NDRG2基因在肾癌中的表达及其抑制肾癌A-498细胞增殖的实验研究
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摘要
目的意义:NDRG2基因是1999年从正常人全脑cDNA文库中克隆到的新基因[GenBank登录号AF159092.],染色体定位于14q12.1,基因组中由15个内含子,16个外显子构成。NDRG2基因的cDNA全长为2024 bp,编码357个氨基酸的蛋白质,分子量约41kD。目前研究表明,NDRG2基因在多种肿瘤组织或细胞系如结肠癌、胶质瘤、白血病、淋巴瘤和腮腺癌中无表达或低表达,相反在上述肿瘤相应的正常组织细胞中有较高水平的表达。另外在中枢神经系统的大脑皮质、白质、神经核,唾液腺上皮细胞和骨骼肌细胞中高表达。同时基因转染实验表明,NDRG2可抑制胶质瘤BT325细胞由G1期向S期的过渡,因此,推测NDRG2可能是一种新的抑癌基因。本研究的目的意义是探讨NDRG2基因在肾癌发病机制中的作用,确定NDRG2基因的功能,为肾癌的防治提供理论依据。
实验方法:采用RT-PCR、Western blot和免疫组织化学方法确定NDRG2在肾癌细胞系、肾脏细胞系及正常组织和肾癌组织的表达;构建NDRG2的腺病毒表达载体并进行腺病毒的包装及滴度测定,获得高滴度的重组NDRG2腺病毒用于后续的细胞功能实验研究;用NDRG2重组腺病毒转染经证实低表达NDRG2的肾透明细胞癌细胞系A-498中,提高该细胞中NDRG2表达水平,通过流式细胞术分析细胞周期并检测凋亡,并运用蛋白免疫印迹检测细胞周期相关蛋白的变化;通过p53重组腺病毒转染肾癌A-498细胞,观察p53基因对NDRG2基因表达的影响。
实验结果:1、通过免疫组织化学、RT-PCR、蛋白印迹等检测了人肾癌组织和人肾小管上皮细胞系、肾癌细胞系中NDRG2蛋白和mRNA的表达,结果显示:人近端肾小管上皮细胞HK-2细胞系、人胚肾上皮细胞HKC细胞系中NDRG2的mRNA表达水平较高;而在肾癌细胞系786-O、A-498中NDRG2的mRNA表达水平较低;NDRG2 mRNA在18例肾癌患者肿瘤组织中的表达水平较其相应癌旁组织低,且该表达与其病理分级呈正相关趋势;仅有6例患者的肾肿瘤组织中NDRG2的mRNA表达水平高于相对应的自身肿瘤瘤旁组织;另外有14例患者两者NDRG2 mRNA的表达无差异。2、采用美国Novagen公司成熟的pAdTrack-CMV腺病毒表达系统首先构建了含有NDRG2基因的重组穿梭载体,通过脂质体转染法将线性化的重组穿梭载体与骨架蛋白载体转染293细胞获得了原始病毒种,通过挑选单蚀斑进一步感染293细胞获得了单蚀斑病毒裂解液,在此基础上我们建立了原始种子批、主种子批及工作种子批,最后用工作种子批完成了pAd-GFP-NDRG2重组腺病毒的生产、病毒的纯化及病毒滴度的测定,最终获得的重组腺病毒滴度为1.1×1011pfu /ml。3、通过蛋白印迹实验证实重组腺病毒pAd-GFP-NDRG2转染可增加A-498细胞内源性NDRG2的表达水平;随后的MTT检测结果表明:A-498细胞病毒转染组,各检测时间点MTT OD值均低于未转染和空质粒转染组,48h差异具有显著性,说明NDRG2表达对A-498细胞生长具有抑制作用;进一步的细胞周期分析显示:A-498细胞质粒转染组出现G1期阻滞(G1期捕获),提示NDRG2通过细胞周期阻滞来抑制A-498细胞增殖;凋亡检测实验结果显示:与未转染的A-498细胞(1.0%)和不含插段的重组腺病毒感染的A-498细胞(2.0%)相比,含NDRG2插段的重组腺病毒组感染的A-498细胞中凋亡细胞明显增多,感染48h后达12.0%;最后通过蛋白印迹实验检测发现:人NDRG2基因转染A-498细胞后,周期素蛋白cyclinD1、cyclinE表达减少,cyclinD2、cyclinD3和cdk2表达无明显变化,由此推测NDRG2基因抑制细胞增殖可能是通过影响周期素蛋白D1和E起作用的。4、通过蛋白印迹和RT-PCR发现A-498细胞表达没有活性的p53,但是基本检测不到NDRG2的表达;通过转染活化型p53基因到A-498细胞后观察到了NDRG2表达水平的升高,且呈剂量依赖关系,该结果说明NDRG2可受到p53基因的调控,提示NDRG2也是p53的一个下游基因。
实验结论:NDRG2在正常肾组织中高表达,而在肾癌组织中出现低表达或无表达;NDRG2能够抑制肾癌A-498细胞的增殖,并能产生G1期阻滞,抑制细胞周期素D1、E的表达;p53的表达能够上调NDRG2的表达,本研究的结果进一步证实NDRG2基因是一种新的抑癌基因,以其为靶点的基因治疗策略可为肾癌的基因治疗提供新思路。
Aim: NDRG2 is a new gene cloned from the healthy human brain cDNA library in 1999 [GenBank accession number: AF159092], and the chromosomal localization is 14q12.1 which consists of 15 introns and 16 extrons. The total length of the cDNA of NDRG2 is 2024 bp which encodes the protein composed of 357 amino acid residues and the molecular weight is about 41 kD. The studies indicated that the expressions of NDRG2 in many tumorous tissues or cell lines, such as colon carcinoma, neurogliocytoma, leukemia, leucoma and carcinoma of parotid gland, are negative or very low, on the contrary, this gene in those corresponding normal tissues of the above mentioned tumors is of high level expression. Moreover, it is highly expressed in the cerebral cortex, substantia alba and nerve corpuscle of the central nervous system as well as in the endothelial cells of salivary gland and skeletal muscle cells. Meanwhile, the gene transfection researches indicated that NDRG2 can inhibit the transition of neurogliocytoma cell line BT325 from G1 phase to S phase. Thus, it is postulated that NDRG2 may be a new anti-oncogene. The purpose of this study is to discuss the role of NDRG2 in the pathogenesy of renal carcinoma and to ascertain the function of NDRG2 in order to provide the theoretical bases for the prevention and treatment of renal carcinoma.
Methods: the RT-PCT, Western blot and immunohistochemistry are applied to determine the expressions of NDRG2 in the renal carcinoma cell line, the renal cell line; normal tissue and tissue of renal carcinoma; construct the recombinant adenovirus expression vector of NDRG2 and perform the packaging of the recombinant adenovirus and titer determination to obtain the recombinant adenovirus vector of NDRG2 with high titer for the subsequent study of cell function; transfect the recombinant adenovirus vector of NDRG2 into the suprarenal epithelioma cell line A-498 which has been confirmed to be with low expression of NDRG2 to increase the expression level of NDRG2 in this cell line, and analyze the cell cycle, detect the apoptosis by flow cytometry and use the Western blot to detect the changes of cell cycle-related protein; observe the effect of p53 gene to the expression of NDRG2 through the transfection of the recombinant adenovirus vector of p53 into the renal carcinoma cell line A-498. Results: 1. The NDRG2 protein and expression of the mRNA in human tissue of renal carcinoma and the renal carcinoma cell lines were detected and the results indicated that the renal cell lines HKC and HK-2 are of high expression levels of NDRG2 mRNA, however, the expression levels of NDRG2 mRNA in the renal cell carcinoma cell lines 786-O and A-498 were comparatively lower; the expression levels of NDRG2 mRNA in the tissue of carcinoma were lower than those in the tissue beside the carcinoma in 18 cases of patients with renal carcinoma and the expression level was directly correlated with the pathological grade; in this study, there were only 6 cases of patients with renal carcinoma in which the expression levels of NDRG2 mRNA in the tissue of carcinoma were higher than those in the corresponding tissue beside carcinoma; otherwise, there were 14 cases in which there was no difference between the expression levels of NDRG2 mRNA between the tissues. 2. The recombinant shuttle vector containing NDRG2 was firstly constructed by the mature adenovirus expression system pAdTrack-CMV from American Novagen company, the linearized recombinant shuttle vector and the vector containing the gene of skelemin were transfected into the cell line HEK293 through the liposomal transfection to obtain the prime viral strains, the single plaque bacteriophages were selected to further infect HEK293 cells to acquire the viral lysate of single plaque bacteriophage. Based on these operations, we established the prime seed lot, major seed lot and working seed lot and finally accomplished the production, purification and titer determination of the recombinant adenovirus pAd-GFP-NDRG2, and the final virus titer of the recombinant adenovirus was 1.1×1011 pfu/ml. 3. The results of Western blot confirmed that the transfection of the recombinant adenovirus pAd-GFP-NDRG2 increased the expression level of the endogenous NDRG2 in the cell line A-498. The subsequent results of MTT detection indicated that the OD values of MTT at different detected time points within 48 h in the A-498 group transfected by the recombinant adenovirus were all significantly lower than those corresponding values in the groups without transfection and transfected by the blank plasmid, which suggested that the expression of NDRG2 inhibited the growth of the cell line A-498. The further analysis of cell cycle showed that the plasmid transfection induced the G1 phase arrest in A-498, which implied that NDRG2 might inhibit the proliferation of A-498 through the cell cycle arrest. The results of the apoptosis analysis indicated that the proportion of the apoptosis cells in the A-498 cells infected by the recombinant adenovirus containing NDRG2, which was 12.% at 48 h after infection, was significantly higher that those in the A-498 cells without transfection (1.0%) and transfected by the recombinant adenovirus without NDRG2 (2.0%). Finally, the results of the Western blot indicated that, in the A-498 cells tranfected by human NDRG2, the expression levels of cyclin D1 and cyclin E decreased, but there was no significant change on the expression levels of cyclin D2, cyclin D3 and cdk2. Thus, it is postulated that NDRG2 may inhibit the cell proliferation through the effect on cyclin D1 and cyclin E. 4. It was discovered that A-498 cells express inactive p53 through the Western blot and RT-PCR, however, the expression of NDRG2 was almost undetectable. The increased expression level of NDRG2 was observed when the A-498 cells were transfected by the p53 gene in activated form, which was dose-dependent. This result suggested that NDRG2 is regulated by p53 gene, thus, NDRG2 is also a downstream gene of p53.
Conclusions: NDRG2 is of high expression level in normal renal tissue but of low expression level or without expression in the tissue of renal carcinoma. NDRG2 can inhibit the proliferation of the renal carcinoma cell line A-498 cells, induce arrest at G1 phase and inhibit the expressions of cyclin D1 and cyclin E. The expression of p53 can up-regulate the expression of NDRG2. Thus, the result in this study further confirmed that NDRG2 is a novel anti-oncogene and the strategy of gene therapy taking this gene as the target might provide a new way to the gene therapy of renal carcinoma.
实验方法:采用RT-PCR、Western blot和免疫组织化学方法确定NDRG2在肾癌细胞系、肾脏细胞系及正常组织和肾癌组织的表达;构建NDRG2的腺病毒表达载体并进行腺病毒的包装及滴度测定,获得高滴度的重组NDRG2腺病毒用于后续的细胞功能实验研究;用NDRG2重组腺病毒转染经证实低表达NDRG2的肾透明细胞癌细胞系A-498中,提高该细胞中NDRG2表达水平,通过流式细胞术分析细胞周期并检测凋亡,并运用蛋白免疫印迹检测细胞周期相关蛋白的变化;通过p53重组腺病毒转染肾癌A-498细胞,观察p53基因对NDRG2基因表达的影响。
实验结果:1、通过免疫组织化学、RT-PCR、蛋白印迹等检测了人肾癌组织和人肾小管上皮细胞系、肾癌细胞系中NDRG2蛋白和mRNA的表达,结果显示:人近端肾小管上皮细胞HK-2细胞系、人胚肾上皮细胞HKC细胞系中NDRG2的mRNA表达水平较高;而在肾癌细胞系786-O、A-498中NDRG2的mRNA表达水平较低;NDRG2 mRNA在18例肾癌患者肿瘤组织中的表达水平较其相应癌旁组织低,且该表达与其病理分级呈正相关趋势;仅有6例患者的肾肿瘤组织中NDRG2的mRNA表达水平高于相对应的自身肿瘤瘤旁组织;另外有14例患者两者NDRG2 mRNA的表达无差异。2、采用美国Novagen公司成熟的pAdTrack-CMV腺病毒表达系统首先构建了含有NDRG2基因的重组穿梭载体,通过脂质体转染法将线性化的重组穿梭载体与骨架蛋白载体转染293细胞获得了原始病毒种,通过挑选单蚀斑进一步感染293细胞获得了单蚀斑病毒裂解液,在此基础上我们建立了原始种子批、主种子批及工作种子批,最后用工作种子批完成了pAd-GFP-NDRG2重组腺病毒的生产、病毒的纯化及病毒滴度的测定,最终获得的重组腺病毒滴度为1.1×1011pfu /ml。3、通过蛋白印迹实验证实重组腺病毒pAd-GFP-NDRG2转染可增加A-498细胞内源性NDRG2的表达水平;随后的MTT检测结果表明:A-498细胞病毒转染组,各检测时间点MTT OD值均低于未转染和空质粒转染组,48h差异具有显著性,说明NDRG2表达对A-498细胞生长具有抑制作用;进一步的细胞周期分析显示:A-498细胞质粒转染组出现G1期阻滞(G1期捕获),提示NDRG2通过细胞周期阻滞来抑制A-498细胞增殖;凋亡检测实验结果显示:与未转染的A-498细胞(1.0%)和不含插段的重组腺病毒感染的A-498细胞(2.0%)相比,含NDRG2插段的重组腺病毒组感染的A-498细胞中凋亡细胞明显增多,感染48h后达12.0%;最后通过蛋白印迹实验检测发现:人NDRG2基因转染A-498细胞后,周期素蛋白cyclinD1、cyclinE表达减少,cyclinD2、cyclinD3和cdk2表达无明显变化,由此推测NDRG2基因抑制细胞增殖可能是通过影响周期素蛋白D1和E起作用的。4、通过蛋白印迹和RT-PCR发现A-498细胞表达没有活性的p53,但是基本检测不到NDRG2的表达;通过转染活化型p53基因到A-498细胞后观察到了NDRG2表达水平的升高,且呈剂量依赖关系,该结果说明NDRG2可受到p53基因的调控,提示NDRG2也是p53的一个下游基因。
实验结论:NDRG2在正常肾组织中高表达,而在肾癌组织中出现低表达或无表达;NDRG2能够抑制肾癌A-498细胞的增殖,并能产生G1期阻滞,抑制细胞周期素D1、E的表达;p53的表达能够上调NDRG2的表达,本研究的结果进一步证实NDRG2基因是一种新的抑癌基因,以其为靶点的基因治疗策略可为肾癌的基因治疗提供新思路。
Aim: NDRG2 is a new gene cloned from the healthy human brain cDNA library in 1999 [GenBank accession number: AF159092], and the chromosomal localization is 14q12.1 which consists of 15 introns and 16 extrons. The total length of the cDNA of NDRG2 is 2024 bp which encodes the protein composed of 357 amino acid residues and the molecular weight is about 41 kD. The studies indicated that the expressions of NDRG2 in many tumorous tissues or cell lines, such as colon carcinoma, neurogliocytoma, leukemia, leucoma and carcinoma of parotid gland, are negative or very low, on the contrary, this gene in those corresponding normal tissues of the above mentioned tumors is of high level expression. Moreover, it is highly expressed in the cerebral cortex, substantia alba and nerve corpuscle of the central nervous system as well as in the endothelial cells of salivary gland and skeletal muscle cells. Meanwhile, the gene transfection researches indicated that NDRG2 can inhibit the transition of neurogliocytoma cell line BT325 from G1 phase to S phase. Thus, it is postulated that NDRG2 may be a new anti-oncogene. The purpose of this study is to discuss the role of NDRG2 in the pathogenesy of renal carcinoma and to ascertain the function of NDRG2 in order to provide the theoretical bases for the prevention and treatment of renal carcinoma.
Methods: the RT-PCT, Western blot and immunohistochemistry are applied to determine the expressions of NDRG2 in the renal carcinoma cell line, the renal cell line; normal tissue and tissue of renal carcinoma; construct the recombinant adenovirus expression vector of NDRG2 and perform the packaging of the recombinant adenovirus and titer determination to obtain the recombinant adenovirus vector of NDRG2 with high titer for the subsequent study of cell function; transfect the recombinant adenovirus vector of NDRG2 into the suprarenal epithelioma cell line A-498 which has been confirmed to be with low expression of NDRG2 to increase the expression level of NDRG2 in this cell line, and analyze the cell cycle, detect the apoptosis by flow cytometry and use the Western blot to detect the changes of cell cycle-related protein; observe the effect of p53 gene to the expression of NDRG2 through the transfection of the recombinant adenovirus vector of p53 into the renal carcinoma cell line A-498. Results: 1. The NDRG2 protein and expression of the mRNA in human tissue of renal carcinoma and the renal carcinoma cell lines were detected and the results indicated that the renal cell lines HKC and HK-2 are of high expression levels of NDRG2 mRNA, however, the expression levels of NDRG2 mRNA in the renal cell carcinoma cell lines 786-O and A-498 were comparatively lower; the expression levels of NDRG2 mRNA in the tissue of carcinoma were lower than those in the tissue beside the carcinoma in 18 cases of patients with renal carcinoma and the expression level was directly correlated with the pathological grade; in this study, there were only 6 cases of patients with renal carcinoma in which the expression levels of NDRG2 mRNA in the tissue of carcinoma were higher than those in the corresponding tissue beside carcinoma; otherwise, there were 14 cases in which there was no difference between the expression levels of NDRG2 mRNA between the tissues. 2. The recombinant shuttle vector containing NDRG2 was firstly constructed by the mature adenovirus expression system pAdTrack-CMV from American Novagen company, the linearized recombinant shuttle vector and the vector containing the gene of skelemin were transfected into the cell line HEK293 through the liposomal transfection to obtain the prime viral strains, the single plaque bacteriophages were selected to further infect HEK293 cells to acquire the viral lysate of single plaque bacteriophage. Based on these operations, we established the prime seed lot, major seed lot and working seed lot and finally accomplished the production, purification and titer determination of the recombinant adenovirus pAd-GFP-NDRG2, and the final virus titer of the recombinant adenovirus was 1.1×1011 pfu/ml. 3. The results of Western blot confirmed that the transfection of the recombinant adenovirus pAd-GFP-NDRG2 increased the expression level of the endogenous NDRG2 in the cell line A-498. The subsequent results of MTT detection indicated that the OD values of MTT at different detected time points within 48 h in the A-498 group transfected by the recombinant adenovirus were all significantly lower than those corresponding values in the groups without transfection and transfected by the blank plasmid, which suggested that the expression of NDRG2 inhibited the growth of the cell line A-498. The further analysis of cell cycle showed that the plasmid transfection induced the G1 phase arrest in A-498, which implied that NDRG2 might inhibit the proliferation of A-498 through the cell cycle arrest. The results of the apoptosis analysis indicated that the proportion of the apoptosis cells in the A-498 cells infected by the recombinant adenovirus containing NDRG2, which was 12.% at 48 h after infection, was significantly higher that those in the A-498 cells without transfection (1.0%) and transfected by the recombinant adenovirus without NDRG2 (2.0%). Finally, the results of the Western blot indicated that, in the A-498 cells tranfected by human NDRG2, the expression levels of cyclin D1 and cyclin E decreased, but there was no significant change on the expression levels of cyclin D2, cyclin D3 and cdk2. Thus, it is postulated that NDRG2 may inhibit the cell proliferation through the effect on cyclin D1 and cyclin E. 4. It was discovered that A-498 cells express inactive p53 through the Western blot and RT-PCR, however, the expression of NDRG2 was almost undetectable. The increased expression level of NDRG2 was observed when the A-498 cells were transfected by the p53 gene in activated form, which was dose-dependent. This result suggested that NDRG2 is regulated by p53 gene, thus, NDRG2 is also a downstream gene of p53.
Conclusions: NDRG2 is of high expression level in normal renal tissue but of low expression level or without expression in the tissue of renal carcinoma. NDRG2 can inhibit the proliferation of the renal carcinoma cell line A-498 cells, induce arrest at G1 phase and inhibit the expressions of cyclin D1 and cyclin E. The expression of p53 can up-regulate the expression of NDRG2. Thus, the result in this study further confirmed that NDRG2 is a novel anti-oncogene and the strategy of gene therapy taking this gene as the target might provide a new way to the gene therapy of renal carcinoma.
引文
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