DADS上调miR-7靶向XIAP抑制人胃癌细胞增殖和侵袭

发布时间：2018-06-25 02:15

本文选题：二烯丙基二硫 + 人胃癌细胞　；参考：《南华大学》2015年博士论文

【摘要】：胃癌是最常见的恶性肿瘤之一,严重危害人类的健康和生命。随着社会经济发展与生活水平的提高,在欧美等发达国家其发病率和死亡率呈明显下降趋势,然而在亚洲、拉丁美洲仍然高发。由于其发病机制非常复杂,其预后仍不容乐观。二烯丙基二硫(diallyl disulfide,DADS)是大蒜中的脂溶性有效成分,可抑制多种肿瘤发生发展。我们前期研究证实,DADS能在体内外抑制人胃癌细胞增殖,其机制与G2/M期阻滞、p38激活、ERK抑制及其他多条信号转导通路异常有关。MicroRNAs是一类小分子非编码RNA,近年研究表明,其与胃癌的关系密切,参与胃癌的发生发展,在胃癌的增殖、侵袭和转移等过程中发挥了重要作用。miR-7是23个核苷酸miRNA,在多种肿瘤中低表达,具有潜在的抑瘤功能。XIAP(X-linked inhibitor of apoptosis)作为凋亡抑制因子家族(inhibitor of apoptosis protein,IAP)成员之一,在肿瘤发生发展中起着癌基因作用,与肿瘤发生、发展、凋亡、迁移、侵袭和转移密切相关。我们前期采用mirna芯片技术筛选dads处理人胃癌细胞的差异mirna表达,发现dads处理后人胃癌细胞mir-7明显上调。同时,qrt-pcr验证mir-7在胃癌组织与胃癌细胞株中显著下调。蛋白质组学发现xiap等24种差异表达的蛋白质,并验证显示,多株人胃癌细胞xiap高表达,而dads处理胃癌细胞后,xiap明显下调。本实验分三部分探讨mir-7和xiap对人胃癌细胞增殖侵袭的影响及二者相互关系。第一部分:dads上调mir-7抑制人胃癌细胞增殖和侵袭目的:探讨dads和mir-7对人胃癌细胞增殖和侵袭的影响。方法:构建mir-7高表达和沉默病毒载体,通过细胞转染,建立mir-7稳定高表达和沉默的sgc-7901和hgc-27细胞系,同时各组细胞经30mg/ldads处理,然后采用qrt-pcr、cck-8、平板克隆、流式细胞术、划痕实验、迁移实验、侵袭实验和裸鼠移植等分析dads和mir-7对胃癌细胞增殖和侵袭的作用。结果:qrt-pcr证实成功构建mir-7稳定高表达和沉默的sgc-7901和hgc-27细胞系,且dads能上调各组细胞mir-7的表达。dads和mir-7高表达分别可显著抑制sgc-7901和hgc-27细胞增殖,诱导sgc-7901细胞凋亡和细胞周期阻滞,抑制sgc-7901细胞克隆形成。划痕实验和transwell迁移和侵袭实验表明,dads和mir-7高表达分别可显著抑制sgc-7901和hgc-27细胞迁移和侵袭能力。裸鼠成瘤实验也表明dads组和mir-7高表达组肿瘤生长较慢,瘤体较小,较对照组有显著性差异(p0.05)。而mir-7沉默后表现出与mir-7高表达相反的结果。各组实验均表明,mir-7高表达可协同dads抑制细胞增殖、迁移、侵袭、凋亡、移植瘤生长,诱导细胞凋亡和周期阻滞,反之,mir-7沉默可拮抗dads的作用。第二部分:dads下调xiap抑制人胃癌细胞增殖和侵袭目的:探讨dads和xiap对人胃癌细胞增殖和侵袭的影响。方法:免疫组织化学检测组织芯片中胃癌组织xiap的表达情况,构建xiap高表达病毒载体和沉默质粒,通过细胞转染,建立xiap稳定高表达和沉默的sgc-7901和hgc-27细胞系,同时各组细胞分别经30mg/ldads处理,然后采用qrt-pcr、westernblot、细胞免疫荧光、cck-8、平板克隆、流式细胞术、划痕实验、迁移实验、侵袭实验以及裸鼠移植等分析dads和xiap对人胃癌细胞增殖和侵袭的作用。结果:xiap在胃癌中呈强阳性表达,阳性部位主要在细胞质中,而正常上皮及不典型增生上皮呈阴性或部分阳性表达,阳性表达率差异具有统计学意义(p0.05)。qrt-pcr证实成功构建xiap稳定高表达和沉默的sgc-7901和hgc-27细胞系,且dads能下调各组细胞xiap的表达。westernblot实验也表明xiap沉默的sgc-7901和hgc-27细胞xiap表达下降,而xiap高表达组则xiap表达上升,dads可使各组xiap表达均下降。细胞免疫荧光亦发现,xiap阳性表达主要在细胞质中,xiap表达水平与westernblot实验一致。dads和xiap沉默分别可显著抑制sgc-7901和hgc-27细胞增殖,诱导sgc-7901细胞凋亡和细胞周期阻滞,抑制sgc-7901细胞克隆形成。划痕实验和transwell迁移和侵袭实验表明,dads和xiap沉默可分别显著抑制sgc-7901和hgc-27细胞迁移和侵袭能力。裸鼠成瘤实验也表明dads组和xiap沉默组肿瘤生长较慢,肿瘤瘤体较小,较对照组有显著性差异(p0.05)。这些实验结果与mir-7高表达一致,而xiap高表达后表现出与xiap沉默相反的结果。而且,各组实验均表明,xiap沉默可协同dads抑制细胞增殖、迁移、侵袭、凋亡和细胞周期阻滞以及抑制移植瘤生长的作用,同时,亦可拮抗xiap过表达的作用。第三部分:mir-7靶向负性调控xiap的表达目的:探讨mir-7和xiap的靶向关系。方法:从mirnabase数据库查找成熟mir-7序列,同时从genbank查询xiap3’utr序列;利用在线数据库diana-microt-cds、targetscan和miranda进行靶基因预测,然后构建包含xiap-3’utr-wt和xiap-3’utr-mt双荧光素酶报告基因系统,与mir-7高表达载体共转染sgc-7901细胞,检测荧光素酶报告基因活性;最后通过qrt-pcr和westernblot检查mir-7稳定高表达和沉默的sgc-7901和hgc-27细胞xiapmrna和蛋白的表达,最终确定mir-7与xiap的靶向关系。结果:在获取miR-7成熟序列和XIAP 3’UTR序列基础上,利用生物信息学分析发现miR-7与XIAP 3’UTR存在多个结合位点,三个在线软件均预测XIAP为miR-7的靶基因之一;双荧光素酶报告基因系统证实miR-7与XIAP 3’UTR野生型共转染SGC-7901细胞,其荧光值与对照组比较,明显下降;miR-7稳定高表达的SGC-7901和HGC-27细胞XIAP mRNA和蛋白表达水平均下调,反向实验验证miR-7稳定沉默的SGC-7901和HGC-27细胞XIAP mRNA和蛋白水平均上调。结论:1.DADS可上调miR-7抑制人胃癌细胞增殖、迁移侵袭与移植瘤生长和诱导凋亡与周期阻滞。2.DADS可下调XIAP抑制人胃癌细胞增殖、迁移侵袭与移植瘤生长和诱导凋亡与周期阻滞。3.miR-7靶向负性调控XIAP mRNA和蛋白的表达。
[Abstract]:Gastric cancer is one of the most common malignant tumors and seriously endangers human health and life. With the development of social economy and the improvement of living standard, the incidence and mortality of the developed countries in Europe and America are obviously decreasing. However, in Asia, Latin America is still high. The prognosis is still not optimistic because its pathogenesis is very complex. Two Allyl two (diallyl disulfide, DADS) is a fat soluble active ingredient in garlic, which inhibits a variety of tumor development. Our previous study confirmed that DADS could inhibit the proliferation of human gastric cancer cells in vivo and in vivo, and its mechanism was associated with G2/M phase block, p38 activation, ERK inhibition, and other multiple signal transduction pathways associated with.MicroRNAs as a class of small fractions. Subcoding RNA, which is closely related to gastric cancer in recent years, is involved in the development of gastric cancer and plays an important role in the proliferation, invasion and metastasis of gastric cancer..miR-7 is 23 nucleotides miRNA, low expression in a variety of tumors and potential inhibition of.XIAP (X-linked inhibitor of apoptosis) as a inhibition of apoptosis One of the members of inhibitor of apoptosis protein (IAP) plays a oncogene role in the development of tumor, which is closely related to the occurrence, development, apoptosis, migration, invasion and metastasis of tumor. We used miRNA chip technique to screen the differential miRNA expression of human gastric cancer cells in dads, and found miR-7 for the miR-7 of the human gastric cancer cells after dads treatment. At the same time, qRT-PCR showed that miR-7 was significantly down regulated in gastric cancer tissue and gastric cancer cell lines. The proteomics found 24 different proteins such as XIAP and other proteins, and demonstrated that XIAP was highly expressed in many human gastric cancer cells, and XIAP was obviously down regulated after dads treatment of gastric cancer cells. This experiment was divided into three parts to discuss miR-7 and XIAP to human gastric cancer cells. The effect of proliferation and invasion and the relationship between the two. Part one: Dads up-regulation miR-7 inhibits the proliferation and invasion of human gastric cancer cells: To explore the effect of dads and miR-7 on the proliferation and invasion of human gastric cancer cells. Methods: to construct miR-7 high expression and silent virus vector, and to establish miR-7 stable and silent SGC-7901 and hgc-27 through cell transfection. Cell lines were treated by 30mg/ldads, and then the effects of dads and miR-7 on the proliferation and invasion of gastric cancer cells were analyzed by qRT-PCR, CCK-8, flat clones, flow cytometry, scratching experiments, migration experiments, invasive experiments and nude mice. Results: qRT-PCR confirmed the successful construction of miR-7 stable high expression and silent SGC-7901 and hgc-27. The proliferation of SGC-7901 and hgc-27 cells was significantly inhibited by dads, and the proliferation of SGC-7901 and hgc-27 cells could be inhibited by the higher expression of.Dads and miR-7 in each group of cells, inducing apoptosis and cell cycle arrest and inhibiting the formation of SGC-7901 cells. The scratch test and Transwell migration and invasion showed that the high expression of dads and miR-7 could significantly inhibit the expression of dads and miR-7. The proliferation and invasion ability of SGC-7901 and hgc-27 cells in nude mice showed that the tumor growth of dads group and miR-7 high expression group was slower, and the tumor body was smaller than that of the control group (P0.05). The miR-7 silence showed the contrary results with the high expression of miR-7. All the experiments showed that the high expression of miR-7 could inhibit the proliferation of the cells with dads. Migration, invasion, apoptosis, tumor growth, induction of cell apoptosis and cycle arrest, and conversely, miR-7 silencing can antagonize the effect of dads. The second part: Dads down XIAP inhibits the proliferation and invasion of human gastric cancer cells: explore the effects of dads and XIAP on the proliferation and invasion of human gastric cancer cells. Methods: immunohistochemical detection of gastric cancer group in tissue microarray The expression of XIAP, XIAP high expression virus vector and silent plasmid were constructed, SGC-7901 and hgc-27 cell lines were stable and highly expressed and silent through cell transfection, and the cells were treated by 30mg/ldads, and then qRT-PCR, Westernblot, cell immunofluorescence, CCK-8, flat clones, flow cytometry, scratching experiments, and migrated experiments were carried out. The effect of dads and XIAP on the proliferation and invasion of human gastric cancer cells was analyzed by migration experiments, invasive experiments and nude mice. Results: the positive expression of XIAP in gastric cancer was strongly positive, the positive parts were mainly in the cytoplasm, while the normal epithelium and atypical hyperplasia epithelium were negative or partial positive, and the difference of positive expression rate was statistically significant (P0.05). QRT-PCR confirmed the successful construction of XIAP and hgc-27 cell lines with high expression and silence, and dads could downregulate the expression of XIAP in each group of cells.Westernblot experiments also showed that XIAP silenced SGC-7901 and hgc-27 cells XIAP expression decreased, while XIAP high expression group increased expression. It was also found that the positive expression of XIAP was mainly in the cytoplasm. The expression level of XIAP and the XIAP silence could significantly inhibit the proliferation of SGC-7901 and hgc-27 cells, induce apoptosis and cell cycle arrest of SGC-7901 cells and inhibit the formation of SGC-7901 cell clones. The scratch test and Transwell migration and invasion experiments showed that dads and XIAP were used. XIAP silencing could significantly inhibit the migration and invasion of SGC-7901 and hgc-27 cells. The tumorigenicity test in nude mice also showed that the tumor growth of the dads group and the XIAP silent group was slower, and the tumor tumor body was smaller than the control group (P0.05). These experimental results were consistent with the high expression of miR-7, and the high expression of XIAP showed the opposite knot with the XIAP silence. Moreover, all experiments showed that XIAP silencing could cooperate with dads to inhibit cell proliferation, migration, invasion, apoptosis and cell cycle arrest, and inhibit the growth of xenografts. At the same time, it can also antagonize the role of XIAP overexpression. The third part: miR-7 targeting the negative regulation of XIAP expression: explore the targeting relationship between miR-7 and XIAP. Method: mirn The abase database searches the mature miR-7 sequence and queries the xiap3 'UTR sequence from the GenBank, uses the online database diana-microt-cds, targetscan and Miranda to predict the target gene, then constructs the xiap-3' utr-wt and xiap-3 'utr-mt double luciferase reporter gene system, and CO transfects the cells with the high expression vector and detects the fluorescent cells. In the end, qRT-PCR and Westernblot were used to detect the expression of xiapmrna and protein in SGC-7901 and hgc-27 cells with high expression and silence by qRT-PCR and Westernblot. Finally, the target relationship between miR-7 and XIAP was determined. Results: on the basis of obtaining miR-7 mature sequence and XIAP 3 'UTR sequence, bioinformatics analysis found miR-7 and minerals. 3 'UTR had multiple binding sites and three online software predicted that XIAP was one of the target genes of miR-7, and the dual luciferase reporter gene system confirmed that miR-7 and XIAP 3' UTR wild-type co transfected SGC-7901 cells, and the fluorescence values were significantly lower than those of the control group; miR-7 stable and high apparent SGC-7901 and HGC-27 cells XIAP mRNA and protein expression water. The average down-regulation of SGC-7901 and HGC-27 cells XIAP mRNA and protein in HGC-27 cells all up. Conclusion: 1.DADS can up regulate the proliferation of human gastric cancer cells with miR-7 inhibition. Migration and invasion and growth of transplanted tumor and induction of apoptosis and cycle arrest.2.DADS can reduce the proliferation of XIAP suppressing human gastric cancer cells, migration and invasion and xenografts. Long and induced apoptosis and cycle arrest.3.miR-7 target negatively regulate XIAP mRNA and protein expression.
【学位授予单位】：南华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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