本文選題：非小細(xì)胞肺癌 + EGFR　； 參考：《山東大學(xué)》2016年博士論文

【摘要】：肺癌是我國(guó)發(fā)病率和死亡率均最高的惡性腫瘤。非小細(xì)胞肺癌(non-small cell lung cancer, NSCLC)約占肺癌的80%-85%,目前主要治療手段包括手術(shù)治療、化學(xué)治療、放射治療及靶向治療。盡管治療方式眾多,肺癌的5年生存率仍不足17%,多數(shù)病人最終死于遠(yuǎn)處轉(zhuǎn)移。研究NSCLC發(fā)生發(fā)展的分子機(jī)制將有助于找尋更好的治療靶點(diǎn),從而設(shè)計(jì)更好的靶向治療藥物。表皮生長(zhǎng)因子受體(Epidermal growth factor receptor, EGFR)是1型酪氨酸激酶ErbB家族成員之一,該家族成員還包括ErbB2/Her2、Her3和Her4。EGFR的過(guò)表達(dá)及異�；罨瘏⑴c多種上皮惡性腫瘤的發(fā)生發(fā)展過(guò)程,NSCLC中亦常出現(xiàn)EGFR過(guò)表達(dá)或是酪氨酸激酶的活化突變。早在1983年就有學(xué)者提出針對(duì)EGFR的靶向治療,目前靶向EGFR的治療已經(jīng)成功應(yīng)用于臨床,其主要方式有兩種：一是針對(duì)EGFR的單克隆抗體,如西妥昔單抗；一是針對(duì)EGFR酪氨酸激酶活化突變的抑制劑(Tyrosine Kinase Inhibitors, TKIs),如吉非替尼、厄洛替尼等。盡管單克隆抗體和TKIs(特別是TKIs)在部分NSCLC病人中療效顯著,但幾乎所有病人最終出現(xiàn)耐藥。如何解決EGFR靶向治療的原發(fā)性及繼發(fā)性耐藥問(wèn)題仍是靶向治療中的難題。microRNAs (miRNAs)是一類(lèi)非編碼的小分子RNA,長(zhǎng)度約在22個(gè)核苷酸左右。miRNAs普遍存在于動(dòng)植物細(xì)胞內(nèi),是基因表達(dá)和調(diào)控的轉(zhuǎn)錄后修飾途徑,主要通過(guò)作用于靶基因mRNA的3'UTR端而發(fā)揮調(diào)節(jié)作用。據(jù)估計(jì),哺乳動(dòng)物中有超過(guò)50%的基因受miRNAs的調(diào)控。niRNAs參與細(xì)胞生命過(guò)程中幾乎所有的重要進(jìn)程,包括細(xì)胞增殖與分化、細(xì)胞周期調(diào)控、細(xì)胞死亡與凋亡、細(xì)胞遷移和侵襲等。miRNAs表達(dá)失調(diào)參與多種疾病包括腫瘤的發(fā)生與發(fā)展,幾乎所有腫瘤中均能檢測(cè)到miRNAs的異常表達(dá),這些異常表達(dá)的miRNAs在腫瘤中發(fā)揮著癌基因或是抑癌基因的作用。近來(lái)研究顯示,基于miRNAs的靶向治療可能是一種有效可行的靶向EGFR的治療手段。目前已知多種miRNAs(如miRNA-7, miRNA-23b/27b和]niRNA-133a等)能直接靶向EGFR,然而,更多靶向EGFR的miRNAs有待發(fā)現(xiàn)。本研究首先通過(guò)靶基因預(yù)測(cè)軟件找尋可能直接靶向EGFR的miRNAs,并從保守的miRNAs中挑選了mirSVR評(píng)分靠前的3個(gè)miRNAs,在NSCLC細(xì)胞系中進(jìn)行進(jìn)一步驗(yàn)證,新發(fā)現(xiàn)一個(gè)能直接靶向EGFR的miRNA, miRNA-134(miR-134)；隨后對(duì)miR-134在NSCLC中的抑癌作用及其機(jī)制進(jìn)行了深入研究,證實(shí)miR-134可能作為NSCLC治療的新的研究靶點(diǎn)。第一部分鑒定直接靶向EGFR的miRNAs[目的]通過(guò)靶基因預(yù)測(cè)軟件找尋直接靶向EGFR但尚未被證實(shí)miRNAs,在NSCLC中驗(yàn)證所選的miRNAs是否能下調(diào)EGFR蛋白的表達(dá),選擇抑制作用最強(qiáng)的miRNA進(jìn)一步研究,最終找到一個(gè)靶向EGFR的新的miRNA.[方法]1.應(yīng)用靶基因預(yù)測(cè)軟件(microrna.org, TargetScan)篩選可能直接靶向EGFR的miRNAs,根據(jù)mirSVR值進(jìn)一步篩選。2.通過(guò)Western blot在NSCLC細(xì)胞系A(chǔ)549和H1299中檢測(cè)轉(zhuǎn)染miRNA模擬劑后EGFR蛋白的表達(dá)情況,選擇抑制作用最強(qiáng)的miRNA:miR-134。3.通過(guò)Western blot在其他4株NSCLC細(xì)胞系H460、H520、H1975和PC9中進(jìn)一步驗(yàn)證miR-134是否能下調(diào)EGFR蛋白表達(dá),并通過(guò)qRT-PCR檢測(cè)轉(zhuǎn)染miRNA模擬劑后EGFR mRNA的表達(dá)情況。4.通過(guò)雙熒光素酶報(bào)告基因?qū)嶒?yàn)驗(yàn)證miR-134是否直接靶向EGFR mRNA的3'UTR端。5.通過(guò)Western blot檢測(cè)miR-134轉(zhuǎn)染后對(duì)EGFR下游信號(hào)通路的影響。[結(jié)果]1.通過(guò)靶基因預(yù)測(cè)軟件,選擇了3個(gè)miRNAs:miR-134、miR-200a和]miR-373進(jìn)行驗(yàn)證,并以miR-7作為陽(yáng)性對(duì)照。2. Western blot結(jié)果顯示,在A549和H1299細(xì)胞中,轉(zhuǎn)染miR-7模擬劑48和72小時(shí)后,EGFR蛋白表達(dá)明顯受到抑制；與miR-200a和miR-373相比,轉(zhuǎn)染miR-134模擬劑組48和72小時(shí)后EGFR下調(diào)更顯著。因而,我們選定miR-134行進(jìn)一步研究。3. Western blot結(jié)果顯示轉(zhuǎn)染miR-134模擬劑能使H520和H1975細(xì)胞中EGFR蛋白表達(dá)下調(diào),而H460和PC9細(xì)胞中的EGFR蛋白表達(dá)無(wú)顯著下調(diào)。在A549、H1299、H520和H1975細(xì)胞中,qRT-PCR進(jìn)一步證實(shí)轉(zhuǎn)染miR-134模擬劑后miR-134表達(dá)顯著上調(diào),而EGFR mRNA水平顯著下調(diào)。4.雙熒光素酶報(bào)告基因?qū)嶒?yàn)證實(shí)miR-134直接作用于EGFR mRNA的3'UTR端,從而抑制EGFR的表達(dá)。5. Western blot結(jié)果顯示,在A549、H1299、H520和H1975細(xì)胞中,轉(zhuǎn)染miR-134模擬劑能使磷酸化EGFR (p-EGFR)表達(dá)顯著下調(diào),且EGFR下游信號(hào)通路亦受到不同程度的抑制。[結(jié)論]1.過(guò)表達(dá)miR-134能下調(diào)NSCLC細(xì)胞系(A549、H1299、H520和H1975)中EGFR的mRNA及蛋白質(zhì)表達(dá)水平。2. EGFR mRNA的3'UTR端是miR-134的直接作用靶點(diǎn)。3.過(guò)表達(dá)miR-134能下調(diào)NSCLC細(xì)胞系(A549、H1299、H520和H1975)中p-EGFR表達(dá),且對(duì)EGFR下游信號(hào)通路有不同程度的抑制作用,說(shuō)明miR-134可能發(fā)揮抑癌基因的作用。第二部分miRNA-134抑制NSCLC增殖、遷移及侵襲的體外實(shí)驗(yàn)研究[目的]第一部分研究顯示miR-134抑制多個(gè)NSCLC細(xì)胞系中EGFR的表達(dá),并不同程度的抑制了EGFR的下游信號(hào)通路,提示miR-134可能發(fā)揮抑癌基因的作用。本部分通過(guò)體外細(xì)胞實(shí)驗(yàn)來(lái)證實(shí)miR-134是否能抑制NSCLC的細(xì)胞增殖、遷移及侵襲,并對(duì)相關(guān)機(jī)制做進(jìn)一步分析。[方法]1.MTT法檢測(cè)過(guò)表達(dá)miR-134對(duì)NSCLC細(xì)胞系(A549、H1299、H520和H1975)增殖的影響。細(xì)胞周期和細(xì)胞凋亡檢測(cè)分析miR-134抑制NSCLC細(xì)胞增殖的可能機(jī)制。2. Transwell小室遷移和侵襲實(shí)驗(yàn)分析過(guò)表達(dá)miR-134對(duì)NSCLC細(xì)胞系(A549和H1299)遷移和侵襲的影響。3.RNA干擾分析沉默EGFR后是否能模擬miR-134過(guò)表達(dá)引起的NSCLC細(xì)胞系(A549和H1299)表型改變。靶基因回復(fù)實(shí)驗(yàn)進(jìn)一步驗(yàn)證EGFR是否為miR-134的功能靶點(diǎn)。4.通過(guò)靶基因預(yù)測(cè)和文獻(xiàn)檢索找尋其他可能的miR-134的功能靶點(diǎn)。5.RNA干擾和靶基因回復(fù)實(shí)驗(yàn)驗(yàn)證ITGB1是否為miR-134的功能靶點(diǎn)。[結(jié)果]1.在NSCLC細(xì)胞系(A549、H1299、H520和H1975)中,過(guò)表達(dá)miR-134顯著抑制細(xì)胞增殖,在轉(zhuǎn)染3天和4天后差異有統(tǒng)計(jì)學(xué)意義。機(jī)制分析顯示,miR-134可能通過(guò)誘導(dǎo)細(xì)胞凋亡和／或誘導(dǎo)G0/G1期細(xì)胞周期阻滯而抑制NSCLC細(xì)胞增殖。2. Transwell小室遷移和侵襲實(shí)驗(yàn)顯示,過(guò)表達(dá)miR-134顯著抑制了A549和H1299細(xì)胞的遷移和侵襲。3.RNA干擾沉默EGFR后,A549和H1299細(xì)胞增殖、遷移和侵襲均受到抑制,與過(guò)表達(dá)miR-134的結(jié)果相似。靶基因回復(fù)實(shí)驗(yàn)顯示,在A549和H1299細(xì)胞中回復(fù)EGFR表達(dá)后部分阻斷了miR-134轉(zhuǎn)染對(duì)細(xì)胞增殖的抑制作用,而對(duì)細(xì)胞遷移和侵襲的抑制影響不大,說(shuō)明miR-134抑制A549和H1299細(xì)胞增殖的機(jī)制與其下調(diào)EGFR有一定關(guān)系,但其抑制A549和H1299細(xì)胞遷移和侵襲的機(jī)制與下調(diào)EGFR無(wú)關(guān),需要鑒定其他可能的靶基因。4.通過(guò)靶基因預(yù)測(cè)軟件和文獻(xiàn)檢索,選擇了3個(gè)可能參與miR-134抑制遷移和侵襲的靶基因：KRAS、FOXM1和ITGB1,進(jìn)行mRNA表達(dá)水平驗(yàn)證。在A549和H1299細(xì)胞中,miR-134對(duì)ITGB1 mRNA的抑制作用均最強(qiáng),于是選擇ITGB1進(jìn)行Western blot檢測(cè)。Western blot結(jié)果證實(shí)miR-134能顯著下調(diào)ITGB1蛋白的表達(dá)。5.RNA干擾沉默ITGB1后,A549和H1299細(xì)胞增殖、遷移和侵襲均受到抑制,與過(guò)表達(dá)miR-134的結(jié)果相似。靶基因回復(fù)實(shí)驗(yàn)顯示,在A549和H11299細(xì)胞中回復(fù)ITGB1表達(dá)后部分阻斷了miR-134轉(zhuǎn)染對(duì)細(xì)胞增殖、遷移和侵襲的抑制作用,說(shuō)明miR-134抑制A549和H1299細(xì)胞增殖、遷移和侵襲的機(jī)制與其下調(diào)ITGB1有一定關(guān)系。[結(jié)論]1.miR-134抑制NSCLC細(xì)胞系(A549、H1299、H520和H1975)的細(xì)胞增殖,抑制A549和H1299細(xì)胞遷移和侵襲,在NSCLC中發(fā)揮抑癌基因作用。2. EGFR是miR-134抑制A549和H1299細(xì)胞增殖的功能靶點(diǎn),但不參與miR-134對(duì)細(xì)胞遷移和侵襲的抑制作用。3. ITGB1是miR-134抑制A549和H1299細(xì)胞增殖、遷移和侵襲的另一功能靶點(diǎn)。4.miR-134通過(guò)下調(diào)癌基因EGFR和ITGB1的表達(dá)而發(fā)揮抑癌基因作用。第三部分1miRNA-134抑制NSCLC生長(zhǎng)和轉(zhuǎn)移的體內(nèi)實(shí)驗(yàn)研究[目的]第二部分通過(guò)體外細(xì)胞實(shí)驗(yàn)證實(shí)miR-134通過(guò)下調(diào)EGFR和ITGB1表達(dá),在NSCLC細(xì)胞中發(fā)揮抑癌基因的作用。本部分進(jìn)一步用裸鼠異種移植瘤模型,驗(yàn)證miR-134能否在體內(nèi)抑制NSCLC的生長(zhǎng)和轉(zhuǎn)移。[方法]1.選擇4-5周雌性裸鼠構(gòu)建A549異種移植瘤模型,待腫瘤直徑達(dá)5-6mm時(shí)隨機(jī)分成2組,分別瘤內(nèi)注射miR-134激動(dòng)劑或是陰性對(duì)照,每3天一次,共5次,并每3天測(cè)量腫瘤直徑,繪制腫瘤生長(zhǎng)曲線(xiàn)。2.末次注射miR-134激動(dòng)劑或是陰性對(duì)照48小時(shí)后處死小鼠,收集腫瘤組織。qRT-PCR檢測(cè)腫瘤中miR-134的表達(dá)情況,Western blot檢測(cè)腫瘤中EGFR和ITGB1蛋白表達(dá)情況。3.腫瘤組織經(jīng)福爾馬林固定、石蠟浸泡制作蠟塊及石蠟切片,應(yīng)用免疫組化分析腫瘤組織切片中EGFR、ITGB1、增殖標(biāo)志Ki-67、凋亡標(biāo)志Cleaved PARP、上皮間質(zhì)轉(zhuǎn)化(epithelial-to-mesenchymal transition, EMT)標(biāo)志E-cadherin和Vimentin的表達(dá)情況。[結(jié)果]1.瘤內(nèi)注射miR-134激動(dòng)劑顯著抑制裸鼠A549異種移植瘤的生長(zhǎng)。2. qRT-PCR證實(shí),瘤內(nèi)注射miR-134激動(dòng)劑組中miR-134顯著上調(diào)。Western blot和免疫組化結(jié)果顯示,瘤內(nèi)注射miR-134激動(dòng)劑組的EGFR和ITGB1的蛋白表達(dá)明顯下調(diào)。3.免疫組化結(jié)果顯示,瘤內(nèi)注射miR-134激動(dòng)劑組中,Ki-67蛋白表達(dá)下調(diào),Cleaved PARP蛋白表達(dá)上調(diào),E-cadherin蛋白表達(dá)上調(diào),而Vimentin蛋白表達(dá)下調(diào)。[結(jié)論]1.miR-134能在體內(nèi)發(fā)揮抑瘤作用,是潛在的腫瘤治療新靶點(diǎn)。2.miR-134可能通過(guò)下調(diào)EGFR和ITGB1表達(dá)、抑制腫瘤細(xì)胞增殖、促進(jìn)腫瘤細(xì)胞凋亡以及抑制EMT,從而抑制A549異種移植瘤的生長(zhǎng)和轉(zhuǎn)移。
[Abstract]:Lung cancer is a malignant tumor with the highest incidence and mortality in China. Non-small cell lung cancer (NSCLC) accounts for about 80%-85% of lung cancer. The main treatments include surgical treatment, chemotherapy, radiotherapy, and targeted therapy. Although the 5 year survival rate of lung cancer is still less than 17%, the majority of patients end up. The molecular mechanism that studies the development of NSCLC will help to find better targets for better targeting therapy. The Epidermal growth factor receptor (EGFR) is one of the members of the 1 tyrosine kinase ErbB family, which also includes ErbB2/Her2, Her3, and Her4.EGFR. Overexpression and abnormal activation are involved in the development of multiple epithelial malignant tumors. EGFR overexpression or activation mutation of tyrosine kinase is often found in NSCLC. A target therapy for EGFR was proposed by scholars in 1983. The target EGFR therapy has been successfully applied to the clinic. There are two main methods: one is E GFR monoclonal antibodies, such as cetuximab; one is an inhibitor of EGFR tyrosine kinase activation mutation (Tyrosine Kinase Inhibitors, TKIs), such as gefitinib, erlotinib, etc.. Although monoclonal antibodies and TKIs (especially TKIs) are effective in some NSCLC patients, almost all patients eventually appear to be resistant. How to solve EGF The problem of primary and secondary drug resistance in R targeting therapy is still a difficult problem in targeting therapy.MicroRNAs (miRNAs) is a class of non coded small molecule RNA, which is about 22 nucleotides in length and is ubiquitous in animal and plant cells. It is a post transcriptional modification approach for gene expression and regulation, mainly through the 3'UTR end of the target gene mRNA. It is estimated that more than 50% of the genes in mammals are regulated by miRNAs in the regulation of.NiRNAs to participate in almost all important processes in cell life processes, including cell proliferation and differentiation, cell cycle regulation, cell death and apoptosis, cell migration and invasion and other.MiRNAs expression disorders involved in a variety of diseases including cancer. The abnormal expression of miRNAs can be detected in almost all tumors, and the abnormal expression of miRNAs plays the role of oncogene or tumor suppressor gene in the tumor. Recent studies have shown that targeting therapy based on miRNAs may be an effective and feasible therapeutic target for targeting EGFR. A variety of miRNAs (such as miRNA-7, miRNA-2) is now known. 3b/27b and]niRNA-133a can directly target EGFR, however, more target to EGFR miRNAs need to be found. Firstly, the target gene prediction software is used to find the miRNAs that may be directly targeted to EGFR, and the 3 miRNAs of the mirSVR score is selected from the conservative miRNAs, which is further verified in the NSCLC cell line, and a new can be found straight. The miRNA, miRNA-134 (miR-134) of the target to EGFR, and the further research on the tumor suppressor effect of miR-134 in NSCLC and its mechanism, confirmed that miR-134 may be a new target for the study of NSCLC therapy. The first part identifies the miRNAs[aim of the direct target to EGFR, and the target gene pretest software seeks the direct target EGFR but has not been confirmed to be miR. NAs, in NSCLC, verify whether the selected miRNAs can downregulate the expression of EGFR protein, select the most powerful miRNA to further study, and finally find a new miRNA.[method for targeting EGFR,]1. application target gene prediction software (microrna.org, TargetScan) screening may directly target EGFR miRNAs. The expression of EGFR protein was detected in NSCLC cell lines A549 and H1299 by Western blot. The strongest inhibitory miRNA:miR-134.3. was selected through Western blot in the other 4 NSCLC cell lines. The expression of EGFR mRNA after NA simulant.4. was tested by the double luciferase reporter gene experiment to verify whether miR-134 directly targets EGFR mRNA in 3'UTR terminal.5. through Western blot detection of the downstream signaling pathway after miR-134. The results of miR-7 as positive control.2. Western blot showed that the expression of EGFR protein was obviously inhibited in A549 and H1299 cells after transfection of miR-7 analogue 48 and 72 hours. Compared with miR-200a and miR-373, miR-134 analogue group was more significant after 48 and 72 hours. The results of.3. Western blot showed that transfection of miR-134 mimic could reduce the expression of EGFR protein in H520 and H1975 cells, but the expression of EGFR protein in H460 and PC9 cells was not significantly down. The.4. double luciferase reporter gene experiment confirmed that miR-134 acted directly on the 3'UTR end of EGFR mRNA, thus inhibiting the.5. Western blot results of EGFR expression, and in A549, H1299, H520, and cells, the transfection of simulant could make phosphorylated expression significantly down, and the downstream signal pathway was also inhibited to varying degrees. [conclusion]1. overexpressed miR-134 can downregulate the mRNA and protein expression level of EGFR in NSCLC cell lines (A549, H1299, H520 and H1975).2. EGFR mRNA. Inhibition, indicating that miR-134 may play the role of tumor suppressor gene. Second part miRNA-134 inhibits the proliferation, migration and invasion of NSCLC in vitro. [Objective] the first part of the study showed that miR-134 inhibits the expression of EGFR in multiple NSCLC cell lines, and inhibits the downstream signal pathway of EGFR in varying degrees, suggesting that miR-134 may play an inhibitory role. The role of oncogene. This part through in vitro cell test to confirm whether miR-134 can inhibit the proliferation, migration and invasion of NSCLC, and further analyze the related mechanisms. [method]1.MTT assay was used to detect the effect of miR-134 on the proliferation of NSCLC cell line (A549, H1299, H520 and H1975). Cell cycle and apoptosis detection and analysis miR-134 The possible mechanism of inhibiting the proliferation of NSCLC cells.2. Transwell cell migration and invasion experiments to analyze the effect of miR-134 on the migration and invasion of NSCLC cell lines (A549 and H1299),.3.RNA interference analysis can simulate the phenotypic changes of NSCLC cell lines (A549 and expressions) induced by miR-134 over expression. Target gene recovery experiment further Verify whether EGFR is a functional target for miR-134,.4. through target gene prediction and literature search to find other possible miR-134 functional targets.5.RNA interference and target gene recovery test to verify whether ITGB1 is the functional target of miR-134. [results]1. in NSCLC cell line (A549, H1299, H520 and absent) significantly inhibits cell proliferation. The difference between 3 days and 4 days was statistically significant. The mechanism analysis showed that miR-134 could inhibit the migration and invasion of.2. Transwell cells by inducing cell apoptosis and / or inducing cell cycle arrest in G0/G1 phase, and that overexpression of miR-134 significantly inhibited the migration of A549 and H1299 cells and the invasion of.3.RNA interference. The proliferation, migration and invasion of A549 and H1299 cells were inhibited after EGFR silencing, similar to the results of overexpressing miR-134. The target gene recovery experiment showed that the inhibitory effect of miR-134 transfection on cell proliferation was partially blocked by EGFR expression in A549 and H1299 cells, but the inhibition of cell migration and invasion was not significant, indicating miR-134. The mechanism of inhibiting the proliferation of A549 and H1299 cells is related to its down-regulation of EGFR, but its mechanism of inhibiting the migration and invasion of A549 and H1299 cells is not related to the downregulation of EGFR. Other possible target genes,.4., should be identified by the target gene prediction software and literature retrieval, and 3 target genes may be selected to inhibit the migration and invasion of miR-134: KRAS The inhibition of miR-134 on ITGB1 mRNA in A549 and H1299 cells were all strongest in both A549 and H1299 cells, and Western blot detection of Western showed that the expression of miR-134 could significantly downregulate the expression of ITGB1 mRNA. Inhibition, similar to the results of overexpressing miR-134. Target gene recovery experiments showed that the response to ITGB1 expression in A549 and H11299 cells partially blocked the inhibitory effects of miR-134 transfection on cell proliferation, migration and invasion, indicating that miR-134 inhibits the proliferation of A549 and H1299 cells, and the mechanism of migration and invasion is related to its down-regulation of ITGB1. [Conclusion] 1.miR-134 inhibits the proliferation of NSCLC cell lines (A549, H1299, H520 and H1975), inhibits the migration and invasion of A549 and H1299 cells, and plays the role of tumor suppressor gene in NSCLC..2. EGFR is the functional target of miR-134 inhibition and proliferation, but it does not participate in the inhibition of cell migration and invasion. Another functional target of H1299 cell proliferation, migration and invasion,.4.miR-134 plays the role of tumor suppressor gene by down-regulation of the expression of oncogene EGFR and ITGB1. Third part of the experimental study on the inhibition of NSCLC growth and metastasis by 1miRNA-134 [Objective] the second part confirmed that miR-134 was expressed by down regulation of EGFR and ITGB1 through in vitro cell experiments, in NSCLC In this part, the tumor suppressor gene is used. This part further uses the xenograft model in nude mice to verify whether miR-134 can inhibit the growth and metastasis of NSCLC in the body. [method]1. selected female nude mice for 4-5 weeks to construct a A549 xenograft model and randomly divided the tumor into 2 groups when the diameter of the tumor was 5-6mm, and the miR-134 agonists were injected into the tumor or were negative respectively. Control, once every 3 days, a total of 5 times, and the tumor diameter was measured every 3 days, the tumor growth curve was plotted for the final injection of miR-134 agonist or negative control for 48 hours, and the tumor tissue.QRT-PCR was collected to detect the expression of miR-134 in the tumor. Western blot was used to detect the expression of EGFR and ITGB1 protein in the tumor. Almarin was fixed, paraffin wax was soaked to make paraffin blocks and paraffin sections. The expression of EGFR, ITGB1, Ki-67, Cleaved PARP, and epithelial-to-mesenchymal transition, EMT (epithelial-to-mesenchymal transition, EMT) were used to analyze the expression of E-cadherin and Vimentin in the tumor tissue sections. [results]1. intratumor injected miR-134 agonists. The growth of.2. qRT-PCR in A549 xenografts in nude mice showed a significant increase in.Western blot and immunohistochemical results of miR-134 in the miR-134 agonist group. The protein expression of EGFR and ITGB1 in the intratumoral miR-134 agonist group was obviously down regulated by.3. immunohistochemical staining, and in the intratumoral injection of the miR-134 agonist group. The expression of protein expression was down regulated, the expression of Cleaved PARP protein was up-regulated, the expression of E-cadherin protein was up, and the expression of Vimentin protein was down regulated. [conclusion]1.miR-134 can play an inhibitory role in the body. It is a potential new target for tumor therapy,.2.miR-134 may reduce the expression of EGFR and ITGB1, inhibit the proliferation of tumor cells, promote the apoptosis of tumor cells and inhibit E. MT, thus inhibiting the growth and metastasis of A549 xenograft tumor.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R734.2
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本文編號(hào)：2115301