發(fā)布時(shí)間：2018-04-23 00:16

  本文選題：PP4C + 膠質(zhì)瘤��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：研究背景及意義腦膠質(zhì)瘤為中樞神經(jīng)系統(tǒng)發(fā)病率最高的惡性腫瘤,約占顱內(nèi)腫瘤的45%,占顱內(nèi)惡性腫瘤的80%左右。腦膠質(zhì)瘤起源于神經(jīng)膠質(zhì)細(xì)胞,生長迅速且具有較強(qiáng)的侵襲能力,給手術(shù)切除造成一定困難。根據(jù)2007年最新的WHO腫瘤分級標(biāo)準(zhǔn),膠質(zhì)瘤可分為Ⅰ-Ⅳ級,其中Ⅰ、Ⅱ級為低級別膠質(zhì)瘤,Ⅲ、Ⅳ級為高級別膠質(zhì)瘤。目前膠質(zhì)瘤的標(biāo)準(zhǔn)治療方案包括手術(shù)聯(lián)合放療和化療,即使經(jīng)過這些綜合治療,高級別膠質(zhì)瘤的預(yù)后仍然不盡人意,尤其是惡性程度最高的多形性膠質(zhì)母細(xì)胞瘤,5年總的生存率低于5%,平均生存期在14-15個(gè)月左右。膠質(zhì)瘤的發(fā)病機(jī)制涉及到多基因、多因素的共同作用。許多相關(guān)基因的異常表達(dá)與膠質(zhì)瘤的發(fā)生密切有關(guān),但仍不能清楚的闡明膠質(zhì)瘤發(fā)生、發(fā)展的全過程。因此,為了進(jìn)一步揭示膠質(zhì)瘤的發(fā)病機(jī)制,尋找膠質(zhì)瘤的生物學(xué)標(biāo)記,探求膠質(zhì)瘤治療的新靶點(diǎn)是當(dāng)前亟待解決的難題。蛋白質(zhì)的磷酸化是蛋白質(zhì)翻譯后修飾的重要組成部分,人體細(xì)胞中約有30%的蛋白質(zhì)被磷酸化修飾,并且這種磷酸化修飾是調(diào)節(jié)細(xì)胞增殖、凋亡、分化和其他細(xì)胞功能的重要機(jī)制之一。蛋白質(zhì)的磷酸化的水平由蛋白激酶和蛋白磷酸酶共同調(diào)節(jié)。蛋白磷酸酶曾被認(rèn)為是伴隨蛋白激酶左右的沉默伴侶,隨機(jī)地逆轉(zhuǎn)蛋白激酶的磷酸化效應(yīng),但越來越多的證據(jù)表明,蛋白磷酸酶組成了一個(gè)與蛋白激酶相平行的結(jié)構(gòu)復(fù)雜多樣的酶家族,能主動(dòng)、特異地對特定底物去磷酸化。蛋白磷酸酶根據(jù)氨基酸序列同源性、結(jié)構(gòu)及催化機(jī)制可分為三個(gè)家族。第一家族是絲/蘇氨酸蛋白磷酸酶,包括磷酸蛋白磷酸酶(Phosphoprotein phosphatase, PPP)及鎂或錳離子依賴性蛋白磷酸酶(Protein phosphatase Mg2+ or Mn2+ dependent, PPM).其中PPP作為其中種類最多并且最為經(jīng)典的家族,又可分為PP1、PP2A、PP、PP5、PP6、PP7。酪氨酸蛋白磷酸酶及基于天冬氨酸伴有DXDXT/V催化的蛋白磷酸酶分別組成第二和第三家族。PP4 (Protein Phosphatase 4)獨(dú)立于其他的磷酸酶,參與多種病理生理過程,越來越受到眾多研究者的關(guān)注。PP4在結(jié)構(gòu)上與PP2A相似,以催化亞基PP4C(Protein Phosphatase 4 catalytic subunit)及不同的調(diào)節(jié)亞基相結(jié)合,組成異源多聚體的形式存在并發(fā)揮功能。因此PP4C作為PP4的核心催化亞基,被認(rèn)為是起重要作用的亞基。PP4C由ppp4c基因編碼,定位于人染色體16p1 1.2區(qū)域,最初由Brewis ND等人從兔肝到cDNA文庫中分離并鑒定,起初稱之為PPX,1993年在哺乳動(dòng)物中克隆出PP4C基因,然后在果蠅、線蟲、酵母中也陸續(xù)發(fā)現(xiàn)PP4C的同源物。PP4C參與多種病理生理過程,如微管的形成、大腦皮質(zhì)的發(fā)育、免疫調(diào)節(jié)、DNA損傷修復(fù)及多個(gè)細(xì)胞信號通路的調(diào)節(jié)。PP4C可以促進(jìn)細(xì)胞增殖、抑制細(xì)胞凋亡,并調(diào)節(jié)NF-κB、JNK和m-TOR等腫瘤相關(guān)信號通路,提示PP4C參與腫瘤的發(fā)生、發(fā)展過程。近年來,有學(xué)者相繼發(fā)現(xiàn)PP4C在乳腺癌、肺癌、胰腺導(dǎo)管癌及結(jié)直腸癌中高表達(dá),并且高表達(dá)PP4C的患者與胰腺導(dǎo)管癌、結(jié)直腸癌患者的預(yù)后差密切相關(guān)。然而,目前國內(nèi)外學(xué)者尚未有關(guān)于PP4C在膠質(zhì)瘤中的報(bào)道。本研究共分為三部分。第一部分,應(yīng)用Western Blot、Real-time PCR和免疫組織化學(xué)檢測PP4C在膠質(zhì)瘤中的表達(dá),并分析PP4C的異常表達(dá)與膠質(zhì)瘤患者臨床病理特征及預(yù)后的相關(guān)性。第二部分,檢測人膠質(zhì)瘤細(xì)胞系A(chǔ)172、U251、U87口正常膠質(zhì)細(xì)胞Astrocyte中PP4C的表達(dá),并探討沉默PP4C基因?qū)θ四X膠質(zhì)瘤細(xì)胞系U251、U87增殖、遷移和侵襲的影響。第三部分,應(yīng)用生物信息軟件預(yù)測調(diào)控PP4C的潛在miRNA,即miR-128-3p,通過熒光素酶報(bào)告基因?qū)嶒?yàn)進(jìn)一步驗(yàn)證其真實(shí)性；并用Western Blot及Real-time PCR檢測miR-128-3p對PP4C蛋白及mRNA的影響;進(jìn)一步在膠質(zhì)瘤組織中檢測miR-128-3p的表達(dá),并分析其與PP4C的相關(guān)性。第一部分PP4C在人腦膠質(zhì)細(xì)胞瘤組織中表達(dá)及其臨床病理特征相關(guān)性研究研究目的：1.探討PP4C蛋白和mRNA在人腦膠質(zhì)細(xì)胞瘤組織和正常腦組織的表達(dá)情況。2.探討PP4C異常表達(dá)與膠質(zhì)瘤患者臨床病理特征及預(yù)后的相關(guān)性。研究方法：1.采用Western Blot和Real-time PCR和檢測PP4C蛋白和mRNA在新鮮膠質(zhì)細(xì)胞瘤組織及正常腦組織中的表達(dá)。2.采用免疫組織化學(xué)方法檢測120例腦膠質(zhì)瘤組織及10例正常腦組織中PP4C蛋白表達(dá)情況及分析其表達(dá)與膠質(zhì)瘤WHO病理級別的相關(guān)性。研究結(jié)果：1.PP4C蛋白和mRNA在腦膠質(zhì)瘤組織中的表達(dá)顯著高于正常腦組織。并且PP4C蛋白和mRNA表達(dá)隨WHO病理級別增高有逐漸升高的趨勢,與膠質(zhì)瘤的惡性程度相關(guān)。2.PP4C陽性表達(dá)為細(xì)胞核和胞漿染成淺黃色、棕黃色或者黃褐色。PP4C蛋白在腦膠質(zhì)瘤組織的高表達(dá)率為60%(72/120),在10例正常腦組織中PP4C均呈低表達(dá)。除此之外,PP4C蛋白在Ⅱ級、Ⅲ級、Ⅳ級膠質(zhì)瘤的高表達(dá)率分別為21.43%(6/28) 、55.56%(20/36)、82.14%(46/56),差異具有統(tǒng)計(jì)學(xué)意義(P0.001)。Kaplan-Meier生存分析表明PP4C高表達(dá)的患者預(yù)后比PP4C低表達(dá)的患者預(yù)后差。Cox比例風(fēng)險(xiǎn)模型進(jìn)行單因素、多因素分析顯示PP4C蛋白高表達(dá)是影響膠質(zhì)瘤患者預(yù)后的獨(dú)立危險(xiǎn)因素。結(jié)論：PP4C蛋白和mRNA在膠質(zhì)瘤中的表達(dá)與正常腦組織相比顯著增高,并與膠質(zhì)瘤病理分級相關(guān)；預(yù)后分析表明PP4C高表達(dá)是膠質(zhì)瘤患者的預(yù)后獨(dú)立危險(xiǎn)因素。第二部分PP4C在人膠質(zhì)瘤細(xì)胞系中的表達(dá)及沉默PP4C基因?qū)?xì)胞增殖、遷移和侵襲的影響研究目的：1.探討人膠質(zhì)瘤細(xì)胞系A(chǔ)172、U251、U87及正常膠質(zhì)細(xì)胞Astrocyte中PP4C的表達(dá)情況。2.探討沉默PP4C基因?qū)θ四X膠質(zhì)瘤細(xì)胞系U251、U87增殖、遷移和侵襲的影口向。研究方法：1. 采用Western Blot和Real-time PCR和檢測PP4C蛋白和mRNA在人膠質(zhì)瘤細(xì)胞系及正常膠質(zhì)細(xì)胞中的表達(dá)情況。2. si-RNA的構(gòu)建及轉(zhuǎn)染：設(shè)計(jì)合成PP4C-siRNA干擾序列,并實(shí)驗(yàn)將U251和U87細(xì)胞株各分成兩組,即實(shí)驗(yàn)組(轉(zhuǎn)染PP4C-siRNA)、陰性對照組(轉(zhuǎn)染非特異性siRNA),用轉(zhuǎn)染試劑進(jìn)行轉(zhuǎn)染。3.PP4C基因沉默效率的檢測：使用Real-time PCR和Western Blot檢測PP4C-siRNA沉默效率。4. 采用CCK-8及EdU實(shí)驗(yàn)檢測沉默PP4C基因后U251、U87細(xì)胞的增殖能力變化。5.采用劃痕實(shí)驗(yàn)檢測沉默PP4C基因后U251、U87細(xì)胞遷移能力的變化。6.采用Transwell實(shí)驗(yàn)檢測沉默PP4C基因后U251、U87細(xì)胞侵襲能力的變化。研究結(jié)果：1.PP4C在腦膠質(zhì)瘤細(xì)胞系中的表達(dá)應(yīng)用Western Blot及Real-time PCR檢測膠質(zhì)瘤細(xì)胞系A(chǔ)172、U251、U87及正常膠質(zhì)細(xì)胞Astrocyte結(jié)果顯示,PP4C蛋白及mRNA在4種細(xì)胞系中均存在表達(dá),在U251、U87細(xì)胞中相對表達(dá)量較高,A172細(xì)胞次之,Astrocyte中相對表達(dá)量最低,因此篩選出U251、U87優(yōu)勢表達(dá)細(xì)胞系進(jìn)行下一步實(shí)驗(yàn)。2. 檢測PP4C-siRNA的沉默效率使用Real-time PCR和Western Blot檢測PP4C-siRNA沉默效率。Real-timePCR及Western Blot結(jié)果顯示,轉(zhuǎn)染48小時(shí)后,相對于陰性對照組,PP4C-siRNA明顯降低膠質(zhì)瘤細(xì)胞系U251、U87中PP4C mRNA及蛋白的表達(dá)(P均0.01)。3.PP4C基因沉默對U251、U87細(xì)胞系增殖能力的影響CCK-8增殖實(shí)驗(yàn)結(jié)果表明,與陰性對照組(NC)相比,P4C-siRNA實(shí)驗(yàn)組U251、U87細(xì)胞增值率顯著降低,分別降低了54%、50%,差異均具有統(tǒng)計(jì)學(xué)意義(P0.01)。 EdU增殖實(shí)驗(yàn)結(jié)果表明,與陰性對照組(NC)相比,PP4C-siRNA實(shí)驗(yàn)組細(xì)胞增殖率顯著降低,U251、U87細(xì)胞分別降低了51%、64%,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。這些結(jié)果表明,PP4C可以促進(jìn)膠質(zhì)瘤細(xì)胞的體外增殖能力,沉默PP4C后膠質(zhì)瘤細(xì)胞的增殖明顯降低。4.PP4C基因沉默對U251、U87細(xì)胞系遷移能力的影響劃痕實(shí)驗(yàn)結(jié)果顯示,相對于陰性對照組,PP4C-siRNA組細(xì)胞遷移數(shù)明顯下降,其中U251細(xì)胞減少了56%,U87細(xì)胞減少了66%,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。5.PP4C基因沉默對U251、U87細(xì)胞系侵襲能力的影響Transwell侵襲實(shí)驗(yàn)結(jié)果表明,與陰性對照組(NC)相比,PP4C-siRNA實(shí)驗(yàn)組穿過小室結(jié)晶紫染色細(xì)胞顯著減少,其中U251細(xì)胞減少了52%,U87細(xì)胞減少了39%,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論：1.PP4C在膠質(zhì)瘤細(xì)胞株中高表達(dá),并篩選出U251、U87優(yōu)勢細(xì)胞株進(jìn)行后續(xù)生物學(xué)實(shí)驗(yàn)。2. PP4C siRNA可以明顯沉默PP4C的表達(dá),并可以抑制U251、U87細(xì)胞的增殖、遷移及侵襲能力。表明PP4C可以促進(jìn)膠質(zhì)瘤細(xì)胞的增殖、遷移和侵襲能力。第三部分miR-128-3p與PP4C基因的靶向調(diào)控關(guān)系研究研究目的：1.預(yù)測可能調(diào)控PP4C的潛在miRNA。2.驗(yàn)證miR-128-3p是否轉(zhuǎn)錄后水平調(diào)控PP4C的表達(dá)。3.檢測miR-128-3p在膠質(zhì)瘤組織中的表達(dá),并分析miR-128-3p與PP4C表達(dá)相關(guān)性。研究方法：1.采用靶基因預(yù)測算法對調(diào)控PP4C的潛在miRNA進(jìn)行生物信息學(xué)預(yù)測,分析PP4C的3,-UTR區(qū)域存在miR-128-3p的結(jié)合位點(diǎn)。2.利用熒光素酶報(bào)告基因?qū)嶒?yàn)在293T細(xì)胞中驗(yàn)證miR-128-3p與PP4C是否存在直接相互作用,進(jìn)一步在膠質(zhì)瘤細(xì)胞系U251、U87中驗(yàn)證miR-128-3p與PP4C是否存在直接相互作用。3. miR-128-3p mimics、miR-NC mimics轉(zhuǎn)染U251、U87細(xì)胞,分別利用VWestern Blot和Real-time PCR檢測PP4C的蛋白和mRNA水平。4. Real-time PCR檢測miR-128-3p在腦膠質(zhì)瘤組織中的表達(dá),并分析miR-128-3p與PP4C表達(dá)相關(guān)性。研究結(jié)果：1. 生物信息學(xué)預(yù)測軟件顯示在PP4C的3,-UTR區(qū)域存在miR-128-3p的結(jié)合位點(diǎn)。2.首先在293T細(xì)胞中,miR-128-3p能直接與PP4C 3'-UTR上的結(jié)合位點(diǎn)結(jié)合從而抑制熒光素酶表達(dá)。其次,miR-128-3p可以在U251和U87細(xì)胞中可以與PP4C 3'-UTR結(jié)合從而抑制熒光素酶活性,從而驗(yàn)證了miR-128-3p與PP4C的直接相互作用,PP4C是miR-128-3p的直接靶基因。3.48小時(shí)后,轉(zhuǎn)染miR-128-3p過表達(dá)miR-128-3p U251、U87細(xì)胞中PP4C蛋白水平相比與轉(zhuǎn)染NC的U251、U87細(xì)胞是顯著降低的,而膠質(zhì)瘤U251和U87細(xì)胞中過表達(dá)miR-128-3p后,PP4C mRNA水平?jīng)]有顯著變化。預(yù)示PP4C作為miR-128-3p的直接靶基因,并被其負(fù)調(diào)控。4. miR-128-3p在膠質(zhì)瘤中的表達(dá)水平顯著低于正常腦組織。并且發(fā)現(xiàn)miR-128-3p的表達(dá)水平隨著膠質(zhì)瘤病理級別的增高逐漸降低。在膠質(zhì)瘤組織中,miR-128-3p的表達(dá)水平與PP4C蛋白表達(dá)水平呈顯著負(fù)相關(guān)。結(jié)論：1. miR-128-3p對膠質(zhì)瘤細(xì)胞系U251、U87中PP4C mRNA的表達(dá)無調(diào)節(jié)作用,對U251、U87細(xì)胞中PP4C蛋白的轉(zhuǎn)錄后表達(dá)具有負(fù)調(diào)控作用,提示PP4C是miR-128-3p直接的靶基因,并被其負(fù)調(diào)控。2. miR-128-3p在膠質(zhì)瘤組織中低表達(dá),PP4C與miR-128-3p在膠質(zhì)瘤組織中的表達(dá)呈負(fù)相關(guān)。
[Abstract]:The background and significance of brain glioma is the highest incidence of the central nervous system, accounting for about 45% of the intracranial tumors, accounting for about 80% of the intracranial malignant tumors. Glioma originated from glial cells, which grew rapidly and had strong invasion ability, which made it difficult to excision. According to the latest WHO tumor classification in 2007 Standard, glioma can be divided into grade I - IV, of which grade I, grade II is low grade glioma, grade III and grade IV is high grade glioma. The standard treatment for glioma currently includes surgery combined with radiotherapy and chemotherapy. Even through these comprehensive treatment, the prognosis of high grade gliomas is still unsatisfactory, especially the highest malignant polyglia. The total survival rate of 5 years is less than 5% and the average survival time is about 14-15 months. The pathogenesis of glioma involves multiple genes and multiple factors. The abnormal expression of many related genes is closely related to the occurrence of glioma. However, the whole process of glioma and the whole process of glioma can not be clearly elucidated. To show the pathogenesis of glioma, to find the biological markers of glioma and to explore new targets for the treatment of glioma, the phosphorylation of protein is an important part of post-translational modification of protein, and about 30% of the proteins in human cells are phosphorylated, and this phosphorylation modification is the regulation of cell proliferation. One of the important mechanisms of apoptosis, differentiation, and other cell functions. The level of phosphorylation of proteins is regulated by protein kinase and protein phosphatase. Protein phosphatase has been considered a silent companion with protein kinase, which reverses the phosphorylation effect of protein kinase at random, but more and more evidence suggests that the protein phosphatase group is a protein phosphatase group. A complex and diverse family of enzymes, parallel to protein kinase, can actively and specifically dephosphorylate specific substrates. Protein phosphatase can be divided into three families according to amino acid sequence homology, structure and catalytic mechanism. The first family is silk / threonine protein phosphatase, including phosphate phosphatase (Phosphoprotein phosph). ATase, PPP) and magnesium or manganese ion dependent protein phosphatase (Protein phosphatase Mg2+ or Mn2+ dependent, PPM), in which PPP is the largest and most classic family, and can be divided into PP1, PP2A, tyrosine phosphatase and protein phosphatase based on aspartate catalyzed by aspartic acid, respectively. The two and third family.PP4 (Protein Phosphatase 4) is independent of other phosphatase and participates in a variety of pathophysiological processes. More and more researchers are concerned that.PP4 is structurally similar to PP2A to catalyze the combination of subunit PP4C (Protein Phosphatase 4 catalytic subunit) and different regulatory subunits to form a heterogeneous polymer. PP4C, as the core catalytic subunit of PP4, is considered to be an important subunit of the subunit.PP4C, encoded by the ppp4c gene, located in the human chromosome 16p1 1.2 region, originally isolated and identified by Brewis ND and others from rabbit liver to cDNA library, originally called PPX. In 1993, the PP4C gene was cloned in mammals, and then in the mammalian In Drosophila, nematodes, and yeast, PP4C's homologous.PP4C is also found to be involved in a variety of pathophysiological processes, such as microtubule formation, cerebral cortex development, immune regulation, DNA damage repair and multiple cell signaling pathways that promote cell proliferation, inhibit cell withering, and regulate the tumor related signaling pathways such as NF- kappa B, JNK and m-TOR. It is suggested that PP4C is involved in the occurrence and development of tumor. In recent years, some scholars have discovered that PP4C is highly expressed in breast cancer, lung cancer, pancreatic ductal carcinoma and colorectal cancer, and the patients with high expression of PP4C are closely related to pancreatic ductal carcinoma, and the prognosis of colorectal cancer patients is closely related. However, scholars at home and abroad have not yet found that PP4C is in glioma. This study is divided into three parts. In the first part, the expression of PP4C in glioma was detected by Western Blot, Real-time PCR and immunohistochemistry, and the correlation between the abnormal expression of PP4C and the clinicopathological features and prognosis of the patients with glioma was analyzed. The second part, the human glioma cell line A172, U251, and U87 normal glial cells Ast were detected. The expression of PP4C in rocyte and the effect of silent PP4C gene on the proliferation, migration and invasion of human glioma cell line U251, U87. The third part, using bioinformatics software to predict the potential miRNA of PP4C, namely miR-128-3p, further verify its authenticity by luciferase reporter gene experiment, and use Western Blot and Real-time PCR. To detect the effect of miR-128-3p on PP4C protein and mRNA; further detect the expression of miR-128-3p in glioma tissue and analyze its correlation with PP4C. Part 1 PP4C in human glioblastoma tissue and the correlation of clinicopathological features: 1. to explore the expression of PP4C protein and mRNA in human glioma tissue The expression of normal brain tissue and.2. study the correlation between abnormal expression of PP4C and the clinicopathological features and prognosis of patients with glioma. Methods: 1. using Western Blot and Real-time PCR and detecting the expression of PP4C protein and mRNA in fresh glioma tissue and normal brain tissue, the immunohistochemical method was used to detect 120 cases. The expression of PP4C protein in glioma tissues and 10 normal brain tissues and the correlation between the expression of WHO and the pathological grade of glioma. The results showed that the expression of 1.PP4C protein and mRNA in glioma tissues was significantly higher than that of normal brain tissue, and the expression of PP4C protein and mRNA was gradually increased with the increase of the pathological grade of WHO. The positive expression of.2.PP4C in the malignant degree of glioma was light yellow in nucleus and cytoplasm, and the high expression rate of brown or yellowish brown.PP4C protein in glioma tissues was 60% (72/120). The expression of PP4C in the normal brain tissues was low. In addition, the high expression rate of PP4C protein in grade II, grade III and grade IV glioma was respectively For 21.43% (6/28), 55.56% (20/36), 82.14% (46/56), the difference was statistically significant (P0.001).Kaplan-Meier survival analysis showed that the prognosis of the patients with high expression of PP4C was more than that of the.Cox proportional risk model with the poor prognosis of PP4C, and the multifactor analysis showed that the high expression of PP4C protein was an independent risk factor affecting the prognosis of glioma patients. Conclusion: the expression of PP4C protein and mRNA in glioma is significantly higher than that of normal brain tissue, and is related to the pathological classification of glioma. The prognosis analysis shows that the high expression of PP4C is an independent risk factor for the patients with glioma. Second part of PP4C in human glioma cell lines and the silence of PP4C gene for cell proliferation and migration Objective: 1. to investigate the expression of PP4C in human glioma cell lines A172, U251, U87 and normal glial cell Astrocyte.2. to explore the shadow of the silent PP4C gene on the human glioma cell line U251, U87 proliferation, migration and invasion. Research methods: 1. Western Blot and Real-time, and the detection of PP4C The expression of A in human glioma cell line and normal glial cell line.2. si-RNA construction and transfection: design and synthesize PP4C-siRNA interference sequence and divide U251 and U87 cell lines into two groups, that is, experimental group (transfected PP4C-siRNA), negative control group (transfected non-specific siRNA), and transfection reagents to transfect.3.PP4C gene silencing effect Detection of rate: using Real-time PCR and Western Blot to detect the PP4C-siRNA silencing efficiency.4., CCK-8 and EdU tests were used to detect the U251 of the silent PP4C gene and the proliferation ability of the U87 cells. Changes in the invasiveness of U87 cells. Results: the expression of 1.PP4C in glioma cell lines using Western Blot and Real-time PCR to detect glioma cell lines A172, U251, U87 and normal glia Astrocyte showed that PP4C protein and mRNA were expressed in 4 cell lines. The 2 cell was the next, the relative expression in Astrocyte was the lowest, so U251 was screened out. The U87 superiority expressed the cell line for the next experiment of.2. detection of the PP4C-siRNA silence efficiency using Real-time PCR and Western Blot detection PP4C-siRNA silencing efficiency.Real-timePCR and Western results showed, after 48 hours transfection, relative to negative control group. SiRNA significantly reduced the expression of PP4C mRNA and protein in the glioma cell line, PP4C mRNA and protein (P 0.01).3.PP4C gene silencing on the proliferation ability of U251 and U87 cell lines, the proliferation test of CCK-8 showed that, compared with the negative control group (NC), the P4C-siRNA experimental group decreased significantly, and decreased by 54%, 50%, respectively. Study significance (P0.01). The results of EdU proliferation test showed that compared with negative control group (NC), the cell proliferation rate of PP4C-siRNA experimental group decreased significantly, U251, U87 cells decreased by 51%, 64%, respectively, and the difference was statistically significant (P0.01). These results showed that PP4C could promote the proliferation ability of glioma cells in vitro and silenced the glioma cells after PP4C. The effect of.4.PP4C gene silencing on the migration ability of U251 and U87 cell lines was significantly reduced. The results of the scratch test showed that the number of cell migration decreased significantly in the PP4C-siRNA group compared with the negative control group, in which the U251 cells decreased by 56%, and the U87 cells decreased by 66%. The difference was statistically significant (P0.01).5.PP4C gene silencing of U251, U87 cell lines invaded. The influence of the attack ability on the Transwell invasion test showed that compared with the negative control group (NC), the PP4C-siRNA experimental group passed through the chamber crystal violet staining cells significantly decreased, of which U251 cells decreased by 52%, U87 cells decreased by 39%, and the difference was statistically significant (P0.01). Conclusion: 1.PP4C was highly expressed in glioma cell lines, and U251, U was screened. 87 dominant cell lines in subsequent biological experiments.2. PP4C siRNA can clearly silence the expression of PP4C, and can inhibit the proliferation, migration and invasion of U251, U87 cells. It shows that PP4C can promote the proliferation, migration and invasion of glioma cells. The target regulation relationship between the third part of miR-128-3p and the PP4C gene is studied: 1. Prediction of potential miRNA.2. validation of PP4C, miR-128-3p post transcriptional level, PP4C expression,.3. detection of miR-128-3p expression in glioma tissue, and analysis of the correlation between miR-128-3p and PP4C expression. Research methods: 1. use target gene prediction algorithm to predict the potential miRNA for PP4C and analyze PP4C 3, the binding site of miR-128-3p in the -UTR region.2. uses luciferase reporter gene test to verify whether there is a direct interaction between miR-128-3p and PP4C in 293T cells, and further in the glioma cell line U251 and U87 to verify whether there is a direct interaction between miR-128-3p and PP4C. VWestern Blot and Real-time PCR were used to detect the expression of PP4C protein and mRNA level.4. Real-time PCR detection miR-128-3p in brain glioma tissue, and the correlation between miR-128-3p and PP4C expression was analyzed. Results: 1. bioinformatics prediction software showed the 3 of the binding site. In 293T cells, miR-128-3p can bind to the binding site on PP4C 3'-UTR to inhibit luciferase expression. Secondly, miR-128-3p can inhibit the activity of luciferase by binding to PP4C 3'-UTR in U251 and U87 cells, thus verifying the direct interaction between miR-128-3p and PP4C. PP4C is a direct target gene. After 48 hours, transfected miR-128-3p overexpressed miR-128-3p U251, and the level of PP4C protein in U87 cells was significantly lower than that of NC transfected U251, but there was no significant change in the PP4C levels after the expression of miR-128-3p in the glioma U251 and U87 cells. The expression level of miR-128-3p in glioma was significantly lower than that in normal brain tissue, and the expression level of glioma was increased with pathological grade of glioma.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R739.41

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