本文關(guān)鍵詞：探討K1f4在鼻咽癌增殖、EMT和侵襲轉(zhuǎn)移中的調(diào)控作用　出處：《南方醫(yī)科大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： NPC Klf4 細(xì)胞增殖 MET 侵襲轉(zhuǎn)移

【摘要】：鼻咽癌(nasopharyngeal carcinoma, NPC)是我國(guó)南方和東南亞常見的惡性腫瘤之一,是指發(fā)生在鼻咽粘膜的惡性腫瘤。其發(fā)病與環(huán)境、飲食、遺傳以及EBV(Epstein Barr Virus)感染等因素有關(guān),發(fā)病年齡大多為中年人,流行病學(xué)研究發(fā)現(xiàn),鼻咽癌發(fā)病有地域、民族和家族聚集現(xiàn)象。目前NPC的治療方法有放療和化療,以放射治療為主,早期治愈率較高,但因其惡性程度高,早期既有淋巴結(jié)轉(zhuǎn)移,因此就診時(shí)往往已達(dá)中晚期,此時(shí)其治愈率卻不理想。而且目前,對(duì)于NPC的具體發(fā)病機(jī)制還不是十分清楚,因此對(duì)于進(jìn)一步了解NPC的發(fā)生發(fā)展以及與之相關(guān)的治療手段值得我們深入研究。 Klf4(Kruppel-like factor4)屬于Klf家族,為一種真核生物中具有鋅指樣結(jié)構(gòu)的蛋白轉(zhuǎn)錄因子。Klf4首先由Shields JM等于1996年被描述,他們從小鼠的NIH3T3細(xì)胞cDNA文庫(kù)中分離得到該基因,因其主要在消化道表達(dá),故又被稱之為胃腸富集Klf(gut-enriched Kruppel-like factor, GKlf)。Klf4蛋白含有多個(gè)功能區(qū)域,包括N末端的富含酸性氨基酸的轉(zhuǎn)錄激活區(qū),能夠參與蛋白-蛋白之間的相互作用；以及c-端能與DNA結(jié)合區(qū)域結(jié)合的鋅指結(jié)構(gòu)區(qū)域(易與CACCC序列元件和(或)富含GC序列的靶基因調(diào)節(jié)序列結(jié)合),另外還有臨近N-端鋅指結(jié)構(gòu)區(qū)域的轉(zhuǎn)錄抑制區(qū)。Klf4主要在皮膚、口腔、胃腸道上皮、血管內(nèi)皮等處表達(dá)豐富。 Klf4在體內(nèi)多個(gè)方面發(fā)揮著重要的作用,參與正常組織細(xì)胞的增殖、分化以及凋亡等多種生物學(xué)行為,并能夠維持干細(xì)胞的自我更新。Klf4通過與不同的靶基因結(jié)合,可以激活或抑制轉(zhuǎn)錄。在結(jié)腸上皮中,Klf4可以通過抑制細(xì)胞增殖和促進(jìn)其分化來維持結(jié)腸上皮的體內(nèi)平衡,另外,Klf4還是皮膚上皮分化的一個(gè)重要分子；研究發(fā)現(xiàn),在血管損傷后,Klf4可以在平滑肌細(xì)胞中迅速表達(dá)上調(diào)。 除此之外,在腫瘤組織中,Klf4可以通過與不同靶基因的作用,發(fā)揮癌基因或抑癌基因的作用,從而多角度的參與腫瘤的發(fā)生發(fā)展。在結(jié)腸癌中,因Klf4的甲基化和雜合性的丟失,證明Klf4在結(jié)腸癌為低表達(dá),并且Klf4通過與APC基因結(jié)合來抑制Wnt通路從而進(jìn)一步抑制結(jié)腸癌的發(fā)生；另外,更多的證據(jù)表明,Klf4同樣可以抑制胃癌的發(fā)生。研究發(fā)現(xiàn),Klf4在食管癌、膀胱癌和肺癌的組織中均表達(dá)降低,起抑癌基因的作用,而在乳腺癌組織中,其表達(dá)升高,發(fā)揮癌基因的作用。研究證實(shí),Klf4表達(dá)升高為喉鱗癌的一個(gè)早期事件。研究表明,無論Klf4作為癌基因或抑癌基因,可能與不同的細(xì)胞環(huán)境、其他基因的表達(dá)模式和單個(gè)細(xì)胞染色質(zhì)環(huán)境有關(guān)。 迄今,Klf4在NPC發(fā)病中所發(fā)揮的作用尚不清楚,那么其在NPC中的表達(dá)情況是怎樣的,又是如何參與NPC的發(fā)生發(fā)展呢?這些都值得我們進(jìn)一步探討。 鑒于Klf4目前的研究現(xiàn)狀,我們檢測(cè)了NPC細(xì)胞及組織中Klf4的表達(dá)譜,基于攜帶Klf4的慢病毒載體建立穩(wěn)定過表達(dá)Klf4的NPC細(xì)胞株,并通過檢測(cè)NPC細(xì)胞株的生物學(xué)行為及EMT相關(guān)基因的變化,來闡明Klf4在NPC發(fā)生發(fā)展中所發(fā)揮的功能,同時(shí)我們還合成了下調(diào)Klf4表達(dá)的siRNA,反面驗(yàn)證Klf4在NPC中的作用。為此我們進(jìn)行了如下研究。 方法： 第一章NPC細(xì)胞株和組織標(biāo)本中Klf4的表達(dá)譜 1.通過qRT-PCR檢測(cè)CNE1、CNE2、C666-1、 HNE1、5-8F、6-1OB、SUNE1和HONE18種NPC細(xì)胞株中Klf4表達(dá)譜(以NP69作為對(duì)照)。 2.收集NPC組織標(biāo)本21例和慢性鼻咽炎癥組織標(biāo)本6例,應(yīng)用qRT-PCR檢測(cè)Klf4的表達(dá)譜,結(jié)果分析運(yùn)用2-ΔΔCt方法進(jìn)行比較。 第二章Klf4對(duì)NPC細(xì)胞增殖的影響 1.建立穩(wěn)定過表達(dá)Klf4的NPC細(xì)胞株 利用慢病毒表達(dá)載體pLenti-Klf4生產(chǎn)攜帶Klf4的慢病毒,收集病毒上清分別感染NPC細(xì)胞株5-8F、HONE1和SUNE1,48-72h后于倒置熒光顯微鏡下檢測(cè)EGFP表達(dá)以觀察感染情況,若感染效率低于90%,應(yīng)用流式細(xì)胞儀分選EGFP+細(xì)胞,大量擴(kuò)增細(xì)胞并保種；提取細(xì)胞RNA及蛋白,通過qRT-PCR、 Western blot檢測(cè)穩(wěn)定細(xì)胞株中Klf4的表達(dá)水平。 2.Klf4過表達(dá)對(duì)NPC細(xì)胞體外增殖能力的影響 通過CCK8實(shí)驗(yàn)、平板克隆和細(xì)胞周期檢測(cè)細(xì)胞增殖能力改變。 3.下調(diào)Klf4表達(dá)對(duì)NPC細(xì)胞體外增殖能力的影響 3.1.化學(xué)合成兩條Klf4siRNA,瞬時(shí)轉(zhuǎn)染NPC細(xì)胞(5-8F),48h收集細(xì)胞并提取RNA及蛋白,通過qRT-PCR、Western blot檢測(cè)兩條Klf4siRNA的抑制效果,選取抑制效率高的一條進(jìn)行后續(xù)實(shí)驗(yàn)； 3.2.Klf4siRNA瞬時(shí)轉(zhuǎn)染5-8F. HONE1,待細(xì)胞生長(zhǎng)狀態(tài)良好時(shí),通過CCK8實(shí)驗(yàn)檢測(cè)細(xì)胞增殖能力。 4.Klf4過表達(dá)對(duì)NPC細(xì)胞體內(nèi)增殖能力的影響 4.1培養(yǎng)擴(kuò)增5-8F-Klf4、5-8F-con細(xì)胞； 4.2選取生長(zhǎng)狀態(tài)良好的細(xì)胞接種于裸鼠皮下,建立NPC異位移植瘤模型,進(jìn)一步研究Klf4過表達(dá)對(duì)鼻咽癌細(xì)胞體內(nèi)成瘤能力的影響。待腫瘤長(zhǎng)徑為0.5cm時(shí)隔天測(cè)量瘤徑,最后計(jì)算腫瘤體積：腫瘤組織取材、固定、染色、并運(yùn)用免疫組織化學(xué)方法檢測(cè)腫瘤組織中Ki-67. BrdU和p21表達(dá),最后統(tǒng)計(jì)分析。 第三章Klf4對(duì)NPC細(xì)胞EMT和遷移能力的影響 1.培養(yǎng)過表達(dá)Klf4的細(xì)胞株,Transwell小室檢測(cè)細(xì)胞遷移能力改變,qRT-PCR、Western blot檢測(cè)E-cadherin、a-catenin、Fibronectin、N-caherin及vimentin等EMT相關(guān)基因的變化。 2. Klf4siRNA瞬時(shí)轉(zhuǎn)染NPC細(xì)胞,Western blot檢測(cè)EMT相關(guān)基因的變化,Transwell小室實(shí)驗(yàn)評(píng)價(jià)下調(diào)Klf4后細(xì)胞體外遷移能力的變化。 結(jié)果： 第一章Klf4在NPC細(xì)胞株和組織標(biāo)本中均低表達(dá) (1) qRT-PCR檢測(cè)結(jié)果顯示：與NP69相比,Klf4在8株NPC細(xì)胞中的表達(dá)下調(diào)(F=34.891,p0.001)。 (2) qRT-PCR檢測(cè)NPC組織標(biāo)本中Klf4表達(dá),結(jié)果顯示：Klf4在NPC組織中的表達(dá)低于鼻咽慢性炎癥組織(t=-2.599,p=0.015)。 第二章Klf4抑制NPC細(xì)胞的增殖 1.成功構(gòu)建穩(wěn)定過表達(dá)Klf4的NPC細(xì)胞株 利用攜帶Klf4的慢病毒感染NPC細(xì)胞株,48-72h后于倒置熒光顯微鏡下檢測(cè)有EGFP表達(dá)確認(rèn)感染成功,隨后提取細(xì)胞RNA及蛋白,qRT-PCR檢測(cè)證實(shí)攜帶有Klf4轉(zhuǎn)基因的NPC細(xì)胞株中Klf4表達(dá)升高(p＜0.01)(表2-1,圖2-3)；Werstern blot結(jié)果顯示,與對(duì)照組相比,Klf4在HONE1、5-8F兩株細(xì)胞中表達(dá)均升高,但SUNE1細(xì)胞中Klf4的表達(dá)升高不明顯,結(jié)果如圖(2-3),上述結(jié)果提示已成功構(gòu)建穩(wěn)定過表達(dá)Klf4的NPC細(xì)胞(5-8F-Klf4、HONE1-Klf4)。 2.Klf4過表達(dá)后NPC細(xì)胞體外增殖能力減弱 穩(wěn)定過表達(dá)Klf4細(xì)胞株構(gòu)建完成后,通過細(xì)胞周期、平板克隆、CCK8檢測(cè)細(xì)胞增殖能力改變。CCK8結(jié)果：過表達(dá)Klf4使HONE1、5-8F細(xì)胞的增殖速率均減慢(p＜0.001,圖2-4)；平板克隆結(jié)果：Klf4過表達(dá)使HONE1、5-8F細(xì)胞的克隆形成能力降低,其克隆形成率分別降低了67%(p＜0.05)和32.17%(p＜0.01)；細(xì)胞周期分布顯示：與對(duì)照組相比,Klf4過表達(dá)使S期細(xì)胞比例減少15.43%(p＜0.05),G1期細(xì)胞比例顯著增高(p＜0.01),該結(jié)果表明Klf4過表達(dá)可誘導(dǎo)細(xì)胞周期S期阻滯(圖2-6)。 3.下調(diào)Klf4表達(dá)后NPC細(xì)胞體外增殖能力增強(qiáng) 3.1將化學(xué)合成的兩條Klf4siRNA分別轉(zhuǎn)染至NPC細(xì)胞中,48h后收集細(xì)胞RNA和蛋白,通過qRT-PCR、Western blot檢測(cè)兩條Klf4siRNA的抑制效果,結(jié)果顯示：5-8F轉(zhuǎn)染Klf4siRNA#1后,與對(duì)照組相比,Klf4的抑制效率為93.71%(p＜0.001,圖2-7),而轉(zhuǎn)染Klf4siRNA#2后Klf4的干擾效率為87.3%(p＜0.001,圖2-7),提示Klf4siRNA#1的抑制效率較高,故選擇Klf4siRNA#1做后續(xù)實(shí)驗(yàn)。 3.2瞬時(shí)轉(zhuǎn)染Klf4siRNA細(xì)胞培養(yǎng)48h后,通過CCK8實(shí)驗(yàn)檢測(cè)下調(diào)Klf4表達(dá)后細(xì)胞的體外增殖能力,結(jié)果顯示：下調(diào)Klf4后,細(xì)胞增殖能力增強(qiáng)(p＜0.01,圖2-8)。 4.過表達(dá)Klf4可抑制NPC細(xì)胞體內(nèi)增殖能力 裸鼠皮下成瘤實(shí)驗(yàn)結(jié)果顯示：接種5-8F-Klf4細(xì)胞的6只裸鼠中,有5只小鼠成瘤,而對(duì)照組細(xì)胞6只小鼠均成瘤,且5-8F-Klf4細(xì)胞所成腫瘤體積及瘤重明顯小于對(duì)照組(p0.05),此外,免疫組化結(jié)果發(fā)現(xiàn),Klf4過表達(dá)組BrdU和Ki-67陽性的細(xì)胞的顯著降低(p＜0.01),而p21陽性的細(xì)胞則顯著升高p＜0.01)。 第三章Klf4對(duì)NPC細(xì)胞EMT和遷移能力的影響 1.Klf4對(duì)NPC細(xì)胞EMT的影響 通過qRT-PCR、Werstern blot檢測(cè)過表達(dá)Klf4細(xì)胞株中EMT相關(guān)基因的變化。qRT-PCR結(jié)果顯示：在5-8F、HONE1細(xì)胞中過表達(dá)Klf4后,與對(duì)照組比較,兩株細(xì)胞中E-cadherin均表達(dá)上調(diào)(p＜0.05,圖3-1),而在HONE1、5-8F中間質(zhì)細(xì)胞標(biāo)志物N-cadherin表達(dá)水平下降(p＜0.05,圖3-1),HONE1細(xì)胞中間質(zhì)細(xì)胞標(biāo)志物Fibronectin表達(dá)下降(p＜0.05)。 Werstern blot實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),與對(duì)照組相比,在5-8F、HONE1細(xì)胞中過表達(dá)Klf4后,E-cadherin表達(dá)上調(diào),而N-cadherin和vimentin表達(dá)水平下降(圖3-1)。 下調(diào)NPC細(xì)胞株中內(nèi)源性Klf4表達(dá)水平后,Werstern blot檢測(cè)細(xì)胞中EMT相關(guān)基因的變化。結(jié)果顯示,與對(duì)照組細(xì)胞相比(HONE1-Klf4、5-8F-Klf4), E-cadherin表達(dá)下調(diào),而N-cadherin和vimentin表達(dá)水平升高(圖3-2)。 2.Klf4對(duì)NPC細(xì)胞遷移能力的影響 Transwell實(shí)驗(yàn)結(jié)果顯示：在5-8F、HONE1細(xì)胞中,過表達(dá)Klf4后,與空載細(xì)胞相比,細(xì)胞穿膜數(shù)量明顯減少(p＜0.01,表3-1和圖3-3)。下調(diào)Klf4表達(dá)后,細(xì)胞穿膜數(shù)量比對(duì)照組增加(p＜0.05,圖3-4) 結(jié)論：Klf4抑制NPC細(xì)胞增殖、誘導(dǎo)MET；并抑制NPC細(xì)胞體外遷移。
[Abstract]:Nasopharyngeal carcinoma (nasopharyngeal carcinoma NPC) is one of South China and the Southeast Asia common malignant tumors that occur in nasopharyngeal mucosa malignant tumors. The incidence of genetic and environment, diet, and EBV (Epstein Barr Virus) infection and other factors, the age of onset mostly middle-aged, epidemiological studies have found that the incidence of nasopharyngeal carcinoma regional, ethnic and familial aggregation. Current treatment of NPC with radiotherapy and chemotherapy, radiation therapy, early cure rate is high, but because of the high degree of malignancy, early lymph node metastasis, the treatment often has reached advanced stage, the cure rate is not ideal. But at present, for NPC the specific pathogenesis is still not very clear, so for the further understanding of the occurrence and development of NPC and treatment related worthy of our in-depth study.
Klf4 (Kruppel-like factor4) belongs to the Klf family, is a kind of eukaryotes with zinc finger structure protein transcription factor.Klf4 by Shields JM in 1996 was the first to describe, they isolated the gene from the cDNA Library of NIH3T3 cells in mice, mainly because of its expression in the digestive tract, so it is also called gastrointestinal the enrichment of Klf (gut-enriched Kruppel-like factor, GKlf).Klf4 protein contains a plurality of functional areas, rich in acidic amino acids including N transcription at the end of the active region, can be involved in protein-protein interaction; and the c- terminal can be combined with the DNA region binding zinc finger domain (with CACCC sequence elements and (or) target gene regulatory sequences with GC rich sequences), in addition to N- near the end of the zinc finger transcription inhibition zone of.Klf4 structure in the region is mainly in the skin, oral cavity, gastrointestinal epithelium, the expression of rich vascular endothelial and so on.
Klf4 plays an important role in many aspects of the body, in normal tissue cell proliferation, differentiation, apoptosis and other biological behavior, and be able to maintain stem cell self-renewal by.Klf4 and target genes of different combination, can activate or inhibit transcription in the colon in the skin, Klf4 can inhibit and promote their differentiation cell proliferation to maintain homeostasis, colonic epithelium in Klf4 or skin epithelial differentiation is an important molecule; found in vascular injury, Klf4 can rapidly up-regulated expression in smooth muscle cells.
In addition, in tumor tissues, Klf4 can be obtained with different target genes play a role in oncogenes or tumor suppressor genes, which participate in the occurrence and development of tumor multi angle. In colon cancer, due to Klf4 methylation and loss of heterozygosity, that low expression of Klf4 in colon cancer and, combined with the APC gene to inhibit the Wnt pathway to inhibit colon cancer by Klf4; in addition, more evidence that Klf4 can also inhibit the occurrence of gastric cancer. The study found that Klf4 in esophageal cancer, bladder cancer and lung cancer tissues were lower expression of tumor suppressor gene plays a role in breast cancer tissues, its expression increased, play the role of oncogenes. The study confirmed that the increased expression of Klf4 is an early event in laryngeal squamous cell carcinoma. The results show that both Klf4 as oncogenes or tumor suppressor genes, with different cell environment, gene expression The model is related to the chromatin environment of a single cell.
So far, the role of Klf4 in the pathogenesis of NPC is not clear. What is its expression in NPC and how to participate in the occurrence and development of NPC? All these are worthy of further discussion.
In view of the present situation of Klf4, we detected the expression of Klf4 NPC cells and tissue spectrum, lentivirus vector based on Klf4 to establish a stable NPC expressing Klf4 cell line, and by detecting the changes of NPC cell lines and biological behavior of EMT related genes, to elucidate the Klf4 in the NPC play in the development of at the same time, we also synthesized the siRNA expression of Klf4, negative verification of Klf4 in NPC. We carried out the following research.
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