本文選題：丙戊酸鈉　切入點：SMAD4　出處：《山東大學》2016年博士論文　論文類型：學位論文

【摘要】：研究背景前列腺癌作為一種男性惡性腫瘤,在歐美等國非常常見。近年來隨著我國經(jīng)濟生活水平的提高,飲食及文化相關的發(fā)展,社會的老齡化及查體的普及,前列腺癌在我國的發(fā)病率呈逐年上升的趨勢。前列腺癌患者發(fā)病時一般較晚,許多患者發(fā)現(xiàn)時已發(fā)生轉(zhuǎn)移,其最常見的轉(zhuǎn)移為骨轉(zhuǎn)移。而研究發(fā)現(xiàn)前列腺癌患者的高病死率與其高轉(zhuǎn)移率密切相關,因此,人們越來越關注針對前列腺癌患者轉(zhuǎn)移相關的治療。上皮間質(zhì)轉(zhuǎn)化(EMT)是指原位腫瘤細胞,在受到物理或化學因素的作用下,從而失去了細胞極性、細胞間黏附等上皮細胞的特性,并獲得間質(zhì)細胞的特性時,轉(zhuǎn)化成為能夠向細胞外基質(zhì)侵襲并向遠處轉(zhuǎn)移的細胞。上皮間質(zhì)轉(zhuǎn)化(EMT)已被證實在前列腺癌轉(zhuǎn)移過程中發(fā)揮著重要作用,其調(diào)控機制由多個信號通路綜合參與引導,包括GF-β、Notch、Wnt以及ERK/MAP K等通路。其中TGF-β/SMAD4通路對腫瘤特別是晚期時的EMT發(fā)展密切相關。SMADs是哺乳動物TGF-β超家族信號傳導的下游信號蛋白,它們可將GF-β信號由細胞膜轉(zhuǎn)導進入細胞核,并與DNA及一些轉(zhuǎn)錄因子相互作用。SMAD4即在TGF-β/SMAD4信號通路中起到關鍵的“渡船”的作用。因此當抑制SMAD4的表達時,可以抑制TGF-β通路信號傳遞,阻止上皮間質(zhì)轉(zhuǎn)化(EMT)在腫瘤細胞中發(fā)生。近年來發(fā)現(xiàn)的miRNA是一組非編碼小RNA,其作用涉及眾多生理、病理過程,主要表現(xiàn)為抑制mRNA的翻譯或誘導其降解,從而改變靶蛋白的表達水平。關于miRNA的研究成為當前的熱點,大量研究證明miRNA在不同的腫瘤中均有不同的表達,且發(fā)揮著重要的作用：例如部分miRNA對腫瘤細胞中的EMT起促進作用,而部分miRNA對腫瘤細胞中的EMT起抑制作用。因此將來治療腫瘤通過研究針對miRNA靶向治療藥物會有很大的發(fā)展前景。目前大量的研究表明,丙戊酸鈉(VPA)可抑制前列腺癌(PCa)細胞轉(zhuǎn)移并且相應的下調(diào)SMAD4蛋白表達水平。然而VPA對前列腺癌細胞中SMAD4表達產(chǎn)生調(diào)控的過程中,是否存在miRNA的參與,這其中涉及的具體機制目前尚不清楚。如果通過實驗找到影響SMAD4表達的核心miRNA,就可以針對該miRNA的作用機制研究相關的藥物治療。這種針對性更強的治療可能會效果更好且可以減少并發(fā)癥,對前列腺癌的治療將有著重要的意義。目的前期研究已證實丙戊酸鈉(VPA)可抑制前列腺癌(PCa)細胞轉(zhuǎn)移,并且能通過下調(diào)SMAD4表達水平阻斷EMT的發(fā)生。通過本實驗,我們研究丙戊酸鈉(VPA)通過何種途徑下調(diào)SMAD4蛋白表達水平,尋找是否存在相關的miRNA與其調(diào)控密切相關,找到這種miRNA并探索能否通過該miRNA的調(diào)控,改變SMAD4的表達水平,從而確定其是否在SMAD4的表達過程中起到關鍵作用,為前列腺癌的治療尋找新的治療靶點。方法我們通過使用www.targetscan.com查找可以與SMAD4互補結(jié)合miRNAs,然后通過在PubMed上搜索及進行文獻復習確定與VPA相關的miRNA,將同時與SMAD4及VPA相關的miRNA作為我們的研究對象。實驗設計首先培養(yǎng)兩種前列腺癌細胞株(PC3、LNCaP),在應用VPA處理后,檢測SMAD4的蛋白和mRNA水平有無變化,變化有無統(tǒng)計學差異。再次用VPA處理前列腺癌細胞株,觀察是否對作為研究對象的miRNA表達水平產(chǎn)生影響。選擇應用VPA處理后的變化有統(tǒng)計學差異的miRNA進行下一步實驗。通過質(zhì)粒轉(zhuǎn)染技術,分別植入轉(zhuǎn)染相應miRNA前體及miRNA抑制劑后,檢測其對前列腺癌細胞中SMAD4表達的影響。選出對SMAD4表達產(chǎn)生有統(tǒng)計學意義影響的miRNA作為最終目標。最后將通過相應的miRNA抑制劑將選出的目標miRNA的表達進行抑制,觀察其能否消除VPA對SMAD4的影響,以評價其是否在VPA抑制SMAD4的表達過程中起到關鍵的作用。結(jié)果通過使用www.targetscan.com查找可以與SMAD4互補結(jié)合miRNAs,然后通過在PubMed上搜索及文獻復習確定與VPA相關的miRNA,我們將同時與SMAD4及VPA相關的5種miRNA作為我們的研究對象進行實驗,分別是miR-20a, miR-34a, miR-124a,miR-144和miR-449a。我們通過實驗再次驗證在經(jīng)過VPA處理后,前列腺癌細胞株中SMAD4的mRNA和蛋白的水平均明顯下調(diào),且具有明顯的濃度依賴性。之后我們用VPA處理前列腺癌細胞株,從初步篩選的5種miRNA中,發(fā)現(xiàn)miR-20a、miR-34a與miR-449a的表達水平在VPA處理后增加且具有統(tǒng)計學意義,而對miR-124a, miR-144的表達則無明顯變化。因此我們將miR-20a、miR-34a與miR-449a作為下一步的研究目標。我們通過質(zhì)粒轉(zhuǎn)染技術,在兩種前列腺癌細胞株(LNCaP、PC3 cells)中分別植入轉(zhuǎn)染miR-20a、miR-34a與miR-449a前體及miR-20a、miR-34a與miR-449a抑制劑后,發(fā)現(xiàn)通過轉(zhuǎn)染pre-miR-34a降低SMAD4的表達,而轉(zhuǎn)染pre-miR-20a或者pre-miR-449a則均增加了SMAD4的表達,且SMAD4表達的變化均有統(tǒng)計學差異；而通過轉(zhuǎn)染miR-34a抑制劑,增加了SMAD4的表達；而轉(zhuǎn)染miR-20a或者miR-449a抑制劑,則均減少了SMAD4的表達。在前列腺癌細胞株LNCaP中,通過轉(zhuǎn)染miR-34a抑制劑后SMAD4表達的變化有統(tǒng)計學差異,而轉(zhuǎn)染miR-20a抑制劑或者miR-449a抑制劑后SMAD4表達的變化無統(tǒng)計學差異。在前列腺癌細胞株PC3 cells中,通過轉(zhuǎn)染pre-miR-34a抑制劑、pre-miR-20a抑制劑、pre-miR-449a抑制劑后SMAD4表達的變化均有統(tǒng)計學差異。通過實驗我們發(fā)現(xiàn)轉(zhuǎn)染miR-34a的前體及抑制物均能對SMAD4的蛋白及mRNA表達水平有顯著的影響,miR-34a在VPA影響SMAD4表達過程中應該起到重要作用,因此我們選擇miR-34a作為研究對象進入下一組實驗。在兩種前列腺癌細胞株(LNCaP、PC3 cells)中,通過轉(zhuǎn)染pre-miR-34a抑制劑來增加SMAD4蛋白水平的表達,然后再加用VPA處理,SMAD4蛋白水平并沒有明顯改變,無統(tǒng)計學差異。同時在兩種前列腺癌細胞株(LNCaP、PC3 cells)中,應用轉(zhuǎn)染pre-miR-34a抑制劑后再應用VPA處理,SMAD4的mRNA水平的變化也無統(tǒng)計學差異。結(jié)果表明降低miR-34a的表達可消除VPA對SMAD4的抑制效果。即上調(diào)miR-34a水平可模擬SMAD4被VPA抑制時的效果,而下調(diào)miR-34a的水平消除VPA在前列腺癌細胞的影響。結(jié)論丙戊酸鈉(VPA)可上調(diào)miR-20a, miR34a, miR449a在前列腺癌細胞株LNCaP和PC3中的表達水平�？拱﹎iRNA中的miR-449a在經(jīng)過VPA治療后其表達明顯上調(diào),這提示miR-449a可能參與VPA的其它復雜的作用機制。pre-miR-20a或pre-miR-449a轉(zhuǎn)染誘導了SMAD4蛋白水平上調(diào),這可能主要是由于間接的影響。VPA作用下miR-34a表達增強可隱藏miR-20a和miR-449a對SMAD4的表達效果。miR-34a是在VPA抑制SMAD4的表達過程的一個關鍵調(diào)節(jié)因子。VPA通過上調(diào)rmiR-34a的表達,從而抑制了SMAD4的表達。
[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.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.25

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本文編號：1580233