miRNA-134抑制非小细胞肺癌增殖、迁移及侵袭的机制研究

发布时间：2018-07-11 13:07

  本文选题：非小细胞肺癌 + EGFR　； 参考：《山东大学》2016年博士论文

【摘要】：肺癌是我国发病率和死亡率均最高的恶性肿瘤。非小细胞肺癌(non-small cell lung cancer, NSCLC)约占肺癌的80%-85%,目前主要治疗手段包括手术治疗、化学治疗、放射治疗及靶向治疗。尽管治疗方式众多,肺癌的5年生存率仍不足17%,多数病人最终死于远处转移。研究NSCLC发生发展的分子机制将有助于找寻更好的治疗靶点,从而设计更好的靶向治疗药物。表皮生长因子受体(Epidermal growth factor receptor, EGFR)是1型酪氨酸激酶ErbB家族成员之一,该家族成员还包括ErbB2/Her2、Her3和Her4。EGFR的过表达及异常活化参与多种上皮恶性肿瘤的发生发展过程,NSCLC中亦常出现EGFR过表达或是酪氨酸激酶的活化突变。早在1983年就有学者提出针对EGFR的靶向治疗,目前靶向EGFR的治疗已经成功应用于临床,其主要方式有两种：一是针对EGFR的单克隆抗体,如西妥昔单抗；一是针对EGFR酪氨酸激酶活化突变的抑制剂(Tyrosine Kinase Inhibitors, TKIs),如吉非替尼、厄洛替尼等。尽管单克隆抗体和TKIs(特别是TKIs)在部分NSCLC病人中疗效显著,但几乎所有病人最终出现耐药。如何解决EGFR靶向治疗的原发性及继发性耐药问题仍是靶向治疗中的难题。microRNAs (miRNAs)是一类非编码的小分子RNA,长度约在22个核苷酸左右。miRNAs普遍存在于动植物细胞内,是基因表达和调控的转录后修饰途径,主要通过作用于靶基因mRNA的3'UTR端而发挥调节作用。据估计,哺乳动物中有超过50%的基因受miRNAs的调控。niRNAs参与细胞生命过程中几乎所有的重要进程,包括细胞增殖与分化、细胞周期调控、细胞死亡与凋亡、细胞迁移和侵袭等。miRNAs表达失调参与多种疾病包括肿瘤的发生与发展,几乎所有肿瘤中均能检测到miRNAs的异常表达,这些异常表达的miRNAs在肿瘤中发挥着癌基因或是抑癌基因的作用。近来研究显示,基于miRNAs的靶向治疗可能是一种有效可行的靶向EGFR的治疗手段。目前已知多种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有待发现。本研究首先通过靶基因预测软件找寻可能直接靶向EGFR的miRNAs,并从保守的miRNAs中挑选了mirSVR评分靠前的3个miRNAs,在NSCLC细胞系中进行进一步验证,新发现一个能直接靶向EGFR的miRNA, miRNA-134(miR-134)；随后对miR-134在NSCLC中的抑癌作用及其机制进行了深入研究,证实miR-134可能作为NSCLC治疗的新的研究靶点。第一部分鉴定直接靶向EGFR的miRNAs[目的]通过靶基因预测软件找寻直接靶向EGFR但尚未被证实miRNAs,在NSCLC中验证所选的miRNAs是否能下调EGFR蛋白的表达,选择抑制作用最强的miRNA进一步研究,最终找到一个靶向EGFR的新的miRNA.[方法]1.应用靶基因预测软件(microrna.org, TargetScan)筛选可能直接靶向EGFR的miRNAs,根据mirSVR值进一步筛选。2.通过Western blot在NSCLC细胞系A549和H1299中检测转染miRNA模拟剂后EGFR蛋白的表达情况,选择抑制作用最强的miRNA:miR-134。3.通过Western blot在其他4株NSCLC细胞系H460、H520、H1975和PC9中进一步验证miR-134是否能下调EGFR蛋白表达,并通过qRT-PCR检测转染miRNA模拟剂后EGFR mRNA的表达情况。4.通过双荧光素酶报告基因实验验证miR-134是否直接靶向EGFR mRNA的3'UTR端。5.通过Western blot检测miR-134转染后对EGFR下游信号通路的影响。[结果]1.通过靶基因预测软件,选择了3个miRNAs:miR-134、miR-200a和]miR-373进行验证,并以miR-7作为阳性对照。2. Western blot结果显示,在A549和H1299细胞中,转染miR-7模拟剂48和72小时后,EGFR蛋白表达明显受到抑制；与miR-200a和miR-373相比,转染miR-134模拟剂组48和72小时后EGFR下调更显著。因而,我们选定miR-134行进一步研究。3. Western blot结果显示转染miR-134模拟剂能使H520和H1975细胞中EGFR蛋白表达下调,而H460和PC9细胞中的EGFR蛋白表达无显著下调。在A549、H1299、H520和H1975细胞中,qRT-PCR进一步证实转染miR-134模拟剂后miR-134表达显著上调,而EGFR mRNA水平显著下调。4.双荧光素酶报告基因实验证实miR-134直接作用于EGFR mRNA的3'UTR端,从而抑制EGFR的表达。5. Western blot结果显示,在A549、H1299、H520和H1975细胞中,转染miR-134模拟剂能使磷酸化EGFR (p-EGFR)表达显著下调,且EGFR下游信号通路亦受到不同程度的抑制。[结论]1.过表达miR-134能下调NSCLC细胞系(A549、H1299、H520和H1975)中EGFR的mRNA及蛋白质表达水平。2. EGFR mRNA的3'UTR端是miR-134的直接作用靶点。3.过表达miR-134能下调NSCLC细胞系(A549、H1299、H520和H1975)中p-EGFR表达,且对EGFR下游信号通路有不同程度的抑制作用,说明miR-134可能发挥抑癌基因的作用。第二部分miRNA-134抑制NSCLC增殖、迁移及侵袭的体外实验研究[目的]第一部分研究显示miR-134抑制多个NSCLC细胞系中EGFR的表达,并不同程度的抑制了EGFR的下游信号通路,提示miR-134可能发挥抑癌基因的作用。本部分通过体外细胞实验来证实miR-134是否能抑制NSCLC的细胞增殖、迁移及侵袭,并对相关机制做进一步分析。[方法]1.MTT法检测过表达miR-134对NSCLC细胞系(A549、H1299、H520和H1975)增殖的影响。细胞周期和细胞凋亡检测分析miR-134抑制NSCLC细胞增殖的可能机制。2. Transwell小室迁移和侵袭实验分析过表达miR-134对NSCLC细胞系(A549和H1299)迁移和侵袭的影响。3.RNA干扰分析沉默EGFR后是否能模拟miR-134过表达引起的NSCLC细胞系(A549和H1299)表型改变。靶基因回复实验进一步验证EGFR是否为miR-134的功能靶点。4.通过靶基因预测和文献检索找寻其他可能的miR-134的功能靶点。5.RNA干扰和靶基因回复实验验证ITGB1是否为miR-134的功能靶点。[结果]1.在NSCLC细胞系(A549、H1299、H520和H1975)中,过表达miR-134显著抑制细胞增殖,在转染3天和4天后差异有统计学意义。机制分析显示,miR-134可能通过诱导细胞凋亡和／或诱导G0/G1期细胞周期阻滞而抑制NSCLC细胞增殖。2. Transwell小室迁移和侵袭实验显示,过表达miR-134显著抑制了A549和H1299细胞的迁移和侵袭。3.RNA干扰沉默EGFR后,A549和H1299细胞增殖、迁移和侵袭均受到抑制,与过表达miR-134的结果相似。靶基因回复实验显示,在A549和H1299细胞中回复EGFR表达后部分阻断了miR-134转染对细胞增殖的抑制作用,而对细胞迁移和侵袭的抑制影响不大,说明miR-134抑制A549和H1299细胞增殖的机制与其下调EGFR有一定关系,但其抑制A549和H1299细胞迁移和侵袭的机制与下调EGFR无关,需要鉴定其他可能的靶基因。4.通过靶基因预测软件和文献检索,选择了3个可能参与miR-134抑制迁移和侵袭的靶基因：KRAS、FOXM1和ITGB1,进行mRNA表达水平验证。在A549和H1299细胞中,miR-134对ITGB1 mRNA的抑制作用均最强,于是选择ITGB1进行Western blot检测。Western blot结果证实miR-134能显著下调ITGB1蛋白的表达。5.RNA干扰沉默ITGB1后,A549和H1299细胞增殖、迁移和侵袭均受到抑制,与过表达miR-134的结果相似。靶基因回复实验显示,在A549和H11299细胞中回复ITGB1表达后部分阻断了miR-134转染对细胞增殖、迁移和侵袭的抑制作用,说明miR-134抑制A549和H1299细胞增殖、迁移和侵袭的机制与其下调ITGB1有一定关系。[结论]1.miR-134抑制NSCLC细胞系(A549、H1299、H520和H1975)的细胞增殖,抑制A549和H1299细胞迁移和侵袭,在NSCLC中发挥抑癌基因作用。2. EGFR是miR-134抑制A549和H1299细胞增殖的功能靶点,但不参与miR-134对细胞迁移和侵袭的抑制作用。3. ITGB1是miR-134抑制A549和H1299细胞增殖、迁移和侵袭的另一功能靶点。4.miR-134通过下调癌基因EGFR和ITGB1的表达而发挥抑癌基因作用。第三部分1miRNA-134抑制NSCLC生长和转移的体内实验研究[目的]第二部分通过体外细胞实验证实miR-134通过下调EGFR和ITGB1表达,在NSCLC细胞中发挥抑癌基因的作用。本部分进一步用裸鼠异种移植瘤模型,验证miR-134能否在体内抑制NSCLC的生长和转移。[方法]1.选择4-5周雌性裸鼠构建A549异种移植瘤模型,待肿瘤直径达5-6mm时随机分成2组,分别瘤内注射miR-134激动剂或是阴性对照,每3天一次,共5次,并每3天测量肿瘤直径,绘制肿瘤生长曲线。2.末次注射miR-134激动剂或是阴性对照48小时后处死小鼠,收集肿瘤组织。qRT-PCR检测肿瘤中miR-134的表达情况,Western blot检测肿瘤中EGFR和ITGB1蛋白表达情况。3.肿瘤组织经福尔马林固定、石蜡浸泡制作蜡块及石蜡切片,应用免疫组化分析肿瘤组织切片中EGFR、ITGB1、增殖标志Ki-67、凋亡标志Cleaved PARP、上皮间质转化(epithelial-to-mesenchymal transition, EMT)标志E-cadherin和Vimentin的表达情况。[结果]1.瘤内注射miR-134激动剂显著抑制裸鼠A549异种移植瘤的生长。2. qRT-PCR证实,瘤内注射miR-134激动剂组中miR-134显著上调。Western blot和免疫组化结果显示,瘤内注射miR-134激动剂组的EGFR和ITGB1的蛋白表达明显下调。3.免疫组化结果显示,瘤内注射miR-134激动剂组中,Ki-67蛋白表达下调,Cleaved PARP蛋白表达上调,E-cadherin蛋白表达上调,而Vimentin蛋白表达下调。[结论]1.miR-134能在体内发挥抑瘤作用,是潜在的肿瘤治疗新靶点。2.miR-134可能通过下调EGFR和ITGB1表达、抑制肿瘤细胞增殖、促进肿瘤细胞凋亡以及抑制EMT,从而抑制A549异种移植瘤的生长和转移。
[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.
【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】：R734.2
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本文编号：2115301