ENO1基因在鼻咽癌发病中的作用研究
摘要
研究背景和目的：
鼻咽癌好发于中国南方,特别是广东省,发病率最高因此又被称为“广东瘤”,是一种具有地域性和家族聚集性的高度恶性肿瘤。鼻咽癌的发生是一个多阶段、多途径、多机制的过程。流行病学调查显示,鼻咽癌的发生涉及到遗传倾向性和环境致癌因素,很可能是遗传易感性个体,接受了致癌物的作用而发生的。其中包括各种环境因素、EBV感染和遗传及表遗传学的改变导致瘤基因过表达及抑瘤基因表达下调或缺失,经过癌前病变,最终发生鼻咽癌。
肿瘤的生成涉及多种基因和基因以外的变化,任何单独一种基因的改变不足以致瘤,多种基因变化的积累才能引起控制细胞生长和分化的机制紊乱,使细胞的生长失控而瘤变。在这些基因的变化中,最常发生两类基因的异常变化,即瘤基因及肿瘤抑制基因。鼻咽癌的生成同样涉及多基因的表达改变。在过去几十年中,虽然我们对于鼻咽癌发生的分子基础认识已经有了很大的提高,但这些认识还是远远不能完全揭示鼻咽癌的发病机理。因此,鼻咽癌相关新基因的发现和研究仍将有助于进一步揭示鼻咽癌的发病机理。
Enolasel基因的表达随细胞病理生理、代谢、发育状况的不同而改变,其主要编码两种蛋白。多数情况下,a-enolase(即ENO1)是烯醇化酶家族中3种同功酶之一,是一个高度保守的胞浆糖酵解酶,既可催化糖酵解过程中2-磷酸-D-甘油酸(PGA)向磷酸-烯醇式丙酮酸(PEP)的转化,又可在糖原合成过程中,催化逆向反应,即作为磷酸丙酮酸水合酶,使PEP向PGA转化,在细胞能量代谢过程中起重要的作用。另一种称为c—myc启动子结合蛋白(MBP—1),不具有酶活性,可表达于细胞核,与核DNA c—myc启动子结合,负向调控c—myc的表达。c-myc基因是一种细胞核内癌基因,它编码的核内转化蛋白p62对正常细胞生长和分化起重要调节作用。c-myc表达活性与细胞生长分裂关系密切,据认为它是诱导增殖和凋亡双重作用的基因。正常情况下C-myc基因不显示转录活性或低水平表达。c-myc基因被激活后可致其产物p62过度表达而堆积在细胞内,使细胞获得永生。近年来多项研究表明c-myc基因在肿瘤的发生发展中起重要作用,其在肿瘤组织中的表达率与患者的生存期呈显著相关。这也提示ENO1除了作为糖酵解反应的限速酶外,还参与细胞的其他生物学活动。
在早期对ENO1研究中发现,ENO1能够抑制肿瘤细胞的生长。但有趣的是,与前述ENO1抑制肿瘤细胞增生的作用截然不同,近年来越来越多的研究表明,ENO1在肿瘤发生及发展中所发挥的作用并非如早年研究所指出的,是一个具有潜在抑癌能力的基因。与之相反,近年来的研究结果不断表明,ENO1很可能是一个在肿瘤生发发展和转移等多个环节过程中均发挥作用的重要癌基因。目前提出ENO1可能具有癌基因功能的多项研究主要集中在肝癌、非小细胞肺癌、甲状腺嗜酸细胞瘤以及黑色素细胞瘤等肿瘤上。通过蛋白质组学,western blot和免疫组化等技术发现,ENO1在这些肿瘤的细胞系、活体组织中有较高水平表达。并且,多篇文献指出,在多种肿瘤患者的外周血中,检测出的ENO1自身抗体的水平与该类肿瘤的分化、分期及预后显示出明显的正相关关系。在对ENO1促进肿瘤细胞生长机制的探讨中,大多数认为ENO1在肿瘤细胞生长过程中的能量代谢过程中起到积极作用,尤其是大多数肿瘤在高代谢情况下,其生长对于糖酵解作用获能的依赖成为目前研究的重点。在增殖较快的肿瘤细胞中,ENO1有相对高水平的表达；而在非增殖的角质化细胞中,ENO1的合成明显减少。由于糖酵解过程的变化与细胞增殖有关,故推测,在肿瘤细胞增殖过程中,ENO1的表达可以通过影响糖酵解,三羧酸循环,以及随后的氧化磷酸化酶作用物的水平,进而影响ATP的合成,这对肿瘤细胞的生长十分重要。
但目前有关ENO1在鼻咽癌中的作用的研究还未见报道。在本研究所方唯意等研究中,利用基因芯片技术对8组NPC病人标本及对照,两组细胞株进行了基因筛选,结果显示,在病理标本及细胞株中,ENO1的表达均显著提高。因此,本课题旨在研究ENO1在鼻咽癌细胞及组织中的表达特性,并进一步通过改变ENO1表达水平来观察其对鼻咽癌细胞生物学特性的影响。为进一步明确鼻咽癌的发病机制奠定良好的基础。
方法：
1.鼻咽癌中ENO1表达特性的鉴定及临床意义。
利用免疫组化检测120例鼻咽癌与40例正常鼻咽中ENO1基因的表达情况。
2.ENO1表达沉默对鼻咽癌细胞HONE1生物学特性的影响。
荧光定量PCR检测鼻咽癌细胞系(6个细胞系,5-8F,6-10B,SUNE1,CNE2,CNE1,HONE1)中ENO1的表达情况,筛选高表达ENO1的细胞株。设计并体外化学合成针对ENO1基因的小干扰RNA序列,以脂质体(lipofectamineTM 2000)为载体转染鼻咽癌细胞株HONE1,采用荧光定量PCR法测定转染后HONE1细胞ENO1及内参GAPDH的mRNA表达,测定干扰效率,以筛选出最有效干扰序列。后将最有效的干扰片段转染至HONE1 NPC细胞。荧光定量PCR法检测ENO1在干扰前后mRNA的变化。以转染细胞系为样本,利用transwell小室实验检测ENO 1被沉默后HONE1细胞体外迁移运动能力的改变,利用Boyden小室实验检测ENO1沉默对NPC细胞的侵袭迁移能力的影响；MTT、流式细胞仪检测ENO1表达水平改变后对NPC细胞增殖、细胞周期分布、细胞凋亡和停泊非依赖性生长能力的影响。
结果：
1.鼻咽癌中ENO1表达特性的鉴定及临床意义
(1)免疫组织化学检测ENO1蛋白在鼻咽癌组织中的表达：SP免疫组化结果表明,ENO1表达主要定位在鼻咽癌细胞胞浆中,阳性表达结果在镜下呈棕黄色。而在慢性鼻咽炎组织及肿瘤周围相对正常组织则为弱阳性或阴性表达。我们利用ENO1兔抗人多克隆抗体对1 20例鼻咽癌标本和40例正常鼻咽组织进行了免疫组化检测,ENO1的高表达率在鼻咽癌组织中为60.0%(72/120),而在正常组织中为27.5%(11/40)(χ2=11.424,p=0.001,)。两组ENO1的表达具有显著性差异。而在对临床分期与淋巴结转移、远处转移的分析中,结果显示ENO1的表达与临床分期显著相关(χ2=13.919,P=0.003)。临床T4期组要低于临床T1-T3期组,即ENO1阳性表达者,多为临床早中期；ENO1阴性表达者转移发生率低,多为临床晚期。但ENO1蛋白蛋白表达与颈淋巴结转移及远处转移无关(P>0.05)。上述实验结果提示我们ENO1在鼻咽癌表达上调,且与转移及临床分期显著相关。但其表达上调对于鼻咽癌细胞生物学特性的影响以及调控的具体机制仍不明确。
2.ENO1表达沉默对鼻咽癌细胞CNE2生物学特性的影响
(1)荧光定量PCR检测：ENO1在鼻咽癌细胞中的表达采用QPCR检测ENO1基因在鼻咽癌细胞株中的表达情况,结果单因素方差分析显示6种细胞株中ENO1的表达差异具有显著性(F=13.067,P=0.000)。其中筛选出HONE1为高表达ENO1的细胞株,作为下一步实验的干扰对象。
(2)瞬时转染siRNA-ENO1-2片段入HONE1细胞系。采用MTT法检测ENO1基因沉默后细胞的体外增殖情况,结果显示,与HONE1-NC和HONE1细胞相比,HONE1-siENO1细胞的增殖速度显著减慢,并且呈时间依赖关系(F=4.764,P=0.001)。细胞仪检测两组细胞的细胞周期分布,结果显示两组细胞的细胞周期分布并无显著性差异。综上所述,ENO1表达沉默显著抑制了鼻咽癌细胞的体外增殖能力。
(3)瞬时转染siRNA-ENO1-2片段入HONE1细胞系。采用Transwell小室检测ENO1基因被沉默后HONE1细胞体外迁徙运动能力的变化,结果显示：结果发现两组细胞运动能力的差异具有显著性,(t=-3.163,P=0.004)。与空载体对照HONE-NC细胞相比,干扰ENO1后的HONE1细胞穿过膜的细胞数显著增加,其运动能力明显增强。
Boyden小室检测ENO1基因沉默后HONE1细胞体外侵袭运动能力的改变,结果显示,两组细胞侵袭能力的差异具有显著性(t=-13.758,P=0.000),与HONE1-NC细胞相比,HONE1-siENO1细胞穿过聚碳酸酯膜的细胞数增加,HONE1-siENO1细胞的侵袭能力显著增强。以上结果表明,ENO1基因沉默后显著提高了鼻咽癌细胞的体外迁移和侵袭能力。
结论：
1.较正常鼻咽组织,ENO1在鼻咽癌组织中表达明显上调,且与鼻咽癌的侵袭与转移密切相关；
2.ENO1基因沉默后显著抑制了鼻咽癌细胞的体外增殖,但促进了迁移和侵袭能力。ENO1与鼻咽癌的发生发展密切相关；
Nasopharyngeal carcinoma(NPC) is one of the commonest carcinomas with a high degree of malignant Phenotype in Southern China.especially those of Cantonese origin. Both the geographic pattern and familial aggregation of incidence are two main characteristics of NPC. The carcinogenesis of nasopharyngeal epithelium is a multi-stage, multi-path and multi-mechanism process, which is involved in the effects of several factors including genetic predisposition, environmental carcinogen and Epstein barr virus (EBV).The changes of various environmental factors, EBV infection, genetics and epigenetics cause the overexpression of oncogenes and downregulated or null expression of tumor suppressors in nasopharyngeal epithelium, Which will lead to the transition of epithelium to Precancerous lesion, and finally be transformed to NPC.
Carcinogenesis is associated with the participation of expression alteration of multiple genes.The expression change of any single gene does not have the ability todrive the malignant transformation of normal epithelium.In the past few years, although the knowledge of molecular mechanisms about NPC has greatly inereased. Its pathogenesis remains to be clarified.
ENO1 gene primarily encodes one of three enolase isoenzymes found in mammals. it is alpha-enolase,an enzyme in the glycolytic pathway,catalysing the formation of phosphoenolpyruvate from 2-phosphoGly-cerate. This gene also encodes a shorter monomeric structural lens protein, was earlier known as the Myc-binding protein-1 (MBP-1). This shorter protein is localized to the cell nuclei, and has been found to bind to an element in the c-myc promoter. Recent researches show that the c-myc protooncogene is a DNA-binding phosphoprotein that plays an important role in cell growth regulation and differentiation. It's expression level is independent predictive factor for suviver time of patients with tumors.
In the early study found that of ENO1, ENO1 could inhibit the growth of tumor. But it is interesting that, recent researches show that ENO1 gene is not, as earlier pointed out,an tumor suppressor gene.In contrast, ENO1 is likely to be play a important role in pathogenesis and progression of tumor.Currently many studies about ENO1 gene function focused on liver cancer, non-small cell lung cancer, hurthle cell cancer,melanoma and other tumors.Through proteomics,western blot and immu-nohistochemical techniques that, ENO1 in these tumors cell lines and tissue have high levels of expression. fuhermore, many papers noted that there was significant relationship between levels of autoantibodies ENO1 in the peripheral blood of patients with multiple tumors and such tumor's stage and prognosis. In the study of the mechanism of ENO1, majority studies suggest that ENO1 play a positive role in the energy metabolism of tumor cells, especially in high metabolic cases.compared with non-proliferation cell lines, the rapid proliferation cell lines have high level of ENO1 expression significantly.So speculate that in the process of cell proliferation, ENO1 expression can affect glycolysis, Krebs cycle and the subsequent oxidative phosphorylation enzyme substrate level, thereby affecting the synthesis of ATP, which is importantant for the growth of tumor cells.
But till now the relationship between the ENO1 gene and NPC has not been reported. In Professor Fang Weiyi previous work, we used gene chip technology to analyse 8 groups of NPC patients and control samples, two cell lines, results showed that in pathological specimens and cell lines, ENO1 expression were significantly increased. Based on these results, we want to study that the characteristics of ENO1 in NPC cell, and further by changing the expression levels of ENO1 to observe its biological characteristics of NPC cells. To further define the pathogenesis of nasopharyngeal carcinoma lay a good foundation.
CONTENTS AND METHODS
1.Identification of expression characteristics of ENO1 in NPC and its significance.
The expression of ENO1 protein in NPC tissues was detected by immune-ohistochemistry (IHC).
2.Effects of ENO1 knock-down on biological behaviors of HONE1 cells.
The expression of ENO1 mRNA in 6 NPC cell lines (5-8F,6-1 OB, SUNE1,CNE2,CNE1,HONE1) was detected by Quantitative real time-PCR respectively. The high-expressing ENO1 clones were screened.
Structure of ENO1 mRNA was analysed by software to select targeted interference sites. Three siRNA and one negative control siRNA were constructed and chemically synthesized,then be transfected into NPC cell line Phonel by positive ion liposome Lipofectamine2000.Use Quantitative real time-PCR to detect the expression of ENOl mRNA,then estimate the interference efficieny,screening the most effective siRNA.
Specific anti-ENO1 short RNA and control vector were constructed respectively, and then were transfected into HONE1 cells. Quantitative real time PCR was used to detect the efficiency of interference of cell clone.after the inhibition of the endogenous ENO1, the growth, cell cycle and invasion of cells were detected by MTT, Transwell, FACS and Boyden chamber assay in vitro respectively.
RESULTS
1. Identification of expression characteristics of ENO1 in NPC tissues.
In an immunohistochemical study, ENO1 staining was mostly observed in the nucleus of carcinoma cells. No specific ENO1 staining was observed in normal adjacent nasopharyngeal epithelial cells and stroma cells in surrounding tissues. ENO1 was found to express in 60.0%(70/120) cases of NPC, higher then 27.5% (11/40) cases of chronic nasopharyngitis tissue samples (χ2=11.424, P=0.001).
The relationship between clinicopathological features and ENO1 expression in NPC was analyzed with Pearson Chi-Square test. But there was no significant relationship between expression of ENO1 and lymph node metastasis and postradiotherapy distant metastasis (P> 0.05). Interestingly, we observed that The positive expression rate of ENO1 protein in stage T1+T2+T3was higher than that of in patients with T4 (x2=13.919, P=0.003).
2. Effects of ENO1 knock-down on biologieal behaviors of HONE1.
Quantitive real-time PCR was used to detect the expression of ENOl in 6 NPC cell lines (5-8F,6-10B, SUNE1,CNE2,CNE1,HONE1).The result shows that the HONE1 line is a high-exprssing clone.
siRNA-ENO1 was successfully transfected into NPC cancer cell line HONE1, ENO1-siRNA were successfully transfected into HONE1 after 12h detected by QPCR, ENO1 mRNA levels in HONE1 cells was significantly inhibited, siRNA-2 resulted in the highest inhibiting rate.HONE1-siRNA cells showed a significantly reduced proliferation compared with HONE1 and HONE1-NC cells as determined by in vitro MTT assay (F=4.764, P=0.001). However, the cell cycle distribution detected by flow cytometry has no significant differences between each other. These results indicated knock-down of ENO1 could reduce proliferation of HONE1 cells.
The results of in vitro proliferation assay showed that compared to aNegative control HONE1 cell, the proliferation ability of HONE1-siRNA Cells was significantly inereased (F=3.163.P=0.004). Furthermore, invasion assay showed that HONE1-siRNA cells had significantly increased invasiveness as compared with HONE1-NC cells (t=13.758, P=0.000). These results showed knock-down of ENO1 results in a leads to an upregulated invasion of HONE1 cells.
Conclusions:
l.The expression of ENO1 is upregulated in NPC and correlates statistically with the malignant status of NPC.
2. ENO1 promotes the growth of NPC cells in vitro,but inhibited invasion. These results indicated that ENO1 might play a pivotal role in the tumorigenesis and progression of NPC.
鼻咽癌好发于中国南方,特别是广东省,发病率最高因此又被称为“广东瘤”,是一种具有地域性和家族聚集性的高度恶性肿瘤。鼻咽癌的发生是一个多阶段、多途径、多机制的过程。流行病学调查显示,鼻咽癌的发生涉及到遗传倾向性和环境致癌因素,很可能是遗传易感性个体,接受了致癌物的作用而发生的。其中包括各种环境因素、EBV感染和遗传及表遗传学的改变导致瘤基因过表达及抑瘤基因表达下调或缺失,经过癌前病变,最终发生鼻咽癌。
肿瘤的生成涉及多种基因和基因以外的变化,任何单独一种基因的改变不足以致瘤,多种基因变化的积累才能引起控制细胞生长和分化的机制紊乱,使细胞的生长失控而瘤变。在这些基因的变化中,最常发生两类基因的异常变化,即瘤基因及肿瘤抑制基因。鼻咽癌的生成同样涉及多基因的表达改变。在过去几十年中,虽然我们对于鼻咽癌发生的分子基础认识已经有了很大的提高,但这些认识还是远远不能完全揭示鼻咽癌的发病机理。因此,鼻咽癌相关新基因的发现和研究仍将有助于进一步揭示鼻咽癌的发病机理。
Enolasel基因的表达随细胞病理生理、代谢、发育状况的不同而改变,其主要编码两种蛋白。多数情况下,a-enolase(即ENO1)是烯醇化酶家族中3种同功酶之一,是一个高度保守的胞浆糖酵解酶,既可催化糖酵解过程中2-磷酸-D-甘油酸(PGA)向磷酸-烯醇式丙酮酸(PEP)的转化,又可在糖原合成过程中,催化逆向反应,即作为磷酸丙酮酸水合酶,使PEP向PGA转化,在细胞能量代谢过程中起重要的作用。另一种称为c—myc启动子结合蛋白(MBP—1),不具有酶活性,可表达于细胞核,与核DNA c—myc启动子结合,负向调控c—myc的表达。c-myc基因是一种细胞核内癌基因,它编码的核内转化蛋白p62对正常细胞生长和分化起重要调节作用。c-myc表达活性与细胞生长分裂关系密切,据认为它是诱导增殖和凋亡双重作用的基因。正常情况下C-myc基因不显示转录活性或低水平表达。c-myc基因被激活后可致其产物p62过度表达而堆积在细胞内,使细胞获得永生。近年来多项研究表明c-myc基因在肿瘤的发生发展中起重要作用,其在肿瘤组织中的表达率与患者的生存期呈显著相关。这也提示ENO1除了作为糖酵解反应的限速酶外,还参与细胞的其他生物学活动。
在早期对ENO1研究中发现,ENO1能够抑制肿瘤细胞的生长。但有趣的是,与前述ENO1抑制肿瘤细胞增生的作用截然不同,近年来越来越多的研究表明,ENO1在肿瘤发生及发展中所发挥的作用并非如早年研究所指出的,是一个具有潜在抑癌能力的基因。与之相反,近年来的研究结果不断表明,ENO1很可能是一个在肿瘤生发发展和转移等多个环节过程中均发挥作用的重要癌基因。目前提出ENO1可能具有癌基因功能的多项研究主要集中在肝癌、非小细胞肺癌、甲状腺嗜酸细胞瘤以及黑色素细胞瘤等肿瘤上。通过蛋白质组学,western blot和免疫组化等技术发现,ENO1在这些肿瘤的细胞系、活体组织中有较高水平表达。并且,多篇文献指出,在多种肿瘤患者的外周血中,检测出的ENO1自身抗体的水平与该类肿瘤的分化、分期及预后显示出明显的正相关关系。在对ENO1促进肿瘤细胞生长机制的探讨中,大多数认为ENO1在肿瘤细胞生长过程中的能量代谢过程中起到积极作用,尤其是大多数肿瘤在高代谢情况下,其生长对于糖酵解作用获能的依赖成为目前研究的重点。在增殖较快的肿瘤细胞中,ENO1有相对高水平的表达；而在非增殖的角质化细胞中,ENO1的合成明显减少。由于糖酵解过程的变化与细胞增殖有关,故推测,在肿瘤细胞增殖过程中,ENO1的表达可以通过影响糖酵解,三羧酸循环,以及随后的氧化磷酸化酶作用物的水平,进而影响ATP的合成,这对肿瘤细胞的生长十分重要。
但目前有关ENO1在鼻咽癌中的作用的研究还未见报道。在本研究所方唯意等研究中,利用基因芯片技术对8组NPC病人标本及对照,两组细胞株进行了基因筛选,结果显示,在病理标本及细胞株中,ENO1的表达均显著提高。因此,本课题旨在研究ENO1在鼻咽癌细胞及组织中的表达特性,并进一步通过改变ENO1表达水平来观察其对鼻咽癌细胞生物学特性的影响。为进一步明确鼻咽癌的发病机制奠定良好的基础。
方法：
1.鼻咽癌中ENO1表达特性的鉴定及临床意义。
利用免疫组化检测120例鼻咽癌与40例正常鼻咽中ENO1基因的表达情况。
2.ENO1表达沉默对鼻咽癌细胞HONE1生物学特性的影响。
荧光定量PCR检测鼻咽癌细胞系(6个细胞系,5-8F,6-10B,SUNE1,CNE2,CNE1,HONE1)中ENO1的表达情况,筛选高表达ENO1的细胞株。设计并体外化学合成针对ENO1基因的小干扰RNA序列,以脂质体(lipofectamineTM 2000)为载体转染鼻咽癌细胞株HONE1,采用荧光定量PCR法测定转染后HONE1细胞ENO1及内参GAPDH的mRNA表达,测定干扰效率,以筛选出最有效干扰序列。后将最有效的干扰片段转染至HONE1 NPC细胞。荧光定量PCR法检测ENO1在干扰前后mRNA的变化。以转染细胞系为样本,利用transwell小室实验检测ENO 1被沉默后HONE1细胞体外迁移运动能力的改变,利用Boyden小室实验检测ENO1沉默对NPC细胞的侵袭迁移能力的影响；MTT、流式细胞仪检测ENO1表达水平改变后对NPC细胞增殖、细胞周期分布、细胞凋亡和停泊非依赖性生长能力的影响。
结果：
1.鼻咽癌中ENO1表达特性的鉴定及临床意义
(1)免疫组织化学检测ENO1蛋白在鼻咽癌组织中的表达：SP免疫组化结果表明,ENO1表达主要定位在鼻咽癌细胞胞浆中,阳性表达结果在镜下呈棕黄色。而在慢性鼻咽炎组织及肿瘤周围相对正常组织则为弱阳性或阴性表达。我们利用ENO1兔抗人多克隆抗体对1 20例鼻咽癌标本和40例正常鼻咽组织进行了免疫组化检测,ENO1的高表达率在鼻咽癌组织中为60.0%(72/120),而在正常组织中为27.5%(11/40)(χ2=11.424,p=0.001,)。两组ENO1的表达具有显著性差异。而在对临床分期与淋巴结转移、远处转移的分析中,结果显示ENO1的表达与临床分期显著相关(χ2=13.919,P=0.003)。临床T4期组要低于临床T1-T3期组,即ENO1阳性表达者,多为临床早中期；ENO1阴性表达者转移发生率低,多为临床晚期。但ENO1蛋白蛋白表达与颈淋巴结转移及远处转移无关(P>0.05)。上述实验结果提示我们ENO1在鼻咽癌表达上调,且与转移及临床分期显著相关。但其表达上调对于鼻咽癌细胞生物学特性的影响以及调控的具体机制仍不明确。
2.ENO1表达沉默对鼻咽癌细胞CNE2生物学特性的影响
(1)荧光定量PCR检测：ENO1在鼻咽癌细胞中的表达采用QPCR检测ENO1基因在鼻咽癌细胞株中的表达情况,结果单因素方差分析显示6种细胞株中ENO1的表达差异具有显著性(F=13.067,P=0.000)。其中筛选出HONE1为高表达ENO1的细胞株,作为下一步实验的干扰对象。
(2)瞬时转染siRNA-ENO1-2片段入HONE1细胞系。采用MTT法检测ENO1基因沉默后细胞的体外增殖情况,结果显示,与HONE1-NC和HONE1细胞相比,HONE1-siENO1细胞的增殖速度显著减慢,并且呈时间依赖关系(F=4.764,P=0.001)。细胞仪检测两组细胞的细胞周期分布,结果显示两组细胞的细胞周期分布并无显著性差异。综上所述,ENO1表达沉默显著抑制了鼻咽癌细胞的体外增殖能力。
(3)瞬时转染siRNA-ENO1-2片段入HONE1细胞系。采用Transwell小室检测ENO1基因被沉默后HONE1细胞体外迁徙运动能力的变化,结果显示：结果发现两组细胞运动能力的差异具有显著性,(t=-3.163,P=0.004)。与空载体对照HONE-NC细胞相比,干扰ENO1后的HONE1细胞穿过膜的细胞数显著增加,其运动能力明显增强。
Boyden小室检测ENO1基因沉默后HONE1细胞体外侵袭运动能力的改变,结果显示,两组细胞侵袭能力的差异具有显著性(t=-13.758,P=0.000),与HONE1-NC细胞相比,HONE1-siENO1细胞穿过聚碳酸酯膜的细胞数增加,HONE1-siENO1细胞的侵袭能力显著增强。以上结果表明,ENO1基因沉默后显著提高了鼻咽癌细胞的体外迁移和侵袭能力。
结论：
1.较正常鼻咽组织,ENO1在鼻咽癌组织中表达明显上调,且与鼻咽癌的侵袭与转移密切相关；
2.ENO1基因沉默后显著抑制了鼻咽癌细胞的体外增殖,但促进了迁移和侵袭能力。ENO1与鼻咽癌的发生发展密切相关；
Nasopharyngeal carcinoma(NPC) is one of the commonest carcinomas with a high degree of malignant Phenotype in Southern China.especially those of Cantonese origin. Both the geographic pattern and familial aggregation of incidence are two main characteristics of NPC. The carcinogenesis of nasopharyngeal epithelium is a multi-stage, multi-path and multi-mechanism process, which is involved in the effects of several factors including genetic predisposition, environmental carcinogen and Epstein barr virus (EBV).The changes of various environmental factors, EBV infection, genetics and epigenetics cause the overexpression of oncogenes and downregulated or null expression of tumor suppressors in nasopharyngeal epithelium, Which will lead to the transition of epithelium to Precancerous lesion, and finally be transformed to NPC.
Carcinogenesis is associated with the participation of expression alteration of multiple genes.The expression change of any single gene does not have the ability todrive the malignant transformation of normal epithelium.In the past few years, although the knowledge of molecular mechanisms about NPC has greatly inereased. Its pathogenesis remains to be clarified.
ENO1 gene primarily encodes one of three enolase isoenzymes found in mammals. it is alpha-enolase,an enzyme in the glycolytic pathway,catalysing the formation of phosphoenolpyruvate from 2-phosphoGly-cerate. This gene also encodes a shorter monomeric structural lens protein, was earlier known as the Myc-binding protein-1 (MBP-1). This shorter protein is localized to the cell nuclei, and has been found to bind to an element in the c-myc promoter. Recent researches show that the c-myc protooncogene is a DNA-binding phosphoprotein that plays an important role in cell growth regulation and differentiation. It's expression level is independent predictive factor for suviver time of patients with tumors.
In the early study found that of ENO1, ENO1 could inhibit the growth of tumor. But it is interesting that, recent researches show that ENO1 gene is not, as earlier pointed out,an tumor suppressor gene.In contrast, ENO1 is likely to be play a important role in pathogenesis and progression of tumor.Currently many studies about ENO1 gene function focused on liver cancer, non-small cell lung cancer, hurthle cell cancer,melanoma and other tumors.Through proteomics,western blot and immu-nohistochemical techniques that, ENO1 in these tumors cell lines and tissue have high levels of expression. fuhermore, many papers noted that there was significant relationship between levels of autoantibodies ENO1 in the peripheral blood of patients with multiple tumors and such tumor's stage and prognosis. In the study of the mechanism of ENO1, majority studies suggest that ENO1 play a positive role in the energy metabolism of tumor cells, especially in high metabolic cases.compared with non-proliferation cell lines, the rapid proliferation cell lines have high level of ENO1 expression significantly.So speculate that in the process of cell proliferation, ENO1 expression can affect glycolysis, Krebs cycle and the subsequent oxidative phosphorylation enzyme substrate level, thereby affecting the synthesis of ATP, which is importantant for the growth of tumor cells.
But till now the relationship between the ENO1 gene and NPC has not been reported. In Professor Fang Weiyi previous work, we used gene chip technology to analyse 8 groups of NPC patients and control samples, two cell lines, results showed that in pathological specimens and cell lines, ENO1 expression were significantly increased. Based on these results, we want to study that the characteristics of ENO1 in NPC cell, and further by changing the expression levels of ENO1 to observe its biological characteristics of NPC cells. To further define the pathogenesis of nasopharyngeal carcinoma lay a good foundation.
CONTENTS AND METHODS
1.Identification of expression characteristics of ENO1 in NPC and its significance.
The expression of ENO1 protein in NPC tissues was detected by immune-ohistochemistry (IHC).
2.Effects of ENO1 knock-down on biological behaviors of HONE1 cells.
The expression of ENO1 mRNA in 6 NPC cell lines (5-8F,6-1 OB, SUNE1,CNE2,CNE1,HONE1) was detected by Quantitative real time-PCR respectively. The high-expressing ENO1 clones were screened.
Structure of ENO1 mRNA was analysed by software to select targeted interference sites. Three siRNA and one negative control siRNA were constructed and chemically synthesized,then be transfected into NPC cell line Phonel by positive ion liposome Lipofectamine2000.Use Quantitative real time-PCR to detect the expression of ENOl mRNA,then estimate the interference efficieny,screening the most effective siRNA.
Specific anti-ENO1 short RNA and control vector were constructed respectively, and then were transfected into HONE1 cells. Quantitative real time PCR was used to detect the efficiency of interference of cell clone.after the inhibition of the endogenous ENO1, the growth, cell cycle and invasion of cells were detected by MTT, Transwell, FACS and Boyden chamber assay in vitro respectively.
RESULTS
1. Identification of expression characteristics of ENO1 in NPC tissues.
In an immunohistochemical study, ENO1 staining was mostly observed in the nucleus of carcinoma cells. No specific ENO1 staining was observed in normal adjacent nasopharyngeal epithelial cells and stroma cells in surrounding tissues. ENO1 was found to express in 60.0%(70/120) cases of NPC, higher then 27.5% (11/40) cases of chronic nasopharyngitis tissue samples (χ2=11.424, P=0.001).
The relationship between clinicopathological features and ENO1 expression in NPC was analyzed with Pearson Chi-Square test. But there was no significant relationship between expression of ENO1 and lymph node metastasis and postradiotherapy distant metastasis (P> 0.05). Interestingly, we observed that The positive expression rate of ENO1 protein in stage T1+T2+T3was higher than that of in patients with T4 (x2=13.919, P=0.003).
2. Effects of ENO1 knock-down on biologieal behaviors of HONE1.
Quantitive real-time PCR was used to detect the expression of ENOl in 6 NPC cell lines (5-8F,6-10B, SUNE1,CNE2,CNE1,HONE1).The result shows that the HONE1 line is a high-exprssing clone.
siRNA-ENO1 was successfully transfected into NPC cancer cell line HONE1, ENO1-siRNA were successfully transfected into HONE1 after 12h detected by QPCR, ENO1 mRNA levels in HONE1 cells was significantly inhibited, siRNA-2 resulted in the highest inhibiting rate.HONE1-siRNA cells showed a significantly reduced proliferation compared with HONE1 and HONE1-NC cells as determined by in vitro MTT assay (F=4.764, P=0.001). However, the cell cycle distribution detected by flow cytometry has no significant differences between each other. These results indicated knock-down of ENO1 could reduce proliferation of HONE1 cells.
The results of in vitro proliferation assay showed that compared to aNegative control HONE1 cell, the proliferation ability of HONE1-siRNA Cells was significantly inereased (F=3.163.P=0.004). Furthermore, invasion assay showed that HONE1-siRNA cells had significantly increased invasiveness as compared with HONE1-NC cells (t=13.758, P=0.000). These results showed knock-down of ENO1 results in a leads to an upregulated invasion of HONE1 cells.
Conclusions:
l.The expression of ENO1 is upregulated in NPC and correlates statistically with the malignant status of NPC.
2. ENO1 promotes the growth of NPC cells in vitro,but inhibited invasion. These results indicated that ENO1 might play a pivotal role in the tumorigenesis and progression of NPC.
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[30]Suzuki A, Iizuka A, Komiyama M, Takikawa M, Kume A, Tai S, Ohshita C, Kurusu A, Nakamura Y, Yamamoto A, Yamazaki N, Yoshikawa S, Kiyohara Y, Akiyama Y. Identification of Melanoma Antigens Using a Serological Proteome Approach (SERPA).Cancer Genomics Proteomics.2010;7(1):17-23
[31]Stieruma R, GaspariM, Dommels Y, et al. Proteome analysis reveals novel proteins associated with p roliferation and differentiation of thecolorectal cancer cell line Caco-2 [J]. Biochim Biophys Acta,2003,1650(122):73-91.
[32]Fang W, Li X, Jiang Q, Liu Z, Yang H, Wang S, Xie S, Liu Q, Liu T, Huang J, Xie W, Li Z, Zhao Y, Wang E, Marincola FM, Yao K. Transcriptional patterns, biomarkers and pathways characterizing nasopharyngeal carcinoma of Southern China.J Transl Med.2008;6:32.
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[3]Takashima M, Kuramitsu Y, Yokoyama Y, Iizuka N, Fujimoto M, Nishisaka T, Okita K, Oka M, Nakamura K.Proteomics. Overexpression of alpha enolase in hepatitis C virus-related hepatocellular carcinoma:association with tumor progression as determined by proteomic analysis.2005;5(6):1686-1692.
[4]Baris O, Savagner F, Nasser V, Loriod B, Granjeaud S, Guyetant S, Franc B, Rodien P, Rohmer V, Bertucci F, Birnbaum D, Malthiery Y, Reynier P, Houlgatte R.Transcriptional profiling reveals coordinated up-regulation of oxidative metabolism genes in thyroid oncocytic tumors[J]. J Clin Endocrinol Metab.2004;89(2):994-1005.
[5]Suzuki A, Iizuka A, Komiyama M, Takikawa M, Kume A, Tai S, Ohshita C, Kurusu A, Nakamura Y, Yamamoto A, Yamazaki N, Yoshikawa S, Kiyohara Y, Akiyama Y. Identification of Melanoma Antigens Using a Serological Proteome Approach (SERPA) [J].Cancer Genomics Proteomics.2010; 7(1):17-23
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[6]Hsu CG, Lin LY, Ko JL, Yang SF, Chang H, Lin CY, Tsai HT, Chen SC, Chen SC, Wang PH. High expression of human nonmetastatic clone 23 type 1 in cancer of uterine cervix and its association with poor cell differentiation and worse overall survival[J].J Surg Oncol.2008 Nov 1;98(6):448-456.
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[8]Okabe-Kado J, Kasukabe T, Honma Y, Kobayashi H, Maseki N, Kaneko Y. Extracellular NM23 protein promotes the growth and survival of primary cultured human acute myelogenous leukemia cells[J].Cancer Sci.2009 Oct; 100(10):1885-1894.
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[10]Wang YF, Chen JY, Chang SY, Chiu JH, Li WY, Chu PY, Tai SK, Wang LS.Nm23-Hl expression of metastatic tumors in the lymph nodes is a prognostic indicator of oral squamous cell carcinoma[J].Int J Cancer.2008 Jan 15;122(2):377-386.
[11]Horak CE, Mendoza A, Vega-Valle E, Albaugh M, Graff-Cherry C, McDermott WG, Hua E, Merino MJ, Steinberg SM, Khanna C, Steeg PS.Nm23-H1 suppresses metastasis by inhibiting expression of the lysophosphatidic acid receptor EDG2[J].Cancer Res.2007 Dec 15;67(24):11751-11759.
[12]Jung S, Paek YW, Moon KS, Wee SC, Ryu HH, Jeong YI, Sun HS, Jin YH, Kim KK, Ahn KY. Expression of Nm23 in gliomas and its effect on migration and invasion in vitro[J].Anticancer Res.2006;26(1A):249-258.
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[29]Baris O, Savagner F, Nasser V, Loriod B, Granjeaud S, Guyetant S, Franc B, Rodien P, Rohmer V, Bertucci F, Birnbaum D, Malthiery Y, Reynier P, Houlgatte R.Transcriptional profiling reveals coordinated up-regulation of oxidative metabolism genes in thyroid oncocytic tumors. J Clin Endocrinol Metab.2004;89(2):994-1005.
[30]Suzuki A, Iizuka A, Komiyama M, Takikawa M, Kume A, Tai S, Ohshita C, Kurusu A, Nakamura Y, Yamamoto A, Yamazaki N, Yoshikawa S, Kiyohara Y, Akiyama Y. Identification of Melanoma Antigens Using a Serological Proteome Approach (SERPA).Cancer Genomics Proteomics.2010;7(1):17-23
[31]Stieruma R, GaspariM, Dommels Y, et al. Proteome analysis reveals novel proteins associated with p roliferation and differentiation of thecolorectal cancer cell line Caco-2 [J]. Biochim Biophys Acta,2003,1650(122):73-91.
[32]Fang W, Li X, Jiang Q, Liu Z, Yang H, Wang S, Xie S, Liu Q, Liu T, Huang J, Xie W, Li Z, Zhao Y, Wang E, Marincola FM, Yao K. Transcriptional patterns, biomarkers and pathways characterizing nasopharyngeal carcinoma of Southern China.J Transl Med.2008;6:32.
[1]Luo J, Xiao J, Tao Z. Detection of c-myc gene expression in nasopharyngeal carcinoma by nonradioactive in situ hybridization and immunehistochemistry. Chin Med J (Engl),1997,110 (3):229.
[2]Chang GC, Liu KJ, Hsieh CL, Hu TS, Charoenfuprasert S, Liu HK, Luh KT, Hsu LH, Wu CW, Ting CC, Chen CY, Chen KC, Yang TY, Chou TY, Wang WH, Whang-Peng J, Shih. NY. Identification of alpha-enolase as an autoantigen in lung cancer:its overexpression is associated with clinical outcomes[J]. Clin Cancer Res.2006 Oct 1;12(19):5746-5754.
[3]Takashima M, Kuramitsu Y, Yokoyama Y, Iizuka N, Fujimoto M, Nishisaka T, Okita K, Oka M, Nakamura K.Proteomics. Overexpression of alpha enolase in hepatitis C virus-related hepatocellular carcinoma:association with tumor progression as determined by proteomic analysis.2005;5(6):1686-1692.
[4]Baris O, Savagner F, Nasser V, Loriod B, Granjeaud S, Guyetant S, Franc B, Rodien P, Rohmer V, Bertucci F, Birnbaum D, Malthiery Y, Reynier P, Houlgatte R.Transcriptional profiling reveals coordinated up-regulation of oxidative metabolism genes in thyroid oncocytic tumors[J]. J Clin Endocrinol Metab.2004;89(2):994-1005.
[5]Suzuki A, Iizuka A, Komiyama M, Takikawa M, Kume A, Tai S, Ohshita C, Kurusu A, Nakamura Y, Yamamoto A, Yamazaki N, Yoshikawa S, Kiyohara Y, Akiyama Y. Identification of Melanoma Antigens Using a Serological Proteome Approach (SERPA) [J].Cancer Genomics Proteomics.2010; 7(1):17-23
[1]Fuchs,U.and A.Borkhardt,The application of siRNA technology to cancer biology discovery. Adv Cancer Res,2007.96:75-102.
[2]2. Aoki,Y,Cioca,D P,Oidaira,H et al.,RNA interference may be more potent than antisense RNA in human cancer cell lines.Clin Exp Pharmacol Physiol,2003.30(1-2):96-102.
[3]3. Vucic,DStennicke,H R Pisabarro M.,et al.,ML-IAP,a novel inhibitor of apoptosis that is prefere-ntially expressed in human melanomas.Curr Biol,2000.10(21):1359-66.
[4]Nykanen,A.,B-Haley and P.D.Zamore,ATP requirements and small interfering RNA structure in the RNA interference pathway.Cell,2001.107(3):309-21.
[5]Katayama M, Nakano H, Ishiuchi A, et al. Protein pattern difference in the colon cancer Protein pattern difference in the colon cancer cell lines examined by two-dimensional differential in-gel electrophoresis and mass spectrometry。 [J]. Surg Today,2006,36 (12):1085-1093.
[6]Hsu CG, Lin LY, Ko JL, Yang SF, Chang H, Lin CY, Tsai HT, Chen SC, Chen SC, Wang PH. High expression of human nonmetastatic clone 23 type 1 in cancer of uterine cervix and its association with poor cell differentiation and worse overall survival[J].J Surg Oncol.2008 Nov 1;98(6):448-456.
[7]Hu BQ, Xiao GM, Luo Y, Zhou SL, Xi XP, Ouyang QC. [Screening of molecular markers of lung squamous cell carcinoma by microarrays[J].Zhonghua Zhong Liu Za Zhi.2004 Nov;26(l 1):660-663.
[8]Okabe-Kado J, Kasukabe T, Honma Y, Kobayashi H, Maseki N, Kaneko Y. Extracellular NM23 protein promotes the growth and survival of primary cultured human acute myelogenous leukemia cells[J].Cancer Sci.2009 Oct; 100(10):1885-1894.
[9]Liu SJ, Sun YM, Tian DF, He YC, Zeng L, He Y, Ling CQ, Sun SH.Downregulated NM23-H1 expression is associated with intracranial invasion of nasopharyngeal carcinoma[J].Br J Cancer.2008 Jan 29;98(2):363-369.
[10]Wang YF, Chen JY, Chang SY, Chiu JH, Li WY, Chu PY, Tai SK, Wang LS.Nm23-Hl expression of metastatic tumors in the lymph nodes is a prognostic indicator of oral squamous cell carcinoma[J].Int J Cancer.2008 Jan 15;122(2):377-386.
[11]Horak CE, Mendoza A, Vega-Valle E, Albaugh M, Graff-Cherry C, McDermott WG, Hua E, Merino MJ, Steinberg SM, Khanna C, Steeg PS.Nm23-H1 suppresses metastasis by inhibiting expression of the lysophosphatidic acid receptor EDG2[J].Cancer Res.2007 Dec 15;67(24):11751-11759.
[12]Jung S, Paek YW, Moon KS, Wee SC, Ryu HH, Jeong YI, Sun HS, Jin YH, Kim KK, Ahn KY. Expression of Nm23 in gliomas and its effect on migration and invasion in vitro[J].Anticancer Res.2006;26(1A):249-258.