端粒酶和PinX1基因在食管癌中的表达及生物学意义
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摘要
目的:食管癌(esophageal carcinoma, EC)是全世界最常见的消化道恶性肿瘤之一,我国的食管癌发病率较高,河北省磁县更是食管癌的高发区,严重影响着人们的生活质量和生命健康。端粒、端粒酶在肿瘤细胞的永生化和演进中扮演重要角色。端粒酶的激活可以导致细胞无限制地增殖和肿瘤的发生。端粒(telomere)作为分子钟控制着人类细胞的复制与衰老,与细胞的寿命密切相关。正常细胞每次分裂,端粒DNA长度会缩短55～200 bp,经历多次分裂后缩短至其下限时,细胞最终凋亡或死亡。端粒酶(telomerase)是一种以自身RNA为模板的逆转录酶,其核心组分之一端粒酶逆转录酶(human telomerase reverse transcriptase , hTERT)是端粒酶发挥活化作用的重要途径,它的表达调控在端粒酶活性调节中起关键作用,是细胞呈现端粒酶活性的决定因素。已有大量研究发现,在恶性肿瘤的发生中存在有端粒DNA长度的缩短,并且端粒酶的激活维持了大多数组织的端粒长度,抵消了细胞分裂导致的端粒DNA消耗。说明端粒缩短在恶性肿瘤的演变中起重要作用。已有大量研究表明,人体正常细胞除少量细胞如精原细胞、胚胎细胞、干细胞外,大多数细胞的端粒酶几乎都是无活性的,而与之相反在绝大多数恶性肿瘤中端粒酶活性高表达,表达率高达90%以上。因此端粒酶的活化可能是恶性肿瘤发生发展的多种基因改变中起到了至关重要的作用,并且可以作为最具普遍性和特异性的肿瘤分子标记物,在肿瘤的诊断和治疗中具有重要的地位。而抑制端粒酶的活性,将有可能阻止肿瘤细胞恶性增殖,这种特性使端粒酶成为当今肿瘤诊治研究的热点。
本实验利用了多项实验方法检测端粒DNA长度、端粒酶hTERT蛋白含量及细胞内DNA含量在食管癌高发区现场人食管癌、异型增生及正常食管的表达,探讨与食管癌发生及生物学行为的关系,试图为食管癌的早期诊断提供客观参考指标,并为揭示食管癌的发生、发展的机制提供理论依据。采用端粒重复序列扩增反应(TRAP)-ELISA和TRAP -银染法半定量技术,检测食管癌及异型增生组织中端粒酶活性的表达,探讨端粒酶活性与食管癌发生发展及其与临床病理因素的关系。
PinX1基因作为端粒酶的内源性抑制基因,近年来被认为是一种潜在的抑癌基因,其表达可能与多种肿瘤的发生发展有关。有实验证实在胃癌、大肠癌及白血病患者中PinX1表达明显下降,端粒酶活性增强。但是也有研究显示PinX1在肝癌细胞中的表达与正常组织无显著差异,而与之相应的是此类肿瘤中大部分端粒酶活性也增强。目前PinX1基因在肿瘤细胞中的作用和对端粒酶活性的调控及在细胞癌变中的机制尚不十分清楚。有关PinX1基因在食管癌中的研究国内外也鲜有报道。PinX1基因的作用越来越引起人们的重视,但到目前为止,PinX1基因在食管癌中生物学意义、作用机理国内外的报道很少,并且没有适合研究PinX1基因的食管癌细胞模型。由于基因转染已经成为治疗肿瘤等基因表达异常的有效手段,那么,通过外源性过表达PinX1基因能否抑制食管癌细胞的增殖?是否可以抑制端粒酶活性?本实验中利用多项技术,应用细胞模型研究PinX1在人食管癌、异型增生组织及正常食管组织中的表达,探讨PinX1与食管癌发生发展及与食管癌临床分期、病理分型及预后的相关性,并分析与端粒酶活性表达的相关性。研究转染PinX1基因前后细胞增殖凋亡等的变化,初步了解该基因对食管癌细胞的生物学影响。同时我们应用TRAP-ELISA和TRAP-银染法检测转染前后Eca109细胞端粒酶活性的变化,进一步探讨PinX1基因与端粒酶的关系。
方法:
1端粒DNA长度及端粒酶hTERT在食管癌高发区人群中的表达及生物学意义
收集采集自河北省磁县食管癌高发区现场的咬检组织1000例,所有患者未接受过化疗和放疗。从中选取130例,根据病情分为3组,第1组为正常食管组织36例,第2组为异型增生组织50例,第3组为食管鳞癌组织44例。经病理组织学诊断,50例异型增生食管粘膜组织中,22例为轻度增生组织,28例为重度增生组织;44例食管鳞状细胞癌中,20例为高分化鳞癌、11例为中分化鳞癌,13例为低分化鳞癌,25例侵及纤维膜及周围软组织,19例未侵及纤维膜;20例为淋巴结转移,24例为无淋巴结转移。采用流式细胞术方法(flow cytometry, FCM)和免疫组织化学方法(immunohistochemistry, IHC)检测测端粒DNA长度、端粒酶hTERT蛋白含量及细胞内DNA含量在食管癌高发区现场人食管癌、异型增生及正常食管的表达,分析与食管癌发生及与临床病理特征的关系。
2食管癌中的端粒酶活性和PinX1的表达及其生物学意义
收集食管鳞状细胞癌手术切除标本130例,每例标本分别取癌组织、癌旁组织(距癌边缘2-5cm)及手术切缘正常食管粘膜(距癌边缘5cm以上)。从中选取50例食管鳞状细胞癌、异型增生及正常食管粘膜组织。经病理组织学诊断,50例食管鳞状细胞癌,39例为高中分化鳞癌,11例为低分化鳞癌,34例侵及纤维膜,16例未达到纤维膜,17例淋巴结转移,33例未转移。采用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)、流式细胞术方法(flow cytometry, FCM)、TRAP-ELISA和TRAP-银染法检测食管癌组织、异型增生、正常粘膜组织中端粒酶活性和PinX1基因和蛋白的表达,分析端粒酶活性和PinX1表达与食管癌临床病理特征的关系。
3基因转染PinX1对食管癌Eca109细胞增殖和凋亡以及端粒酶活性的影响
利用分子生物学技术构建pCDNA3.1(+)-PinX1重组质粒。采用脂质体转染方法将pCDNA3.1(+)-PinX1重组质粒转染Eca109细胞,同时也设置空载体pCDNA3.1转染组。转染72小时后用G418进行稳定转染细胞的筛选,筛选出来的阳性细胞分别命名为Eca109/PinX1、Eca109/pCDNA3.1细胞。
应用激光共聚焦方法测定转染效率,MTS方法检测过表达PinX1对Eca109/ PinX1、Eca109/pCDNA3.1、Eca109细胞生长抑制作用。FCM方法检测细胞生长周期和细胞凋亡。RT-PCR、FCM、Western-blot方法检测Eca109/PinX1、Eca109/pCDNA3.1、Eca109细胞中PinX1基因及蛋白的表达,TRAP-ELISA和TRAP-银染法检测端粒酶活性在细胞中表达。
结果:
1端粒DNA长度及端粒酶hTERT在食管癌高发区人群中的表达及生物学意义
FCM检测端粒长度结果显示,端粒DNA长度在食管癌组织中表达量与正常食管组织相比明显缩短,在食管正常组织、异型增生组织、癌组织的表达量呈现逐渐降低趋势(P<0.01);端粒DNA长度在食管轻度增生、重度增生之间呈逐渐降低趋势,重度增生组与轻度增生组相比明显缩短(P<0.01),轻度增生组与正常组比较无显著性差异(P>0.05)。随着细胞学分级增高,端粒DNA长度Q-FISH值逐渐降低,端粒DNA长度与细胞学分级呈负相关(r=-0.79, P <0.01)。
FCM检测端粒酶hTERT基因蛋白含量,在癌、重度增生、轻度增生和正常食管的FI值分别为1.84±0.21、1.39±0.24、1.13±0.19和0.87±0.18,癌组hTERT含量高于重度增生组(P <0.01)、重度增生组高于轻度增生组(P<0.01),轻度增生组与正常组相比无显著性差别。随着细胞学分级增高, hTERT的FI值随之升高, hTERT蛋白含量与细胞学分级呈正相关关系(r=0.83, P<0.01)。根据FI值>1.0为阳性表达的判断标准,轻度增生组、重度增生组和癌组hTERT蛋白阳性表达率分别为68.18%、92.86%和95.45%,重度增生组和癌组的表达率都高于轻度增生组(P<0.01)。
FCM检测正常组、轻度增生组、重度增生组和癌组细胞DNA含量,DI值分别为1.03±0.34、1.06±0.28、1.17±0.25和1.54±0.37,正常组、轻度增生组和重度增生组的DI值呈上升趋势(P>0.05),癌组的DI值明显高于重度增生组。DI值与细胞学分级正相关(r=0.62, P<0.01);正常组、轻度增生组、重度增生组和癌组DNA异倍体率分别为11.11(4/36)、9.09(2/22)、28.57(8/28)和81.82(36/44),癌组异倍体率高于重度增生组(P<0.01),重度增生组高于轻度增生组(P<0.05);从正常组到癌组PI值分别为10.98±4.11、14.15±4.76、18.97±5.27和28.79±5.47,随着细胞学分级的升高,PI值增大,癌组显著高于重度增生组(P<0.01),重度增生组高于轻度增生组(P<0.05)。PI值与细胞学分级正相关(r=0.64, P<0.01)。
130例食管样本的DI值与PI值高度正相关(r=0.76, P<0.01);端粒长度Q-FISH值与端粒酶hTERT蛋白FI值负相关(r=-7.49, P<0.01);端粒Q-FISH值与DI值负相关(r=-0.41, P<0.01)。
IHC结果显示:端粒酶hTERT蛋白阳性颗粒呈棕黄色,阳性物质全部位于上皮细胞和癌细胞的核内。在异型增生组织,端粒酶hTERT蛋白的阳性表达部位主要集中在基底细胞层及其上方的细胞核内,随着食管黏膜上皮增生程度的加重,hTERT阳性细胞逐渐增多,阳性细胞带上移。正常食管粘膜组织中阳性表达率为11.1%(4/36),异型增生组织和癌组织hTERT蛋白阳性表达率分别为44.0%(22/50)和86.37%(38/44)。三者阳性率比较,癌组织显著高于异型增生组织(P<0.01),异型增生组织高于正常上皮组织(P<0.01)。
2食管癌中的端粒酶活性和PinX1的表达及其生物学意义
端粒酶活性检查结果显示,在食管癌、异型增生和正常食管组织中,阳性表达率分别为84.0%、62.0%及6.0%,食管癌及异型增生组织与正常食管粘膜比较具有显著统计学差异(P<0.05)。端粒酶活性的平均A值分别为食管癌组织1.457±0.838、食管异型增生组织0.429±0.346和正常食管粘膜组织0.073±0.039。三组间两两比较端粒酶活性的平均A值均存在高度显著统计学差异(P<0.05)。按正常食管粘膜组织、食管异型增生组织和食管鳞癌组织的序列,端粒酶阳性表达率和平均A值依次明显增高。食管癌组织中端粒酶阳性表达率与组织学分级和淋巴结转移无关,而端粒酶活性的平均A值与组织学分级和淋巴结转移有关;食管癌组织中端粒酶阳性表达率及端粒酶活性的平均A值与其他临床病例因素如年龄、性别和浸润深度等无关。
RT-PCR及FCM检测PinX1结果显示,PinX1 mRNA及蛋白在食管癌组织中表达均显著低于食管正常粘膜(P <0.01),在食管正常粘膜、异型增生、癌组织之间呈现逐渐降低趋势(P <0.05)。食管癌组织中,PinX1 mRNA和蛋白与分化程度、浸润深度及有无淋巴结转移密切相关(P <0.05),与性别和年龄无明显相关(P >0.05)。食管癌组织中端粒酶活性表达与PinX1蛋白表达呈负相关性(rs =-0.883,P=0.000)。
3基因转染PinX1对食管癌Eca109细胞增殖和凋亡以及端粒酶活性的影响
应用脂质体转染方法,将pCDNA3.1(+)-PinX1重组质粒及空载体pCDNA3.1成功转染入Eca109细胞中,并应用G418成功筛选出稳定转染细胞,转染pCDNA3.1- PinX1重组质粒与空载体pCDNA3.1的阳性克隆细胞分别记为Eca109/ PinX1、Eca109/pCDNA3.1细胞。
MTS法测定增殖情况结果显示,转染PinX1基因后,Eca109细胞生长明显减慢(P<0.05);而Eca109细胞与仅转染入空载体的Eca109细胞生长比较,两者无明显差异(P>0.05)。
FCM测定细胞生长周期实验结果显示,转染PinX1基因的Eca109细胞的G_0/G_1期细胞数(%)为70.58±5.26,明显高于未转染或仅转染空载体的食管癌Eca109细胞的G_0/G_1期细胞数(%)(分别为53.14±4.83及47.27±3.73)(P<0.05)。说明转染PinX1基因后,细胞的增殖明显变慢,细胞生长阻滞于G_0/G_1期(Fig. 3-9)。
FCM测定细胞凋亡实验结果显示,流式细胞仪检测细胞凋亡表明:转染PinX1基因的Eca109细胞的凋亡率(%)为23.28±5.73,明显高于未转染或仅转染空载体的食管癌Eca109细胞的凋亡率(%)(分别为0.27±0.18及3.40±1.09)(P<0.05)。说明转染入PinX1基因后,细胞发生早期凋亡改变。RT-PCR、Western-blot和FCM检测结果显示,Eca109/ PinX1细胞中PinX1 mRNA和蛋白表达水平较Eca109/pCDNA3.1、Eca109细胞显著升高(P <0.05)。
细胞端粒酶活性的检测结果显示,转染PinX1基因后,细胞端粒酶活性明显降低(P<0.05)。
转染前后Eca109中端粒酶活性表达与细胞增殖指数(PI)表达呈正相关性(rs=0.451,P=0.000)。
结论:
1端粒DNA长度在食管癌前细胞及癌细胞中明显缩短,并且随病变程度加重而递减。端粒酶hTERT、细胞内DNA含量和PI值在食管上皮癌变发生中明显递增,提示食管癌变与三者密切相关。端粒DNA长度、端粒酶hTERT在食管癌前病变早期即出现变化,可以做为监测食管癌发生发展的参考指标。端粒酶hTERT与食管癌的临床病理特征无关,不能做为食管癌病人预后的指标。端粒缩短、端粒酶hTERT高表达都与食管癌变密切相关,有望以上述二者为靶点指导抗肿瘤的基因治疗。
2食管癌及异型增生组织中存在端粒酶活性表达,并存在量的差异。端粒酶活性在异型增生即呈现高表达,随着病变的加重,端粒酶活性依次显著增高,提示端粒酶参与了食管癌的发生与发展,端粒酶的激活是食管癌的早期事件。端粒酶可望成为食管癌的病情监测及早期诊断的生物学指标。端粒酶活性A值与食管癌的组织学分级和淋结转移有关,提示食管癌端粒酶活性A值可作为判断疗效和评价预后的指标。PinX1在食管癌组织中表达显著低于食管正常组织,在食管正常组织、异型增生组织、癌组织之间呈现逐渐降低趋势。PinX1在食管癌中表达和病理分级呈负相关,分级越高,PinX1表达越低。浸润深度达到纤维膜者、有淋巴结转移者的PinX1表达要低于浸润深度未达到纤维膜者、无淋巴结转移者。认为PinX1是一种潜在的食管癌相关基因,检测PinX1的表达水平可能对评价食管癌恶性程度、转移潜能和预后评估有重要的临床价值。食管癌组织中端粒酶活性与PinX1表达呈显著负相关性,证实PinX1抑制端粒酶活性。
3首次应用脂质体转染方法建立食管癌细胞株Eca109/PinX1,目前国内外文献尚未见相关报道。Eca109细胞转染PinX1基因后,细胞的增殖明显变慢,细胞生长阻滞于G_0/G_1期,细胞发生早期凋亡改变。Eca109细胞转染PinX1基因后,端粒酶活性明显降低,并且端粒活性与细胞增殖指数呈显著正相关,考虑在食管癌细胞中PinX1基因通过降低端粒的活性,减弱细胞的增殖,有望成为食管癌治疗的分子靶点。
Objectives:Esophageal carcinoma is one of the most common malignant gastrointestinal tumors. Telomeres are repetitive DNA sequences at the ends of linear chromosomes that are essential for chromosome protection and genomic stability, which play the key role in human cell replication and aging. Shortening of telomeres is associated with each round of cell division and eventually results in the cells apoptosis or death. Once telomeres are short, cells enter an irreversible growth arrest state called replicative senescence. So maintenance of telomere length is important in preventing the consumption of telomere DNA during cell division. Telomerase, a cellular reverse transcriptase, helps stabilize telomere length in human stem, reproductive and cancer cells by compensating this progressive telomere attrition through de novo addition of TTAGGG repeats to the chromosome ends. Among telomerase, the human telomerase reverse transcriptase (hTERT) is one of core components and plays a key role in the activation of telomerase. Though telomerase is considered to be necessary for the indefinite proliferation of human cells, several lines of studies show high level expressions of telomerase are found in tumor, which implicate that activation of telomerase plays a key role in the malignant transformation process. Since most cancers express telomerase, this indicates that telomerase-induced telomere length manipulations may have utility for tissue engineering to malignant proliferation and involve in cancer development. Inhibition of telomerase results in gradual erosion of telomeres followed by cessation of proliferation or apoptosis, and thus may be a promising target for cancer therapy.
As the endogenous suppressor genes of telomerase, PinX1 is regarded as one of potential tumor suppressor genes and involves in variety of tumor development. Recent reports showed that expression of PinX1 was obviously decreased and telomerase activity was enhanced in gastric carcinoma, colorectal cancer and leukemia. But it was also reported that there was no significant difference in the expression of PinX1 in the hepatumo tissues compared to that in normal control tissues, while high telomerase activity was found in the hepatumo tissues. Those contradictious researches showed the exact role of PinX1 genes in regulation of telomerase activity in tumor cells is still unclear. So it is also unclear whether PinX1 inhibits telomerase activity and/or suppresses tumor cell proliferation. It is achieved to set up an esophageal cancer cells model to get insight into the functions of PinX1 gene in regulating the telomerase activity. The development of telomerase inhibitors as therapeutic agents has become new method for cancer therapy and the study of PinX1 in esophageal cancer cells will contribute to the advanced therapeutic schemes.
In order to explore the role of telomere and telomerase in carcinogenesis of esophageal epithelium, the telomere length and the expression of hTERT protein in esophageal carcinoma and atypical hyperplasia were measured by FCM and IHC. Meanwhile, telomerase activity of esophageal carcinoma and atypical hyperplasia tissues was further detected by TRAP- argentation. Relationship between telomere length and telomerase activation and esophageal carcinoma clinical pathological features was analyzed to explore the involvement of telomere and telomerase in carcinogenesis of esophageal epithelium.
In order to explore the role of PinX1 in carcinogenesis of esophageal epithelium and in inhibiting telomerase activation, the expression of PinX1 in esophageal carcinoma and atypical hyperplasia were measured by FCM and IHC and the proliferation and apoptosis of Eca 109 cells transfected with Exogenous PinX1 were assessed by RT-PCR, FCM and Western blot. Meanwhile, telomerase activity of Eca 109 cells transfected with PinX1 was further detected by TRAP- argentation.
Methods:
1 Expression of length of telomere DNA and hTERT in high-risk area of esophageal carcinoma and related biology significance
esophageal carcinoma tissues from the patients without chemotherapy and radiotherapy from esophageal carcinoma high-risk area of China were collected in this study. All the specimens were verified by pathologic diagnosis, 44 cases of esophageal squamous cell carcinoma (ESCC), 50 dysplasia of esophageal squamous epithelium and 36 normal esophageal mucosa. 44 ESCC tissues included well (n=20) and moderately differentiated ESCC (n=11), poorly differentiated ESCC (n=13), fibrous membrane invasion (n=25), fibrous membrane untouched (n=19), lymph node metastasis positive (n=20), lymph node metastasis negative (n=24). 50 dysplasia of esophageal squamous epithelium included 22 cases of mild dysplasia and 28 cases of severe dysplasia. The telomere length and the expression of hTERT protein in esophageal carcinoma and atypical hyperplasia were measured by flow cytometry (FCM) and immunohistochemistry (IHC). Relationship between their expressions and clinical pathological features was analyzed.
2 Expression of telomerase activity and PinX1 in esophageal carcinoma and related biology significance
Esophageal carcinoma tissues, paired adjacent mucosa (2~5cm from margin of esophageal carcinoma), and paired normal mucosa (at least 5cm from margin of esophageal carcinoma) were obtained from resected surgical specimens of esophageal squamous cell carcinoma (ESCC). All the specimens were verified by pathologic diagnosis, 50 cases of esophageal squamous cell carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were selected from 130 specimens. 80 ESCC tissues included well and moderately differentiated ESCC (n=39), poorly differentiated ESCC(n=11) ; fibrous membrane invasion(n=34), fibrous membrane untouched(n=16); lymph node metastasis positive(n=17), lymph node metastasis negative(n=33). PinX1 gene and protein expression in 50 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and flow cytometry(FCM). Telomerase activity was detected by transcription-polymerase chain reaction- argentation (TRAP- argentation). Relationship between their expressions and clinical pathological features was analyzed.
3 Effects of PinX1 gene on the proliferation and apoptosis and telomerase activity of esophageal cancer cell
We clone the whole length of PinX1 gene from gene bank and ligate the whole length of PinX1 gene to the pCDNA3.1 vector to construct the custom-crafted plasmid pCDNA3.1- PinX1. The positive clones pCDNA3.1- PinX1 was transfected to Eca109 cells and positive cell clones were selected with G418 after transfected 72h. Eca109 cell transfected with pCDNA3.1 as control group. Eca109 cell transfected with pCDNA3.1- PinX1 and pCDNA3.1 was named Eca109/ PinX1 and Eca109/pCDNA3.1 cell respectively.
Transfected rate was investigated by laser confocalmicroscopy and the effect of PinX1 on the growth of Eca109/ PinX1, Eca109/pCDNA3.1 as well as Eca109 cells was detected by MTS. The expression of PinX1 mRNA and protein in transfection Eca 109 cells were investigated by RT-PCR, FCM as well as Western-blot. The changes of telomerase activity in Eca109/ PinX1, Eca109/pCDNA3.1 as well as Eca109 cells were measured by TRAP- argentation. Results:
1 Expression of length of telomere DNA and hTERT in high-risk area of esophageal carcinoma and related biology significance Value of Q-FISH was detected by FCM in normal esophageal epithelium group, mild dysplasia group, severe dysplasia group and cancinoma group. Value of Q-FISH in cancinoma group was higher than that in severe dysplasia group (P<0.01), severe dysplasia group was higher than mild dysplasia group (P<0.01). There was a negative correlation betwen telomere length and cytologic grade(r=-0.79, P<0.01).
FI value of hTERT in normal esophageal epithelium group, mild dysplasia group, severe dysplasia group and cancinoma group was 0.87±0.18, 1.13±0.19, 1.39±0.24 and 1.84±0.21. FI value of hTERT in cancinoma group was higher than that in severe dysplasia group (P<0.01), severe dysplasia group was higher than mild dysplasia group (P<0.01). FI value of hTERT was positive related with cytologic grade(r=0.84, P<0.01); According to the standard: value of FI>1.0 was positive, expreesing rate of hTERT portain was 68.18%、92.86% and 95.45% in groups of mild dysplasia, severe dyaplsia and carcinoma. expressing rate in cancinoma group and severe dysplasia group were higher than that in mild dysplasia group (P<0.01).
From normal group to cancinoma group value of DI (DNA Index) was 1.03±0.34, 1.06±0.28, 1.17±0.25 and 1.54±0.37. DI value in cancinoma group was higher than that in severe dysplasia group (P<0.01), severe dysplasia group was higher than mild dysplasia group (P<0.01). There was positive correlation betwen value of DI and cytologic grade (r=0.62, P<0.01). With the cytologic grade progressing value of PI (proliferation index) increased, value of PI (28.79±5.47) in cancinoma group was higher than that (18.97±5.27) in severe dysplasia group. Value of PI was positive related with cytologic grade (r=0.64, P<0.01).
There was show significant positive correlation betwen value of DI and PI(r=0.76, P<0.01), and negative correlation between Q-FISH value of telomere and FI value of hTERT(r=-7.49, P<0.01), as well as Q-FISH value of telomere vs. value of DI(r=-0.41, P<0.01).
The expression of hTERT located in nucleus of esophageal epithelial cells and cancerous cells by Immunohistochemistry staining. The positive rate of hTERT in incisal margin normal tissue was 11.1%(4/36), and those in para-tumorous dysplasia tissues and cancinoma were 44.0%(22/50) and 86.37%(38/44), there was instinct difference of positive expression among them (P<0.01). Expression of hTERT had no relationgship observed with age, gender, differentation grade, lymph node matastasis and invasive degree of patients (P>0.05).
2 Expression of telomerase activity and PinX1 in esophageal carcinoma and related biology significance
The positive rate of telomerase activity were 84%,62% and 6% respectively in 50 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal. Telomerase activity in esophageal cancer tissues and dysplasia of esophageal squamous epithelium were significantly higher than that in normal tissues (P<0.05).The A value of telomerase activity were 1.457±0.838, 0.429±0.346 and 0.073±0.039 in 50 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal (P<0.05). The A value of telomerase activity was related to the tumor tissue grade and lymph no demetastasis (P<0.05, P<0.01), but not to other clinicopathological features in ESCCS (P>0.05).
The expression of PinX1 mRNA and protein in esophageal cancer tissues were significantly lower than that in normal tissues (P<0.01). In esophageal cancer tissues, there were no difference in the expression of PinX1 mRNA and protein between age and gender (P>0.05), but the expression of PinX1 in poorly differentiated, fibrous membrane invasion and lymph node metastasis positive ESCC were significantly lower than that in well and moderately differentiated, fibrous membrane untouched and lymph node metastasis negative tissues (P<0.05).
There was significant negative correlation between telomerase activity expression and PinX1 protein expression (rs =-0.883,P=0.000).
3 Transfection of PinX1 gene, its influence to esophageal cancer cell and telomerase activity
Eca109/PinX1 and Eca109/pCDNA3.1 cell were established successfully. PinX1-transfected Eca109 cells grew more slowly than the cells without transfection or transfected with void vectors (P<0.05). The growth of PinX1-transfected cells was retarded and blocked into G0/G1 stage. The apoptosis rate was higher in PinX1-transfected Eca109 than that in PinX1-untransfected cells or transfected with void vectors only by FCM.
The expression of PinX1 mRNA and protein in Eca109/PinX1 cells was significantly higher than that in Eca109/pCDNA3.1 and Eca109 cells (P<0.05). The expression of telomerase activity was lower in PinX1-transfected Eca109 than that in PinX1-untransfected cells or transfected with void vectors only (P<0.05). There was significant positive correlation between telomerase activity expression and PI expression (rs=0.451,P=0.000).
Conclusions:
1 Shortening of telomeres and increased hTERT expression, DNA content and PI value were found in ESC, which showed telomeres and telomerase are associated to carcinogenesis of esophageal epithelium. The difference of telomere length and hTERT expression in tumor of initial period of carcinogenesis might be a useful cancer biomarker for early diagnosis of ESC.
2 Telomerase activity was increased in atypical hyperplasia tissues than that in normal tissues of the esophagus. Upon to the malignant degrade of pathological changes in ESC, the telomerase activity increased more in malignant tumor. The results suggested that telomerase is involved in tumor progression and serves as an early event in ESC, and may contribute to early diagnosis of ESC and supervising the development of ESC. The A value of telomerase activity was related to the tissue grade and lymph node metastasis in ESC, but not to other clinicopathologieal features. It suggested that the A value of telomerase activity may be used as a sign to estimate the curative effect and to appraise the prognosis of ESC.
The down-expression of PinX1 was founded in ESC. The down grade is depended for the clinical stage and pathological grade in ESC. PinX1 gene may be used as one of the molecular markers to determine the malignant degree, metastasis potency and predicting progression of ESC. Telomerase activity expression was significant negative correlation to PinX1 expression, suggested that PinX1 can decrease telomerase activation.
3 Within the successfully established Eca109/PinX1esophageal cancer cell model, the esophageal cancer Eca109 cells growths were inhibited by PinX1 transfenction evidenced by increased apoptosis and an arrest of G0/G1 phase and telomerase activity of Eca 109 cells was decreased by PinX1 gene. The results show PinX1 serves as an inhibitors of telomerase activity to suppress tumor development.
本实验利用了多项实验方法检测端粒DNA长度、端粒酶hTERT蛋白含量及细胞内DNA含量在食管癌高发区现场人食管癌、异型增生及正常食管的表达,探讨与食管癌发生及生物学行为的关系,试图为食管癌的早期诊断提供客观参考指标,并为揭示食管癌的发生、发展的机制提供理论依据。采用端粒重复序列扩增反应(TRAP)-ELISA和TRAP -银染法半定量技术,检测食管癌及异型增生组织中端粒酶活性的表达,探讨端粒酶活性与食管癌发生发展及其与临床病理因素的关系。
PinX1基因作为端粒酶的内源性抑制基因,近年来被认为是一种潜在的抑癌基因,其表达可能与多种肿瘤的发生发展有关。有实验证实在胃癌、大肠癌及白血病患者中PinX1表达明显下降,端粒酶活性增强。但是也有研究显示PinX1在肝癌细胞中的表达与正常组织无显著差异,而与之相应的是此类肿瘤中大部分端粒酶活性也增强。目前PinX1基因在肿瘤细胞中的作用和对端粒酶活性的调控及在细胞癌变中的机制尚不十分清楚。有关PinX1基因在食管癌中的研究国内外也鲜有报道。PinX1基因的作用越来越引起人们的重视,但到目前为止,PinX1基因在食管癌中生物学意义、作用机理国内外的报道很少,并且没有适合研究PinX1基因的食管癌细胞模型。由于基因转染已经成为治疗肿瘤等基因表达异常的有效手段,那么,通过外源性过表达PinX1基因能否抑制食管癌细胞的增殖?是否可以抑制端粒酶活性?本实验中利用多项技术,应用细胞模型研究PinX1在人食管癌、异型增生组织及正常食管组织中的表达,探讨PinX1与食管癌发生发展及与食管癌临床分期、病理分型及预后的相关性,并分析与端粒酶活性表达的相关性。研究转染PinX1基因前后细胞增殖凋亡等的变化,初步了解该基因对食管癌细胞的生物学影响。同时我们应用TRAP-ELISA和TRAP-银染法检测转染前后Eca109细胞端粒酶活性的变化,进一步探讨PinX1基因与端粒酶的关系。
方法:
1端粒DNA长度及端粒酶hTERT在食管癌高发区人群中的表达及生物学意义
收集采集自河北省磁县食管癌高发区现场的咬检组织1000例,所有患者未接受过化疗和放疗。从中选取130例,根据病情分为3组,第1组为正常食管组织36例,第2组为异型增生组织50例,第3组为食管鳞癌组织44例。经病理组织学诊断,50例异型增生食管粘膜组织中,22例为轻度增生组织,28例为重度增生组织;44例食管鳞状细胞癌中,20例为高分化鳞癌、11例为中分化鳞癌,13例为低分化鳞癌,25例侵及纤维膜及周围软组织,19例未侵及纤维膜;20例为淋巴结转移,24例为无淋巴结转移。采用流式细胞术方法(flow cytometry, FCM)和免疫组织化学方法(immunohistochemistry, IHC)检测测端粒DNA长度、端粒酶hTERT蛋白含量及细胞内DNA含量在食管癌高发区现场人食管癌、异型增生及正常食管的表达,分析与食管癌发生及与临床病理特征的关系。
2食管癌中的端粒酶活性和PinX1的表达及其生物学意义
收集食管鳞状细胞癌手术切除标本130例,每例标本分别取癌组织、癌旁组织(距癌边缘2-5cm)及手术切缘正常食管粘膜(距癌边缘5cm以上)。从中选取50例食管鳞状细胞癌、异型增生及正常食管粘膜组织。经病理组织学诊断,50例食管鳞状细胞癌,39例为高中分化鳞癌,11例为低分化鳞癌,34例侵及纤维膜,16例未达到纤维膜,17例淋巴结转移,33例未转移。采用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)、流式细胞术方法(flow cytometry, FCM)、TRAP-ELISA和TRAP-银染法检测食管癌组织、异型增生、正常粘膜组织中端粒酶活性和PinX1基因和蛋白的表达,分析端粒酶活性和PinX1表达与食管癌临床病理特征的关系。
3基因转染PinX1对食管癌Eca109细胞增殖和凋亡以及端粒酶活性的影响
利用分子生物学技术构建pCDNA3.1(+)-PinX1重组质粒。采用脂质体转染方法将pCDNA3.1(+)-PinX1重组质粒转染Eca109细胞,同时也设置空载体pCDNA3.1转染组。转染72小时后用G418进行稳定转染细胞的筛选,筛选出来的阳性细胞分别命名为Eca109/PinX1、Eca109/pCDNA3.1细胞。
应用激光共聚焦方法测定转染效率,MTS方法检测过表达PinX1对Eca109/ PinX1、Eca109/pCDNA3.1、Eca109细胞生长抑制作用。FCM方法检测细胞生长周期和细胞凋亡。RT-PCR、FCM、Western-blot方法检测Eca109/PinX1、Eca109/pCDNA3.1、Eca109细胞中PinX1基因及蛋白的表达,TRAP-ELISA和TRAP-银染法检测端粒酶活性在细胞中表达。
结果:
1端粒DNA长度及端粒酶hTERT在食管癌高发区人群中的表达及生物学意义
FCM检测端粒长度结果显示,端粒DNA长度在食管癌组织中表达量与正常食管组织相比明显缩短,在食管正常组织、异型增生组织、癌组织的表达量呈现逐渐降低趋势(P<0.01);端粒DNA长度在食管轻度增生、重度增生之间呈逐渐降低趋势,重度增生组与轻度增生组相比明显缩短(P<0.01),轻度增生组与正常组比较无显著性差异(P>0.05)。随着细胞学分级增高,端粒DNA长度Q-FISH值逐渐降低,端粒DNA长度与细胞学分级呈负相关(r=-0.79, P <0.01)。
FCM检测端粒酶hTERT基因蛋白含量,在癌、重度增生、轻度增生和正常食管的FI值分别为1.84±0.21、1.39±0.24、1.13±0.19和0.87±0.18,癌组hTERT含量高于重度增生组(P <0.01)、重度增生组高于轻度增生组(P<0.01),轻度增生组与正常组相比无显著性差别。随着细胞学分级增高, hTERT的FI值随之升高, hTERT蛋白含量与细胞学分级呈正相关关系(r=0.83, P<0.01)。根据FI值>1.0为阳性表达的判断标准,轻度增生组、重度增生组和癌组hTERT蛋白阳性表达率分别为68.18%、92.86%和95.45%,重度增生组和癌组的表达率都高于轻度增生组(P<0.01)。
FCM检测正常组、轻度增生组、重度增生组和癌组细胞DNA含量,DI值分别为1.03±0.34、1.06±0.28、1.17±0.25和1.54±0.37,正常组、轻度增生组和重度增生组的DI值呈上升趋势(P>0.05),癌组的DI值明显高于重度增生组。DI值与细胞学分级正相关(r=0.62, P<0.01);正常组、轻度增生组、重度增生组和癌组DNA异倍体率分别为11.11(4/36)、9.09(2/22)、28.57(8/28)和81.82(36/44),癌组异倍体率高于重度增生组(P<0.01),重度增生组高于轻度增生组(P<0.05);从正常组到癌组PI值分别为10.98±4.11、14.15±4.76、18.97±5.27和28.79±5.47,随着细胞学分级的升高,PI值增大,癌组显著高于重度增生组(P<0.01),重度增生组高于轻度增生组(P<0.05)。PI值与细胞学分级正相关(r=0.64, P<0.01)。
130例食管样本的DI值与PI值高度正相关(r=0.76, P<0.01);端粒长度Q-FISH值与端粒酶hTERT蛋白FI值负相关(r=-7.49, P<0.01);端粒Q-FISH值与DI值负相关(r=-0.41, P<0.01)。
IHC结果显示:端粒酶hTERT蛋白阳性颗粒呈棕黄色,阳性物质全部位于上皮细胞和癌细胞的核内。在异型增生组织,端粒酶hTERT蛋白的阳性表达部位主要集中在基底细胞层及其上方的细胞核内,随着食管黏膜上皮增生程度的加重,hTERT阳性细胞逐渐增多,阳性细胞带上移。正常食管粘膜组织中阳性表达率为11.1%(4/36),异型增生组织和癌组织hTERT蛋白阳性表达率分别为44.0%(22/50)和86.37%(38/44)。三者阳性率比较,癌组织显著高于异型增生组织(P<0.01),异型增生组织高于正常上皮组织(P<0.01)。
2食管癌中的端粒酶活性和PinX1的表达及其生物学意义
端粒酶活性检查结果显示,在食管癌、异型增生和正常食管组织中,阳性表达率分别为84.0%、62.0%及6.0%,食管癌及异型增生组织与正常食管粘膜比较具有显著统计学差异(P<0.05)。端粒酶活性的平均A值分别为食管癌组织1.457±0.838、食管异型增生组织0.429±0.346和正常食管粘膜组织0.073±0.039。三组间两两比较端粒酶活性的平均A值均存在高度显著统计学差异(P<0.05)。按正常食管粘膜组织、食管异型增生组织和食管鳞癌组织的序列,端粒酶阳性表达率和平均A值依次明显增高。食管癌组织中端粒酶阳性表达率与组织学分级和淋巴结转移无关,而端粒酶活性的平均A值与组织学分级和淋巴结转移有关;食管癌组织中端粒酶阳性表达率及端粒酶活性的平均A值与其他临床病例因素如年龄、性别和浸润深度等无关。
RT-PCR及FCM检测PinX1结果显示,PinX1 mRNA及蛋白在食管癌组织中表达均显著低于食管正常粘膜(P <0.01),在食管正常粘膜、异型增生、癌组织之间呈现逐渐降低趋势(P <0.05)。食管癌组织中,PinX1 mRNA和蛋白与分化程度、浸润深度及有无淋巴结转移密切相关(P <0.05),与性别和年龄无明显相关(P >0.05)。食管癌组织中端粒酶活性表达与PinX1蛋白表达呈负相关性(rs =-0.883,P=0.000)。
3基因转染PinX1对食管癌Eca109细胞增殖和凋亡以及端粒酶活性的影响
应用脂质体转染方法,将pCDNA3.1(+)-PinX1重组质粒及空载体pCDNA3.1成功转染入Eca109细胞中,并应用G418成功筛选出稳定转染细胞,转染pCDNA3.1- PinX1重组质粒与空载体pCDNA3.1的阳性克隆细胞分别记为Eca109/ PinX1、Eca109/pCDNA3.1细胞。
MTS法测定增殖情况结果显示,转染PinX1基因后,Eca109细胞生长明显减慢(P<0.05);而Eca109细胞与仅转染入空载体的Eca109细胞生长比较,两者无明显差异(P>0.05)。
FCM测定细胞生长周期实验结果显示,转染PinX1基因的Eca109细胞的G_0/G_1期细胞数(%)为70.58±5.26,明显高于未转染或仅转染空载体的食管癌Eca109细胞的G_0/G_1期细胞数(%)(分别为53.14±4.83及47.27±3.73)(P<0.05)。说明转染PinX1基因后,细胞的增殖明显变慢,细胞生长阻滞于G_0/G_1期(Fig. 3-9)。
FCM测定细胞凋亡实验结果显示,流式细胞仪检测细胞凋亡表明:转染PinX1基因的Eca109细胞的凋亡率(%)为23.28±5.73,明显高于未转染或仅转染空载体的食管癌Eca109细胞的凋亡率(%)(分别为0.27±0.18及3.40±1.09)(P<0.05)。说明转染入PinX1基因后,细胞发生早期凋亡改变。RT-PCR、Western-blot和FCM检测结果显示,Eca109/ PinX1细胞中PinX1 mRNA和蛋白表达水平较Eca109/pCDNA3.1、Eca109细胞显著升高(P <0.05)。
细胞端粒酶活性的检测结果显示,转染PinX1基因后,细胞端粒酶活性明显降低(P<0.05)。
转染前后Eca109中端粒酶活性表达与细胞增殖指数(PI)表达呈正相关性(rs=0.451,P=0.000)。
结论:
1端粒DNA长度在食管癌前细胞及癌细胞中明显缩短,并且随病变程度加重而递减。端粒酶hTERT、细胞内DNA含量和PI值在食管上皮癌变发生中明显递增,提示食管癌变与三者密切相关。端粒DNA长度、端粒酶hTERT在食管癌前病变早期即出现变化,可以做为监测食管癌发生发展的参考指标。端粒酶hTERT与食管癌的临床病理特征无关,不能做为食管癌病人预后的指标。端粒缩短、端粒酶hTERT高表达都与食管癌变密切相关,有望以上述二者为靶点指导抗肿瘤的基因治疗。
2食管癌及异型增生组织中存在端粒酶活性表达,并存在量的差异。端粒酶活性在异型增生即呈现高表达,随着病变的加重,端粒酶活性依次显著增高,提示端粒酶参与了食管癌的发生与发展,端粒酶的激活是食管癌的早期事件。端粒酶可望成为食管癌的病情监测及早期诊断的生物学指标。端粒酶活性A值与食管癌的组织学分级和淋结转移有关,提示食管癌端粒酶活性A值可作为判断疗效和评价预后的指标。PinX1在食管癌组织中表达显著低于食管正常组织,在食管正常组织、异型增生组织、癌组织之间呈现逐渐降低趋势。PinX1在食管癌中表达和病理分级呈负相关,分级越高,PinX1表达越低。浸润深度达到纤维膜者、有淋巴结转移者的PinX1表达要低于浸润深度未达到纤维膜者、无淋巴结转移者。认为PinX1是一种潜在的食管癌相关基因,检测PinX1的表达水平可能对评价食管癌恶性程度、转移潜能和预后评估有重要的临床价值。食管癌组织中端粒酶活性与PinX1表达呈显著负相关性,证实PinX1抑制端粒酶活性。
3首次应用脂质体转染方法建立食管癌细胞株Eca109/PinX1,目前国内外文献尚未见相关报道。Eca109细胞转染PinX1基因后,细胞的增殖明显变慢,细胞生长阻滞于G_0/G_1期,细胞发生早期凋亡改变。Eca109细胞转染PinX1基因后,端粒酶活性明显降低,并且端粒活性与细胞增殖指数呈显著正相关,考虑在食管癌细胞中PinX1基因通过降低端粒的活性,减弱细胞的增殖,有望成为食管癌治疗的分子靶点。
Objectives:Esophageal carcinoma is one of the most common malignant gastrointestinal tumors. Telomeres are repetitive DNA sequences at the ends of linear chromosomes that are essential for chromosome protection and genomic stability, which play the key role in human cell replication and aging. Shortening of telomeres is associated with each round of cell division and eventually results in the cells apoptosis or death. Once telomeres are short, cells enter an irreversible growth arrest state called replicative senescence. So maintenance of telomere length is important in preventing the consumption of telomere DNA during cell division. Telomerase, a cellular reverse transcriptase, helps stabilize telomere length in human stem, reproductive and cancer cells by compensating this progressive telomere attrition through de novo addition of TTAGGG repeats to the chromosome ends. Among telomerase, the human telomerase reverse transcriptase (hTERT) is one of core components and plays a key role in the activation of telomerase. Though telomerase is considered to be necessary for the indefinite proliferation of human cells, several lines of studies show high level expressions of telomerase are found in tumor, which implicate that activation of telomerase plays a key role in the malignant transformation process. Since most cancers express telomerase, this indicates that telomerase-induced telomere length manipulations may have utility for tissue engineering to malignant proliferation and involve in cancer development. Inhibition of telomerase results in gradual erosion of telomeres followed by cessation of proliferation or apoptosis, and thus may be a promising target for cancer therapy.
As the endogenous suppressor genes of telomerase, PinX1 is regarded as one of potential tumor suppressor genes and involves in variety of tumor development. Recent reports showed that expression of PinX1 was obviously decreased and telomerase activity was enhanced in gastric carcinoma, colorectal cancer and leukemia. But it was also reported that there was no significant difference in the expression of PinX1 in the hepatumo tissues compared to that in normal control tissues, while high telomerase activity was found in the hepatumo tissues. Those contradictious researches showed the exact role of PinX1 genes in regulation of telomerase activity in tumor cells is still unclear. So it is also unclear whether PinX1 inhibits telomerase activity and/or suppresses tumor cell proliferation. It is achieved to set up an esophageal cancer cells model to get insight into the functions of PinX1 gene in regulating the telomerase activity. The development of telomerase inhibitors as therapeutic agents has become new method for cancer therapy and the study of PinX1 in esophageal cancer cells will contribute to the advanced therapeutic schemes.
In order to explore the role of telomere and telomerase in carcinogenesis of esophageal epithelium, the telomere length and the expression of hTERT protein in esophageal carcinoma and atypical hyperplasia were measured by FCM and IHC. Meanwhile, telomerase activity of esophageal carcinoma and atypical hyperplasia tissues was further detected by TRAP- argentation. Relationship between telomere length and telomerase activation and esophageal carcinoma clinical pathological features was analyzed to explore the involvement of telomere and telomerase in carcinogenesis of esophageal epithelium.
In order to explore the role of PinX1 in carcinogenesis of esophageal epithelium and in inhibiting telomerase activation, the expression of PinX1 in esophageal carcinoma and atypical hyperplasia were measured by FCM and IHC and the proliferation and apoptosis of Eca 109 cells transfected with Exogenous PinX1 were assessed by RT-PCR, FCM and Western blot. Meanwhile, telomerase activity of Eca 109 cells transfected with PinX1 was further detected by TRAP- argentation.
Methods:
1 Expression of length of telomere DNA and hTERT in high-risk area of esophageal carcinoma and related biology significance
esophageal carcinoma tissues from the patients without chemotherapy and radiotherapy from esophageal carcinoma high-risk area of China were collected in this study. All the specimens were verified by pathologic diagnosis, 44 cases of esophageal squamous cell carcinoma (ESCC), 50 dysplasia of esophageal squamous epithelium and 36 normal esophageal mucosa. 44 ESCC tissues included well (n=20) and moderately differentiated ESCC (n=11), poorly differentiated ESCC (n=13), fibrous membrane invasion (n=25), fibrous membrane untouched (n=19), lymph node metastasis positive (n=20), lymph node metastasis negative (n=24). 50 dysplasia of esophageal squamous epithelium included 22 cases of mild dysplasia and 28 cases of severe dysplasia. The telomere length and the expression of hTERT protein in esophageal carcinoma and atypical hyperplasia were measured by flow cytometry (FCM) and immunohistochemistry (IHC). Relationship between their expressions and clinical pathological features was analyzed.
2 Expression of telomerase activity and PinX1 in esophageal carcinoma and related biology significance
Esophageal carcinoma tissues, paired adjacent mucosa (2~5cm from margin of esophageal carcinoma), and paired normal mucosa (at least 5cm from margin of esophageal carcinoma) were obtained from resected surgical specimens of esophageal squamous cell carcinoma (ESCC). All the specimens were verified by pathologic diagnosis, 50 cases of esophageal squamous cell carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were selected from 130 specimens. 80 ESCC tissues included well and moderately differentiated ESCC (n=39), poorly differentiated ESCC(n=11) ; fibrous membrane invasion(n=34), fibrous membrane untouched(n=16); lymph node metastasis positive(n=17), lymph node metastasis negative(n=33). PinX1 gene and protein expression in 50 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and flow cytometry(FCM). Telomerase activity was detected by transcription-polymerase chain reaction- argentation (TRAP- argentation). Relationship between their expressions and clinical pathological features was analyzed.
3 Effects of PinX1 gene on the proliferation and apoptosis and telomerase activity of esophageal cancer cell
We clone the whole length of PinX1 gene from gene bank and ligate the whole length of PinX1 gene to the pCDNA3.1 vector to construct the custom-crafted plasmid pCDNA3.1- PinX1. The positive clones pCDNA3.1- PinX1 was transfected to Eca109 cells and positive cell clones were selected with G418 after transfected 72h. Eca109 cell transfected with pCDNA3.1 as control group. Eca109 cell transfected with pCDNA3.1- PinX1 and pCDNA3.1 was named Eca109/ PinX1 and Eca109/pCDNA3.1 cell respectively.
Transfected rate was investigated by laser confocalmicroscopy and the effect of PinX1 on the growth of Eca109/ PinX1, Eca109/pCDNA3.1 as well as Eca109 cells was detected by MTS. The expression of PinX1 mRNA and protein in transfection Eca 109 cells were investigated by RT-PCR, FCM as well as Western-blot. The changes of telomerase activity in Eca109/ PinX1, Eca109/pCDNA3.1 as well as Eca109 cells were measured by TRAP- argentation. Results:
1 Expression of length of telomere DNA and hTERT in high-risk area of esophageal carcinoma and related biology significance Value of Q-FISH was detected by FCM in normal esophageal epithelium group, mild dysplasia group, severe dysplasia group and cancinoma group. Value of Q-FISH in cancinoma group was higher than that in severe dysplasia group (P<0.01), severe dysplasia group was higher than mild dysplasia group (P<0.01). There was a negative correlation betwen telomere length and cytologic grade(r=-0.79, P<0.01).
FI value of hTERT in normal esophageal epithelium group, mild dysplasia group, severe dysplasia group and cancinoma group was 0.87±0.18, 1.13±0.19, 1.39±0.24 and 1.84±0.21. FI value of hTERT in cancinoma group was higher than that in severe dysplasia group (P<0.01), severe dysplasia group was higher than mild dysplasia group (P<0.01). FI value of hTERT was positive related with cytologic grade(r=0.84, P<0.01); According to the standard: value of FI>1.0 was positive, expreesing rate of hTERT portain was 68.18%、92.86% and 95.45% in groups of mild dysplasia, severe dyaplsia and carcinoma. expressing rate in cancinoma group and severe dysplasia group were higher than that in mild dysplasia group (P<0.01).
From normal group to cancinoma group value of DI (DNA Index) was 1.03±0.34, 1.06±0.28, 1.17±0.25 and 1.54±0.37. DI value in cancinoma group was higher than that in severe dysplasia group (P<0.01), severe dysplasia group was higher than mild dysplasia group (P<0.01). There was positive correlation betwen value of DI and cytologic grade (r=0.62, P<0.01). With the cytologic grade progressing value of PI (proliferation index) increased, value of PI (28.79±5.47) in cancinoma group was higher than that (18.97±5.27) in severe dysplasia group. Value of PI was positive related with cytologic grade (r=0.64, P<0.01).
There was show significant positive correlation betwen value of DI and PI(r=0.76, P<0.01), and negative correlation between Q-FISH value of telomere and FI value of hTERT(r=-7.49, P<0.01), as well as Q-FISH value of telomere vs. value of DI(r=-0.41, P<0.01).
The expression of hTERT located in nucleus of esophageal epithelial cells and cancerous cells by Immunohistochemistry staining. The positive rate of hTERT in incisal margin normal tissue was 11.1%(4/36), and those in para-tumorous dysplasia tissues and cancinoma were 44.0%(22/50) and 86.37%(38/44), there was instinct difference of positive expression among them (P<0.01). Expression of hTERT had no relationgship observed with age, gender, differentation grade, lymph node matastasis and invasive degree of patients (P>0.05).
2 Expression of telomerase activity and PinX1 in esophageal carcinoma and related biology significance
The positive rate of telomerase activity were 84%,62% and 6% respectively in 50 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal. Telomerase activity in esophageal cancer tissues and dysplasia of esophageal squamous epithelium were significantly higher than that in normal tissues (P<0.05).The A value of telomerase activity were 1.457±0.838, 0.429±0.346 and 0.073±0.039 in 50 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal (P<0.05). The A value of telomerase activity was related to the tumor tissue grade and lymph no demetastasis (P<0.05, P<0.01), but not to other clinicopathological features in ESCCS (P>0.05).
The expression of PinX1 mRNA and protein in esophageal cancer tissues were significantly lower than that in normal tissues (P<0.01). In esophageal cancer tissues, there were no difference in the expression of PinX1 mRNA and protein between age and gender (P>0.05), but the expression of PinX1 in poorly differentiated, fibrous membrane invasion and lymph node metastasis positive ESCC were significantly lower than that in well and moderately differentiated, fibrous membrane untouched and lymph node metastasis negative tissues (P<0.05).
There was significant negative correlation between telomerase activity expression and PinX1 protein expression (rs =-0.883,P=0.000).
3 Transfection of PinX1 gene, its influence to esophageal cancer cell and telomerase activity
Eca109/PinX1 and Eca109/pCDNA3.1 cell were established successfully. PinX1-transfected Eca109 cells grew more slowly than the cells without transfection or transfected with void vectors (P<0.05). The growth of PinX1-transfected cells was retarded and blocked into G0/G1 stage. The apoptosis rate was higher in PinX1-transfected Eca109 than that in PinX1-untransfected cells or transfected with void vectors only by FCM.
The expression of PinX1 mRNA and protein in Eca109/PinX1 cells was significantly higher than that in Eca109/pCDNA3.1 and Eca109 cells (P<0.05). The expression of telomerase activity was lower in PinX1-transfected Eca109 than that in PinX1-untransfected cells or transfected with void vectors only (P<0.05). There was significant positive correlation between telomerase activity expression and PI expression (rs=0.451,P=0.000).
Conclusions:
1 Shortening of telomeres and increased hTERT expression, DNA content and PI value were found in ESC, which showed telomeres and telomerase are associated to carcinogenesis of esophageal epithelium. The difference of telomere length and hTERT expression in tumor of initial period of carcinogenesis might be a useful cancer biomarker for early diagnosis of ESC.
2 Telomerase activity was increased in atypical hyperplasia tissues than that in normal tissues of the esophagus. Upon to the malignant degrade of pathological changes in ESC, the telomerase activity increased more in malignant tumor. The results suggested that telomerase is involved in tumor progression and serves as an early event in ESC, and may contribute to early diagnosis of ESC and supervising the development of ESC. The A value of telomerase activity was related to the tissue grade and lymph node metastasis in ESC, but not to other clinicopathologieal features. It suggested that the A value of telomerase activity may be used as a sign to estimate the curative effect and to appraise the prognosis of ESC.
The down-expression of PinX1 was founded in ESC. The down grade is depended for the clinical stage and pathological grade in ESC. PinX1 gene may be used as one of the molecular markers to determine the malignant degree, metastasis potency and predicting progression of ESC. Telomerase activity expression was significant negative correlation to PinX1 expression, suggested that PinX1 can decrease telomerase activation.
3 Within the successfully established Eca109/PinX1esophageal cancer cell model, the esophageal cancer Eca109 cells growths were inhibited by PinX1 transfenction evidenced by increased apoptosis and an arrest of G0/G1 phase and telomerase activity of Eca 109 cells was decreased by PinX1 gene. The results show PinX1 serves as an inhibitors of telomerase activity to suppress tumor development.
引文
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