miR-218通过影响MEF2D表达对非小细胞肺癌的作用研究

发布时间：2018-07-28 17:31

【摘要】：肺癌是一种致死性恶性疾病,因其预后差、发病率还逐年增加,对它的研究也在大量开展。即便如此,其发生、发展的分子机制仍然没有完全被阐明。最近有报导称,肌细胞增强因子2D(myocyte enhancer factor 2D,MEF2D)可以促进肝癌的生长,但是尚不明确其在肺癌中是否也存在相同或类似的作用。同时,根据现有研究所示,MEF2D激活及过表达促进肿瘤进程的机制是多样的。有趣的是,这些研究在证实MEF2D可以促进肿瘤进程的同时,或多或少都发现了一些可以抑制MEF2D基因过表达的微小RNA(micro RNA,mi RNA)。在证实了MEF2D对肺癌细胞的增殖、凋亡和侵袭能力均有影响后,我们想要进一步研究可以作用于MEF2D基因并抑制其表达的mi RNA。为此,我们查阅了大量文献,并求助于mi RNA及目标基因的在线数据库,最终发现,在MEF2Dm RNA的3'UTR上,有一种mi R-218的mi RNA识别元件(micro RNA recognition element,MRE),而mi R-218可能会抑制肺癌的进程。因此,我们将mi R-218对MEF2D表达的影响作为了第二步研究的目标。因此我们设计了既有区别又相互关联的两个子实验研究mi R-218及MEF2D与肺癌的关联。为了探讨在非小细胞肺癌(non-small cell lung cancer,NSCLC)中,MEF2家族的表达特点。我们首先重点研究MEF2D表达对非小细胞肺癌的增殖、凋亡及侵袭能力的影响。我们运用q PCR的方法,研究30例非小细胞肺癌患者肺组织及正常组织中,MEF2家族四种基因的表达情况。使用免疫印迹法比较MEF2D在肺癌组织及正常组织中的表达情况。应用免疫荧光染色,明确MEF2D基因所表达的蛋白在肺癌和正常细胞中的分布情况。在正常肺组织细胞高表达MEF2D及沉默肿瘤细胞MEF2D的表达后,通过transwell试验验证MEF2D对细胞侵袭性的影响;通过Brd U试验及ki67表达情况来验证MEF2D对细胞增殖率的影响;通过流式细胞技术来验证MEF2D对细胞凋亡的影响。最后,建立肺癌细胞异种移植小鼠动物模型,将表达MEF2D发夹结构的慢病毒载体和对照载体注射到小鼠肺癌组织中。通过定期测量肿瘤的大小和重量,来验证MEF2D对肺癌体内试验肿瘤增殖情况的影响。通过免疫组织化学技术验证肿瘤切片中MEF2D和ki67的表达情况,侧面反应MEF2D对肿瘤增殖能力的影响。同时,用TUNEL试验来检测体内实验肿瘤细胞的凋亡情况。q PCR试验证实,与正常组织与正常细胞相比,肺癌组织和肺癌细胞中的MEF2D的m RNA表达量显著提高,而MEF2A和MEF2C的表达水平没有显著差异,实验中没有检测到MEF2B的表达。免疫印迹试验证实,MEF2D蛋白在肺癌组织和细胞中异常高表达。免疫荧光染色提示,MEF2D主要存在于肺癌细胞系A549细胞核内,而在正常肺MRC-5细胞中,MEF2D在细胞核内的表达比A549细胞要低,但在细胞质中的表达却高于A549细胞。Transwell试验表明,MEF2D表达水平的增加可以提高MRC-5细胞的侵袭性;Brd U试验和ki67表达情况均表明,MEF2D的过表达能够提高MRC-5细胞的增殖率。转染靶向抑制MEF2D的si RNA后,Brd U试验和ki67表达情况都表明,肿瘤细胞增殖率明显降低;通过流式细胞检测证实,沉默MEF2D后,肿瘤细胞凋亡率增加;transwell试验表明,转染MEF2Dsi RNA可降低A549和H460细胞的侵袭性。在小鼠肺癌细胞异种移植动物模型的相关实验也证实了,注射Lv-scrambled质粒的组织MEF2D广泛表达,而注射Lv-sh MEF2D质粒组,其表达则受到抑制;用TUNEL试验来检测同一肿瘤的细胞凋亡情况,发现注射了Lv-sh MEF2D质粒组呈现明显的阳性染色。综合以上实验结果,抑制MEF2D能够抑制小鼠体内肺癌的生长。我们进一步研究了mi R-218与MEF2D表达的关系,及其对非小细胞肺癌生物学行为的影响;以及mi R-218是否直接作用于MEF2D。我们分别将野生型和突变型MEF2D的3'UTR插入到一种荧光素酶表达载体中,将其分别转染肺癌细胞系及正常肺细胞系。之后,用mi R-218的mimic转染肺癌细胞并用inhibitor转染正常细胞系,通过其表达荧光素酶的情况,来证实其对目标基因MEF2D表达情况的影响。另外,应用q PCR和免疫印迹方法,分别检测不同mi R-218丰度下,肺癌细胞系及正常肺组织细胞中MEF2D的表达情况,从而协助明确mi R-218对MEF2D表达情况的影响。收集10例临床非小细胞肺癌患者的肺癌样本,同时收集相应患者的血清,对其肺癌中MEF2D表达水平进行q PCR检测,同时测量其血清中mi R-218的水平,用以比较两者之间是否存在线性关系。用pc DNA-MEF2D(表达MEF2D m RNA的载体,不受mi R-218调控)和mi R-218 mimics同时转染A549细胞和正常肺细胞。在不同时间点计数细胞以计算其增殖率。同时,利用流式细胞术测定不同组细胞凋亡情况,并对不同组细胞进行Transwell试验以明确其侵袭能力的变化。我们发现将合成的mi R-218 mimics转染A549细胞后,将极大的抑制野生型荧光素酶表达载体荧光素酶的表达,但不会影响突变型荧光素酶的表达。同样,mi R-218 inhibitor增加了正常肺纤维母细胞中荧光素酶的表达,对突变型无影响。q PCR和蛋白印迹方法也证实,mi R-218 mimic能够降低肺癌细胞中MEF2D的表达水平。而mi R-218 inhibitor可以使正常细胞MEF2D的表达增加。另外,对临床肺癌样本中MEF2D行q PCR检验,同时对比相应患者血清mi R-218水平,结果也发现mi R-218跟MEF2D的表达水平存在负相关。之后,用pc DNA-MEF2D和mi R-218同时转染肺癌细胞系,发现mi R-218并没有抑制MEF2D的过表达。通过流式细胞术也证实,即便已经转染了mi R-218的A549细胞,其细胞凋亡仍然被过表达的MEF2D所抑制。Transwell试验也表明,转染了mi R-218 mimics的肺癌细胞中,MEF2D的过表达仍能显著增强肺癌细胞的侵袭性。通过以上的实验,我们认为在非小细胞肺癌组织和细胞中,存在MEF2D的过表达。MEF2D除了具有促进肺癌细胞增殖和侵袭的能力,其过度表达还能够增强肺癌细胞的存活能力。因此,我们可以推断,MEF2D可能是非小细胞肺癌的一种致癌基因。抑制其表达,可以控制非小细胞肺癌的发生和发展,提示MEF2D可能会成为非小细胞肺癌治疗的新靶点。同时,我们也发现mi R-218跟MEF2D的表达水平存在负相关,MEF2D是肿瘤抑制因子mi R-218的目标基因。同时,单纯转染mi R-218mimic而不抑制MEF2D表达对肿瘤无抑制作用,从而证明了mi R-218是通过减少MEF2D表达来抑制肺癌细胞生长。
[Abstract]:Lung cancer is a fatal malignant disease. Because of its poor prognosis, the incidence of lung cancer is increasing year by year, and its research is also being carried out. Even so, the molecular mechanism of its occurrence and development is still not fully elucidated. Recently, it has been reported that the 2D (myocyte enhancer factor 2D, MEF2D) can promote the growth of liver cancer, but it is still possible to promote the growth of liver cancer. It is not clear whether it has the same or similar role in lung cancer. According to existing research, the mechanisms of MEF2D activation and overexpression are diverse. Interestingly, these studies have found that some of the MEF2D can inhibit the overexpression of MEF2D genes at the same time that the cancer process can be promoted. RNA (micro RNA, MI RNA). After confirming the effect of MEF2D on the proliferation, apoptosis and invasion of lung cancer cells, we want to further study the function of the MEF2D gene and inhibit the MI RNA. of its expression. We have consulted a large number of documents and resorted to the online database of MI RNA and target genes. On the 3'UTR of NA, there is a mi RNA identification element of MI R-218 (micro RNA recognition element, MRE), and MI may inhibit the process of lung cancer. The association with lung cancer. To investigate the expression of the MEF2 family in non-small cell lung cancer (NSCLC), we first focus on the effect of MEF2D expression on the proliferation, apoptosis and invasion of non-small cell lung cancer. We used Q PCR to study lung tissue and normal tissue in 30 patients with non-small cell lung cancer. The expression of four genes in the MEF2 family. The expression of MEF2D in lung cancer tissues and normal tissues was compared by immunoblotting. The distribution of the protein expressed by the MEF2D gene in lung cancer and normal cells was determined by immunoblotting. The expression of MEF2D in normal lung tissue and the expression of MEF2D in the silent tumor cells were expressed in normal lung tissue. The effect of MEF2D on cell invasiveness was verified by Transwell test, and the effect of MEF2D on cell proliferation rate was verified by Brd U test and Ki67 expression, and the effect of MEF2D on cell apoptosis was verified by flow cytometry. Finally, the rat model of xenotransplantation of lung cancer cells was established, and the lentivirus expressing MEF2D hairpin structure would be expressed. The carrier and control vector were injected into the lung cancer tissue in mice. The effect of MEF2D on the tumor proliferation in lung cancer was verified by measuring the size and weight of the tumor. The expression of MEF2D and Ki67 in the tumor slices and the effect of side reaction of MEF2D on the proliferation of tumor were verified by immunohistochemistry. At the same time, TUNE was used. L test to detect the apoptosis of tumor cells in vivo,.Q PCR test confirmed that the expression of M RNA in lung cancer tissues and lung cancer cells increased significantly compared with normal tissues and normal cells, but there was no significant difference in the expression level of MEF2A and MEF2C, and the expression of MEF2B was not detected in the experiment. Western blot test confirmed that MEF2D, MEF2D, MEF2D, and MEF2D. The protein is highly expressed in the tissues and cells of lung cancer. Immunofluorescence staining suggests that MEF2D mainly exists in the nucleus of lung cancer cell line A549, while in normal lung MRC-5 cells, the expression of MEF2D in the nucleus is lower than that of A549 cells, but the expression in cytoplasm is higher than that of the A549 cell.Transwell test that the expression of MEF2D is increased. Addition of addition can improve the invasiveness of MRC-5 cells; Brd U test and Ki67 expression showed that the overexpression of MEF2D could increase the proliferation rate of MRC-5 cells. After transfection of Si RNA to the targeting of MEF2D, Brd U test and Ki67 expression showed that the proliferation rate of the tumor cells decreased obviously; through flow cytometry confirmed that the tumor was fine after silence. The apoptosis rate increased; Transwell test showed that transfection of MEF2Dsi RNA could reduce the invasiveness of A549 and H460 cells. Related experiments in the xenotransplantation animal model of lung cancer cells in mice also confirmed that the tissue MEF2D of the injected Lv-scrambled plasmid was widely expressed, while the expression of the Lv-sh MEF2D plasmid group was inhibited, and the TUNEL test was used to detect the expression of the Lv-scrambled plasmid. It was found that the Lv-sh MEF2D plasmid group showed positive positive staining in the same tumor cell apoptosis. The inhibition of MEF2D could inhibit the growth of lung cancer in mice. We further studied the relationship between MI R-218 and the expression of MEF2D, and the effect on the biological behavior of non small cell lung cancer; and mi R-218 In MEF2D., we inserted the wild and mutant MEF2D 3'UTR into a luciferase expression vector, respectively, and transfected them to lung cancer cell lines and normal lung cell lines respectively. Then, the lung cancer cells were transfected with MI R-218 mimic and transfected with inhibitor to normal cell lines. The expression of luciferase expression was confirmed by the expression of luciferase. The effect on the expression of target gene MEF2D. In addition, Q PCR and immunoblotting were used to detect the expression of MEF2D in lung cancer cell lines and normal lung tissue cells under different mi R-218 abundances, and to help clarify the effect of MI R-218 on MEF2D expression. 10 cases of lung cancer samples from patients with non small cell lung cancer were collected. At the same time, the serum of the corresponding patients was collected, and the expression level of MEF2D in the lung cancer was detected by Q PCR, and the level of MI R-218 in the serum was measured to compare the linear relationship between them. PC DNA-MEF2D (the carrier of MEF2D m RNA, not regulated by Mi R-218) and the normal lung cells were transfected at the same time. The cell proliferation rate was counted at different time points. At the same time, the cell apoptosis in different groups was measured by flow cytometry, and the changes in the invasion ability of different groups were determined by Transwell test. We found that the transfection of synthetic mi R-218 mimics to A549 cells would greatly inhibit the expression of wild type luciferase. The expression of luciferase did not affect the expression of mutant luciferase. Similarly, MI R-218 inhibitor increased the expression of luciferase in normal lung fibroblasts. There was no effect on.Q PCR and Western blotting on mutagenesis. Mi R-218 mimic could reduce the expression level of MEF2D in lung cancer cells. Mi R-218 inhibitor. To increase the expression of MEF2D in normal cells. In addition, the Q PCR test of MEF2D in the clinical lung cancer samples and the serum mi R-218 level of the corresponding patients were compared. The results also showed that there was a negative correlation between the expression level of MI R-218 and MEF2D. Then, the lung cancer cell lines were transfected with PC DNA-MEF2D and MI. Through flow cytometry, it was also confirmed that even if the transfected mi R-218 A549 cells were still transfected with the expressed MEF2D, the.Transwell test also showed that the overexpression of MEF2D still significantly enhanced the invasiveness of lung cancer cells in the transfected mi R-218 mimics lung cancer cells. The overexpression of MEF2D in the tissues and cells of non-small cell lung cancer has the ability to promote the proliferation and invasion of lung cancer cells, and its overexpression can also enhance the viability of lung cancer cells. Therefore, we can deduce that MEF2D may be a oncogene in non-small cell lung cancer. The occurrence and development of lung cancer suggest that MEF2D may be a new target for the treatment of non-small cell lung cancer. At the same time, we also found that there is a negative correlation between the expression level of MI R-218 and MEF2D, MEF2D is the target gene of the tumor suppressor factor mi R-218. Meanwhile, the simple transfection of MI R-218mimic without inhibiting the expression of MEF2D has no inhibitory effect on the tumor, thus proving that the expression of MEF2D has no inhibitory effect on the tumor. Mi R-218 inhibits the growth of lung cancer cells by reducing MEF2D expression.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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