PARP1在β-拉帕醌杀伤胃癌细胞中的作用及机制

发布时间：2018-07-16 16:02

【摘要】：目的探讨PARP1在β-拉帕醌杀伤人AGS胃癌细胞珠中的作用及分子机制。方法1.选择人AGS胃癌细胞株,设计并合成3个针对靶基因PARP1的siRNA寡核苷酸基因片段,以及阴性对照组NC-siRNA,采用常规瞬时转染的方法干扰PARP-1 表达。2.转染后分别对4组加入4μmol/L的β-拉帕醌,采用Western blot实验检测四组PARP1裂解产物蛋白的表达变化,采用方差分析进行比较,筛选出稳定的转染细胞株;3.选取已筛选出的siRNA重新转染AGS细胞珠,同时设置阴性对照组NC-siRNA组;采用MTT实验及平板克隆实验分别检测目的基因转染组(PARP1-siRNA转染组)、NC-siRNA对照组中β-拉帕醌对AGS细胞珠增殖及克隆能力的影响;用划痕实验检测PARP1-siRNA转染组、NC-siRNA对照组中β-拉帕醌对AGS细胞珠迁移能力的影响。结果1.AGS胃癌细胞珠转染后的转染效率:在显微镜下观察转染后48h的细胞形态,与阴性对照组相比,PARP1基因经siRNA抑制表达后在形态上未见明显改变,贴壁生长,呈圆形,可见PARP1基因沉默对细胞的形态生长无显著的影响。Western blot检测转染(已加入4μmol/Lβ-拉帕醌)'后72h目的基因PARP1裂解产物蛋白的表达:与NC-siRNA对照组相比,siRNA组PARP1的裂解产蛋白的表达显著降低,PARP1-siRNA 1 下降了 89%(P0.01),PARP1-siRNA 2 下降了 73%(P0.05),PARP1-siRNA3下降了 83%(P0.05),与另外三组相比,转染后蛋白表达水平较低的是PARP1-siRNA 1组,说明PARP1-siRNA 1是最有效和最特异的序列,可以用于干扰沉默PARP1基因的表达,并用于后续实验。2.PARP1-siRNA转染对AGS细胞珠增殖、克隆及迁移能力的影响:MTT、平板克隆实验、划痕实验可以观察到β-拉帕醌可显著抑制NC-siRNA对照组组细胞的增殖、克隆及迁移能力,而对PARP1-siRNA转染组的抑制效果明显减弱;siRNA沉默PARP1基因表达后可显著降低β-拉帕醌对胃癌细胞的杀伤力。结论1.使用RNA干扰技术可以成功抑制AGS胃癌细胞珠中PARP 1基因的表达。2.过度活化的PARP1在β-拉帕醌杀伤胃癌细胞中发挥着关键作用。
[Abstract]:Objective to investigate the role of PARP1 in killing human AGS gastric cancer cells and its molecular mechanism. Method 1. Three siRNA oligonucleotide fragments targeting the target gene PARP1 and NC-siRNAs of negative control group were designed and synthesized from human AGS gastric cancer cell lines. The expression of PARP-1 was interfered with by conventional transient transfection. After transfection, 4 渭 mol / L 尾 -lappa quinone was added to the four groups. The protein expression of the four groups of PARP1 cleavage products was detected by Western blot assay, and the stable transfected cell line was screened by variance analysis. The selected siRNA was retransfected into AGS cells, and the negative control group was set up in NC-siRNA group. MTT assay and plate cloning assay were used to detect the effects of 尾 -lapraquinone on the proliferation and cloning ability of AGS cells in the target gene transfected group (PARP1-siRNA transfected group). The effect of 尾 -lapraquinone on the migration of AGS cells was detected by scratch test in the control group of PARP1-siRNA transfection group. Results 1. Transfection efficiency after transfection of AGS gastric cancer cell beads: the morphology of the cells was observed under microscope at 48h after transfection. Compared with the negative control group, the expression of PARP1 gene did not change significantly after siRNA inhibition, and the cells grew round and adherent to the wall. 2. It can be seen that paRP1 gene silencing has no significant effect on cell morphological growth. Western blot was used to detect the protein expression of the target gene PARP1 cleavage product 72 h after transfection (4 渭 mol / L 尾 -lapraquinone): compared with NC-siRNA control group, the cleavage of PARP1 produced protein in siRNA group. The expression of PARP1-siRNA1 decreased by 89% (P0.01) and PARP1-siRNA2 decreased by 73% (P0.05), and the expression of PARP1-siRNA3 decreased by 83% (P0.05), compared with the other three groups. The protein expression level of PARP1-siRNA1 group was lower after transfection, indicating that PARP1-siRNA1 was the most effective and specific sequence, which could be used to interfere with the silencing of PARP1 gene expression, and to further experiment .2.PARP1-siRNA transfection on the proliferation of AGS cells. The effects of cloning and migration ability on cell proliferation, cloning and migration of NC-siRNA control group were observed by using 尾 -lapraquinone as control group, and the proliferation, cloning and migration of NC-siRNA control group were significantly inhibited by 尾 -lapraquinone. The inhibitory effect of PARP1-siRNA transfection group on PARP1 gene expression was significantly decreased after silencing PARP1 gene expression by siRNA. The inhibitory effect of 尾 -lapraquinone on gastric cancer cells was significantly decreased. Conclusion 1. The expression of PARP1 gene in AGS gastric cancer cells was successfully inhibited by RNA interference. Overexpression of PARP1 plays a key role in killing gastric cancer cells by 尾-lapanone.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

【参考文献】

相关期刊论文 前10条

1 高羽亭;杜进林;杨慧;唐焕文;;沉默PARP-1对氢醌所致大鼠骨髓间充质干细胞凋亡影响[J];中国职业医学;2017年01期

2 刘悦;焦冰洋;于楠楠;王闯;韩英浩;金成浩;孙虎男;;β-拉帕醌通过下调过氧化物酶V诱导SW480结肠癌细胞凋亡[J];中国细胞生物学学报;2016年12期

3 徐敏;邱雷;郭连峰;赵宁;张宪军;张丽娟;;AIF对人宫颈癌Hela细胞体外生物学活性的影响及机制研究[J];重庆医学;2016年32期

4 侯立春;林黎娟;;NQO1高表达提示宫颈癌预后不良[J];实用肿瘤学杂志;2016年04期

5 潘理会;张平;李春辉;;PI3K/AKT/mTOR信号传导通路在胃癌中的研究进展[J];承德医学院学报;2016年04期

6 陈利俊;马丽;李莉萍;;聚腺苷二磷酸-核糖聚合酶1与DNA双链断裂修复的相关性[J];中国生物化学与分子生物学报;2016年06期

7 宋仕茂;陈宇;张治国;骆志国;;胃癌细胞SGC7901凋亡的PI3K/Akt通路介导作用及雷帕霉素的作用机制分析[J];癌症进展;2016年06期

8 胡中慧;马慧萍;李加恒;贾正平;;线粒体自噬在缺氧条件下适应机制的研究进展[J];现代生物医学进展;2016年08期

9 陈磊;;胃癌患者术前预后营养指数与辅助化疗效果及预后的关系[J];中国医学前沿杂志(电子版);2016年02期

10 王月霞;陈敏;;自噬性细胞死亡及其与细胞凋亡、坏死关系研究进展[J];中国公共卫生;2016年10期

相关会议论文 前3条

1 代淑阳;郭志敏;蔡梦;李薇;赵晓航;;RIP3激酶活性非依赖的细胞DNA损伤修复功能[A];全国肿瘤流行病学和肿瘤病因学学术会议论文集[C];2015年

2 刘慧颖;程学芳;李清然;王洪;王广基;郝海平;;NQO1靶向药物调控NAD+合成代谢及诱导细胞凋亡作用机理研究[A];2014年创新药物成药性评价高层学术论坛报告汇编[C];2014年

3 蒲咏梅;张东才;;细胞凋亡过程中的钙信号研究[A];首届粤港生物物理学术研讨会论文集[C];1999年

相关博士学位论文 前4条

1 王凌波;聚二磷酸腺苷核糖聚合酶1在急性髓系白血病中的作用及机制研究[D];山东大学;2016年

2 郑林杰;JNK在氧化应激导致胶质瘤细胞Parthanatos中的作用及机制研究[D];吉林大学;2016年

3 周宪春;β-拉帕醌通过EMT抑制乳腺癌侵袭转移的机制研究[D];延边大学;2016年

4 秦云植;NQO1及其抑制剂在胃癌预后评估及靶向治疗中的作用研究[D];延边大学;2015年

相关硕士学位论文 前4条

1 樊彩芳;PARP-1/AIF通路介导戊二酸尿症Ⅰ型大鼠大脑皮质损伤的机制研究[D];郑州大学;2015年

2 杨洋;醌氧化还原酶及其抑制剂在乳腺癌增殖与迁移中的作用研究[D];延边大学;2015年

3 姚圆圆;PARP-1抑制剂PJ34对人肺腺癌顺铂耐药细胞A549/DDP生长活性及耐药性的影响[D];安徽医科大学;2015年

4 金丹;宫颈鳞状细胞癌中NQO1蛋白过表达的临床病理学意义[D];延边大学;2014年

，

本文编号：2126900