成人散发性难治性肾病差异基因的筛选
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摘要
目的：筛选及分析成人散发性难治性肾病(RNS)、激素敏感性肾病综合征(SSNS)及正常人间差异表达片段,获取RNS激素耐药相关基因,为认识和阐明原发性肾病综合征(PNS)患者激素耐药的分子机制奠定基础。方法：(1)选择成人散发性RNS患者4例(R1～4)、SSNS患者4例(S1～4)为实验组,正常人2例(H1-2)为对照组,提取患者静脉血中总RNA。利用差异显示PCR技术(DDRT-PCR),扩增总RNA基因表达谱。采用琼脂糖凝胶电泳初步检测PCR产物；变性聚丙烯酰胺凝胶(PAGE)高压电泳进一步分离PCR产物条带,硝酸银染色显示电泳条带；回收RNS患者、SSNS患者及正常人间差异表达片段,重扩增并纯化差异表达片段后送生物公司测序。所得序列在基因库(Genbank)中通过BLAST软件与人类基因已知序列进行同源性比较。用所有样本的RNA为模板进行RT-PCR特异性扩增,验证差异片段假阳性和假阴性。(2)RT-PCR验证MAML2基因的表达。另外选择RNS患者14例(R1'～14)、SSNS患者26例(S1’～26)为实验组、正常人8例(H1’-8)为对照组,提取各组患者静脉血中总RNA。采用RT-PCR以所有样本RNA为模板分组进行特异性扩增,观察各组MAML2表达是否有差异。结果：(1)成功筛选出成人RNS患者、SSNS患者及正常人间差异表达片段,获得1个在RNS患者高表达的差异片段R31,1个在SSNS患者高表达的差异片段S34,与Genbank已知序列同源比较结果：片段R31与一个功能不明确的人已知基因部分序列高度同源；片段S34与人MAML2基因部分序列高度同源。RT-PCR证实筛选出的差异片段在对应患者全血总RNA中均有表达。(2)MAML2在正常对照组未见表达,在SSNS组表达高于RNS组,差异有统计学意义(P<0.01)。
结论：(1)mRNA差异显示技术是一种简单易行的筛选差异基因的技术。(2)RNS、SSNS患者及正常人间存在较明显的差异基因。本实验仅筛选出小部分差异片段。(3)筛选出的在SSNS患者及RNS患者中有差异表达的片段R31、R34在RNS激素耐药相关基因的研究中尚未见报道。Genbank同源分析结果显示其中1条差异片段与MAML2基因部分片段高度同源。(4)MAML2与RNS激素耐药存在相关性,可能是受到激素刺激后活化表达,可能作为P300的上游基因调节P300的表达,并通过P300的激素放大效应,使SSNS患者对激素治疗敏感。
Abstract:Objective:By comparing the differential expression of genes between adult sporadic refractory nephrotic syndrome (RNS), steroid-sensitive nephrotic syndrome (SSNS), and healthy controls, we may identify some specific steroid-resistant genes in RNS,by which we will reveal a molecular basis for the pathogenesis of primary nephrotic syndrome (PNS).Methods:(1). Four adult RNS patients (R1-4) and 4 SSNS patients (S1-4),together with,2 age-matched healthy adults (H1-2), were randomly chosen. The total RNA was extracted from the blood. A RT-PCR differential display technology (DDRT-PCR) was used to determine the gene expression profiling.The RT-PCR products were illuminated by agarose gel electrophoresis, or were separated by high voltage-denaturing urea polyacrylamide gel electrophoresis (PAGE) with silver nitrate staining. The differentially displayed bands were compared between samples from RNS patients, SSNS patients, and healthy controls..The identified fragments were recovered and sent for sequencing by a commercial service. The sequence was compared to human genome by using the BLAST software. In order to ensure the RNA quality,a positive RT-PCR (GAPDH) reaction was evaluated in all samples.(2) Identification of the expression of the MAML2 gene by RT-PCR. In this study,14 adult RNS patients (R1'～14) and 26 SSNS patients (S1'-26) were chosen, in addition to 8 healthy adult controls (H1'-8).The total RNA was extracted from the blood. RT-PCR was used to specifically amplify a fragment of the MAML2 gene, in order to determine whether there is any difference in the expression of this gene between these groups. Results:(1)By careful analyzing the differentially displayed bands, we have identified two unique expression bands.The first one, namely R31,was identified from RNS patients, and second one, namely S34, was from SSNS patients.Via homology comparison with known genes in Genbank, the fragment R31 has a high degree of homology to a known gene with unidentified functions, and the fragment S34 has a high degree of homology to the MAML2 gene. RT-PCR has demonstrate that these identified fragments are consistently expressed in patients with the same diagnosis.(2) The expression of the MAML2 gene is not found in healthy control adults,but showed a higher level in RNS patients, and the highest level in SSNS patients. These differences are all significant (P<0.01).Conclusions:(1).Differential display of mRNAs is an easy and feasible means to screen gene expression. (2).With this technology, we have found that there are differences in the expression of genes between RNS patients, SSNS patients, and healthy adults, while our experiments have only identified a few differences so far. (3).The identified specific expression in SSNS and RNS patients was not previously reported in any publications.One identified fragment has a high homology to the MAML2 gene. (4).MAML2 may play a role in the steroid-resistant of RNS,since this gene could be activated by the use of the steroids, and could enhance the effect of the steroid by activating P300,so that SSNS patients are more sensitive to a steroid treatment.
结论：(1)mRNA差异显示技术是一种简单易行的筛选差异基因的技术。(2)RNS、SSNS患者及正常人间存在较明显的差异基因。本实验仅筛选出小部分差异片段。(3)筛选出的在SSNS患者及RNS患者中有差异表达的片段R31、R34在RNS激素耐药相关基因的研究中尚未见报道。Genbank同源分析结果显示其中1条差异片段与MAML2基因部分片段高度同源。(4)MAML2与RNS激素耐药存在相关性,可能是受到激素刺激后活化表达,可能作为P300的上游基因调节P300的表达,并通过P300的激素放大效应,使SSNS患者对激素治疗敏感。
Abstract:Objective:By comparing the differential expression of genes between adult sporadic refractory nephrotic syndrome (RNS), steroid-sensitive nephrotic syndrome (SSNS), and healthy controls, we may identify some specific steroid-resistant genes in RNS,by which we will reveal a molecular basis for the pathogenesis of primary nephrotic syndrome (PNS).Methods:(1). Four adult RNS patients (R1-4) and 4 SSNS patients (S1-4),together with,2 age-matched healthy adults (H1-2), were randomly chosen. The total RNA was extracted from the blood. A RT-PCR differential display technology (DDRT-PCR) was used to determine the gene expression profiling.The RT-PCR products were illuminated by agarose gel electrophoresis, or were separated by high voltage-denaturing urea polyacrylamide gel electrophoresis (PAGE) with silver nitrate staining. The differentially displayed bands were compared between samples from RNS patients, SSNS patients, and healthy controls..The identified fragments were recovered and sent for sequencing by a commercial service. The sequence was compared to human genome by using the BLAST software. In order to ensure the RNA quality,a positive RT-PCR (GAPDH) reaction was evaluated in all samples.(2) Identification of the expression of the MAML2 gene by RT-PCR. In this study,14 adult RNS patients (R1'～14) and 26 SSNS patients (S1'-26) were chosen, in addition to 8 healthy adult controls (H1'-8).The total RNA was extracted from the blood. RT-PCR was used to specifically amplify a fragment of the MAML2 gene, in order to determine whether there is any difference in the expression of this gene between these groups. Results:(1)By careful analyzing the differentially displayed bands, we have identified two unique expression bands.The first one, namely R31,was identified from RNS patients, and second one, namely S34, was from SSNS patients.Via homology comparison with known genes in Genbank, the fragment R31 has a high degree of homology to a known gene with unidentified functions, and the fragment S34 has a high degree of homology to the MAML2 gene. RT-PCR has demonstrate that these identified fragments are consistently expressed in patients with the same diagnosis.(2) The expression of the MAML2 gene is not found in healthy control adults,but showed a higher level in RNS patients, and the highest level in SSNS patients. These differences are all significant (P<0.01).Conclusions:(1).Differential display of mRNAs is an easy and feasible means to screen gene expression. (2).With this technology, we have found that there are differences in the expression of genes between RNS patients, SSNS patients, and healthy adults, while our experiments have only identified a few differences so far. (3).The identified specific expression in SSNS and RNS patients was not previously reported in any publications.One identified fragment has a high homology to the MAML2 gene. (4).MAML2 may play a role in the steroid-resistant of RNS,since this gene could be activated by the use of the steroids, and could enhance the effect of the steroid by activating P300,so that SSNS patients are more sensitive to a steroid treatment.
引文
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5.Ye J,Yu Z,Ding J,et al.Genetic variations of the NR3C1 gene in children with sporadic nephrotic syndrome[J].Biochem Biophys Res Commun.2006, 348(2):507-513.
6.Sun H, Zhou W, Wang J, et al.A novel mutation in NPHS2 gene identified in a Chinese pedigree with autosomal recessive steroid-resistant nephrotic syndrome[J].Pathology.2009,41(7):661-665.
7.Arias LF, Vieco BE, Arteta AA.Expression of nephrin, podocin and a-actinine-4 in renal tissue of patients with proteinuria[J].Nefrologia.2009, 29(6):569-575.
8.Benoit G, Machuca E, Nevo F, et al.Analysis of recessive CD2AP and ACTN4 mutations in steroid-resistant nephrotic syndrome[J].Pediatr Nephrol.2010,25(3):445-451.
9.Lowik MM, Levtchenko EN, Monnens LA, et al.WT-1 and NPHS2 mutation analysis in patients with non-familial steroid-resistant focal-segmental glomerulosclerosis[J].Clin Nephrol.2003,59(2):143-146.
10.Kim SD, Kim IS,Lee BC, et al.Apolipoprotein E polymorphism and clinical course in childhood nephrotic syndrome[J].Pediatr Nephrol.2003, 18(3):230-233.
11.刘宇健,宋亮年,李保春等.激素抵抗型肾病综合征患者外周白细胞中糖皮质激素受体GR β mRNA的表达.中华内科杂志.2001,40(11)：725-728.
12.Feng HY, Miao XY, Wang XB,et al.Screening and identification of differentially expressed genes in Beijing fatty and broiler breast muscles[J]. Yi Chuan.2009,31(1):83-87.
13.wei W, Bilsborrow P, Hooley P, et al.Variation between two near isogenic barley(Hordeum vulgare) cultivars in expression of the B subunit of the vacuolar ATPase in response to salinity [J].Hereditas,2001,135: 227-231.
14. Miele G, MacRae L, McBride D, et al.Elimination of false positives generated through PCR re-amplification of differfential display cDNA. Biotechniques,1998,25(1):138-144.
15.Lin SE, Oyama T, Nagase T, et al.Identification of new human mastermind proteins defines a family that consists of positive regulators for notch signaling[J].J Biol Chem.2002,277(52):50612-50620
16.Kurihara I, Shibata H, Suzuki T. Expression and regulation of nuclear receptor coactivators in glucocorticoid action[J].Mol Cell Endocrinol, 2002,189(1-2):181-189.
17.张慧琼,易著文,何小解等.P300在肾病综合征糖皮质激素耐药中的作用[J].临床儿科杂志,2005,23(4)：205-209.
18.贾济,候丽宏,陈绍洋.Notch信号通路和Notch相关疾病[J].麻醉与监护论坛,2009,16(5)：224-227.
19.A Fehr, K Roser, K Heidorn, et al.A new type of MAML2 fusion in mucoepidermoid carcinoma. Genes Chromosomes Cancer,2008,47(3): 203-207.
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