沉默核干细胞因子对食管癌细胞生物学特性的影响
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摘要
中国是世界上食管癌发病率和病死率最高的国家,每年全世界新诊断的30万食管癌患者中1/2(16.72万)以上发生在中国。食管癌的浸润转移是引起食管癌患者死亡的主要原因,因此对食管癌发生发展、浸润转移机制的探讨已成为研究的热点之一。
肿瘤的发生发展是多因素、多步骤、多阶段和多基因改变的过程,癌基因和抑癌基因是受累最多的两大类型,各种关键基因的改变是人类肿瘤形成和发展的基础。近年来发现核干细胞因子(nucleostemin,NS)基因是一种新的p53结合蛋白,参与干细胞和肿瘤细胞的增殖调控,能维持细胞的扩增并抑制其向成熟细胞分化。先后发现胃癌、肾癌、乳癌、膀胱癌等肿瘤组织中NS基因表达量与肿瘤的侵袭力呈正相关,NS可能是在细胞周期的S期末和G2期通过已知的肿瘤抑制基因p53发挥作用来调节干细胞和癌细胞的增殖状态,NS可能是干细胞和肿瘤细胞穿越G2/M关键点的特异性调控因子。
表皮生长因子(epidermal growth factor,EGF)是近年来发现的在细胞增殖和分化中起重要作用的因子,可促进正常细胞的恶性转化及刺激肿瘤细胞的增殖。还与肿瘤浸润转移密切相关。研究表明EGF是通过细胞表面的表皮生长因子受体(epidermalgrowth factor receptor,EGFR)发挥其生物学效应。在许多肿瘤组织中均发现EGF及其受体EGFR的过表达且二者具有相关性。
RNA干扰(RNAi)是多种生物体内由双链RNA介导同源mRNA降解的现象。在细胞中,长的dsRNA被类似RNaseIH的Dicer酶切割成21-26核苷酸的小干扰RNA(siRNA)。RNAi基因阻断的有效性和特异性使其可能成为疾病基因疗法的理想工具。
作者拟联合检测食管癌组织中NS、EGF、EGFR的表达,构建针对NS基因的siRNA真核表达载体,转染食管癌EC9706细胞株,观察EC9706细胞生物学特性的变化及NS基因沉默对裸鼠移植瘤生长的影响。全文共分3部分,摘要分述如下。
第一部分食管鳞状细胞癌组织中NS、EGF及EGFR的表达及其相互关系
目的探讨食管鳞状细胞癌组织中NS、EGF及EGFR的表达及三者之间的关系。
方法采集62例食管鳞状细胞癌组织及其相应的31例癌旁不典型增生组织和62例正常食管黏膜组织标本。62例食管鳞状细胞癌组织学分级Ⅰ级15例,Ⅱ级25例,Ⅲ级22例;淋巴结转移20例,无淋巴结转移组42例。浸润浅层7例,肿瘤浸润深度在黏膜层、黏膜下层或浅肌层;深层55例,肿瘤浸润深度在深肌层或纤维膜。分别采用免疫组织化学、原位杂交方法检测上述标本NS、EGF、EGFR蛋白及mRNA的表达情况,RT-PCR、Real-time PCR检测NS mRNA,并分析其与食管鳞状细胞癌临床病理参数的关系。
结果
1)免疫组织化学方法检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中NS蛋白阳性表达率分别为17.7%、41.9%和69.4%,3组间比较差异有统计学意义(P＜0.05);原位杂交检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中NS mRNA阳性表达率分别为21.0%、25.8%和69.4%,3组间比较差异有统计学意义(P＜0.05);RT-PCR检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中NS mRNA相对表达量依次为0.866±0.103、0.913±0.085、0.971±0.121,3组间比较差异有统计学意义(P＜0.05);Real-time PCR检测结果显示:食管鳞状细胞癌组织、癌旁不典型增生组织和正常食管黏膜组织中NS mRNA表达水平分别为(4.53±2.16)、(3.09±2.28)和(2.10±2.34),3组间比较差异有统计学意义(P＜0.05)。食管鳞状细胞癌组织中NS蛋白阳性表达率、NS mRNA阳性表达率及RT-PCR、Real-time PCR检测结果均显示:NS蛋白和NS mRNA的表达与患者的年龄、性别无关(P＞0.05);与组织学分级、浸润深度和淋巴结转移有关(P＜0.05)。组织学分级越差、浸润越深及有淋巴结转移者NS蛋白、NS mRNA阳性表达率及RT-PCR、Real-time PCR检测的NS mRNA表达水平越高。
2)免疫组织化学方法检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中EGF蛋白的阳性表达率分别为27.4%、45.2%和69.4%,3组间比较差异有统计学意义(P＜0.05)。原位杂交检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中EGF mRNA的阳性表达率分别为40.3%、48.3%和77.4%。3组间比较差异有统计学意义(P＜0.05)。食管鳞状细胞癌组织中EGF蛋白阳性表达率、EGF mRNA阳性表达率与患者的年龄、性别无关(P＞0.05);与组织学分级、浸润深度和淋巴结转移有关(P＜0.05),组织学分级越差、浸润越深及有淋巴结转移者NS蛋白阳性表达率、EGF mRNA阳性表达率越高。
3)免疫组织化学方法检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中EGFR蛋白的阳性表达率分别为14.5%、35.5%和71.0%,3组间比较差异有统计学意义(P＜0.05)。原位杂交检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中EGFR mRNA的阳性表达率分别为40.3%、48.3%和75.8%,3组间比较差异有统计学意义(P＜0.05)。食管鳞状细胞癌组织中EGFR蛋白阳性表达率、EGFR mRNA阳性表达率与患者的年龄、性别无关(P＞0.05);与组织学分级、浸润深度和淋巴结转移有关(P＜0.05),组织学分级越差、浸润越深及有淋巴结转移者NS蛋白、EGFR mRNA阳性表达率越高。
4)食管鳞状细胞癌组织中NS蛋白和EGF、EGFR蛋白的表达呈正相关,相关系数分别为0.545、0.731;食管鳞状细胞癌组织中NS mRNA和EGF mRNA、EGFRmRNA的表达呈正相关,相关系数分别为0.394、0.604。
结论NS、EGF及EGFR的表达与食管鳞状细胞癌的发生发展密切相关,并且三者之间关系密切。
第二部分pRNAT-U6.1-siNS干扰表达载体的构建及其对EC9706细胞生物学特性的影响
目的构建针对NS基因的小干扰RNA(siRNA)真核表达载体,筛选出抑制效率最强的表达载体,探讨NS基因siRNA表达载体转染EC9706细胞后对其细胞生物学特性的影响。
方法根据GenBank提供的NS基因AY825265 mRNA序列及siRNA设计原则设计siRNA,筛选得到126-144 nt,199-217 nt和487-505 nt 3个19 bp片段为靶序列;设计包含BamHI和XhoI酶切位点的3对靶向NS发卡样DNA寡核苷酸和一对随机对照发卡样DNA寡核苷酸,经过退火,将此序列克隆至载体pRNAT-U6.1中构建重组干扰表达载体;转化感受态细菌DH5α,提取质粒,应用PCR技术和测序方法鉴定重组克隆重组子。将构建好的4种siRNA表达载体转染EC9706细胞,同时设空载体对照组(转染pRNAT-U6.1空载体的细胞)及空白对照组(未转染的细胞),RT-PCR检测siRNA表达载体转染EC9706细胞后对NS mRNA的抑制作用,从而筛选出干扰效率最强的表达载体,并利用该载体转染EC9706细胞,应用Real-time PCR、原位杂交技术、Western blot和免疫细胞化学法分别检测干扰前后NS和EGFR mRNA及蛋白的表达的变化,进一步应用Boyden Chamber体外侵袭实验观察其对侵袭转移的影响,最后应用流式细胞术观察EC9706细胞周期和细胞凋亡的变化。
结果
1) siRNA发卡DNA的退火结果:siNS1、siNS2、siNS3和siC发卡样siRNA单链DNA寡核苷酸,退火后,电泳可见明亮条带,位于100bp下,约50-60bp处,与设计完全一致。
2)克隆重组子鉴定结果:将转化菌落扩增后提取质粒,以空pRNAT-U6.1为阴性对照,使用pRNAT-U6.1插入鉴定引物进行扩增。空pRNAT-U6.1扩增产物为150 bp,阳性克隆由于插入约50 bp的发卡DNA片段,故扩增产物约为200 bp。所有3种针对NS和1个无关对照重组转化克隆都得到有阳性克隆,阳性重组子pRNAT-U6.1-siNS1、pRNAT-U6.1-siNS2、pRNAT-U6.1-siNS3和pRNAT-U6.1-siC插入片段测序的结果,与设计序列比较结果完全一致。
3) RT-PCR方法分别测定各组EC9706细胞中NS mRNA表达水平显示:转染pRNAT-U6.1-siNS1、pRNAT-U6.1-siNS2和pRNAT-U6.1-siNS3的实验组细胞与3个对照组相比NS基因的mRNA表达水平明显降低,且pRNAT-U6.1-siNS2的沉默抑制作用强于pRNAT-U6.1-siNS1和pRNAT-U6.1-siNS3,几乎完全抑制,而3个对照组(无关siRNA对照组、空载体对照组和空白对照组)细胞中都存在较高水平NS mRNA表达。
4)荧光定量PCR检测显示siRNA干预组NS mRNA水平为0.034±0.008;无关siRNA对照组为0.255±0.049;空白对照组为0.273±0.049;原位杂交法检测各组1000个细胞,siRNA干预组NS mRNA阳性表达细胞数20.8±22.5,无关siRNA对照组138.6±13.8,空白对照组137.6±9.9;免疫细胞化学法检测各组1000个细胞,siRNA干预组NS蛋白阳性表达细胞数60.8±33.5,无关siRNA对照组154.2±16.4,空白对照组154.0±17.2;siRNA干预组EC9706细胞NS mRNA和蛋白表达水平明显低于两个对照组(P＜0.01)。
5)荧光定量PCR检测显示siRNA干预组EGFR mRNA水平为0.071±0.017;无关siRNA对照组为0.094±0.030;空白对照组为0.102±0.030;原位杂交法检测各组1000个细胞,siRNA干预组EGFR mRNA阳性表达细胞数29.4±10.4,无关siRNA对照组64.6±14.9,空白对照组79.4±20.8;免疫细胞化学法检测各组1000个细胞,siRNA干预组EGFR蛋白阳性表达细胞数38.2±15.6,无关siRNA对照组92.6±29.2,空白对照组94.8±19.4;siRNA干预组EC9706细胞EGFR mRNA和蛋白表达水平明显低于2个对照组(P＜0.01)。
6) Boyden chamber体外侵袭实验结果:siRNA干预组穿越Matrigel胶的细胞数69.0±13.6,无关siRNA对照组为126.6±10.8,空白对照组119.4±20.9,与2对照组相比,siRNA干预组穿越Matrigel胶的细胞数减少(P＜0.05)。
7)流式细胞术结果显示干扰NS基因的表达能明显促使细胞静止在G0/G1期,并且能诱导细胞凋亡。
结论成功构建了一组针对NS基因的siRNA表达载体,并筛选出最强的干扰表达载体,NS的下调能直接影响到EGFR的表达水平的下调,此外抑制NS的表达能降低EC9706细胞侵袭转移能力,并促使细胞周期静止在G0/G1期和诱导细胞凋亡。这些数据为后续利用该基因靶向治疗食管鳞状细胞癌提供理论依据。
第三部分NS基因沉默对裸鼠移植瘤生长的抑制作用
目的靶向NS基因的RNAi技术沉默EC9706细胞的NS表达,探讨将其接种裸鼠体内后对EC9706细胞的生长抑制作用。
方法分别将转染pRNAT-U6.1-siNS2的EC9706细胞(siRNA干预组),转染pRNAT-U6.1-siC的EC9706细胞(无关siRNA对照组)和未转染的EC9706(空白对照组)种植到裸鼠皮下,每组5只,比较裸鼠成瘤大小。分别应用RT-PCR、原位杂交、Western-blot及免疫组织化学方法检测裸鼠肿瘤组织中NS、EGF及EGFR基因mRNA和蛋白的表达情况。
结果3组细胞接种裸鼠背部皮下,经过5～10 d的成瘤潜伏期后,在其接种部位形成肉眼可见的肿瘤块,5周后3组裸鼠皮下均形成瘤体,成瘤率均为100%。5周后,空白对照组移植瘤大小为(1 806.40±77.75)mm~3,无关siRNA对照组为(1702.20±88.60)mm~3,siRNA干预组为(847.00±82.25)mm~3。RT-PCR、原位杂交、Western-blot及免疫组织化学方法检测裸鼠肿瘤组织中NS基因mRNA和蛋白的表达情况均显示,siRNA干预组NS基因的mRNA及蛋白表达均较两对照组降低(P＜0.05),免疫组化和原位杂交显示siRNA干预组EGF和EGFR基因的mRNA及蛋白表达均较两对照组降低(P＜0.05)。
结论EC9706细胞NS基因沉默可抑制裸鼠移植瘤生长,降低裸鼠体内NS、EGF及EGFR基因的表达。NS基因沉默可能成为食管癌治疗的新策略。
China is a country with the highest morbidity and mortality of esophageal carcinoma in the world.Each year,300 thousand people develop esophageal carcinoma,while more than half of them are Chinese people(167.2 thousand).Most patients suffered from esophageal carcinoma die of tumor infiltration and metastasis.So how to control tumors, especially esophageal carcinoma,to develop,infiltration,and metastasis has been the hotspot.
Tumorigenesis arises from the acquisition of multi-genetic alterations,combined with multiple factors and develops in consecutive progression.Oncogenes and tumor suppressor genes are the two kinds of genes most involved in tumorigenesis,among which the alterations of multiple key genes lie the basis to carcinogenesis.The production of nucleostemin gene,nucleostemin(NS),a novel p53 binding protein,is found to play a role in controlling the cell cycle progression in stem cells and malignant cells,predominantly maintain the cell proliferation and lead to the cell cycle withdrawal before maturation.NS gene expression is positively associated with tumor invasion,which has been confirmed in gastric cancer,renal cancer,breast cancer,and bladder cancer,etc.NS regulates the stem cell and malignant cell proliferation by p53 gene in the late S stage and G2 stage during the cell cycle.That's why NS is supposed to be the pivotal regulator specifically controlling the cell cycle from G2 to M.
Epidermal growth factor(EGF) is another vital factor in the cell proliferation and differentiation,which participates in transforming the normal cell to the neoplastic cell,and stimulates malignant cell to overgrowth,and which contributes to the neoplastic infiltration and metastasis.The excessive expression of EGF,together with the epidermal growth factor receptor(EGFR) has been indentified in many neoplasms.
RNA interference(RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequence-specific manner.RNAi has been observed in all eukaryotes,from yeast to mammals.In the eukaryotes,the long dsRNAs get processed into 21-26 nucleotide small interfering RNAs(siRNAs) by an RNaseⅢ-like enzyme called Dicer.The power and utility of RNAi for specifically silencing the expression of any gene for which sequence is available has driven its incredibly rapid adoption as a tool for gene research.To demonstrate the application of RNAi on neoplasm,especially esophageal carcinoma,we designed the present research.We detected the NS,EGF,and EGFR in malignant esophagus tissues,constructed the siRNA expression system targeting NS, transfected the vector to esophageal carcinoma cell lines(EC9706),and finally,assessed the NS,EGF,and EGFR in the malignant cells as well as examined the neoplasm growth in vivo in the nude mice inoculated with tumor cells.The following was the abstract of the full article.
Part One Expressions of NS,EGF and EGFR in esophageal squamous cell carcinoma and their associations with malignancy
Objective To demonstrate the expressions of NS,EGF and EGFR in esophageal squamous cell carcinoma(ESCC) and their associations with neoplastic progression.
Methods Sixty-two ESCC specimens,thirty-one adjacent dysplasia specimens and sixty-two normal mucosa specimens were used for determination.Histologically,fifteen neoplastic lesions were stageⅠ,twenty-five were stageⅡ,and twenty-two were stageⅢ. On metastasis,twenty had lymph node metastasis,and the other forty-two had not. According to invasion depth,seven cases were superficial,confined to the mucosa, submucosa,and superficial muscularis propria;while other fifty-five were deep to muscularis propria or adventitia.Immunohistochemi-stry was adopted to determine NS, EGF and EGFR proteins,and in situ hybridization was applied to measure NS,EGF and EGFR mRNA.RT-PCR and Real-time PCR were used to examine the expression levels of NS mRNA.The correlations of these proteins and mRNA with ESCC clinical data were statistically analyzed.
Results
1) The NS was expressed more(69.4%) in ESCC than in the adjacent hyperplasia and normal mucosa(41.9%and 17.7%,respectively) by immunohistochemisty(P＜0.05), and NS mRNA was more positively detected in ESCC(69.4%) than in the other two groups (25.8%and 21.0%,respectively,P＜0.05) by in situ hybridization.The following RT-PCR and Real-time PCR further confirmed that NS mRNA level in ESCC were higher than the other groups(P＜0.05).Correlation analysis revealed that NS and its mRNA were unrelated with age or gender(P＞0.05),but positively related with the histological evaluation, invasion depth and the lymph node metastasis.
2) More EGF protein was expressed in ESCC(69.4%) than in adjacent hyperplasia and normal mucosa(45.2%and 27.4%respectively,P＜0.05),and the adjacent hyperplasia had more EGF than the normal mucosa(P＜0.05).In situ hybridization demonstrated the similar results:EGF mRNA in ESCC was expressed the most(77.4%) among the three groups,and the adjacent hyperplasia was less than the ESCC but more than the normal tissue(45.2%vs.27.4%,P＜0.05).It could not be confirmed that the EGF and its mRNA was associated with age or gender(P＞0.05),but what has been confirmed was that they were related to the tumor malignancy as well as the NS and NS mRNA(P＜0.05).
3) EGFR and EGFR mRNA expression from the most to the least were ESCC(71.0% and 75.8%),adjacent hyperplasia(35.5%and 48.3%) and normal mucosa(14.5%and 40.3%)(P＜0.05).EGFR and EGFR mRNA were not relative to the age or gender but to the tumor stages,invasion depth,and lymph node metastasis.
4) The NS in neoplastic tissues was positively correlated with the EGF and EGFR, with the correlation coefficients were 0.545 and 0.731 respectively.And so did their mRNA, with the coefficients were 0.394 and 0.604 respectively.
Conclusion NS,EGF and EGFR are closely related with the carcinogenesis and development of ESCC.
Part Two The biobehaviour of the EC9706 cells transfected with the constructed siRNA expression system targeting against NS gene
Objective To construct and select an effective eukaryotic expression system of short interfering RNA(siRNA) suppressing NS gene,and evaluate the biobehaviour of EC9706 cells transfected with this expression system.
Methods The sequences of siRNA for NS were taken from GenBank,and three 19 bp oligonucleotides were selected as targeting sequences(accession numbers:AY825265 126-144 nt,199-217 nt and 487-505 nt).The three pairs of siRNA duplex for NS were inserted with BamHI and XhoI sites and a pair of nonspecific siRNA duplex was as control. After annealing,the designed oligonucleotides were cloned into plasmid pRNAT-U6.1 as the expression vector.The plasmid was transformed into the host E.coli DH5αand extracted for identification by PCR and sequencing.Transfected the four siRNA expression system to EC9706 cell line and set the empty vector as control and cells without transfection as blank control.The most effective siRNA expression system was selected by RT-PCR and transfected into the tumor cell line EC9706.The NS mRNA,EGFR mRNA and their proteins in the transfected EC9706 cells were detected by Real-time PCR,in situ hybridization and immunocytochemistry.The cell migration was examined by Boyden chamber assay.The cell cycle and apoptosis were assayed by flow cytometry.
Results
1) The bands of designed siRNA duplex of siNS1,siNS2,siNS3 and siC were visualized at 50-60 bp by gel electrophoresis,identical in size to the designed.
2) The recombinant plasmids were extracted from the proliferated host and amplified. The empty vector was 150 bp and the recombinant plasmids were 200 bp,suggesting the 50 bp hairpin DNA oligonucleotides were inserted.The sequences of inserted fragments in the recombinant plasmid pRNAT-U6.1-siNS1,pRNAT-U6.1-siNS2,pRNAT-U6.1-siNS3 and pRNAT-U6.1-siC were identical to the designed sequences.
3) RT-PCR showed that the NS mRNA was down-regulated in the cells transfected with pRNAT-U6.1-siNS1,pRNAT-U6.1-siNS2 and pRNAT-U6.1-siNS3,compared with the three control groups.The NS mRNA in cells transfected with pRNAT-U6.1-siNS2 was almost suppressed completely,with the least NS produced in all the transfected cells. Contrary to the cells transfected with pRNAT-U6.1-siNS1,pRNAT-U6.1-siNS2,and pRNAT-U6.1-siNS3,the cells transfectd with nonspecific siRNA plasmid,empty plasmid and blank cells produced high levels of NS mRNA.
4) NS mRNAs in siRNA silencing group,nonspecific group and blank group were 0.034±0.008,0.255±0.049,0.273±0.049,respectively,by Real-time PCR.The in situ hybridization demonstrated that(2.08±2.25)%cells in siRNA silencing group expressed NS mRNA,(13.86±1.38)%cells in nonspecific siRNA group expressed NS mRNA and (13.76±0.99)%cells expressed NS mRNA in blank group.Immunocytochemistry detected the NS protein in(6.08±3.35)%cells of siRNA silencing group,in(15.42±1.64)%cells of nonspecific siRNA group and(15.40±1.72)%cells of blank control.The NS gene was significantly suppressed by siRNA,compared with the nonspecific group and the blank control group(P＜0.01).
5) EGFR mRNAs in siRNA silencing group,nonspecific group and blank group were 0.071±0.017,0.094±0.030 and 0.102±0.030 respectively,by Real-time PCR.The in situ hybridization demonstrated that(2.94±1.04)%cells in siRNA group expressed EGFR mRNA,(7.94±2.08)%cells in nonspecific siRNA group expressed EGFR mRNA and (6.46±1.49)%cells expressed EGFR mRNA in blank group,Immunocytochemistry detected the EGFR protein in(3.82±1.56)%cells of siRNA silencing group,in (9.26±2.92)%cells of nonspecific siRNA group and(9.48±1.94)%cells of blank control. The EGFR gene was significantly suppressed by siRNA,compared with the nonspecific group and the blank control group(P＜0.01).
6) The invasion test showed that less EC9706 cells with NS knocked down migrated across the Matrigel compared with the nonspecific siRNA cells and blank cells(69.0±13.6 vs.126.6±10.8 and 69.0±13.6 vs.119.4±20.9,respectively,P＜0.05).
7) The flow cytometry showed that NS suppression arrested the cell cycle in G0/G1 phase,and induced the cell apoptosis.
Conclusion The NS specific siRNA expression system is established.The most effective expression system is selected and transfected into EC9706 cells.NS downregulation decreases the expression of EGFR and inhibites the aggression of EC9706. Furthermore,the NS suppression halts the cell cycle in G0/G1 phase and induces cell apoptosis.These data provide the foundation for the further gene therapy to esophageal cancer.
Part Three The inhibition of siRNA-mediated NS gene silencing on the nude murine tumor xenograft model
Objective To assess the inhibition of siRNA induced NS gene suppression on growth of EC9706 cells in the nude murine tumor xenograft model.
Methods Inoculated the EC9706 cells transfected with pRNAT-U6.1-siNS2 to five nude mice in each group subcutaneously,injected pRNAT-U6.1-siC as nonspecific siRNA control,and untransfected cells as blank control.The volume of the xenograft was measured.RT-PCR,in situ hybridization and Western-blot were applied for detecting NS, EGF and EGFR mRNAs and their products.
Results The tumors were visible five to ten days after inoculation,and five weeks later,the tumor xenografts were formed in all the animals.The volumes of tumor xenografts were(1806.40±77.75) mm~3 in blank group,(1702.20±88.60) mm~3 in nonspecific siRNA group,and(847.00±82.25) mm~3 in NS silencing group.Further assays showed that the targeted NS gene had been effectively inhibited and NS mRNA and protein had been knocked down in the NS specific siRNA group.The EGF mRNA and EGFR mRNA and proteins were also downregulated(P＜0.05).
Conclusion siRNA duplex targeting against NS delays the tumor xenograft growth, and reduces the expression of NS,EGF,and EGFR genes in the tumor xenograft models. The highly efficient siRNA mediated NS silence provides a novel gene analysis tool for therapeutic applications in malignant diseases.
肿瘤的发生发展是多因素、多步骤、多阶段和多基因改变的过程,癌基因和抑癌基因是受累最多的两大类型,各种关键基因的改变是人类肿瘤形成和发展的基础。近年来发现核干细胞因子(nucleostemin,NS)基因是一种新的p53结合蛋白,参与干细胞和肿瘤细胞的增殖调控,能维持细胞的扩增并抑制其向成熟细胞分化。先后发现胃癌、肾癌、乳癌、膀胱癌等肿瘤组织中NS基因表达量与肿瘤的侵袭力呈正相关,NS可能是在细胞周期的S期末和G2期通过已知的肿瘤抑制基因p53发挥作用来调节干细胞和癌细胞的增殖状态,NS可能是干细胞和肿瘤细胞穿越G2/M关键点的特异性调控因子。
表皮生长因子(epidermal growth factor,EGF)是近年来发现的在细胞增殖和分化中起重要作用的因子,可促进正常细胞的恶性转化及刺激肿瘤细胞的增殖。还与肿瘤浸润转移密切相关。研究表明EGF是通过细胞表面的表皮生长因子受体(epidermalgrowth factor receptor,EGFR)发挥其生物学效应。在许多肿瘤组织中均发现EGF及其受体EGFR的过表达且二者具有相关性。
RNA干扰(RNAi)是多种生物体内由双链RNA介导同源mRNA降解的现象。在细胞中,长的dsRNA被类似RNaseIH的Dicer酶切割成21-26核苷酸的小干扰RNA(siRNA)。RNAi基因阻断的有效性和特异性使其可能成为疾病基因疗法的理想工具。
作者拟联合检测食管癌组织中NS、EGF、EGFR的表达,构建针对NS基因的siRNA真核表达载体,转染食管癌EC9706细胞株,观察EC9706细胞生物学特性的变化及NS基因沉默对裸鼠移植瘤生长的影响。全文共分3部分,摘要分述如下。
第一部分食管鳞状细胞癌组织中NS、EGF及EGFR的表达及其相互关系
目的探讨食管鳞状细胞癌组织中NS、EGF及EGFR的表达及三者之间的关系。
方法采集62例食管鳞状细胞癌组织及其相应的31例癌旁不典型增生组织和62例正常食管黏膜组织标本。62例食管鳞状细胞癌组织学分级Ⅰ级15例,Ⅱ级25例,Ⅲ级22例;淋巴结转移20例,无淋巴结转移组42例。浸润浅层7例,肿瘤浸润深度在黏膜层、黏膜下层或浅肌层;深层55例,肿瘤浸润深度在深肌层或纤维膜。分别采用免疫组织化学、原位杂交方法检测上述标本NS、EGF、EGFR蛋白及mRNA的表达情况,RT-PCR、Real-time PCR检测NS mRNA,并分析其与食管鳞状细胞癌临床病理参数的关系。
结果
1)免疫组织化学方法检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中NS蛋白阳性表达率分别为17.7%、41.9%和69.4%,3组间比较差异有统计学意义(P＜0.05);原位杂交检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中NS mRNA阳性表达率分别为21.0%、25.8%和69.4%,3组间比较差异有统计学意义(P＜0.05);RT-PCR检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中NS mRNA相对表达量依次为0.866±0.103、0.913±0.085、0.971±0.121,3组间比较差异有统计学意义(P＜0.05);Real-time PCR检测结果显示:食管鳞状细胞癌组织、癌旁不典型增生组织和正常食管黏膜组织中NS mRNA表达水平分别为(4.53±2.16)、(3.09±2.28)和(2.10±2.34),3组间比较差异有统计学意义(P＜0.05)。食管鳞状细胞癌组织中NS蛋白阳性表达率、NS mRNA阳性表达率及RT-PCR、Real-time PCR检测结果均显示:NS蛋白和NS mRNA的表达与患者的年龄、性别无关(P＞0.05);与组织学分级、浸润深度和淋巴结转移有关(P＜0.05)。组织学分级越差、浸润越深及有淋巴结转移者NS蛋白、NS mRNA阳性表达率及RT-PCR、Real-time PCR检测的NS mRNA表达水平越高。
2)免疫组织化学方法检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中EGF蛋白的阳性表达率分别为27.4%、45.2%和69.4%,3组间比较差异有统计学意义(P＜0.05)。原位杂交检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中EGF mRNA的阳性表达率分别为40.3%、48.3%和77.4%。3组间比较差异有统计学意义(P＜0.05)。食管鳞状细胞癌组织中EGF蛋白阳性表达率、EGF mRNA阳性表达率与患者的年龄、性别无关(P＞0.05);与组织学分级、浸润深度和淋巴结转移有关(P＜0.05),组织学分级越差、浸润越深及有淋巴结转移者NS蛋白阳性表达率、EGF mRNA阳性表达率越高。
3)免疫组织化学方法检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中EGFR蛋白的阳性表达率分别为14.5%、35.5%和71.0%,3组间比较差异有统计学意义(P＜0.05)。原位杂交检测结果显示:正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中EGFR mRNA的阳性表达率分别为40.3%、48.3%和75.8%,3组间比较差异有统计学意义(P＜0.05)。食管鳞状细胞癌组织中EGFR蛋白阳性表达率、EGFR mRNA阳性表达率与患者的年龄、性别无关(P＞0.05);与组织学分级、浸润深度和淋巴结转移有关(P＜0.05),组织学分级越差、浸润越深及有淋巴结转移者NS蛋白、EGFR mRNA阳性表达率越高。
4)食管鳞状细胞癌组织中NS蛋白和EGF、EGFR蛋白的表达呈正相关,相关系数分别为0.545、0.731;食管鳞状细胞癌组织中NS mRNA和EGF mRNA、EGFRmRNA的表达呈正相关,相关系数分别为0.394、0.604。
结论NS、EGF及EGFR的表达与食管鳞状细胞癌的发生发展密切相关,并且三者之间关系密切。
第二部分pRNAT-U6.1-siNS干扰表达载体的构建及其对EC9706细胞生物学特性的影响
目的构建针对NS基因的小干扰RNA(siRNA)真核表达载体,筛选出抑制效率最强的表达载体,探讨NS基因siRNA表达载体转染EC9706细胞后对其细胞生物学特性的影响。
方法根据GenBank提供的NS基因AY825265 mRNA序列及siRNA设计原则设计siRNA,筛选得到126-144 nt,199-217 nt和487-505 nt 3个19 bp片段为靶序列;设计包含BamHI和XhoI酶切位点的3对靶向NS发卡样DNA寡核苷酸和一对随机对照发卡样DNA寡核苷酸,经过退火,将此序列克隆至载体pRNAT-U6.1中构建重组干扰表达载体;转化感受态细菌DH5α,提取质粒,应用PCR技术和测序方法鉴定重组克隆重组子。将构建好的4种siRNA表达载体转染EC9706细胞,同时设空载体对照组(转染pRNAT-U6.1空载体的细胞)及空白对照组(未转染的细胞),RT-PCR检测siRNA表达载体转染EC9706细胞后对NS mRNA的抑制作用,从而筛选出干扰效率最强的表达载体,并利用该载体转染EC9706细胞,应用Real-time PCR、原位杂交技术、Western blot和免疫细胞化学法分别检测干扰前后NS和EGFR mRNA及蛋白的表达的变化,进一步应用Boyden Chamber体外侵袭实验观察其对侵袭转移的影响,最后应用流式细胞术观察EC9706细胞周期和细胞凋亡的变化。
结果
1) siRNA发卡DNA的退火结果:siNS1、siNS2、siNS3和siC发卡样siRNA单链DNA寡核苷酸,退火后,电泳可见明亮条带,位于100bp下,约50-60bp处,与设计完全一致。
2)克隆重组子鉴定结果:将转化菌落扩增后提取质粒,以空pRNAT-U6.1为阴性对照,使用pRNAT-U6.1插入鉴定引物进行扩增。空pRNAT-U6.1扩增产物为150 bp,阳性克隆由于插入约50 bp的发卡DNA片段,故扩增产物约为200 bp。所有3种针对NS和1个无关对照重组转化克隆都得到有阳性克隆,阳性重组子pRNAT-U6.1-siNS1、pRNAT-U6.1-siNS2、pRNAT-U6.1-siNS3和pRNAT-U6.1-siC插入片段测序的结果,与设计序列比较结果完全一致。
3) RT-PCR方法分别测定各组EC9706细胞中NS mRNA表达水平显示:转染pRNAT-U6.1-siNS1、pRNAT-U6.1-siNS2和pRNAT-U6.1-siNS3的实验组细胞与3个对照组相比NS基因的mRNA表达水平明显降低,且pRNAT-U6.1-siNS2的沉默抑制作用强于pRNAT-U6.1-siNS1和pRNAT-U6.1-siNS3,几乎完全抑制,而3个对照组(无关siRNA对照组、空载体对照组和空白对照组)细胞中都存在较高水平NS mRNA表达。
4)荧光定量PCR检测显示siRNA干预组NS mRNA水平为0.034±0.008;无关siRNA对照组为0.255±0.049;空白对照组为0.273±0.049;原位杂交法检测各组1000个细胞,siRNA干预组NS mRNA阳性表达细胞数20.8±22.5,无关siRNA对照组138.6±13.8,空白对照组137.6±9.9;免疫细胞化学法检测各组1000个细胞,siRNA干预组NS蛋白阳性表达细胞数60.8±33.5,无关siRNA对照组154.2±16.4,空白对照组154.0±17.2;siRNA干预组EC9706细胞NS mRNA和蛋白表达水平明显低于两个对照组(P＜0.01)。
5)荧光定量PCR检测显示siRNA干预组EGFR mRNA水平为0.071±0.017;无关siRNA对照组为0.094±0.030;空白对照组为0.102±0.030;原位杂交法检测各组1000个细胞,siRNA干预组EGFR mRNA阳性表达细胞数29.4±10.4,无关siRNA对照组64.6±14.9,空白对照组79.4±20.8;免疫细胞化学法检测各组1000个细胞,siRNA干预组EGFR蛋白阳性表达细胞数38.2±15.6,无关siRNA对照组92.6±29.2,空白对照组94.8±19.4;siRNA干预组EC9706细胞EGFR mRNA和蛋白表达水平明显低于2个对照组(P＜0.01)。
6) Boyden chamber体外侵袭实验结果:siRNA干预组穿越Matrigel胶的细胞数69.0±13.6,无关siRNA对照组为126.6±10.8,空白对照组119.4±20.9,与2对照组相比,siRNA干预组穿越Matrigel胶的细胞数减少(P＜0.05)。
7)流式细胞术结果显示干扰NS基因的表达能明显促使细胞静止在G0/G1期,并且能诱导细胞凋亡。
结论成功构建了一组针对NS基因的siRNA表达载体,并筛选出最强的干扰表达载体,NS的下调能直接影响到EGFR的表达水平的下调,此外抑制NS的表达能降低EC9706细胞侵袭转移能力,并促使细胞周期静止在G0/G1期和诱导细胞凋亡。这些数据为后续利用该基因靶向治疗食管鳞状细胞癌提供理论依据。
第三部分NS基因沉默对裸鼠移植瘤生长的抑制作用
目的靶向NS基因的RNAi技术沉默EC9706细胞的NS表达,探讨将其接种裸鼠体内后对EC9706细胞的生长抑制作用。
方法分别将转染pRNAT-U6.1-siNS2的EC9706细胞(siRNA干预组),转染pRNAT-U6.1-siC的EC9706细胞(无关siRNA对照组)和未转染的EC9706(空白对照组)种植到裸鼠皮下,每组5只,比较裸鼠成瘤大小。分别应用RT-PCR、原位杂交、Western-blot及免疫组织化学方法检测裸鼠肿瘤组织中NS、EGF及EGFR基因mRNA和蛋白的表达情况。
结果3组细胞接种裸鼠背部皮下,经过5～10 d的成瘤潜伏期后,在其接种部位形成肉眼可见的肿瘤块,5周后3组裸鼠皮下均形成瘤体,成瘤率均为100%。5周后,空白对照组移植瘤大小为(1 806.40±77.75)mm~3,无关siRNA对照组为(1702.20±88.60)mm~3,siRNA干预组为(847.00±82.25)mm~3。RT-PCR、原位杂交、Western-blot及免疫组织化学方法检测裸鼠肿瘤组织中NS基因mRNA和蛋白的表达情况均显示,siRNA干预组NS基因的mRNA及蛋白表达均较两对照组降低(P＜0.05),免疫组化和原位杂交显示siRNA干预组EGF和EGFR基因的mRNA及蛋白表达均较两对照组降低(P＜0.05)。
结论EC9706细胞NS基因沉默可抑制裸鼠移植瘤生长,降低裸鼠体内NS、EGF及EGFR基因的表达。NS基因沉默可能成为食管癌治疗的新策略。
China is a country with the highest morbidity and mortality of esophageal carcinoma in the world.Each year,300 thousand people develop esophageal carcinoma,while more than half of them are Chinese people(167.2 thousand).Most patients suffered from esophageal carcinoma die of tumor infiltration and metastasis.So how to control tumors, especially esophageal carcinoma,to develop,infiltration,and metastasis has been the hotspot.
Tumorigenesis arises from the acquisition of multi-genetic alterations,combined with multiple factors and develops in consecutive progression.Oncogenes and tumor suppressor genes are the two kinds of genes most involved in tumorigenesis,among which the alterations of multiple key genes lie the basis to carcinogenesis.The production of nucleostemin gene,nucleostemin(NS),a novel p53 binding protein,is found to play a role in controlling the cell cycle progression in stem cells and malignant cells,predominantly maintain the cell proliferation and lead to the cell cycle withdrawal before maturation.NS gene expression is positively associated with tumor invasion,which has been confirmed in gastric cancer,renal cancer,breast cancer,and bladder cancer,etc.NS regulates the stem cell and malignant cell proliferation by p53 gene in the late S stage and G2 stage during the cell cycle.That's why NS is supposed to be the pivotal regulator specifically controlling the cell cycle from G2 to M.
Epidermal growth factor(EGF) is another vital factor in the cell proliferation and differentiation,which participates in transforming the normal cell to the neoplastic cell,and stimulates malignant cell to overgrowth,and which contributes to the neoplastic infiltration and metastasis.The excessive expression of EGF,together with the epidermal growth factor receptor(EGFR) has been indentified in many neoplasms.
RNA interference(RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequence-specific manner.RNAi has been observed in all eukaryotes,from yeast to mammals.In the eukaryotes,the long dsRNAs get processed into 21-26 nucleotide small interfering RNAs(siRNAs) by an RNaseⅢ-like enzyme called Dicer.The power and utility of RNAi for specifically silencing the expression of any gene for which sequence is available has driven its incredibly rapid adoption as a tool for gene research.To demonstrate the application of RNAi on neoplasm,especially esophageal carcinoma,we designed the present research.We detected the NS,EGF,and EGFR in malignant esophagus tissues,constructed the siRNA expression system targeting NS, transfected the vector to esophageal carcinoma cell lines(EC9706),and finally,assessed the NS,EGF,and EGFR in the malignant cells as well as examined the neoplasm growth in vivo in the nude mice inoculated with tumor cells.The following was the abstract of the full article.
Part One Expressions of NS,EGF and EGFR in esophageal squamous cell carcinoma and their associations with malignancy
Objective To demonstrate the expressions of NS,EGF and EGFR in esophageal squamous cell carcinoma(ESCC) and their associations with neoplastic progression.
Methods Sixty-two ESCC specimens,thirty-one adjacent dysplasia specimens and sixty-two normal mucosa specimens were used for determination.Histologically,fifteen neoplastic lesions were stageⅠ,twenty-five were stageⅡ,and twenty-two were stageⅢ. On metastasis,twenty had lymph node metastasis,and the other forty-two had not. According to invasion depth,seven cases were superficial,confined to the mucosa, submucosa,and superficial muscularis propria;while other fifty-five were deep to muscularis propria or adventitia.Immunohistochemi-stry was adopted to determine NS, EGF and EGFR proteins,and in situ hybridization was applied to measure NS,EGF and EGFR mRNA.RT-PCR and Real-time PCR were used to examine the expression levels of NS mRNA.The correlations of these proteins and mRNA with ESCC clinical data were statistically analyzed.
Results
1) The NS was expressed more(69.4%) in ESCC than in the adjacent hyperplasia and normal mucosa(41.9%and 17.7%,respectively) by immunohistochemisty(P＜0.05), and NS mRNA was more positively detected in ESCC(69.4%) than in the other two groups (25.8%and 21.0%,respectively,P＜0.05) by in situ hybridization.The following RT-PCR and Real-time PCR further confirmed that NS mRNA level in ESCC were higher than the other groups(P＜0.05).Correlation analysis revealed that NS and its mRNA were unrelated with age or gender(P＞0.05),but positively related with the histological evaluation, invasion depth and the lymph node metastasis.
2) More EGF protein was expressed in ESCC(69.4%) than in adjacent hyperplasia and normal mucosa(45.2%and 27.4%respectively,P＜0.05),and the adjacent hyperplasia had more EGF than the normal mucosa(P＜0.05).In situ hybridization demonstrated the similar results:EGF mRNA in ESCC was expressed the most(77.4%) among the three groups,and the adjacent hyperplasia was less than the ESCC but more than the normal tissue(45.2%vs.27.4%,P＜0.05).It could not be confirmed that the EGF and its mRNA was associated with age or gender(P＞0.05),but what has been confirmed was that they were related to the tumor malignancy as well as the NS and NS mRNA(P＜0.05).
3) EGFR and EGFR mRNA expression from the most to the least were ESCC(71.0% and 75.8%),adjacent hyperplasia(35.5%and 48.3%) and normal mucosa(14.5%and 40.3%)(P＜0.05).EGFR and EGFR mRNA were not relative to the age or gender but to the tumor stages,invasion depth,and lymph node metastasis.
4) The NS in neoplastic tissues was positively correlated with the EGF and EGFR, with the correlation coefficients were 0.545 and 0.731 respectively.And so did their mRNA, with the coefficients were 0.394 and 0.604 respectively.
Conclusion NS,EGF and EGFR are closely related with the carcinogenesis and development of ESCC.
Part Two The biobehaviour of the EC9706 cells transfected with the constructed siRNA expression system targeting against NS gene
Objective To construct and select an effective eukaryotic expression system of short interfering RNA(siRNA) suppressing NS gene,and evaluate the biobehaviour of EC9706 cells transfected with this expression system.
Methods The sequences of siRNA for NS were taken from GenBank,and three 19 bp oligonucleotides were selected as targeting sequences(accession numbers:AY825265 126-144 nt,199-217 nt and 487-505 nt).The three pairs of siRNA duplex for NS were inserted with BamHI and XhoI sites and a pair of nonspecific siRNA duplex was as control. After annealing,the designed oligonucleotides were cloned into plasmid pRNAT-U6.1 as the expression vector.The plasmid was transformed into the host E.coli DH5αand extracted for identification by PCR and sequencing.Transfected the four siRNA expression system to EC9706 cell line and set the empty vector as control and cells without transfection as blank control.The most effective siRNA expression system was selected by RT-PCR and transfected into the tumor cell line EC9706.The NS mRNA,EGFR mRNA and their proteins in the transfected EC9706 cells were detected by Real-time PCR,in situ hybridization and immunocytochemistry.The cell migration was examined by Boyden chamber assay.The cell cycle and apoptosis were assayed by flow cytometry.
Results
1) The bands of designed siRNA duplex of siNS1,siNS2,siNS3 and siC were visualized at 50-60 bp by gel electrophoresis,identical in size to the designed.
2) The recombinant plasmids were extracted from the proliferated host and amplified. The empty vector was 150 bp and the recombinant plasmids were 200 bp,suggesting the 50 bp hairpin DNA oligonucleotides were inserted.The sequences of inserted fragments in the recombinant plasmid pRNAT-U6.1-siNS1,pRNAT-U6.1-siNS2,pRNAT-U6.1-siNS3 and pRNAT-U6.1-siC were identical to the designed sequences.
3) RT-PCR showed that the NS mRNA was down-regulated in the cells transfected with pRNAT-U6.1-siNS1,pRNAT-U6.1-siNS2 and pRNAT-U6.1-siNS3,compared with the three control groups.The NS mRNA in cells transfected with pRNAT-U6.1-siNS2 was almost suppressed completely,with the least NS produced in all the transfected cells. Contrary to the cells transfected with pRNAT-U6.1-siNS1,pRNAT-U6.1-siNS2,and pRNAT-U6.1-siNS3,the cells transfectd with nonspecific siRNA plasmid,empty plasmid and blank cells produced high levels of NS mRNA.
4) NS mRNAs in siRNA silencing group,nonspecific group and blank group were 0.034±0.008,0.255±0.049,0.273±0.049,respectively,by Real-time PCR.The in situ hybridization demonstrated that(2.08±2.25)%cells in siRNA silencing group expressed NS mRNA,(13.86±1.38)%cells in nonspecific siRNA group expressed NS mRNA and (13.76±0.99)%cells expressed NS mRNA in blank group.Immunocytochemistry detected the NS protein in(6.08±3.35)%cells of siRNA silencing group,in(15.42±1.64)%cells of nonspecific siRNA group and(15.40±1.72)%cells of blank control.The NS gene was significantly suppressed by siRNA,compared with the nonspecific group and the blank control group(P＜0.01).
5) EGFR mRNAs in siRNA silencing group,nonspecific group and blank group were 0.071±0.017,0.094±0.030 and 0.102±0.030 respectively,by Real-time PCR.The in situ hybridization demonstrated that(2.94±1.04)%cells in siRNA group expressed EGFR mRNA,(7.94±2.08)%cells in nonspecific siRNA group expressed EGFR mRNA and (6.46±1.49)%cells expressed EGFR mRNA in blank group,Immunocytochemistry detected the EGFR protein in(3.82±1.56)%cells of siRNA silencing group,in (9.26±2.92)%cells of nonspecific siRNA group and(9.48±1.94)%cells of blank control. The EGFR gene was significantly suppressed by siRNA,compared with the nonspecific group and the blank control group(P＜0.01).
6) The invasion test showed that less EC9706 cells with NS knocked down migrated across the Matrigel compared with the nonspecific siRNA cells and blank cells(69.0±13.6 vs.126.6±10.8 and 69.0±13.6 vs.119.4±20.9,respectively,P＜0.05).
7) The flow cytometry showed that NS suppression arrested the cell cycle in G0/G1 phase,and induced the cell apoptosis.
Conclusion The NS specific siRNA expression system is established.The most effective expression system is selected and transfected into EC9706 cells.NS downregulation decreases the expression of EGFR and inhibites the aggression of EC9706. Furthermore,the NS suppression halts the cell cycle in G0/G1 phase and induces cell apoptosis.These data provide the foundation for the further gene therapy to esophageal cancer.
Part Three The inhibition of siRNA-mediated NS gene silencing on the nude murine tumor xenograft model
Objective To assess the inhibition of siRNA induced NS gene suppression on growth of EC9706 cells in the nude murine tumor xenograft model.
Methods Inoculated the EC9706 cells transfected with pRNAT-U6.1-siNS2 to five nude mice in each group subcutaneously,injected pRNAT-U6.1-siC as nonspecific siRNA control,and untransfected cells as blank control.The volume of the xenograft was measured.RT-PCR,in situ hybridization and Western-blot were applied for detecting NS, EGF and EGFR mRNAs and their products.
Results The tumors were visible five to ten days after inoculation,and five weeks later,the tumor xenografts were formed in all the animals.The volumes of tumor xenografts were(1806.40±77.75) mm~3 in blank group,(1702.20±88.60) mm~3 in nonspecific siRNA group,and(847.00±82.25) mm~3 in NS silencing group.Further assays showed that the targeted NS gene had been effectively inhibited and NS mRNA and protein had been knocked down in the NS specific siRNA group.The EGF mRNA and EGFR mRNA and proteins were also downregulated(P＜0.05).
Conclusion siRNA duplex targeting against NS delays the tumor xenograft growth, and reduces the expression of NS,EGF,and EGFR genes in the tumor xenograft models. The highly efficient siRNA mediated NS silence provides a novel gene analysis tool for therapeutic applications in malignant diseases.
引文
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