miRNA介导GSPT与Survivin蛋白互作及功能调控_肖瑞琳_9787524500858

GSPT(G1 to S phase transition)是一种翻译终止因子,它通过结合eRF1识别终止密码子，参与蛋白质翻译终止过程中肽链的释放，对细胞周期有调控作用。Survivin是凋亡抑制蛋白IAP（inhibitors of apoptosis）家族的最小成员，结构独特，功能多样，并在细胞分化、细胞周期G2/M以及细胞凋亡等途径中起调控作用。miRNA是内源性非编码小RNA，参与细胞分化、增殖、凋亡等一系列生理活动。由于miRNA存在的普遍性、功能的多样性，可以作为研究多功能蛋白的一种新型干扰手段。本书利用miRNA干扰技术首次研究了eRF3a/ GSPT1对细胞增殖、迁移的调控作用，同时分析了Survivin突变体对细胞增殖、迁移的分子调控机制，又对eRF3a/ GSPT1、eRF3b/ GSPT2与Survivin的相互作用位点进行系统的细化分析，这为今后研究GSPT与Survivin相互作用的功能研究提供理论支持。

第 1 章国内外研究概览································································· 1 1.1 eRF3/GSPT 蛋白 ······························································· 1 1.1.1 eRF3 的来源 ···························································· 4 1.1.2 eRF3 的结构特征 ······················································ 7 1.1.3 eRF3a 的功能 ·························································12 1.2 Survivin 蛋白 ··································································24 1.2.1 Survivin 的来源及其家族 ···········································24 1.2.2 Survivin 的定位和结构特征 ········································25 1.2.3 Survivin 的功能 ·······················································28 1.3 miRNAs ·········································································34 1.3.1 miRNAs 的来源 ······················································34 1.3.2 miRNAs 的形成及作用原理 ·······································39 1.3.3 miRNAs 的功能 ······················································41 1.4 本书研究目的、意义及创新点 ············································56 1.4.1 研究目的及意义 ······················································56 1.4.2 研究创新点 ····························································59 第 2 章 miR-144 通过调控 eRF3a/GSPT1 对细胞增殖、迁移的影响研究 ········································································63 2.1 实验材料 ·······································································65 2.1.1 实验细胞及菌株 ······················································65 miRNA 介导 GSPT 与 Survivin 蛋白互作及功能调控 II 2.1.2 细胞与菌株的质量控制 ············································ 65 2.1.3 质粒及 microRNA 模拟物 ········································· 65 2.1.4 主要的酶及试剂 ····················································· 67 2.2 实验方法 ······································································· 68 2.2.1 细胞培养 ······························································ 68 2.2.2 双荧光素酶报告质粒的构建 ······································ 70 2.2.3 质粒转染 ······························································ 71 2.2.4 RNA 干扰 ····························································· 71 2.2.5 RNA 提取及实时荧光定量 qPCR ································ 72 2.2.6 miRNA 的提取及实时荧光定量 qPCR ·························· 73 2.2.7 细胞增殖活性 MTT 检测 ·········································· 74 2.2.8 划痕实验 ······························································ 76 2.2.9 Western blot ··························································· 77 2.3 结果 ············································································· 78 2.3.1 GSPT1 和 miR-144 在结肠癌细胞 HCT116 细胞中的表达水平检测 ························································ 78 2.3.2 GSPT1 3′UTR 是 miR-144 的靶物 ······························· 79 2.3.3 miR-144 通过下调 GSPT1 抑制细胞增殖 ······················ 81 2.3.4 miR-144 通过下调 GSPT1 抑制细胞迁移 ······················ 83 2.4 讨论 ············································································· 85 第 3 章 Survivin 70 、71 位氨基酸突变体对结肠癌细胞增殖、迁移的分子调控 ······························································· 87 3.1 材料 ············································································· 89 3.1.1 实验细胞株、菌株与质粒 ········································· 89 3.1.2 试剂及工具酶 ························································ 90 3.2 方法 ············································································· 91 3.2.1 点突变 ································································· 91 3.2.2 pull-down 技术 ······················································· 91 3.2.3 Survivin 突变体与 eRF3 的共定位 ······························· 92 3.3 结果 ············································································· 93 目录 III 3.3.1 Survivin 70、71 位氨基酸突变测序图 ···························93 3.3.2 Survivin 70、71 位氨基酸的突变体重组蛋白的表达 ·········94 3.3.3 Survivin 70、71 位氨基酸的突变对与 eRF3a/GSPT1 相互作用的影响 ······················································95 3.3.4 Survivin 70、71 位氨基酸的突变对 Survivin/Survivin 二聚体形成的影响 ···················································96 3.3.5 Survivin 及其 70、71 位氨基酸突变体与 GSPT1 的定位 ·····································································97 3.3.6 Survivin 70、71 位氨基酸的突变抑制了结肠癌细胞 HCT116 增殖 ··························································98 3.3.7 Survivin 70、71 位氨基酸突变抑制了结肠癌细胞 HCT116 迁移 ··························································99 3.3.8 Survivin 70、71 位氨基酸的突变促进了细胞凋亡 ·········· 100 3.3.9 Survivin 70、71 位氨基酸的突变提高了其对顺铂药物的敏感性 ····························································· 101 3.4 讨论 ··········································································· 102 3.5 结论与展望 ·································································· 106 第 4 章人类截短型 GSPT1 蛋白与生存素 Survivin 在体内相互作用及区域分析 ························································ 111 4.1 材料 ··········································································· 112 4.1.1 菌株与质粒 ·························································· 112 4.1.2 寡聚核苷酸 ·························································· 112 4.1.3 工具酶及生化试剂 ················································· 113 4.1.4 主要试剂的配制 ···················································· 113 4.2 方法 ··········································································· 114 4.2.1 eRF3 截短体的酵母双杂交重组质粒构建 ···················· 114 4.2.2 eRF3 截短体酵母双杂交重组质粒的共转化 ················· 114 4.2.3 β-半乳糖苷酶活性分析 ··········································· 115 4.2.4 阳性克隆的严谨型筛选 ··········································· 118 4.2.5 荧光共定位 ·························································· 120 miRNA 介导 GSPT 与 Survivin 蛋白互作及功能调控 IV 4.3 结果 ············································································ 123 4.3.1 eRF3 N 末端区域截短设计 ······································· 123 4.3.2 截短体构建方案 ···················································· 123 4.3.3 实验设计原理 ······················································· 125 4.3.4 截短体功能预测 ···················································· 125 4.3.5 截短型 eRF3a/GSPT1、eRF3b/GSPT2 酵母双杂交重组质粒的构建和鉴定 ·············································· 125 4.3.6 β-半乳糖苷酶活性分析 ············································ 129 4.3.7 eRF3 与 Survivin 共定位 ·········································· 130 4.4 讨论 ············································································ 133 第 5 章人类肽链释放因子 eRF3 与生存素 Survivin 在体外相互作用及区域分析 ························································· 136 5.1 材料 ············································································ 136 5.1.1 菌株与质粒 ·························································· 138 5.1.2 寡聚核苷酸 ·························································· 139 5.1.3 主要酶及生化试剂 ················································· 139 5.1.4 主要试剂的配制 ···················································· 140 5.2 方法 ············································································ 141 5.2.1 原核重组表达质粒的构建 ········································ 141 5.2.2 eRF3a 截短体原核表达重组质粒的转化与量诱导表达 ···· 143 5.2.3 重组蛋白的大量表达 ·············································· 145 5.2.4 pull-down 分析 ······················································ 148 5.2.5 Western blot 分析 ··················································· 150 5.3 结果 ············································································ 150 5.3.1 eRF3 截短体原核表达重组质粒的构建 ························ 150 5.3.2 eRF3 不同截短体蛋白的原核表达 ······························ 152 5.3.3 体外相互作用的 pull-down 分析 ································ 153 5.4 讨论 ············································································ 156 5.4.1 相互作用研究的系统验证 ········································ 156 5.4.2 细胞周期调控的协同作用假说 ·································· 156 目录 V 5.4.3 待解决的关键科学问题 ··········································· 157 5.4.4 未来研究方向 ······················································· 157 5.5 总结与展望 ·································································· 158 5.5.1 总结 ··································································· 158 5.5.2 展望 ··································································· 160

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