本文關(guān)鍵詞：miR-9對(duì)鼻咽癌增殖、腫瘤干細(xì)胞“干性”、EMT和轉(zhuǎn)移的調(diào)控作用及機(jī)制，，由筆耕文化傳播整理發(fā)布。

        背景和目的鼻咽癌是一種發(fā)生于鼻咽粘膜的惡性腫瘤,其惡性程度較高,且具有極強(qiáng)的轉(zhuǎn)移能力。我國(guó)是鼻咽癌發(fā)病率最高的國(guó)家,以廣東、廣西、湖南、福建等南方地區(qū)為著。早在80年代初,姚開(kāi)泰就提出了鼻咽癌發(fā)病的三擊／多步假說(shuō),認(rèn)為EB病毒（EBV）、化學(xué)致癌物和胚性擊中（即遺傳因素或自發(fā)突變）是構(gòu)成NPC的主要病因,不僅為一系列后續(xù)研究提供了理論基礎(chǔ),而且對(duì)鼻咽癌的防治具有重要的實(shí)踐指導(dǎo)意義。生活方式已被證實(shí)和鼻咽癌的發(fā)病有關(guān),尤其吸煙不僅是個(gè)體鼻咽癌發(fā)病的危險(xiǎn)因素,也與EB病毒血清陽(yáng)性的健康男性的發(fā)病相關(guān),吸煙能誘導(dǎo)EB病毒的活動(dòng)。因此,鼻咽癌的發(fā)生是多因素、多基因、多階段、多途徑的。鼻咽癌治療方法包括放射治療、外科手術(shù)治療、化學(xué)藥物治療、免疫和生物治療,基因靶向治療也為鼻咽癌的治療提供新的前景。2011年Weinberg指出腫瘤具有自給自足的生長(zhǎng)信號(hào)、抗生長(zhǎng)信號(hào)的不敏感、抵抗細(xì)胞死亡、潛力無(wú)限的復(fù)制能力、持續(xù)的血管生成、組織浸潤(rùn)和轉(zhuǎn)移、避免免疫摧毀、促進(jìn)腫瘤的炎癥、細(xì)胞能量異常、基因組不穩(wěn)定和突變十種特征,并綜述了針對(duì)以上特征的靶向治療方法。大多數(shù)實(shí)體瘤的腫瘤微環(huán)境中存在各種類(lèi)型的細(xì)胞,如腫瘤細(xì)胞、炎癥細(xì)胞、腫瘤干細(xì)胞等。腫瘤干細(xì)胞（CSCs）是指可以自我更新和分化成為不同表型的腫瘤細(xì)胞。腫瘤干細(xì)胞假說(shuō)認(rèn)為,腫瘤細(xì)胞中有一小部分細(xì)胞,導(dǎo)致腫瘤發(fā)生和生長(zhǎng),具有無(wú)限的自我更新和復(fù)制能力,并對(duì)放療和化療產(chǎn)生抵抗。不同腫瘤的腫瘤干細(xì)胞表面標(biāo)記物不同�；贑SCs的特性,有效殺滅清除CSCs被認(rèn)為是根治腫瘤的有效途徑之一。目前針對(duì)CSCs的靶向治療主要有靶向抑制其表面標(biāo)志物,調(diào)節(jié)CSCs通路,誘導(dǎo)CSCs向腫瘤細(xì)胞分化,細(xì)胞治療（如誘導(dǎo)生成y8T cells殺傷CSC）等。轉(zhuǎn)移是惡性腫瘤最基本的生物學(xué)特征,腫瘤的轉(zhuǎn)移是影響患者生存期的首要因素,已成為腫瘤研究領(lǐng)域的熱點(diǎn)和難點(diǎn),亦是迫切要解決的重大問(wèn)題之一。腫瘤轉(zhuǎn)移最突出的特征是不同的腫瘤能在人體內(nèi)相同或不同的部位形成轉(zhuǎn)移,即腫瘤轉(zhuǎn)移的組織特異性。上皮-間充質(zhì)轉(zhuǎn)化（Epithelial-mesenchymal transition, EMT）是指上皮細(xì)胞通過(guò)特定程序轉(zhuǎn)化為具有間充質(zhì)表型細(xì)胞的生物學(xué)過(guò)程,是上皮細(xì)胞來(lái)源的惡性腫瘤細(xì)胞獲得遷移和侵襲能力的重要生物學(xué)過(guò)程,是腫瘤轉(zhuǎn)移的重要階段。miRNAs是一種小的內(nèi)源性非編碼RNA分子,大約由21—25個(gè)核苷酸組成,在動(dòng)物和植物,它們是重要的調(diào)節(jié)分子,每個(gè)miRNA可以有多個(gè)靶基因,而幾個(gè)miRNAs也可以調(diào)節(jié)同一個(gè)基因,它們通過(guò)與其靶基因的相互作用來(lái)調(diào)節(jié)基因表達(dá),進(jìn)而調(diào)控生物體的生長(zhǎng)發(fā)育。最近的研究表明,一些miRNA調(diào)節(jié)細(xì)胞增殖和凋亡的過(guò)程,在腫瘤的形成中是很重要的,一些miRNAs可作為癌基因或抑癌功能,miRNA表達(dá)譜可能成為腫瘤診斷的有用的生物標(biāo)志物,而miRNAs可能是一個(gè)功能強(qiáng)大的工具,用于腫瘤的預(yù)防和治療。研究表明,miR-9在多種人體腫瘤組織中異常表達(dá),如miR-9在乳腺癌、胃癌、卵巢癌等中表達(dá)下調(diào),但在神經(jīng)系統(tǒng)腫瘤中表達(dá)上調(diào)。miR-9在腫瘤細(xì)胞增殖、凋亡、EMT、侵襲、轉(zhuǎn)移等方面發(fā)揮重要作用。有研究人員的基因芯片結(jié)果顯示,miR-9在人NPC組織標(biāo)本上表達(dá)下調(diào),但功能不詳。我們的預(yù)實(shí)驗(yàn)結(jié)果顯示,miR-9在人NPC細(xì)胞株中表達(dá)下調(diào),但在NPC組織標(biāo)本上表達(dá)卻上調(diào)：同時(shí),miR-9過(guò)表達(dá)抑制NPC細(xì)胞體內(nèi)外增殖,而miR-9過(guò)表達(dá)卻促進(jìn)NPC細(xì)胞發(fā)生EMT和遷移。以上這些似乎自相矛盾的實(shí)驗(yàn)結(jié)果促使我們?nèi)ド钊虢馕鰉iR-9在人NPC發(fā)病中的作用及其機(jī)制。方法第一章miR-9靶向調(diào)控CCNG1抑制鼻咽癌細(xì)胞的增殖1.質(zhì)粒提取試劑盒提取質(zhì)粒后,NaAC、乙醇沉淀法純化質(zhì)粒。2.穩(wěn)定細(xì)胞株建立包裝載體PMD2G和穿梭載體PPAX2與目的質(zhì)粒lipotemin2000共轉(zhuǎn)染至293FT細(xì)胞,得到的病毒上清以濃度梯度方式感染細(xì)胞,嘌呤霉素2μg/ml （GIBCO）作用約2周殺死未感染細(xì)胞,或者流式細(xì)胞儀分選出成功感染的細(xì)胞。3. TRIzol--氯仿抽提法提取RNA,按照Bio-Rad逆轉(zhuǎn)錄試劑盒操作說(shuō)明書(shū),采用兩步法逆轉(zhuǎn)錄,實(shí)時(shí)熒光定量PCR（Realtime qPCR）檢測(cè)miRNA和基因mRNA水平的表達(dá)。Western blot檢測(cè)基因蛋白水平表達(dá)。免疫組化檢測(cè)組織中基因表達(dá)位置和含量。4.CCK-8法繪制細(xì)胞生長(zhǎng)曲線(xiàn),平板克隆實(shí)驗(yàn)觀(guān)測(cè)細(xì)胞增殖情況。5.流式細(xì)胞儀細(xì)胞周期檢測(cè)。6.熒光素酶活性檢測(cè)：螢火蟲(chóng)熒光素酶測(cè)定得到的RLU值除以Renilla熒光素酶測(cè)定得到的RLU值驗(yàn)證3’-UTR靶向結(jié)合位點(diǎn)活性。7.皮下成瘤觀(guān)測(cè)細(xì)胞在體內(nèi)的成瘤和增殖情況,肝包膜異位成瘤觀(guān)測(cè)細(xì)胞的成瘤和轉(zhuǎn)移情況。8.統(tǒng)計(jì)學(xué)分析采用SPSS13.0統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)分析。熒光定量PCR各細(xì)胞株2-△△Ct比較采用單因素方差分析（One-way ANONA）;轉(zhuǎn)染后miR-9表達(dá)、細(xì)胞周期、平板克隆的兩組間比較及免疫組化結(jié)果采用兩獨(dú)立樣本t檢驗(yàn)；CCK-8生長(zhǎng)曲線(xiàn)及動(dòng)物實(shí)驗(yàn)中皮下成瘤體積比較采用析因設(shè)計(jì)的方差分析。P<0.05為有統(tǒng)計(jì)學(xué)差異,數(shù)值大小以均值±標(biāo)準(zhǔn)差（X±S）來(lái)表示。第二章miR-9靶向調(diào)控Hesl在鼻咽癌腫瘤干細(xì)胞干性維持中作用及機(jī)制1. TRIzol-氯仿抽提法提取RNA,按照Bio-Rad逆轉(zhuǎn)錄試劑盒操作說(shuō)明書(shū),采用兩步法逆轉(zhuǎn)錄,實(shí)時(shí)熒光定量PCR（Realtime qPCR）檢測(cè)miRNA和基因mRNA水平的表達(dá)。2. Western blot檢測(cè)基因蛋白水平表達(dá)。3.細(xì)胞免疫熒光觀(guān)察基因在細(xì)胞中表達(dá)的定位和表達(dá)水平。4.免疫組化檢測(cè)組織中基因表達(dá)位置和含量。5.腫瘤球培養(yǎng)、SP細(xì)胞比例檢測(cè)觀(guān)測(cè)腫瘤細(xì)胞的“干性”。6.熒光素酶活性檢測(cè)：螢火蟲(chóng)熒光素酶測(cè)定得到的RLU值除以Renilla熒光素酶測(cè)定得到的RLU值驗(yàn)證3’-UTR靶向結(jié)合位點(diǎn)活性。7.miR-9的治療作用：皮下成瘤后,miR-9mimics通過(guò)轉(zhuǎn)染試劑注射至瘤體內(nèi),觀(guān)察瘤體生長(zhǎng)情況。8.統(tǒng)計(jì)學(xué)分析采用SPSS13.0統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)分析。腫瘤球計(jì)數(shù)及免疫組化結(jié)果采用兩獨(dú)立樣本t檢驗(yàn)。P<0.05為有統(tǒng)計(jì)學(xué)差異。第三章c-Myc通過(guò)miR-9靶向調(diào)控Klf4促進(jìn)鼻咽癌細(xì)胞EMT、侵襲和轉(zhuǎn)移的作用1.質(zhì)粒擴(kuò)增提取和純化、穩(wěn)定細(xì)胞株建立、熒光素酶活性檢測(cè)、Western blotting、免疫組化、細(xì)胞免疫熒光見(jiàn)第一、第二章。2. Transwell遷移試驗(yàn)、Boyden侵襲試驗(yàn)和劃痕試驗(yàn)檢測(cè)細(xì)胞的轉(zhuǎn)移能力。3.熒光標(biāo)記鬼筆環(huán)肽染色試驗(yàn)和激光共聚焦觀(guān)察細(xì)胞骨架變化。4.掃描電鏡觀(guān)察細(xì)胞形態(tài)。5.細(xì)胞粘附試驗(yàn)和倒置顯微鏡觀(guān)測(cè)細(xì)胞粘附能力。6.肝包膜下移植后觀(guān)測(cè)移植位點(diǎn)的成瘤情況及遠(yuǎn)處轉(zhuǎn)移情況。7.統(tǒng)計(jì)學(xué)分析：采用SPSS13.0統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)分析。熒光定量PCR2-ΔΔCt兩兩比較比較采用兩獨(dú)立樣本t檢驗(yàn)；Transwell和Boyden小室結(jié)果多組間比較采用單因素方差分析（One-way ANONA）,兩組間比較采用兩獨(dú)立樣本t檢驗(yàn)；細(xì)胞粘附能力比較采用析因設(shè)計(jì)的方差分析。P<0.05為有統(tǒng)計(jì)學(xué)差異,數(shù)值大小以均值±標(biāo)準(zhǔn)差（X±S）來(lái)表示。第四章miR-9調(diào)控鼻咽癌細(xì)胞中免疫和炎癥相關(guān)基因的表達(dá)1.人類(lèi)基因U133Plus2.0陣列分析基因表達(dá)譜。2.RNA提取、逆轉(zhuǎn)、實(shí)時(shí)熒光定量驗(yàn)證芯片結(jié)果。3.統(tǒng)計(jì)學(xué)分析采用SPSS13.0統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)分析。熒光定量PCR各細(xì)胞株2-△△Ct比較采用兩獨(dú)立樣本t檢驗(yàn)。P<0.05為有統(tǒng)計(jì)學(xué)差異,數(shù)值大小以均值±標(biāo)準(zhǔn)差（X±S）來(lái)表示。結(jié)果第一章miR-9靶向調(diào)控CCNG1抑制鼻咽癌細(xì)胞的增殖1.miR-9在鼻咽癌細(xì)胞株中的表達(dá)我們采用實(shí)時(shí)熒光定量PCR （Real-time qPCR）檢測(cè)了miR-9在NP-69、6-10B、5-8F、CNE2、HNE1、HONE1、SUNE1中的表達(dá)水平。結(jié)果顯示：miR-9在鼻咽癌細(xì)胞株上表達(dá)較NP-69明顯下降（F=370.010,P=0.000）。2.構(gòu)建穩(wěn)定過(guò)表達(dá)miR-9細(xì)胞株將三質(zhì)粒包裝系統(tǒng)（LV-con或LV-miR-9, PMD2G和PPAX2）共轉(zhuǎn)染至293T細(xì)胞,產(chǎn)生兩種病毒,即空白對(duì)照（LV-con）病毒和過(guò)表達(dá)miR-9（LV-miR-9）的病毒。轉(zhuǎn)染72h后,收病毒上清感染細(xì)胞CNE2、HONE1、SUNE1,48h后觀(guān)察綠色熒光,嘌呤霉素殺死未感染的細(xì)胞后,用qRT-PCR檢測(cè)miR-9表達(dá)情況,結(jié)果示各種細(xì)胞較對(duì)照細(xì)胞miR-9表達(dá)均顯著升高（t=7.355、5.451、5.794,P=0.018、0.032、0.029）。3. qRT-PCR檢測(cè)鼻咽癌細(xì)胞株瞬時(shí)轉(zhuǎn)染miRNAs后miR-9的表達(dá)采用脂質(zhì)體法將miR-9mimics和inhibitor及其對(duì)照mimics con和inhibitor con轉(zhuǎn)染鼻咽癌細(xì)胞株。轉(zhuǎn)染48h后,提取RNA,逆轉(zhuǎn)錄后qRT-PCR檢測(cè)miR-9的表達(dá)。結(jié)果示,miR-9mimics成功使CNE2、HONE1、SUNE1中miR-9過(guò)表達(dá)（t=7.619、11.224、5.228, P=0.017、0.000、0.035）而inhibitor降低CNE2和5-8F中miR-9的表達(dá)水平（t=10.924、14.95,P=0.000、0.004）。4.miR-9抑制鼻咽癌細(xì)胞增殖4.1CCK-8和平板克隆形成實(shí)驗(yàn)表明miR-9抑制鼻咽癌細(xì)胞增殖我們采用CCK-8法檢測(cè)細(xì)胞生長(zhǎng)曲線(xiàn),發(fā)現(xiàn)CNE2細(xì)胞過(guò)表達(dá)miR-9后細(xì)胞生長(zhǎng)變慢,而抑制miR-9后細(xì)胞生長(zhǎng)加快（P=0.000）。平板克隆形成實(shí)驗(yàn)示,miR-9可顯著減少CNE2、HONE1、SUNE1細(xì)胞克隆形成（t=7.721、2.744、16.545,P=0.000、0.034、0.000）。以上結(jié)果表明,miR-9過(guò)表達(dá)可抑制鼻咽癌細(xì)胞增殖。4.2miR-9影響鼻咽癌細(xì)胞的周期分布為研究miR-9抑制增殖的機(jī)制,我們進(jìn)一步檢測(cè)了miR-9對(duì)鼻咽癌細(xì)胞周期的影響。結(jié)果顯示,miR-9過(guò)表達(dá)后,CNE2和HONE1細(xì)胞呈現(xiàn)G0-G1期阻滯,而miR-9抑制后,G0-G1期的細(xì)胞較對(duì)照組顯著減少。5.miR-9過(guò)表達(dá)抑制CNE2細(xì)胞的體內(nèi)成瘤能力為研究miR-9對(duì)鼻咽癌細(xì)胞在體內(nèi)增殖的影響,我們首先將過(guò)表達(dá)miR-9的CNE2細(xì)胞及其對(duì)照細(xì)胞接種至4只裸鼠背部皮下,細(xì)胞數(shù)為1×106個(gè),接種后第34天取材。結(jié)果對(duì)照組均成瘤,而miR-9過(guò)表達(dá)組均未成瘤。接著,我們將過(guò)表達(dá)miR-9的CNE2細(xì)胞及其對(duì)照細(xì)胞的皮下接種細(xì)胞數(shù)提高至1.5x106個(gè),接種后第21天取材。結(jié)果示,對(duì)照組均成瘤,而miR-9過(guò)表達(dá)組僅一只成瘤。此外,我們進(jìn)行了肝包膜下CNE2細(xì)胞異位移植成瘤,實(shí)驗(yàn)組與對(duì)照組移植細(xì)胞數(shù)均為1.2x106個(gè),接種第23天取材,發(fā)現(xiàn)miR-9過(guò)表達(dá)組在肝包膜下均未成瘤,而對(duì)照組全部成瘤。6.miR-9過(guò)表達(dá)抑制SUNE1細(xì)胞裸鼠皮下成瘤能力如上所述,miR-9過(guò)表達(dá)的CNE2細(xì)胞皮下成瘤能力及肝包膜下異位移植能力均顯著下降。接下來(lái)我們研究了miR-9過(guò)表達(dá)的SUNE1細(xì)胞的皮下成瘤能力。將過(guò)表達(dá)miR-9的SUNE1細(xì)胞及其對(duì)照細(xì)胞接種至5只裸鼠背部皮下,細(xì)胞數(shù)為1×106個(gè),接種后第19天取材。結(jié)果發(fā)現(xiàn)miR-9過(guò)表達(dá)的SUNE1細(xì)胞成瘤后瘤體較對(duì)照組顯著減�。╰=4.279,P=0.003）,且生長(zhǎng)速度顯著減慢（F=34.112,P=0.000）。此外,miR-9過(guò)表達(dá)組組織中BrdU+和Ki67+的細(xì)胞均較對(duì)照組的少。7. CCNGl為miR-9的靶基因我們發(fā)現(xiàn)在miR-9過(guò)表達(dá)的鼻咽癌細(xì)胞株中CCNG1表達(dá)下降,而抑制miR-9后,CCNG1表達(dá)升高,在皮下成瘤的瘤體組織中,miR-9過(guò)表達(dá)組的組織中CCNG1表達(dá)下降。生物信息學(xué)軟件預(yù)測(cè)發(fā)現(xiàn),miR-9序列5’端2-9位核苷酸與CCNG1mRNA3’端-UTR完全互補(bǔ)。雙熒光素酶報(bào)告基因系統(tǒng)的檢測(cè)結(jié)果表明,重組質(zhì)粒wt3’-UTR和miR-9mimics共轉(zhuǎn)染可顯著降低熒光素酶活性,而將wt3’-UTR和miR-9inhibitor共轉(zhuǎn)染可顯著增強(qiáng)熒光素酶活性（t=32.542、6.238,P=0.000、0.003）。突變質(zhì)粒mut3’-UTR和miR-9mimics或miR-9inhibitor共轉(zhuǎn)染均未明顯改變熒光素酶活性（t=0.455、0.241,P=0.673、0.821）。以上數(shù)據(jù)表明,CCNG1為miR-9的靶基因。8.miR-9靶向抑制CCNG1表達(dá)而使細(xì)胞增殖降低為探討CCNG1是否介導(dǎo)了miR-9抑制鼻咽癌細(xì)胞增殖的功能,我們首先把HONE1細(xì)胞株中CCNG1沉默,以明確CCNG1表達(dá)下調(diào)是否亦抑制了細(xì)胞增殖;Western blot顯示,miR-9mimics和siCCNG1均能下調(diào)CCNGl表達(dá),而siCCNG1和miR-9mimics均能抑制細(xì)胞增殖,二者均能使細(xì)胞周期G0-G1期細(xì)胞增多（F=14.134,P=0.002）,S期減少（F=24.175,P=0.000）。為研究CCNG1是否有拮抗miR-9的周期阻滯作用,我們?cè)谶^(guò)表達(dá)miR-9的HONE1細(xì)胞的基礎(chǔ)上進(jìn)一步過(guò)表達(dá)CCNG1（LV-miR-9+pCl-CCNG1）,結(jié)果示,CCNG1表達(dá)升高,細(xì)胞增殖增快,G0-G1期的細(xì)胞減少（F=93.810,P=0.000）,S期和(G2-M期細(xì)胞顯著增多（F=39.597、25.712,P=0.000、0.001）。綜上,CCNG1介導(dǎo)了miR-9抑制鼻咽癌細(xì)胞增殖的功能。第二章miR-9靶向調(diào)控Hesl在鼻咽癌腫瘤干細(xì)胞干性維持中作用及機(jī)制1.miR-9過(guò)表達(dá)顯著抑制NPC CSCs的自我更新miR-9過(guò)表達(dá)可顯著下調(diào)CNE2和SUNE1細(xì)胞中干性相關(guān)基因（如Nanog、Oct4和ABCG2）表達(dá),而下調(diào)NPC細(xì)胞中miR-9表達(dá)則導(dǎo)致干性基因表達(dá)上調(diào)；miR-9過(guò)表達(dá)可顯著降低NPC細(xì)胞中SP細(xì)胞含量,并極顯著抑制NPC細(xì)胞形成腫瘤球,且過(guò)表達(dá)組所形成腫瘤球的直徑小于對(duì)照細(xì)胞的（t-39.436、31.577,P=0.000、0.000）。以上結(jié)果表明,miR-9至少抑制了NPC CSCs的自我更新。2.腫瘤細(xì)胞和腫瘤細(xì)胞所培養(yǎng)出的腫瘤球間miR-9和Hesl表達(dá)差異為了明確Hesl與鼻咽癌腫瘤干細(xì)胞的潛在關(guān)系,我們對(duì)miR-9和Hesl在鼻咽癌細(xì)胞（如CNE2和SUNE1細(xì)胞）和由其培養(yǎng)出的腫瘤球間基因表達(dá)差異進(jìn)行了分析。結(jié)果顯示,miR-9的表達(dá)在腫瘤球和腫瘤細(xì)胞間無(wú)明顯差異（t-0.765、0.653,P=0.487、0.533）,而腫瘤球中Hes1、Sox2、Oct4、Nanog和ABCG2的表達(dá)均較腫瘤細(xì)胞的高。這預(yù)示Hesl在鼻咽癌腫瘤干細(xì)胞上發(fā)揮一定的功能。3.Hesl過(guò)表達(dá)顯著促進(jìn)了NPC CSCs的自我更新Hesl過(guò)表達(dá)可顯著上調(diào)CNE2和SUNE1細(xì)胞中干性相關(guān)基因（即Nanog和ABCG2）表達(dá),而下調(diào)NPC細(xì)胞中Hesl表達(dá)則導(dǎo)致干性基因表達(dá)下調(diào)；流式細(xì)胞儀分析表明,Hesl過(guò)表達(dá)可顯著增加NPC細(xì)胞（CNE2和SUNE1細(xì)胞）中SP細(xì)胞含量,而下調(diào)Hes1則顯著下調(diào)NPC細(xì)胞（CNE2和SUNE1細(xì)胞）中SP細(xì)胞含量；腫瘤球形成實(shí)驗(yàn)表明,過(guò)表達(dá)Hes1的CNE2和SUNE1細(xì)胞較對(duì)照組的成球能力顯著增強(qiáng)（t=14.902、20.412,P=0.003、0.000）,而抑制Hesl組的成球能力顯著下降（t=14.248、18.251,P=0.000、0.000）。以上數(shù)據(jù)提示,Hes1參與了NPC CSCs自我更新的調(diào)控。4.Hesl是miR-9的靶基因既然miR-9參與了NPC CSCs自我更新的調(diào)控,其通過(guò)下游哪一個(gè)靶基因?qū)崿F(xiàn)?我們的研究發(fā)現(xiàn),miR-9過(guò)表達(dá)降低NPC細(xì)胞（即CNE2和SUNE1細(xì)胞）中Hesl表達(dá),而下調(diào)miR-9表達(dá)則導(dǎo)致Hesl表達(dá)上調(diào)；CNE2和SUNE1細(xì)胞的皮下成瘤組織中,miR-9過(guò)表達(dá)組的Hes1表達(dá)下調(diào)。生物信息學(xué)軟件預(yù)測(cè)發(fā)現(xiàn),miR-9序列5’端2-7位核苷酸與Hes1mRNA3’端-UTR完全互補(bǔ)。雙熒光素酶報(bào)告基因系統(tǒng)的檢測(cè)結(jié)果表明,重組質(zhì)粒wt3’-UTR和miR-9mimics共轉(zhuǎn)染可顯著降低熒光素酶活性,而將wt3’-UTR和miR-9inhibitor共轉(zhuǎn)染可顯著增強(qiáng)熒光素酶活性（t-=23.089、11.573,P=0.000、0.000）；突變質(zhì)粒mut3’-UTR和miR-9mimics或miR-9inhibitor共轉(zhuǎn)染均未明顯改變熒光素酶活性（t==1.075、0.218,P=0.343、0.838）。以上數(shù)據(jù)表明,Hes1為miR-9的靶基因。5.Hesl介導(dǎo)了miR-9抑制鼻咽癌腫瘤干細(xì)胞自我更新的功能在明確Hesl是miR-9的靶基因基礎(chǔ)上,擬進(jìn)一步闡明miR-9是否通過(guò)下調(diào)Hesl表達(dá)抑制了鼻咽癌腫瘤干細(xì)胞的自我更新。Western blot結(jié)果顯示,miR-9和Hesl共同過(guò)表達(dá)組中Nanog和ABCG2的表達(dá)水平較miR-9組的高,SP細(xì)胞含量較miR.-9組的高,且Hesl過(guò)表達(dá)后有效阻斷了miR-9抑制腫瘤球形成的能力（F=162.277,P=0.000）。綜上,miR-9抑制鼻咽癌腫瘤干細(xì)胞自我更新的功能可由其靶基因Hes1介導(dǎo)。6.探討miR-9潛在的治療價(jià)值為研究miR-9對(duì)鼻咽癌的治療作用,我們進(jìn)行了miR-9的裸鼠皮下瘤內(nèi)注射實(shí)驗(yàn),首次預(yù)實(shí)驗(yàn)中,注射miR-9Agomir組的三只裸鼠均發(fā)現(xiàn)沿進(jìn)針?lè)较蛐纬煽斩辞疫w延不愈,而對(duì)照組未見(jiàn)空洞（數(shù)據(jù)未展示）。第二次重復(fù)實(shí)驗(yàn)中使用miR-9mimics與MaxSuppressorTM In vivo RNA-LANCErⅡ共注射,結(jié)果發(fā)現(xiàn)miR-9可抑制瘤體生長(zhǎng),且部分瘤體中心出現(xiàn)空洞。兩組分別提取組織RNA和蛋白進(jìn)行分析,結(jié)果顯示,實(shí)驗(yàn)組中miR-9成功過(guò)表達(dá)；且其靶基因CCNG1和Hes1均被抑制。此部分結(jié)果正在進(jìn)行第三次實(shí)驗(yàn)驗(yàn)證。第三章c-Myc通過(guò)miR-9靶向調(diào)控Klf4促進(jìn)鼻咽癌細(xì)胞EMT、侵襲和轉(zhuǎn)移的作用1. c-Myc在鼻咽癌細(xì)胞中上調(diào)miR-9表達(dá)c-Myc過(guò)表達(dá)上調(diào)鼻咽癌細(xì)胞株中miR-9表達(dá),而下調(diào)c-Myc則抑制miR-9表達(dá)�？梢�(jiàn),c-Myc正向調(diào)控了鼻咽癌細(xì)胞中miR-9的表達(dá)。2. c-Myc靶向上調(diào)miR-9表達(dá)引起EMT相關(guān)基因表達(dá)變化為明確c-Myc對(duì)EMT相關(guān)基因是否有影響,我們首先行qRT-PCR檢測(cè)EMT相關(guān)基因表達(dá)情況,結(jié)果顯示,c-Myc過(guò)表達(dá)顯著下調(diào)HONE1細(xì)胞中E-cadherin表達(dá)（t=-44.880,P=0.000）,而通過(guò)RNAi抑制c-Myc表達(dá)則升高E-cadherin表達(dá)（t=3.149,P=0.035）;此外,c-Myc無(wú)論過(guò)表達(dá)還是通過(guò)RNAi抑制c-Myc表達(dá)均在mRNA水平對(duì)N-cadherin和Vimentin表達(dá)無(wú)顯著影響（P>0.05）。Western blot檢測(cè)結(jié)果表明,c-Myc過(guò)表達(dá)顯著下調(diào)HONE1細(xì)胞中E-cadherin表達(dá),上調(diào)Vimentin表達(dá),而抑制c-Myc表達(dá)則上調(diào)E-cadherin表達(dá),并下降Vimentin表達(dá)。此外,miR-9inhibitor能阻斷c-Myc對(duì)E-cadherin和Vimentin的影響,并在CNE2細(xì)胞中得到進(jìn)一步驗(yàn)證。3. c-Myc通過(guò)上調(diào)miR-9表達(dá)促進(jìn)了NPC細(xì)胞遷移和侵襲如上所述,鼻咽癌細(xì)胞中miR-9介導(dǎo)了c-Myc對(duì)E-cadherin和Vimentin表達(dá)的影響,接下來(lái)我們研究了c-Myc對(duì)鼻咽癌細(xì)胞遷移和侵襲的影響及miR-9在其中所起的作用。Transwell結(jié)果顯示,c-Myc促進(jìn)細(xì)胞遷移（t=12.231,P=0.000）, Boyden小室結(jié)果顯示c-Myc可促進(jìn)細(xì)胞侵襲（t=10.000,P=0.000）,而miR-9inhibitor可阻斷c-Myc促遷移和侵襲的作用（F=147.016、59.900,P=0.001、0.001）。4.miR-9對(duì)鼻咽癌細(xì)胞中EMT相關(guān)基因表達(dá)的調(diào)控作用在培養(yǎng)穩(wěn)定過(guò)表達(dá)miR-9的細(xì)胞株過(guò)程中,發(fā)現(xiàn)CNE2和HONE1細(xì)胞形態(tài)發(fā)生了變化,部分細(xì)胞變得細(xì)長(zhǎng)且觸角增多,細(xì)胞失去原有的鋪路石樣或規(guī)整的形態(tài),這提示miR-9過(guò)表達(dá)可能誘導(dǎo)了EMT。隨后,在基因?qū)用孢M(jìn)一步明確miR-9是否確實(shí)誘發(fā)了EMT。結(jié)果表明,miR-9過(guò)表達(dá)導(dǎo)致SUNE1、CNE2、 HONE1、HNE1細(xì)胞中上皮相關(guān)基因E-cadherin和a-catenin表達(dá)下降,而間充質(zhì)相關(guān)基因N-cadherin和Vimentin表達(dá)升高；下調(diào)miR-9表達(dá)則引起CNE2和HONE1細(xì)胞中E-cadherin表達(dá)升高,而N-cadherin和Vimentin表達(dá)下降。以上數(shù)據(jù)提示,miR-9過(guò)表達(dá)在鼻咽癌細(xì)胞上誘導(dǎo)了EMT。5.miR-9促進(jìn)鼻咽癌細(xì)胞的遷移和侵襲我們進(jìn)一步采用Transwell和Boyden小室明確miR-9過(guò)表達(dá)對(duì)CNE2和HONE1細(xì)胞的遷移和侵襲能力的影響。結(jié)果顯示,過(guò)表達(dá)miR-9的CNE2和HONE1細(xì)胞的遷移和侵襲能力較對(duì)照組的顯著增強(qiáng)（t=6.533、10.983：13.131、12.351,P=0.000、0.000；0.000、0.000）。劃痕實(shí)驗(yàn)亦顯示,miR-9過(guò)表達(dá)可促進(jìn)CNE2和HONE1細(xì)胞遷移。綜上,miR-9可促進(jìn)鼻咽癌細(xì)胞遷移和侵襲。6.miR-9過(guò)表達(dá)降低鼻咽癌細(xì)胞的粘附能力為研究miR-9對(duì)腫瘤細(xì)胞的粘附能力的影響,我們用0.05nM EDTA處理過(guò)表達(dá)miR-9的CNE2和HONE1細(xì)胞及其對(duì)照細(xì)胞后,miR-9過(guò)表達(dá)的鼻咽癌細(xì)胞較對(duì)照細(xì)胞粘附能力下降（F=846.528、751.057,P=0.000、0.000）,提示過(guò)表達(dá)miR-9的鼻咽癌細(xì)胞粘附降低。7.miR-9對(duì)細(xì)胞骨架的影響我們用鬼筆環(huán)肽標(biāo)記法檢測(cè)了miR-9過(guò)表達(dá)對(duì)CNE2和HONE1細(xì)胞細(xì)胞骨架的影響,并用掃描電鏡觀(guān)測(cè)了細(xì)胞的微觀(guān)形態(tài)。結(jié)果顯示,miR-9過(guò)表達(dá)的細(xì)胞表面纖維增粗增多,偽足增多。Western blot檢測(cè)Rho酶和Rac的表達(dá)較對(duì)照的增多。提示miR-9與細(xì)胞形態(tài)改變和偽足形成等轉(zhuǎn)移起始步驟相關(guān)。8.Klf4為miR-9的靶基因miR-9過(guò)表達(dá)降低NPC細(xì)胞（即CNE2和SUNE1細(xì)胞）中Klf4表達(dá),而下調(diào)miR-9表達(dá)則導(dǎo)致K1f4表達(dá)上調(diào)；CNE2和SUNE1細(xì)胞的皮下成瘤組織中,miR-9過(guò)表達(dá)組的Klf4表達(dá)下調(diào)。生物信息學(xué)軟件預(yù)測(cè)發(fā)現(xiàn),miR-9序列5’端2-7位核苷酸與Klf4mRNA3’端-UTR完全互補(bǔ)。雙熒光素酶報(bào)告基因系統(tǒng)的檢測(cè)結(jié)果表明,重組質(zhì)粒wt3’-UTR和miR-9mimics共轉(zhuǎn)染可顯著降低熒光素酶活性,而將wt3’-UTR和miR-9inhibitor共轉(zhuǎn)染可顯著增強(qiáng)熒光素酶活性（t=4.380、13.777,P=0.012、0.000）；突變質(zhì)粒mut3’-UTR和miR-9mimics或miR-9inhibitor共轉(zhuǎn)染均未明顯改變熒光素酶活性（t=0.921、0.302,P=0.409、0.777）。以上數(shù)據(jù)表明,Klf4為miR-9的靶基因。9.miR-9靶向抑制Klf4進(jìn)而增強(qiáng)鼻咽癌細(xì)胞的遷移和侵襲能力我們的前期研究表明,Klf4在鼻咽癌細(xì)胞中具有抑制EMT、遷移和侵襲的功能,為研究Klf4是否具有拮抗miR-9促進(jìn)遷移和侵襲的作用,我們?cè)贖oNE1細(xì)胞過(guò)表達(dá)miR-9的基礎(chǔ)上過(guò)表達(dá)Klf4,結(jié)果過(guò)表達(dá)Klf4組（LV-miR-9+LV-Klf4）的Klf4表達(dá)升高,同時(shí)遷移和侵襲能力較LV-miR-9組顯著增強(qiáng)（F=137.015、12.438,P=0.000、0.001）。以上提示,Klf4拮抗了miR-9促遷移和侵襲的作用,即miR-9通過(guò)靶向抑制Klf4促進(jìn)細(xì)胞遷移和侵襲。10. c-Myc過(guò)表達(dá)促進(jìn)鼻咽癌細(xì)胞體內(nèi)轉(zhuǎn)移為明確c-Myc對(duì)鼻咽癌細(xì)胞體內(nèi)轉(zhuǎn)移的影響,我們把過(guò)表達(dá)c-Myc的CNE2細(xì)胞及對(duì)照細(xì)胞（細(xì)胞劑量：1×106/只）分別接種于裸鼠肝包膜下,每組7只裸鼠,結(jié)果示接種處全部成瘤,而發(fā)生淋巴結(jié)轉(zhuǎn)移的過(guò)表達(dá)組有5/7只,而對(duì)照2/7只。第四章miR-9調(diào)控鼻咽癌細(xì)胞中免疫和炎癥相關(guān)基因的表達(dá)1.miR-9引起鼻咽癌細(xì)胞中干擾素相關(guān)基因表達(dá)變化微陣列分析顯示,miR-9過(guò)表達(dá)的CNE2細(xì)胞中多種IFN調(diào)節(jié)的基因如IFI44L、PSMB8、IRF5、PSMB10、IFI27、PSB9HUMAN、IFIT2、TRAIL、IFIT1、 PSB8HUMAN、IRF1和B2M的表達(dá)都發(fā)生了變化。qRT-PCR進(jìn)一步驗(yàn)證了基因芯片結(jié)果（P<0.05或0.01）。為了進(jìn)一步證實(shí)miR-9對(duì)IFN調(diào)節(jié)因子的作用,我們又進(jìn)行了miR-9mimics和inhibitor的瞬時(shí)轉(zhuǎn)染,qRT-PCR發(fā)現(xiàn)miR-9mimics組中IFI44L、PSMB8、IRF5、 PSMB10、IFI27、IFIT2、TRAIL、IFIT1、IRF1、B2M和GBP1表達(dá)升高,而ISG20和AIM2表達(dá)下降。相反,miR-9inhibitor組中IFI44L、PSMB8、IRF5、PSMB10、 IFI27、IFIT2、TRAIL、IFIT1、IRF1、B2M和GBP1表達(dá)下降,而ISG20和AIM2表達(dá)則升高（P<0.05或0.01）。2.miR-9引起鼻咽癌細(xì)胞中主要組織相容性復(fù)合體Ⅰ型（MHCI）分子表達(dá)變化微陣列分析顯示,miR-9過(guò)表達(dá)的CNE2細(xì)胞中多種主要組織相容性復(fù)合體Ⅰ型（MHC Ⅰ）分子如HLA-B、HLA-H、HLA-C、HLA-F、Q8WW48HUMAN、 NP001004349.1、Q6ZUW0HUMAN、O19682HUMAN和TAP1表達(dá)升高。qRT-PCR證實(shí)了HLA-B、HLA-F和TAP1表達(dá)的變化（P<0.05或0.01）。為了進(jìn)一步證實(shí)miR-9對(duì)MHC1分子的作用,我們又進(jìn)行了miR-9mimics和inhibitor的瞬時(shí)轉(zhuǎn)染,qRT-PCR發(fā)現(xiàn)HLA-B、HLA-F和TAP1在miR-9mimics組中的表達(dá)升高,而在miR-9inhibitor組中表達(dá)下降（P<0.05或0.01）。3.miR-9引起鼻咽癌細(xì)胞白介素（IL）相關(guān)基因表達(dá)變化微陣列分析顯示,miR-9過(guò)表達(dá)的CNE2細(xì)胞中多種IL相關(guān)基因IL20RB、 GALT、IL7、IL1B、IL11、IL1F8、IL1A、IL6和IL7R等表達(dá)發(fā)生變化,結(jié)果用qRT-PCR證實(shí)（P<0.05或0.01）。為了進(jìn)一步證實(shí)miR-9對(duì)IL相關(guān)基因的作用,我們又進(jìn)行了miR-9mimics和inhibitor的瞬時(shí)轉(zhuǎn)染,qRT-PCR發(fā)現(xiàn)miR-9mimics可以顯著上調(diào)一些IL相關(guān)基因（IL20RB、GALT、IL7）（P<0.05或0.01）,而下調(diào)另一些IL相關(guān)基因（IL1B、 IL11、IL1F8、IL1A、IL6、IL7R）（P<0.05或0.01）。相反,miR-9inhibitor下調(diào)IL20RB、GALT和IL7（P<0.05或0.01）,而上調(diào)IL1B、IL11、IL1F8、ILIA、IL6和IL7R（P<0.05或0.01）。結(jié)論1.miR-9靶向下調(diào)CCNG1抑制NPC細(xì)胞增殖；2.miR-9的靶基因Hesl介導(dǎo)了miR-9抑制鼻咽癌腫瘤干細(xì)胞自我更新的功能；3. c-Myc通過(guò)miR-9靶向調(diào)控Klf4促進(jìn)了鼻咽癌細(xì)胞EMT、侵襲和轉(zhuǎn)移；4.miR-9可調(diào)節(jié)NPC細(xì)胞中免疫和炎癥相關(guān)基因表達(dá),提示miR-9可能在炎癥和腫瘤間起橋梁作用；5.miR-9靶向調(diào)控CCNG1表達(dá)抑制了NPC細(xì)胞增殖,而miR-9靶向下調(diào)Klf4表達(dá)則促進(jìn)了鼻咽癌細(xì)胞EMT、侵襲和遷移,這些預(yù)示miR-9可通過(guò)靶向調(diào)控不同的靶基因在腫瘤細(xì)胞的增殖以及EMT、侵襲和轉(zhuǎn)移中發(fā)揮不同的功能,這也預(yù)示了miRNAs功能的多樣性和復(fù)雜性。

    Background and objectiveNasopharyngeal carcinoma （NPC） is the most common cancer arising from the mucosal epithelium of the nasopharynx, and is extremely common in southern regions of China, particularly in Guangdong, Guangxi, Hunan, Fujian. NPC frequently metastasizes to regional lymph nodes. The etiology of NPC seems to follow a multi-step process, in which Epstein-Barr virus infection, dietary, smoking, genetic factors and environmental carcinogens seem to play important roles. Typical conventional treatments of NPC include radiotherapy, surgery and chemotherapy. Recently, molecular targeted therapy has become the hotspot and focus of comprehensive treatment of NPC.As was shown by Weinberg in2011, The hallmarks of cancer comprise ten biological capabilities acquired during the multistep development of human tumors including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, genome instability, inflammation, reprogramming of energy metabolism and evading immune destruction. There are many kinds of cells in "tumor microenvironment", such as tumor cells, inflammatory cells, cancer stem cells （CSCs）. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis, moreover, they can resistant to chemotherapy and radiotherapy. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival cancer patients.Metastasis is a basic biological characteristics of malignant tumor, and the primary factors affecting patient survival, which has become a hot and difficult field of cancer research. The most prominent feature of tumor metastasis is the tissue specificity, which is different tumor can form metastases in the same or different parts of the human body. Epithelial-mesenchymal transition or transformation （EMT） is a process characterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell motility. EMT is an important contributor to the invasion and metastasis of epithelial-derived cancers.miRNAs are small non-coding RNA molecules, and important regulatory molecules in animals and plants. Each miRNA may have a plurality of target genes, while several miRNAs can also target the same gene. Recent studies show that some miRNAs regulate cell proliferation and apoptosis processes in cancer. In addition, some miRNAs may function as oncogenes or tumor suppressors. miRNA expression profiles may become useful biomarkers for cancer diagnostics. Moreover, miRNA therapy could be a powerful tool for cancer prevention and therapeutics. Therefore, aberrant expression of miRNAs has been implicated in numerous disease states, and miRNA-based therapies are under investigation.miR-9, initially demonstrated to function in neurogenesis, has been confirmed to be implicated in cancer. miR-9is under-expressed in many types of cancers, including breast cancer, hepatocellular carcinomas, gastric cancer, ovarian cancer, colon cancer, melanoma, clear cell renal cell carcinoma and medulloblastoma, all of which is indicative of a tumor suppressor potential, whereas miR-9is over-expressed in brain cancer and Hodgkin’s lymphoma, suggesting oncomir activity for miR-9in these two cancers. Some evidence indicates that the functions of miR-9in cancer cells are recently implicated in regulating proliferation, EMT, metastasis, apoptosis and tumor angiogenesis, etc. However, the contribution of miR-9which is commonly down-regulated in NPC to the pathogenesis of NPC remains to be characterized.MethodsPart I miR-9inhibits cell growth and tumorigenesis through repressing CCNG1in NPC1Establishment of miR-9stably over-expressed NPC cells:Particles expressing vector along with packaging plasmids psPAX2and pMD2.G were transfected into HEK293T cells （maintained in10%FBS） using Lipofectamine2000reagent （Invitrogen） according to the manufacturers’instruction.48h after transfection, virus supernatant was harvested from these cells, and then used to infect NPC cells. To attain100%percentage of infected NPC cells based on EGFP assay and FACS analysis, lentiviral infected cells were selected by2μg/ml puromycin （GIBCO） for2weeks to kill non-infected cells.2RNA isolation, reverse transcription and qRT-PCR:For miRNA and mRNA analyses, total RNA from NPC cells was extracted with Trizol Reagent （TaKaRa） according to the protocol provided by the manufacturer. Total RNA was reversely transcribed with the PrimeScript RT reagent Kit （TaKaRa）. The expression levels of mature miRNA were determined by SYBR Green quantitative PCR amplifications performed on the Stratagene Mx3000P Real-Time PCR system （Agilent Technologies, Inc., USA）. U6was used for normalization. Expression of mRNA analysis was performed using SYBR Green Master Mix （TaKaRa） as described using GAPDH for normalization on a Stratagene Mx3000P qRT-PCR System. The primers used for the amplification of the indicated genes were listed in Table S1-4. All samples were normalized to internal controls and fold changes were calculated through relative quantification （2-ΔΔCt）.3CCK-8assay:A total of800-1000cells were seeded in a96-well plate and then allowed to grow in normal medium for96hours. Cells were incubated in100ul normal+10ul CCK-8. The absorbance in each well was measured at450nm by a microplate reader. 4Colony formation assay:200cells were seeded in6-well plates and grown for2weeks. Then washed with PBS, fixed with methanol for10min, and stained with hematoxylin for10min.5Cell-cycle analysis:Pipet cell suspension （EtOH70%） over night at-20℃. Treated with RNase A to remove RNAs from the cells, cell-cycle distribution was analyzed by propidium iodide staining and flow cytometry.6Luciferase activities detection:Insert the3’-UTR into3’downstream to the luciferase reporter in the pGL3vector which carries activated promoter sequences. The association of miRNA with3’-UTR will cause the attenuation of translation of luciferase mRNA, which shows the decline of luciferase activities.7Western blot and high sensitivity chemiluminescence imaging system were used to survey the proteic level of genes.8Animal experiment:Cells were injected subcutaneously or into hepatic subcapsular of nude mice, tumors in subcutaneous were measured every2-3days, tumor volunes were calculated as follows:D×d×d/2（D meant the longest diameter and d meant the shortest diameter）. Tumors were used to extract RNA and proptein, and fixed with4%paraformaldehyde for HE staining and Immunohistochemistry.9Statistical analysis:Data were presented as mean±SEM unless otherwise indicated of at least3independent experiments. Statistical analysis was performed using a SPSS13.0software package. Two-tailed Student’s t test was used for comparisons of2independent groups. Statistical significance was assessed by the Student’s t-test （*p<0.5;#p<0.01）.Part II miR-9impairs cancer stem cells through negative regulation of Hesl in NPC1Establishment of Hes1and shHes1stably over-expressed NPC cells, Western blot and Cell Immunofluorescence were used to analyze protein expression of "stemness "genes.2Luciferase activities were detected to verify Hesl was a direct target of miR-9in NPC cells after Hes1wt3’UTR （mut3’UTR） and miRNAs cotransfection.3Tumor sphere formation and ratio of stem cell populations （SP） detected by flow cytometry and hoechst33342were performed to evaluate the "sternness" of NPC cells.4Intratumoral delivery of miR-9in a nasopharyngeal cancer xenograft model: Nude mice were subcutaneously inoculated with CNE2cells and maintained until the tumor cells had formed solid, palpable tumors with an average volume of80to100mm3,7days following inoculation, miR-9or a negative control miRNA was repeatedly administered by intratumoral injections every3days. All mice were killed on day16.5Statistical analysis:Data were presented as mean±SEM unless otherwise indicated of at least3independent experiments. Statistical analysis was performed using a SPSS13.0software package, Two-tailed Student’s t test was used for comparisons of2independent groups.Part Ⅲ miR-9triggers EMT and migration by repressing Klf4in NPC1Establishment of c-Myc and miR-9stably over-expressed NPC cells, Western blot and Cell Immunofluorescence were used to analyze protein expression of genes, Immunohistochemistry was used to investigate the protein expression and the definite localization of proteins.2Transwell, Scratching test and Boyden were used for migration and invasion of cells.3Fluorescently labeled phalloidin staining test and Scanning electron microscopy were used to survey the Skeleton and morphological variation of NPC cells.4PBS with0.05nM EDTA and inverted fluorescence microscope were used for adhesion ability of cells.5Statistical analysis:Data were presented as mean±SEM unless otherwise indicated of at least3independent experiments. Statistical analysis was performed using a SPSS13.0software package.Two-tailed Student’s t test was used for comparisons of2independent groups; One-way ANONA was used for comparisons of several groups; Factorial ANOVA performed an analysis of variance for multiple classification variables. Part IV miR-9modulates the expression of interferon-regulated genes and MHC class I molecules in NPC1RNA extract and qRT-PCR were carried out to detect the mRNA expression of genes。2mRNA microarray analysis:Expression microarray analysis was carried out with commercially available Affymetrix Human Gene U133Plus2.0array according to the Affymetrix standard protocol, which carried47,000transcripts representing38,500well-characterized human genes. All the hybridization procedures and data analysis were performed by Capital Bio Corp.（Bejing, China）. Total RNA samples were isolated from NPC cells （CNE2cells） using Trizol reagent （Invitrogen）. Briefly, total RNA was used to synthesize cDNA in an in vitro transcription reaction, and then cDNA was fluorescently labeled by Cy5or Cy3-CPT with Klenow enzyme. Labeled cDNA was then hybridized to Affymetrix Human Gene U133Plus2.0arrays. Hybridization was processed at45℃, with rotation for16h （Affymetrix GeneChip Hybridization Oven640）. Chips were then washed and stained in the Affymetrix Fluidics Station450. Hybridization signals were scanned with a Lux-Scan3.0scanner （Capital Bio. Corporation, Beijing, China）. The resultant images were digitized with Genepix Pro6.0software （Axon Instruments, Foster City, CA, USA）.3Statistical analysis:Data were presented as mean±SEM unless otherwise indicated of at least3independent experiments. Statistical analysis was performed using a SPSS13.0software package. Two-tailed Student’s t test was used for comparisons of2independent groups.ResultsPart I miR-9inhibits cell growth and tumorigenesis through repressing CCNG1in NPC1. qRT-PCR data demonstrated the miR-9expression was significantly lower in NPC cells （i.e.,6-10B,5-8F, CNE2, HNE1, HONE1, SUNE1cells） than that in NP-69cells （F=370.010, P=0.000）.2. We established miR-9-expressing NPC cell lines （i.e., CNE2, HONE1and SUNE1cells）.3. The levels of miR-9in CNE2, HONE1and SUNE1cells transfected with miR-9mimics was much higher than that in CNE2, HONE1and SUNE1cells transfected with control RNA （t=7.619,11.224,5.228, P=0.017,0.000,0.035） Moreover, the levels of miR-9in CNE2and5-8F cells transfected with miR-9inhibitor was much lower than that in CNE2and5-8F cells transfected with inhibitor control （t=10.924,14.95, P=0.000,0.004）, indicating that miR-9inhibitor efficiently down-regulated the endogenous miR-9expression in CNE2and5-8F cells.4. miR-9inhibits cell growth and affect cell-cycle distribuionCCK-8assay showed that miR-9-expressing NPC cells inhibited cell growth, whereas the proliferation of NPC cells transfected with miR-9inhibitor was enhanced （P=0.000）. Flat cloning formation experiments also indicated that the ability of proliferation was significantly inhibited in miR-9-expressing groups （t=7.721,2.744,16.545, P=0.000,0.034,0.000）. In sum, the above two experiments illustrates that miR-9overexpression suppresses the proliferation of NPC cells in vitro.We studied the effects of miR-9on cell cycle using FACS. The results showed that compare to the control group, the ratio of G0-G1phase in miR-9-expressing cells were significantly increased （P<0.05）, while the ratio of G0-G1phase in NPC cells transfected with miR-9inhibitor were significantly decreased （P<0.05）, indicating that miR-9could cease cell cycle at phase G0/G1.5. The suppressive effects of miR-9overexpression on tumorigenicity of NPC cellsTo explore the effects of miR-9on NPC tumorigenicity, miR-9-expressing CNE2cells （1×106） we firstly subcutaneously injected into the dorsal flank of nude mice. At34days after implantation, mice injected with vector-expressing CNE2cells carried large tumor burdens, while mice injected with miR-9-expressing CNE2cells did not developed tumor. Then, we increased the number of injected cells from1×106to1.5×106. At21days after implantation, all the mice injected with vector-expressing CNE2cells developed tumors, but only one of four mice injected with miR-9-expressing CNE2cells developed tumor which is much smaller than the controls. Additionally, miR-9-expressing CNE2cells （1.2×106） were transplanted subcapsularly into the livers of nude mice. At23days after implantation, all the mice transplanted with vector-expressing CNE2cells formed tumors in livers, while none of those mice injected with miR-9-expressing CNE2cells developed tumor.Nextly, miR-9-expressing SUNE1cells （1×106） cells were subcutaneously transplanted into the dorsal flank nude mice. The tumor became palpable between7and9days after inoculation, and all the mice injected with vector-expressing cells and4/5mice injected with miR-9-expressing CNE2cells formed tumors at the end of the experiment （t=4.279, P=0.003）. As early as9days postimplantation, the growth of transplanted tumors between2groups became statistically significant （P=0.021）. At19days after implantation, those mice injected with vector-expressing cells carried larger burdens. We also showed that both the staining intensity and the number of hyperproliferative BrdU and Ki67tumor cells were significantly decreased compared with control.6. CCNG1is a direct target gene of miR-9To explore the mechanism of growth inhibition induced by miR-9, we want to idenfity the target gene of miR-9. Firstly, we found that miR-9overexpression down-regulated CCNG1expression in NPC cells, while miR-9inhibitor up-regulated CCNG1expression in NPC cells. Secondly, we performed luciferase reporter assay to determine whether miR-9could directly target the3’-UTR of CCNG1in NPC cells. CNE2cells were transfected with wt or mt3’-UTR vector and miR-9mimics. The results showed a significant decrease of luciferase activity when compared with miR control （t=32.542, P=0.000）. The activity of mt3’-UTR vector was unaffected by a simultaneous transfection with miR-9mimics or miR-9inhibitor （t=0.455,0.241, P=0.673,0.821）. Moreover, cotransfection with miR-9inhibitor and wt3’-UTR vector in CNE2cells led to an increase of luciferase activity （t=6.238, P=0.003） Taken together, all these results strongly suggest that CCNG1is a direct target gene of miR-9in NPC cells. 7. miR-9affects cell proliferation and cell cycle via CCNG1in NPC cells.To elucidate whether the growth-suppressive effect of miR-9could be mediated by CCNG1in NPC cells, we firstly silenced CCNG1to investigate whether the reduced expression of CCNG1could mimic the suppressive effect of miR-9. HONE1cells were transfected with si-CCNG1, and then we examined cell proliferation rate and cell cycle distribution （F=14.134, P=0.002）. CCNG1knockdown led to significant cell growth inhibition and cell-cycle arrest, similar to those induced by miR-9（P<0.01）. Subsequently, we evaluated whether ectopic expression of CCNG1could rescue the suppressive effect of miR-9. HONE1cells overexpressing miR-9were transfected with pC3-CCNG1. We observed that the ectopic expression of CCNG1in miR-9-expressing NPC cells significantly rescued miR-9-induced cell growth inhibition and cell-cycle arrest （F=93.810, P=0.000）. In summary, the growth-suppressive effect of miR-9could be mediated by CCNG1in NPC cells.Part II miR-9impairs cancer stem cells through negative regulation of Hesl in NPC1. miR-9overexpresssion down-regulates the expression of stemness-related genes in NPC cellsWestern blot showed that Nanog, Oct4and ABCG2was down-expressed in miR-9-expressing CNE2and SUNE1cells, and up-expressed in5-8F cells transfected with miR-9inhibitor. Immunohistochemistry also showed a low-expression of Nanog and Sox2in subcutaneous xenotransplanted tumors overexpressing miR-9. We found that the ability of tumor spheres of miR-9-expressing cells reduced （t=39.436,31.577, P=0.000,0.000）, and the ratio of SP cells was decreased in miR-9-expressing cells.2. The expression of stemness-related genes between adherent cells and tumor spheresFor further study, we tested the expression of stemness-related genes between adherent cells and tumor spheres （the second generation）. Western blot data resulte indicated that the expression of Hesl, Sox2, Oct4, Nanog and ABCG2was higher in tumor spheres than in adherent cells, while miR-9expression （detected by qRT-PCR） showed no disparity between adherent cells and tumor spheres （t=0.765,0.653, P=0.487,0.533）3. Effects of Hesl on "sternness" of NPC cellsWe found a high expression of Nanog and ABCG2in Hesl-expressing （LV-Hes1） NPC cells, and low expression in Hesl-silenced （shHesl） cells. The SP cells in SUNE1overexpressing Hes1could up to8.6%compared to the control5.3%, while1.0%in Hes1-silenced cells; the SP cells in CNE2overexpressing Hes1could be up to5.3%compared to the control2.0%, while0.4%in Hesl-silenced CNE2cells. Moreover, the ability of tumor sphere formation was enhanced in Hes1-expressing CNE2and SUNE1cells （t=14.902,20.412, P=0.003,0.000）4. Hes1is a direct target of miR-9in NPC cellsAccording to the targeted gene prediction performed by TargetScan and studies reported by Siok-Lay Tan, to explore the mechanism of "sternness" inhibition induced by miR-9, we investigated whether miR-9could regulate Hesl expression in NPC cells, and we found that miR-9overexpression down-regulated Hesl expression in NPC cells, while miR-9inhibitor up-regulated Hesl expression in NPC cells.We performed luciferase reporter assay to determine whether miR-9could directly target the3’-UTR of Hesl in NPC cells. CNE2cells were then transfected with wt or mt3’-UTR vector and miR-9mimics. The results showed a significant decrease of luciferase activity when compared with miR control （t=23.089, P=0.000）. The activity of mt3’-UTR vector was unaffected by a simultaneous transfection with miR-9mimics or miR-9inhibitor （t=1.075,0.218, P=0.343,0.838）. Moreover, cotransfection with miR-9inhibitor and wt3’-UTR vector in CNE2cells led to an increase of luciferase activity （t=11.573, P=0.000）. Taken together, these results strongly suggest that Hesl is a direct target gene of miR-9in NPC cells.5. Hesl rescues the suppressive effect of miR-9on "sternness"Subsequently, we evaluated whether over-expression of Hes1could rescue the suppressive effect of miR-9. miR-9-expressing CNE2cells were infected with LV-Hes1. We showed that ectopic expression of Hesl significantly increased expression of Hesl and other genes related to "sternness", and rescued miR-9-induced reduction of SP cells and tumor spheres （F=162.277, P=0.000）6. Intraturnoral delivery of miR-9leads to regression of tumors in a NPC xenograft modelAs the in vitro and in vivo data showed an antitumorigenic role for miR-9in NPC, we examined the therapeutic potential of synthetic miR-9Agomir or mimics in vivo. In the first pre-experiment, we found that the tumor injected with miR-9Agomir formed cavities along the direction of the needles after injection with a delayed healing, but the controls healed rapidly （data not shown）. In the repeated test, we injected miR-9mimics into two tumors with MaxSuppressorTM In vivo RNA-LANCErⅡ, and found that intratumoral delivery of synthetic miR-9induced a specific inhibitory response and it formed a hollow in the center of one tumor which was interesting. Now, we are carrying out a third time repeat.Part III miR-9triggers EMT and migration by repressing Klf4in NPC1. c-Myc activates miR-9expressionWe established c-Myc-expressing NPC cells, levels of c-Myc and miR-9expression were evaluated by qRT-PCR. The result showed that c-Myc and miR-9levels in HONE1and SUNE1cells harboring c-Myc transgene was significantly higher than that in cells harboring vector control （t=5.199,4.769, P=0.035,0.041） and （t=2.536,4.690, P=0.039,0.005）. Moreover, the levels of c-Myc and miR-9in HONE1and SUNE1cells transfected with sic-Myc was much lower than that transfected with inhibitor control （t=11.545,11.744, P=0.000,0.000） and （t=6.585,10.473, P=0.003,0.000）. In summary, c-Myc efficiently up-regulated the endogenous miR-9expression in NPC cells.2. miR-9-mediated c-Myc induced EMTTo study whether c-Myc can affect EMT process in NPC cells, we detected EMT-related genes using qRT-PCR. The level of E-cadherin seemed to be variable and became higher when c-Myc was highly expressed（t=-44.880, P=0.000）, whereas there was no such relationship with N-cadherin and Vimentin （P>0.05）. Interestingly, we found expreesion of vimentin at protein level was higher when c-Myc was highly expressed, suggesting that c-Myc may have some correlation with E-cadherin and vimentin.3. miR-9-mediated c-Myc enhanced motilityAs shown above, miR-9-mediated c-Myc induced E-cadherin and vimentin alteration. Transwell assay and Boyden chamber assay displayed the enhanced ability of migration and invasion in c-Myc-expressing cells （t=12.231,10.000; P=0.000,0.000）, while miR-9inhibitor could rescue c-Myc-induced enhancement of motility （F=147.016,59.900, P=0.001,0.001）4. miR-9induces EMT-like changes in NPC cells.During culture of miR-9-expressing cells, we were surprised to find that the cells gradually lose the original cobblestone-like or neat form and appeard morphological diversification, some cells became elongated and the antennas of cells were increased. Previous studies suggested that miR-9was associated with EMT in breast cancer, further study on the role of miR-9in EMT was carried out. Western blot showed that E-cadherin and a-catenin which represent epithelial factors were down-regulated in NPC cells overexpressing miR-9, however, up-regulated by miR-9inhibitor. While the expression of N-cadherin and vimentin were up-regulated in miR-9-expressing cells, but down-regulated by miR-9inhibitor, indicating that the ectopic expression of miR-9lead to an EMT-like conversion.5. miR-9promotes cell migration and invasion in NPC.Transwell assay and Boyden chamber assay demonstrated that miR-9overexpression increased the migration and invasion of CNE2and HONE1cells （t=6.533,10.983,13.131,12.351,P=0.000,0.000,0.000,0.000）. Scratch-Migration assay also showed the increased migration of cells overexpressing miR-9as compared with controls.6. miR-9reduces the ability of cell adhesionCell adhesion was performed and analysed by counting the adherent cells, experiments showed that miR-9-expressing cells had lower rates of adhesion than the control cells （F=846.528,751.057, P=0.000,0.000） 7. Effect of miR-9on cytoskeletonWe investigated whether or not miR-9induces changes in cytoskeleton by detect ing F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin, and we also observed the microscopic morphology of the cells using scanning electron microscopy. We found an increased expression of F-actin in miR-9overexpressed cells, with increased pseudopodia.As is known, Rac and Rho regulate the formation of distinct actin filament-based structures, and Cdc42and Rac are also required for the assembly of adhesion sites to the extracellular matrix. We detected an increased expression of Rho and Rac in miR-9-expressing cells. All above results suggested that miR-9was correlated with changes in shape of cells and pseudopodia formation, which is the initial step of tumor metastasis.8. Klf4is a direct target gene of miR-9in NPC cellsWe found that Klf4promoted mesenchymal-epithelial transition （MET） and inhibited migration and metastasis of NPC cells in other studies of our group. According to the targeted gene prediction performed by microrna.org, we investigated whether miR-9could regulate Klf4expression in NPC cells. Western blot suggested a decreased expression of Klf4in miR-9-expressing NPC cells and subcutaneous xenotransplanted tumors, whereas the expression of Klf4was up-regulated by miR-9inhibitor.We performed luciferase reporter assay to determine whether miR-9could directly target the3’-UTR of Klf4in NPC cells. The target sequence of Klf43’-UTR （wt3’-UTR） or the mutant sequence （mt3’-UTR） was cloned into a luciferase reporter vector. CNE2cells were then transfected with wt or mt3’-UTR vector and miR-9mimics. The results showed a significant decrease of luciferase activity when compared with miR control （t=4.380, P=0.012）. The activity of mt3’-UTR vector was unaffected by a simultaneous transfection with miR-9mimics or miR-9inhibitor （t=0.921,0.302, P=0.409,0.777）. Moreover, cotransfection with miR-9inhibitor and wt3’-UTR vector in CNE2cells led to a increase of luciferase activity （t=13.777, P=0.000）. Taken together, all these results strongly suggest that Klf4 was a direct target gene of miR-9in NPC cells.9. miR-9-induced EMT and enhanced motility are mediated by Klf4.Subsequently, we evaluated whether over-expression of Klf4could rescue the improvement of miR-9in motility. HONE1cells overexpressing miR-9were infected with LV-Klf4. Western blot showed that ectopic expression of Klf4significantly increased expression of Klf4and E-cadherin, but decreased expression of N-cadherin. Furthermore, Transwell assay and Boyden chamber assay showed that Klf4rescued the increased ability of cell migration and invasion induced by miR-9（F=137.015, P=0.000）. In sum, we suggest that miR-9promotes EMT and cell motility by targeting Klf4.10. c-Myc promotes tumor metastasis in vivoTo fully explore the effects of c-Myc on metastasis in vivo, we injected1×106CNE2cells infected with LV-c-Myc or the control plasmid （LV-con） subcapsularly into the livers of nude mice,7mice for each group,23days after implantation, all the mice developed tumors in liver at the end of the experiment. The5per7mice in LV-c-Myc group developed lymph node metastases, while2per7mice in control group developed lymph node metastases, suggesting c-Myc can promote tumor metastasis in vivo.In summary, we have found that miR-9triggers EMT and migration in NPC by repressing Klf4.Part IV miR-9modulates the expression of interferon-regulated genes and MHC class I molecules in NPC1. miR-9altered IFN-regulated gene expression in NPC cellsMicroarray analysis of CNE2cells infected with LV-miR-9showed the induction of many IFN-regulated target genes （e.g., IFI44L, PSMB8, IRF5, PSMB10, IFI27, PSB9HUMAN, IFIT2, TRAIL, IFIT1, PSB8HUMAN, IRF1and B2M）, which was further confirmed by qRT-PCR analyses.To fully explore the effects of miR-9on IFN-regulated target genes, CNE2cells were transiently transfected with miR-9mimics or anti-miR-9, respectively. The results of qRT-PCR demonstrated that miR-9mimic upregulated the expression of some IFN-regulated genes （such as IFI44L, PSMB8, IRF5, PSMB10, IFI27, IFIT2, TRAIL, IFIT1, IRF1, B2M and GBP1） in CNE2cells （P<0.05or0.01）, and downregulated the expression of some IFN-regulated genes （such as ISG20and AIM2） in CNE2cells （P<0.05or0.01）, respectively, while anti-miR-9correspondingly decreased the expression of miR-9mimics-upregulated IFN-related genes （i.e., IFI44L, PSMB8, IRF5, PSMB10, IFI27, IFIT2, TRAIL, IFIT1, IRF1, B2M and GBP1） in CNE2cells （P<0.05or0.01）, and increased the expression of miR-9mimics-downregulated gene （i.e., ISG20and AIM2） in CNE2cells （P<0.01）, separately. Collectively, the most significant alteration after miR-9overexpression in NPC cells was the increased expression of genes involved in IFN induction, including IFI44L, PSMB8, IRF5, PSMB10, IFI27, IFIT2, TRAIL, IFIT1, IRF1, B2M and GBP1.2. Induction of the expression of MHC class I molecules by miR-9in NPC cellsMicroarray analysis of CNE2cells infected with LV-miR-9showed the up-regulated expression of MHC class I molecules （HLA-B, HLA-H, HLA-C, HLA-F, Q8WW48HUMAN, NP001004349.1, Q6ZUW0HUMAN and O19682HUMAN） and TAP1gene （encoding antigen peptide transporter1, as ATP-binding cassette （ABC） transporter）. qRT-PCR of HLA-B, HLA-F and TAP1confirmed the microarray changes.Furthermore, when CNE2cells were transiently transfected with miR-9mimics, the expression of MHC Class I molecules （such as HLA-B and HLA-F） and TAP1was significantly upregulated, while CNE2cells transfected with anti-miR-9indicated the decreased expression of HLA-B, HLA-F and TAP1（P<0.05or0.01）. In summary, miR-9plays a significant role in regulating the expression of MHC Class I molecules.3. miR-9overexpression enhanced or reduced IL-related gene expression in NPC cellsThe microarray data derived from CNE2cells infected with LV-miR-9demonstrated the significantly altered expression in human IL-related genes （for example, IL20RB, GALT, IL7, IL1B, IL11, IL1F8, ILIA, IL6and IL7R）, which was further confirmed by qRT-PCR analyses. To fully explore the effects of both miR-9upregulation and endogenous miR-9downregulation on IL-related genes, CNE2cells were transiently transfected with miR-9mimics or anti-miR-9, respectively. The results of qRT-PCR illustrated that miR-9mimics upregulated the expression of some IL-related genes （i.e., IL20RB, GALT, IL7） in CNE2cells （P<0.05or0.01）, while other interleukin-related genes （i.e., IL1B, IL11, IL1F8, ILIA, IL6and IL7R） were remarkably downregulated by miR-9mimics in CNE2cells （P<0.05or0.01）. Conversely, anti-miR-9correspondingly decreased the expression of IL20RB, GALT and IL7in CNE2cells （P<0.05or0.01）, and increased the expression of IL1B, IL11, IL1F8, IL1A, IL6and IL7R in CNE2cells （P<0.05or0.01）, separately. Collectively, the most significant alteration after miR-9overexpression was the decreased expression of IL-related genes, including IL1B, IL11, IL1F8, IL1A, IL6and IL7R.Conclusions1. miR-9inhibits cell proliferation and tumorigenesis by targeting CCNG1, while miR-9promotes EMT and migration by repressing Klf4, suggesting that miR-9plays different roles in modulating cell proliferation, and EMT, migration&metastasis by directly targets CCNG1and Klf4in NPC, respectively;2. miR-9significantly suppresses cell proliferation and tumorigenesis of NPC by directly downregulating Hesl to deplete cancer stem cells;3. miR-9modulates the expression of interferon-regulated genes and MHC class I molecules in NPC.

miR-9對(duì)鼻咽癌增殖、腫瘤干細(xì)胞“干性”、EMT和轉(zhuǎn)移的調(diào)控作用及機(jī)制

摘要3-17ABSTRACT17-32前言35-44    參考文獻(xiàn)40-44第一章 MIR-9靶向調(diào)控CCNG1抑制鼻咽癌細(xì)胞的增殖44-74    一 材料與方法44-55    二 結(jié)果55-62    三 討論62-63    參考文獻(xiàn)63-65    附圖65-74第二章 M[R-9對(duì)鼻咽癌腫瘤干細(xì)胞的調(diào)控作用及機(jī)制研究74-91    一 材料與方法74-76    二 結(jié)果76-80    三 討論80-82    參考文獻(xiàn)82-84    附圖84-91第三章 C-MYC通過(guò)MIR-9靶向調(diào)控KLF4促進(jìn)鼻咽癌細(xì)胞EMT、侵襲和轉(zhuǎn)移的作用及機(jī)制研究91-114    一 材料與方法91-94    二 結(jié)果94-100    三 討論100-102    參考文獻(xiàn)102-104    附圖104-114第四章 MIR-9調(diào)控鼻咽癌細(xì)胞中免疫和炎癥相關(guān)基因的表達(dá)114-133    一 材料與方法114-115    二 結(jié)果115-124    三 討論124-126    參考文獻(xiàn)126-129    附圖129-133全文小結(jié)133-134附錄134-135攻讀學(xué)位期間成果135-136致謝136-138統(tǒng)計(jì)學(xué)合格證明138

  本文關(guān)鍵詞：miR-9對(duì)鼻咽癌增殖、腫瘤干細(xì)胞“干性”、EMT和轉(zhuǎn)移的調(diào)控作用及機(jī)制，由筆耕文化傳播整理發(fā)布。