-144和miR-449a。我们通过实验再次验证在经过VPA处理后,前列腺癌细胞株中SMAD4的mRNA和蛋白的水平均明显下调,且具有明显的浓度依赖性。之后我们用VPA处理前列腺癌细胞株,从初步筛选的5种miRNA中,发现miR-20a、miR-34a与miR-449a的表达水平在VPA处理后增加且具有统计学意义,而对miR-124a, miR-144的表达则无明显变化。因此我们将miR-20a、miR-34a与miR-449a作为下一步的研究目标。我们通过质粒转染技术,在两种前列腺癌细胞株(LNCaP、PC3 cells)中分别植入转染miR-20a、miR-34a与miR-449a前体及miR-20a、miR-34a与miR-449a抑制剂后,发现通过转染pre-miR-34a降低SMAD4的表达,而转染pre-miR-20a或者pre-miR-449a则均增加了SMAD4的表达,且SMAD4表达的变化均有统计学差异；而通过转染miR-34a抑制剂,增加了SMAD4的表达；而转染miR-20a或者miR-449a抑制剂,则均减少了SMAD4的表达。在前列腺癌细胞株LNCaP中,通过转染miR-34a抑制剂后SMAD4表达的变化有统计学差异,而转染miR-20a抑制剂或者miR-449a抑制剂后SMAD4表达的变化无统计学差异。在前列腺癌细胞株PC3 cells中,通过转染pre-miR-34a抑制剂、pre-miR-20a抑制剂、pre-miR-449a抑制剂后SMAD4表达的变化均有统计学差异。通过实验我们发现转染miR-34a的前体及抑制物均能对SMAD4的蛋白及mRNA表达水平有显著的影响,miR-34a在VPA影响SMAD4表达过程中应该起到重要作用,因此我们选择miR-34a作为研究对象进入下一组实验。在两种前列腺癌细胞株(LNCaP、PC3 cells)中,通过转染pre-miR-34a抑制剂来增加SMAD4蛋白水平的表达,然后再加用VPA处理,SMAD4蛋白水平并没有明显改变,无统计学差异。同时在两种前列腺癌细胞株(LNCaP、PC3 cells)中,应用转染pre-miR-34a抑制剂后再应用VPA处理,SMAD4的mRNA水平的变化也无统计学差异。结果表明降低miR-34a的表达可消除VPA对SMAD4的抑制效果。即上调miR-34a水平可模拟SMAD4被VPA抑制时的效果,而下调miR-34a的水平消除VPA在前列腺癌细胞的影响。结论丙戊酸钠(VPA)可上调miR-20a, miR34a, miR449a在前列腺癌细胞株LNCaP和PC3中的表达水平。抗癌miRNA中的miR-449a在经过VPA治疗后其表达明显上调,这提示miR-449a可能参与VPA的其它复杂的作用机制。pre-miR-20a或pre-miR-449a转染诱导了SMAD4蛋白水平上调,这可能主要是由于间接的影响。VPA作用下miR-34a表达增强可隐藏miR-20a和miR-449a对SMAD4的表达效果。miR-34a是在VPA抑制SMAD4的表达过程的一个关键调节因子。VPA通过上调rmiR-34a的表达,从而抑制了SMAD4的表达。
[Abstract]:Background prostate cancer is a malignant tumor of male, is very common in Europe and the United States. In recent years, with China's economic and the improvement of living standards, the development of cultural and diet related, the aging of society and check the popularity of prostate cancer incidence in China is a rising trend year by year. The incidence of patients with prostate cancer generally late, many patients found metastasis has occurred, the most common metastasis to bone metastasis in patients with prostate cancer. The study found that the high mortality rate and high transfer rate are closely related, therefore, people pay more and more attention to treatment for metastatic prostate cancer patients. The epithelial mesenchymal transition (EMT) refers to the in situ in tumor cells, by physical or chemical factors, the loss of cell polarity, cell adhesion characteristics of epithelial cells, and interstitial cell properties, can be transformed to the extracellular matrix. Mass invasion and metastasis to the distant cells. Epithelial mesenchymal transition (EMT) has been demonstrated to play an important role in prostate cancer metastasis process, its regulation mechanism by multiple signaling pathways in comprehensive guide, including GF- Notch, Wnt ERK/MAP and beta K pathway. Which TGF- beta /SMAD4 pathway on tumor especially the late EMT.SMADs is closely related to the development of the downstream signal protein of mammalian TGF- beta superfamily signaling, which can be GF- beta signaling by cell membrane transduction into the nucleus, and some transcription factors DNA and.SMAD4 interaction to play a key role in the "ferry" TGF- beta /SMAD4 signal pathway. So when inhibition of the expression of SMAD4, TGF- signaling pathway can inhibit beta, prevent epithelial mesenchymal transition (EMT) occurs in tumor cells. In recent years, found that miRNA is a group of non small RNA encoding, which involves many physiological diseases. Process, mainly for inhibition of mRNA translation or inducing its degradation, thus changing the expression level of target protein. The research on miRNA has become the hot spot, a large number of studies have shown that miRNA in different tumors have different expression, and play an important role: for example part miRNA to promote tumor cells EMT play a role, and some of the miRNA inhibitory effect on tumor cells in EMT. So the future treatment of cancer through the study of miRNA targeted therapy will have great prospects for development. At present a large number of studies show that sodium valproate (VPA) can inhibit the metastasis of prostate cancer (PCa) cells and down-regulation of SMAD4 protein expression of the corresponding level. However, the VPA of SMAD4 expression in prostate cancer cells produced in the process of regulation, the existence of miRNA participation, which relates to the specific mechanism is still unclear. If found by the experiments SMAD The core of the expression of miRNA 4, you can according to the mechanism of the medical treatment of miRNA related research. This may be for more treatment effect is better and can reduce complications, will have important significance in the treatment of prostate cancer. Previous studies have confirmed that the purpose of valproic acid sodium (VPA) can inhibit prostate cancer (PCa) cell transfer, and it can also decrease the expression level of SMAD4 by EMT. Through this experiment, we studied valproate (VPA) reduced the expression level of SMAD4 protein in what way, looking for the existence of related miRNA and its regulation are closely related, and to explore the possibility of finding such a miRNA through the regulation of the miRNA, the level of the expression change of SMAD4 in order to determine whether the expression of SMAD4 plays a key role, to find a new therapeutic target for the treatment of prostate cancer. Methods we use www.targetscan.com to find the The combination of miRNAs and SMAD4 can complement each other, and then through the search and review of the literature to determine the related VPA miRNA in PubMed, and correlated with SMAD4 and VPA miRNA as our research object. Experimental design first cultivate two prostate cancer cell lines (PC3, LNCaP), the application of VPA after treatment, detection of SMAD4 protein the levels of mRNA and there is no change, change has no significant difference. Prostate cancer cell lines treated with VPA again, to observe whether the expression of miRNA as the research object. The influence level changes after treated with VPA had a significant difference miRNA the next step of the experiment. Through plasmid transfection, transfection were implanted into the corresponding miRNA precursor and miRNA inhibitor, to detect the influence on the expression of SMAD4 in prostate cancer cells. Selected had significant effect on the expression of SMAD4 miRNA as the ultimate goal. Finally, through MiRNA inhibitors will be selected target inhibition of miRNA expression, to observe the effect of VPA on SMAD4 can be eliminated, to assess whether the inhibition in VPA expression plays a key role in the process of SMAD4. By using the www.targetscan.com search results can be complementary with SMAD4 combined with miRNAs, in the PubMed search and review of the literature and determine VPA miRNA then by 5, we will also miRNA with SMAD4 and VPA related as our research object. In the experiment, including miR-20a, miR-34a, miR-124a, miR-144 and miR-449a. by experiment we verified again after VPA treatment, mRNA and SMAD4 protein in prostate cancer cell lines were significantly reduced and a clear dose-dependent manner. After we treated with VPA prostate cancer cell line, miR-20a 5 miRNA from the initial screening, the expression of miR-34a and miR-449a. At the level of VPA after treatment increased and was statistically significant, but for miR-124a, the expression of miR-144 had no obvious change. So we will be miR-20a, miR-34a and miR-449a as the research target in the next step. We through plasmid transfection, in two prostate cancer cell lines (LNCaP, PC3, cells) were implanted into miR-20a. MiR-34a and miR-449a precursor and miR-20a, miR-34a and miR-449a inhibitors, found that the decreased expression of SMAD4 by transfection of pre-miR-34a, pre-miR-20a or pre-miR-449a were transfected and increased the expression of SMAD4 and SMAD4 expression changes were statistically significant; and by transfection of miR-34a inhibitor, increased the expression of SMAD4 and miR-20a or miR-449a inhibitors; transfection then, reduced the expression of SMAD4 in prostate cancer cell line LNCaP, the expression of SMAD4 after transfection of miR-34a inhibitor has statistical differences, and There was no significant difference in changes of transfection of miR-20a inhibitor or miR-449a inhibitor SMAD4. Expression of PC3 in prostate cancer cell line cells, transfected by pre-miR-34a inhibitors, pre-miR-20a inhibitors, were statistically difference SMAD4 expression after pre-miR-449a inhibitor. Through the experiment we found that the precursor and inhibitor transfection of miR-34a protein and mRNA of SMAD4 can have the expression level of miR-34a significantly influence, should play an important role in the expression of SMAD4 VPA in effect, so we choose miR-34a as the research object in the experimental group. In two prostate cancer cell lines (LNCaP, PC3, cells) by transfection of pre-miR-34a inhibitor to increase the expression level of SMAD4 protein, and then added VPA, SMAD4 protein levels did not change significantly, the difference was not statistically significant. At the same time in two prostate cancer cell lines (LNCaP, PC3, cells) In the application of pre-miR-34a inhibitor transfection after the application of VPA treatment, there was no statistically significant difference in the change of SMAD4 mRNA level. The results show that reducing the expression of miR-34a can eliminate the inhibitory effect of VPA on SMAD4. That increase the level of miR-34a can be inhibited by VPA SMAD4 simulation of the effect, and the down-regulation of miR-34a level to eliminate the influence of VPA in prostate cancer cells. Conclusion valproate (VPA) could increase miR-20a, miR34a, LNCaP and PC3 expression level of miR449a in prostate cancer cell lines. MiRNA miR-449a in cancer after VPA after treatment was obviously up-regulated, suggesting that miR-449a may be involved in the VPA of the other complex mechanism of.Pre-miR-20a or pre-miR-449a transfection induced upregulation of SMAD4 protein levels, which may be mainly due to the indirect effects of.VPA under the enhanced expression of miR-34a miR-20a and miR-449a SMAD4 can be hidden on the expression of.MiR-34a in the VPA effect A key regulatory factor that inhibits the expression of SMAD4,.VPA, inhibits the expression of SMAD4 by up regulation of the expression of rmiR-34a.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

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