GSPT(G1 to S phase transition)是一種翻譯終止因子,它通過結(jié)合eRF1識別終止密碼子，參與蛋白質(zhì)翻譯終止過程中肽鏈的釋放，對細胞周期有調(diào)控作用。Survivin是凋亡抑制蛋白IAP（inhibitors of apoptosis）家族的最小成員，結(jié)構(gòu)獨特，功能多樣，并在細胞分化、細胞周期G2/M以及細胞凋亡等途徑中起調(diào)控作用。miRNA是內(nèi)源性非編碼小RNA，參與細胞分化、增殖、凋亡等一系列生理活動。由于miRNA存在的普遍性、功能的多樣性，可以作為研究多功能蛋白的一種新型干擾手段。 本書利用miRNA干擾技術(shù)首次研究了eRF3a/ GSPT1對細胞增殖、遷移的調(diào)控作用，同時分析了Survivin突變體對細胞增殖、遷移的分子調(diào)控機制，又對eRF3a/ GSPT1、eRF3b/ GSPT2與Survivin的相互作用位點進行系統(tǒng)的細化分析，這為今后研究GSPT與Survivin相互作用的功能研究提供理論支持。

肖瑞琳，博士，畢業(yè)于山西大學(xué)生物化學(xué)與分子生物學(xué)專業(yè)，現(xiàn)就職于太原理工大學(xué)。一直致力于miRNA功能及其相關(guān)分子靶點相互作用的研究，運用基因編輯技術(shù)、蛋白質(zhì)組學(xué)分析等生物技術(shù)和實驗方法，深入探究 miRNA 調(diào)控分子靶點的具體機制，進一步揭示 miRNA 調(diào)控細胞功能的分子基礎(chǔ)。在該領(lǐng)域成果豐碩，已發(fā)表相關(guān) SCI 論文十余篇，主持多項省級科研項目，并參與多項國家級科研項目。

第 1 章 國內(nèi)外研究概覽································································· 1 1.1 eRF3/GSPT 蛋白 ······························································· 1 1.1.1 eRF3 的來源 ···························································· 4 1.1.2 eRF3 的結(jié)構(gòu)特征 ······················································ 7 1.1.3 eRF3a 的功能 ·························································12 1.2 Survivin 蛋白 ··································································24 1.2.1 Survivin 的來源及其家族 ···········································24 1.2.2 Survivin 的定位和結(jié)構(gòu)特征 ········································25 1.2.3 Survivin 的功能 ·······················································28 1.3 miRNAs ·········································································34 1.3.1 miRNAs 的來源 ······················································34 1.3.2 miRNAs 的形成及作用原理 ·······································39 1.3.3 miRNAs 的功能 ······················································41 1.4 本書研究目的、意義及創(chuàng)新點 ············································56 1.4.1 研究目的及意義 ······················································56 1.4.2 研究創(chuàng)新點 ····························································59 第 2 章 miR-144 通過調(diào)控 eRF3a/GSPT1 對細胞增殖、遷移的 影響研究 ········································································63 2.1 實驗材料 ·······································································65 2.1.1 實驗細胞及菌株 ······················································65 miRNA 介導(dǎo) GSPT 與 Survivin 蛋白互作及功能調(diào)控 II 2.1.2 細胞與菌株的質(zhì)量控制 ············································ 65 2.1.3 質(zhì)粒及 microRNA 模擬物 ········································· 65 2.1.4 主要的酶及試劑 ····················································· 67 2.2 實驗方法 ······································································· 68 2.2.1 細胞培養(yǎng) ······························································ 68 2.2.2 雙熒光素酶報告質(zhì)粒的構(gòu)建 ······································ 70 2.2.3 質(zhì)粒轉(zhuǎn)染 ······························································ 71 2.2.4 RNA 干擾 ····························································· 71 2.2.5 RNA 提取及實時熒光定量 qPCR ································ 72 2.2.6 miRNA 的提取及實時熒光定量 qPCR ·························· 73 2.2.7 細胞增殖活性 MTT 檢測 ·········································· 74 2.2.8 劃痕實驗 ······························································ 76 2.2.9 Western blot ··························································· 77 2.3 結(jié)果 ············································································· 78 2.3.1 GSPT1 和 miR-144 在結(jié)腸癌細胞 HCT116 細胞中的 表達水平檢測 ························································ 78 2.3.2 GSPT1 3′UTR 是 miR-144 的靶物 ······························· 79 2.3.3 miR-144 通過下調(diào) GSPT1 抑制細胞增殖 ······················ 81 2.3.4 miR-144 通過下調(diào) GSPT1 抑制細胞遷移 ······················ 83 2.4 討論 ············································································· 85 第 3 章 Survivin 70 、71 位氨基酸突變體對結(jié)腸癌細胞增殖、 遷移的分子調(diào)控 ······························································· 87 3.1 材料 ············································································· 89 3.1.1 實驗細胞株、菌株與質(zhì)粒 ········································· 89 3.1.2 試劑及工具酶 ························································ 90 3.2 方法 ············································································· 91 3.2.1 點突變 ································································· 91 3.2.2 pull-down 技術(shù) ······················································· 91 3.2.3 Survivin 突變體與 eRF3 的共定位 ······························· 92 3.3 結(jié)果 ············································································· 93 目 錄 III 3.3.1 Survivin 70、71 位氨基酸突變測序圖 ···························93 3.3.2 Survivin 70、71 位氨基酸的突變體重組蛋白的表達 ·········94 3.3.3 Survivin 70、71 位氨基酸的突變對與 eRF3a/GSPT1 相互作用的影響 ······················································95 3.3.4 Survivin 70、71 位氨基酸的突變對 Survivin/Survivin 二聚體形成的影響 ···················································96 3.3.5 Survivin 及其 70、71 位氨基酸突變體與 GSPT1 的 定位 ·····································································97 3.3.6 Survivin 70、71 位氨基酸的突變抑制了結(jié)腸癌細胞 HCT116 增殖 ··························································98 3.3.7 Survivin 70、71 位氨基酸突變抑制了結(jié)腸癌細胞 HCT116 遷移 ··························································99 3.3.8 Survivin 70、71 位氨基酸的突變促進了細胞凋亡 ·········· 100 3.3.9 Survivin 70、71 位氨基酸的突變提高了其對順鉑藥物 的敏感性 ····························································· 101 3.4 討論 ··········································································· 102 3.5 結(jié)論與展望 ·································································· 106 第 4 章 人類截短型 GSPT1 蛋白與生存素 Survivin 在體內(nèi) 相互作用及區(qū)域分析 ························································ 111 4.1 材料 ··········································································· 112 4.1.1 菌株與質(zhì)粒 ·························································· 112 4.1.2 寡聚核苷酸 ·························································· 112 4.1.3 工具酶及生化試劑 ················································· 113 4.1.4 主要試劑的配制 ···················································· 113 4.2 方法 ··········································································· 114 4.2.1 eRF3 截短體的酵母雙雜交重組質(zhì)粒構(gòu)建 ···················· 114 4.2.2 eRF3 截短體酵母雙雜交重組質(zhì)粒的共轉(zhuǎn)化 ················· 114 4.2.3 β-半乳糖苷酶活性分析 ··········································· 115 4.2.4 陽性克隆的嚴(yán)謹型篩選 ··········································· 118 4.2.5 熒光共定位 ·························································· 120 miRNA 介導(dǎo) GSPT 與 Survivin 蛋白互作及功能調(diào)控 IV 4.3 結(jié)果 ············································································ 123 4.3.1 eRF3 N 末端區(qū)域截短設(shè)計 ······································· 123 4.3.2 截短體構(gòu)建方案 ···················································· 123 4.3.3 實驗設(shè)計原理 ······················································· 125 4.3.4 截短體功能預(yù)測 ···················································· 125 4.3.5 截短型 eRF3a/GSPT1、eRF3b/GSPT2 酵母雙雜交重 組質(zhì)粒的構(gòu)建和鑒定 ·············································· 125 4.3.6 β-半乳糖苷酶活性分析 ············································ 129 4.3.7 eRF3 與 Survivin 共定位 ·········································· 130 4.4 討論 ············································································ 133 第 5 章 人類肽鏈釋放因子 eRF3 與生存素 Survivin 在體外 相互作用及區(qū)域分析 ························································· 136 5.1 材料 ············································································ 136 5.1.1 菌株與質(zhì)粒 ·························································· 138 5.1.2 寡聚核苷酸 ·························································· 139 5.1.3 主要酶及生化試劑 ················································· 139 5.1.4 主要試劑的配制 ···················································· 140 5.2 方法 ············································································ 141 5.2.1 原核重組表達質(zhì)粒的構(gòu)建 ········································ 141 5.2.2 eRF3a 截短體原核表達重組質(zhì)粒的轉(zhuǎn)化與量誘導(dǎo)表達 ···· 143 5.2.3 重組蛋白的大量表達 ·············································· 145 5.2.4 pull-down 分析 ······················································ 148 5.2.5 Western blot 分析 ··················································· 150 5.3 結(jié)果 ············································································ 150 5.3.1 eRF3 截短體原核表達重組質(zhì)粒的構(gòu)建 ························ 150 5.3.2 eRF3 不同截短體蛋白的原核表達 ······························ 152 5.3.3 體外相互作用的 pull-down 分析 ································ 153 5.4 討論 ············································································ 156 5.4.1 相互作用研究的系統(tǒng)驗證 ········································ 156 5.4.2 細胞周期調(diào)控的協(xié)同作用假說 ·································· 156 目 錄 V 5.4.3 待解決的關(guān)鍵科學(xué)問題 ··········································· 157 5.4.4 未來研究方向 ······················································· 157 5.5 總結(jié)與展望 ·································································· 158 5.5.1 總結(jié) ··································································· 158 5.5.2 展望 ··································································· 160