NDRG-2在肿瘤细胞中的初步功能研究
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摘要
人NDRG2(human n-myc downstream regulated gene2, hNDRG2)基因为我国第四军医大学首次发现，已被Genbank收录，登陆号为AFI59092，经原位杂交实验证实，NDRG2 mRNA在正常组织内广泛表达，并在部分肿瘤存在表达差异，推断其可能是抑癌基因，但是对这个基因的准确的作用机理和方式还有待于研究。
本实验将NDRG2基因克隆进PQE30／M15表达载体，经DNA测序证明构建正确，IPTG诱导表达，SDS-PAGE证实表达蛋白分子量符合预期。采用快速稀释方式进行蛋白复性，采用脱盐柱脱盐后，采用亲和层析纯化制备高纯度的蛋白。测定纯化产物的等电点为6.3。蛋白N端测序证实表达蛋白N端氨基酸序列正确。通过设置不同浓度的蛋白剂量并设置不同对照组，采用细胞MTT比色法和细胞计数检测考察NDRG2蛋白对7901、HHCC肿瘤细胞的抑制效果，流式细胞仪检测细胞周期的变化，体内实验采用裸鼠抑瘤试验，实验证实重组人NDRG2对肿瘤细胞有较强的抑制作用，并引起细胞G1期变化，体外能抑制裸鼠肿瘤的生成。
此外，本实验还构建了表达NDRG2的真核表达载体，以及含有针对NDRG2的shRNA的表达质粒，目的是通过增加或抑制NDRG2的表达研究NDRG2表达变化后细胞功能的变化，我们采用PCR方法从人基因组DNA中扩增出336bp的U_6启动子，与29bp的NDRG2靶序列的反向重复序列和pSNAV质粒相连，连接后的pSNAVU_6质粒转染入HHCC细胞，检测它对NDRG2表达的影响，结果显示针对NDRG2的短的发夹RNA能抑制NDRG2在HHCC细胞中的表达，并引起被转染细胞G1期的延迟，通过软琼脂集落生成试验和裸鼠抑瘤实验证实被转染细胞集落生成能力减弱，裸鼠肿瘤生长速度比对照组也明显降低。
第四军医大学博士学位论文
通过本研究发现，重组NDRGZ蛋白能够影响肿瘤细胞细胞周期，抑
制细胞增殖，而抑制内源性NDRGZ的表达获得相似的结果，推测NDRGZ
序列中可能存在一种核锚定序列引起蛋白在细胞内外功能的差异。
最后，我们还针对NDRGZ的表达和抑制进行了肿瘤发现者和凋亡相
关基因的基因芯片实验研究，结果发现NDRGZ的表达高低没有出现肿
瘤细胞凋亡现象，而与肿瘤发生通路的基因存在一定关系，NDRGZ与
P21呈负调控作用，可能通过促进血管生成因子受体Tie一2上调而促进癌
生长，NDRGZ与CD4OL可能是负调控关系。在细胞内NDRGZ一定程
度上表现出癌基因的特征，我们还需要更多实验去阐明其中机理。
Human NDRG2(N-myc downstream regulated gene 2) was first discovered by FMMU of China, which was registrated in Genbank with No. AFI59092. NDRG2 is expressed widely in normal tissues and differently between normal and tumor tissues through in-site hybridization. But the mechanism and process of the gene in regulation on cells is still not clear.
In this study, the NDRG2 gene was cloned into PQE30/M15, and DNA Sequencing proved the construction of the plasmids was correct. After expressed by the IPTG induction, MW of the proteins expressed by the vector is correct. We refold the protein through quick dilution.After desalt,we purified the protein by the affinity resin.The isoelectric point (PI) of the protein was 6.3.N-terminal protein sequencing proved the protein N- terminal amino acid construction was correct.We investigated inhibitory activity of the NDRG2 protein to the HHCC and 7901 cells by setting up the different protein concentration and control groups. The cell cycle were analysed by two-parameter flow cytometry. The in vivo effects were evaluated by tumor -inhibition test on nude mice. These results indicated that the recombinant NDRG2 proteins had some inhibited effects on tumor cells, causing G1 phase arrested and inhibiting tumor formation on nude mice in vivo.
Also we have constructed a plasmid containg the shRNA of NDRG2 to suppress the expression of NDRG2 in HHCC cell, and a plasmid containing NDRG2 to improve the expression of NDRG2. Our purpose was to test the cells properties of improving or reducing the NDRG2 content. A 336 bp human U6 snRNA promoter was amplified from human genomic DNA by PCR and ligased with a 29 bp reverse repeated motif of NDRG2 target
sequence with 9 bp spacer and plasmid pSNAV. The recombinant pSNAVPU6 plasmid transfected with HHCC cell lines to detect the effects of NDRG2 expression separately. The analysed results indicated that pSNAVPU6 suppressed the NDRG2 expression successfully and caused Gl phase arrested. The transfected cells had less ability of colony formation and tumor formation on nude mice in vivo, than the control ones. The above results implicates that there may be a kind of nuclear translocation sequence in NDRG2.
Finally, we did some genechip analysis about cell apoptosis and human cancer pathwayfinder on the cells of adding and reducing NDRG2 expression. The results indicated that there was no direct relationships between expression level of NDRG2 and cell apoptosis. However, NDRG2 had some effects on the cancer pathwayfinder genes. NDRG2 has negative control on p21.lt may stimulate growth of cancer cells by upregulating vascular endothelial growth factor receptor Tie-2. Similary, it has negative control on CD40L. We concluded that NDRG2 put up some characters of cancer genes in some cells. It need more tests to make it clear about the mechanism.
本实验将NDRG2基因克隆进PQE30／M15表达载体，经DNA测序证明构建正确，IPTG诱导表达，SDS-PAGE证实表达蛋白分子量符合预期。采用快速稀释方式进行蛋白复性，采用脱盐柱脱盐后，采用亲和层析纯化制备高纯度的蛋白。测定纯化产物的等电点为6.3。蛋白N端测序证实表达蛋白N端氨基酸序列正确。通过设置不同浓度的蛋白剂量并设置不同对照组，采用细胞MTT比色法和细胞计数检测考察NDRG2蛋白对7901、HHCC肿瘤细胞的抑制效果，流式细胞仪检测细胞周期的变化，体内实验采用裸鼠抑瘤试验，实验证实重组人NDRG2对肿瘤细胞有较强的抑制作用，并引起细胞G1期变化，体外能抑制裸鼠肿瘤的生成。
此外，本实验还构建了表达NDRG2的真核表达载体，以及含有针对NDRG2的shRNA的表达质粒，目的是通过增加或抑制NDRG2的表达研究NDRG2表达变化后细胞功能的变化，我们采用PCR方法从人基因组DNA中扩增出336bp的U_6启动子，与29bp的NDRG2靶序列的反向重复序列和pSNAV质粒相连，连接后的pSNAVU_6质粒转染入HHCC细胞，检测它对NDRG2表达的影响，结果显示针对NDRG2的短的发夹RNA能抑制NDRG2在HHCC细胞中的表达，并引起被转染细胞G1期的延迟，通过软琼脂集落生成试验和裸鼠抑瘤实验证实被转染细胞集落生成能力减弱，裸鼠肿瘤生长速度比对照组也明显降低。
第四军医大学博士学位论文
通过本研究发现，重组NDRGZ蛋白能够影响肿瘤细胞细胞周期，抑
制细胞增殖，而抑制内源性NDRGZ的表达获得相似的结果，推测NDRGZ
序列中可能存在一种核锚定序列引起蛋白在细胞内外功能的差异。
最后，我们还针对NDRGZ的表达和抑制进行了肿瘤发现者和凋亡相
关基因的基因芯片实验研究，结果发现NDRGZ的表达高低没有出现肿
瘤细胞凋亡现象，而与肿瘤发生通路的基因存在一定关系，NDRGZ与
P21呈负调控作用，可能通过促进血管生成因子受体Tie一2上调而促进癌
生长，NDRGZ与CD4OL可能是负调控关系。在细胞内NDRGZ一定程
度上表现出癌基因的特征，我们还需要更多实验去阐明其中机理。
Human NDRG2(N-myc downstream regulated gene 2) was first discovered by FMMU of China, which was registrated in Genbank with No. AFI59092. NDRG2 is expressed widely in normal tissues and differently between normal and tumor tissues through in-site hybridization. But the mechanism and process of the gene in regulation on cells is still not clear.
In this study, the NDRG2 gene was cloned into PQE30/M15, and DNA Sequencing proved the construction of the plasmids was correct. After expressed by the IPTG induction, MW of the proteins expressed by the vector is correct. We refold the protein through quick dilution.After desalt,we purified the protein by the affinity resin.The isoelectric point (PI) of the protein was 6.3.N-terminal protein sequencing proved the protein N- terminal amino acid construction was correct.We investigated inhibitory activity of the NDRG2 protein to the HHCC and 7901 cells by setting up the different protein concentration and control groups. The cell cycle were analysed by two-parameter flow cytometry. The in vivo effects were evaluated by tumor -inhibition test on nude mice. These results indicated that the recombinant NDRG2 proteins had some inhibited effects on tumor cells, causing G1 phase arrested and inhibiting tumor formation on nude mice in vivo.
Also we have constructed a plasmid containg the shRNA of NDRG2 to suppress the expression of NDRG2 in HHCC cell, and a plasmid containing NDRG2 to improve the expression of NDRG2. Our purpose was to test the cells properties of improving or reducing the NDRG2 content. A 336 bp human U6 snRNA promoter was amplified from human genomic DNA by PCR and ligased with a 29 bp reverse repeated motif of NDRG2 target
sequence with 9 bp spacer and plasmid pSNAV. The recombinant pSNAVPU6 plasmid transfected with HHCC cell lines to detect the effects of NDRG2 expression separately. The analysed results indicated that pSNAVPU6 suppressed the NDRG2 expression successfully and caused Gl phase arrested. The transfected cells had less ability of colony formation and tumor formation on nude mice in vivo, than the control ones. The above results implicates that there may be a kind of nuclear translocation sequence in NDRG2.
Finally, we did some genechip analysis about cell apoptosis and human cancer pathwayfinder on the cells of adding and reducing NDRG2 expression. The results indicated that there was no direct relationships between expression level of NDRG2 and cell apoptosis. However, NDRG2 had some effects on the cancer pathwayfinder genes. NDRG2 has negative control on p21.lt may stimulate growth of cancer cells by upregulating vascular endothelial growth factor receptor Tie-2. Similary, it has negative control on CD40L. We concluded that NDRG2 put up some characters of cancer genes in some cells. It need more tests to make it clear about the mechanism.
引文
[1] Lachat P, Shaw P, Gcbhard S, Van Belzen N, Chaubert P, Bosman
FT. Expression of NDRG1, a differentiation-related gene, in human tissues. Histochem Cell Biol., 2002,118(5):399-408.
[2] Zhou RH, Kokame K, Tsukamoto Y, Yutani C, Kato H, Miyata T, Characterization of the human NDRG gene family: a newly identified member, NDRG4, is specifically expressed in brain and heart. Genomics 2001 Apr 1; 73(1):86～97
[3] Qu X, Zhai Y, Wei H, Zhang C, Xing G, Yu Y, He F。 Characterization and expression of three novel differentiation-related genes belong to the human NDRG gene family. Mol Cell Biochem 2002 Jan; 229(1-2):35～44
[4] Kalaydjieva L, Gresham D, Gooding R, Heather L, Baas F, de Jonge R, Blechschmidt K, Angelicheva D, Chandler D, Worsley P, Rosenthal A, King RH, Thomas PK, N-myc downstream-regulated gene 1 is mutated in hereditary motor and sensory neuropathy-Lom. Am J Hum Genet 2000 Jul;67(1):47～58
[5] Sheerazed Boulkroun, Michel Fay, Maria-Christina Zennaro, Brigitte Escoubet, Frederic Jaisser, Marcel Blot-Chabaud, Nicolette Farman, Nathalie Courtois-Coutry. Characterization of Rat NDRG2(N-Myc Downstream regulated gene2), a novel early mineralocorticoid-specific induced gene. The J Biological chemistry, 2002,277(35):31506-15.
[6] Choi SC, Kim KD, Kim JT, Kim JW, Yoon DY, Choe YK, Chang YS, Paik SG, Lim JS. Expression and regulation of NDRG2(N-myc downstream regulated gene 2) during the differentiation of dentric cells. FEBS Lett, 2003, 553(3):413-8.
[7] 张文红，刘新平，王吉村，韩月恒，邓艳春，周洁，药立波。兔抗人NDR2高效价抗血清的制备、纯化及鉴定。第四军医大学学报，2002：23 (8)：716～19
[8] 李剑，刘新平，林树新，邓艳春，孟庆军，张文红，李树钧，聂晓燕，药立波，蒋慧君。一种新的抑癌侯选基因——NDR2在
人类正常组织及相应肿瘤中的表达。生物化学与生物物理进展，2002：29(2)：223～27
[9] 刘新平，邓艳春，韩炯，李剑，王吉村，李莹，药立波。NDRG2基因表达对胃癌细胞增殖调控及其机理的研究。生物化学与生物物理进展，2003；30(1)：116～21
[10] 邓艳春，药立波，刘新平，苏成芝。一种含有ACP样结构域新基因的发现[J]．生物化学与生物物理进展，2001，28(1)：72～76
[11] Aravin AA, Klenov MS, Vagin VV, et al. Role of Double-Stranded RNA in Eukaryotic Gene Silencing [J]. Mol Biol, 2002, 36(2): 180-188.
[12] Elbashir SM, Lendeckel W, Tuschel T. RNA interference ismediated by 21-and 22-nucleotide RNAs[J]. Genes Dev, 2001,15(2):188-200.
[13] Phillip AS. RNA interference-2001 [J]. Genes Dev, 2001,15(5):485-489.
[14] Yanehun Deng, Libo Yao, Ling Chua, et al.. N-myc downstream-regulated gene2 inhibits glioblastoma cell proliferation.Int. J. Cancer. 2003, 106,342-347.
[15] Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAi in mammalian cells.Science, 2002, 296: 5502553.
[16] Sui G, Soohoo C, Affar EIB, et al. A DNA vector based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci, 2002, 99: 551525520.
[17] Yu J Y, DeRuiter SL, Turner DL. RNA interference by expression of short2interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci, 2002, 99:604726052.
FT. Expression of NDRG1, a differentiation-related gene, in human tissues. Histochem Cell Biol., 2002,118(5):399-408.
[2] Zhou RH, Kokame K, Tsukamoto Y, Yutani C, Kato H, Miyata T, Characterization of the human NDRG gene family: a newly identified member, NDRG4, is specifically expressed in brain and heart. Genomics 2001 Apr 1; 73(1):86～97
[3] Qu X, Zhai Y, Wei H, Zhang C, Xing G, Yu Y, He F。 Characterization and expression of three novel differentiation-related genes belong to the human NDRG gene family. Mol Cell Biochem 2002 Jan; 229(1-2):35～44
[4] Kalaydjieva L, Gresham D, Gooding R, Heather L, Baas F, de Jonge R, Blechschmidt K, Angelicheva D, Chandler D, Worsley P, Rosenthal A, King RH, Thomas PK, N-myc downstream-regulated gene 1 is mutated in hereditary motor and sensory neuropathy-Lom. Am J Hum Genet 2000 Jul;67(1):47～58
[5] Sheerazed Boulkroun, Michel Fay, Maria-Christina Zennaro, Brigitte Escoubet, Frederic Jaisser, Marcel Blot-Chabaud, Nicolette Farman, Nathalie Courtois-Coutry. Characterization of Rat NDRG2(N-Myc Downstream regulated gene2), a novel early mineralocorticoid-specific induced gene. The J Biological chemistry, 2002,277(35):31506-15.
[6] Choi SC, Kim KD, Kim JT, Kim JW, Yoon DY, Choe YK, Chang YS, Paik SG, Lim JS. Expression and regulation of NDRG2(N-myc downstream regulated gene 2) during the differentiation of dentric cells. FEBS Lett, 2003, 553(3):413-8.
[7] 张文红，刘新平，王吉村，韩月恒，邓艳春，周洁，药立波。兔抗人NDR2高效价抗血清的制备、纯化及鉴定。第四军医大学学报，2002：23 (8)：716～19
[8] 李剑，刘新平，林树新，邓艳春，孟庆军，张文红，李树钧，聂晓燕，药立波，蒋慧君。一种新的抑癌侯选基因——NDR2在
人类正常组织及相应肿瘤中的表达。生物化学与生物物理进展，2002：29(2)：223～27
[9] 刘新平，邓艳春，韩炯，李剑，王吉村，李莹，药立波。NDRG2基因表达对胃癌细胞增殖调控及其机理的研究。生物化学与生物物理进展，2003；30(1)：116～21
[10] 邓艳春，药立波，刘新平，苏成芝。一种含有ACP样结构域新基因的发现[J]．生物化学与生物物理进展，2001，28(1)：72～76
[11] Aravin AA, Klenov MS, Vagin VV, et al. Role of Double-Stranded RNA in Eukaryotic Gene Silencing [J]. Mol Biol, 2002, 36(2): 180-188.
[12] Elbashir SM, Lendeckel W, Tuschel T. RNA interference ismediated by 21-and 22-nucleotide RNAs[J]. Genes Dev, 2001,15(2):188-200.
[13] Phillip AS. RNA interference-2001 [J]. Genes Dev, 2001,15(5):485-489.
[14] Yanehun Deng, Libo Yao, Ling Chua, et al.. N-myc downstream-regulated gene2 inhibits glioblastoma cell proliferation.Int. J. Cancer. 2003, 106,342-347.
[15] Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAi in mammalian cells.Science, 2002, 296: 5502553.
[16] Sui G, Soohoo C, Affar EIB, et al. A DNA vector based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci, 2002, 99: 551525520.
[17] Yu J Y, DeRuiter SL, Turner DL. RNA interference by expression of short2interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci, 2002, 99:604726052.