摘要
前言
WNK4 (with-no-lysine kinase-4)是新近克隆的一种丝氨酸/苏氨酸蛋白激酶的编码基因,该基因第7外显子和第17外显子错义突变可导致常染色体显性遗传疾病-Ⅱ型假性低醛固酮血症(Pseudohypoaldosteronism typeⅡ, PHAⅡ),主要表现为高血压和高血钾。研究表明WNK4主要作用于肾脏远曲小管和集合管上的多个离子转运体和离子通道,比如Na-Cl共转运体(Na-Cl co-transporter, NCCT)、髓外钾离子通道(Renal Outer Medullary Potassium channel, ROMK)、上皮钠通道(Epithelial Sodium Channel, ENaC),从而起到对体液离子平衡和血压的调控作用。目前大量的研究集中在WNK4的功能上,而WNK4基因调控机制仍不清楚。
基因组包含编码序列和非编码序列,其中编码序列在人类基因组中所占比例不到5%,非编码序列超过95%。过去很长一段时间内认为这些非编码序列是“垃圾DNA (junk DNA)",现在人们逐渐认识到这些非编码序列对基因的表达发挥着重要的调控作用,尤其是基因的5'和3'侧翼的非编码区。本研究组前期对hWNK4基因的5'启动子区进行了研究,发现hWNK4的启动子并不含有TATA盒,它包含多个潜在的转录因子结合位点,比如Sp1、GR、GATA-1、AP1、AP2、NF-κB和C/EBPα等。李春义等证实在启动子区的-285和-337位存在两个糖皮质激素反应元件(glucocorticoid response element, GRE),地塞米松(dexamethasone, Dex)可通过这两个GRE抑制hWNK4的转录活性。李苗等发现在启动子区的-426位存在一个GATA-1反应元件,应用组蛋白去乙酰化酶抑制剂曲古抑菌素A(trichostatin A,TSA)可以增加GATA-1的乙酰化,增强其与反应元件的亲和力,从而影响启动子的活性,上调hWNK4的表达。这些关于hWNK4基因的5'启动子的表达调控研究对阐明高血压的发病机制有重要意义；然而,hWNK4基因的3'非翻译区(3'-untranslated region,3'-UTR)的调控作用迄今未见报道。
微小RNA (microRNA, miRNA)是近年来生物学研究的一个热点,是一类长度约19-25nt内源性单链非编码RNA分子。研究表明miRNA通过调控基因表达参与了生命中的一系列重要过程,包括早期发育、细胞分化、细胞增殖、细胞凋亡以及细胞死亡等。大部分实验证明动物miRNA通过与靶基因mRNA的3'-UTR不完全配对结合,在转录后水平负调控基因表达。因此,本研究拟重点探讨hWNK4基因3'-UTR的作用和功能。通过生物信息学、报告基因载体构建、real-time PCR、Western Blot等实验技术验证hWNK4 3'-UTR的作用以及其上的潜在miRNA结合位点,从而探讨hWNK4 3'-UTR参与基因表达调控的机制。
材料与方法
在萤光素酶报告基因载体pGL3-Basic基础上,利用基因重组技术构建多种质粒,包括pSV40-luc、pSV40-luc-UTR、p484-luc、p484-luc-UTR、p337-luc、p337-luc-UTR、p216-luc、p216-luc-UTR。利用Lipofectamine 2000分别转染人胚肾细胞系HEK293、人胃腺癌细胞系BGC823和人宫颈腺癌细胞系Hela,应用DUAL-GLOTM双荧光素酶检测系统检测荧光素酶的活性。应用染色体构象捕获技术观察hWNK4 3'-UTR与启动子的远隔交互作用。
利用生物信息学软件对hWNK4基因3'-UTR进行miRNA靶向预测。在pcDNA3.1(+)基础上构建miR-296的表达载体pmiR-296,并购买了miR-296的抑制剂(Ambion)。利用瞬时转染技术过表达miR-296或抑制miR-296表达,通过萤光素酶报告基因、Real-time PCR及Western印迹杂交等实验进行验证。
结果
1、通过UCSC Genome Bioinformatic (http://genome.ucsc.edu/)对多个哺乳动物的比较,WNK4序列具有高度保守性,其3'-UTR区一致性达95%。
2、在HEK293、BGC823和Hela中分别转入pSV40-luc或pSV40-luc-UTR,发现hWNK4 3'-UTR具有增强子作用,将异源SV40启动子换为同源的484-bp启动子后增强子作用仍存在,但是在各细胞中的作用强度不一致。
3、应用染色体构象捕获技术观察到hWNK4启动子和3'-UTR之间存在远隔交互作用,彼此可能在空间上相互靠近形成DNA-袢结构。
4、将一系列含有5'截短的hWNK4启动子和／或3'-UTR的报告载体分别转染HEK293、BGC823和Hela,发现3'-UTR对不同长度启动子的影响不同：对全长484-bp启动子有增强作用；但是对337-bp启动子基本没有作用；对216-bp启动子呈明显的抑制作用,提示3'增强子和5'启动子区顺式反应元件对hWNK4转录有协同调节作用,启动子-484至-337区可能是3'-UTR发挥增强子作用的关键区域。
5、对转染细胞施与hWNK4启动子的刺激剂(TSA)或抑制剂(DEX),不改变对hWNK4 3'-UTR增强子的作用趋势,同时TSA还可提高3'-UTR对484启动子增强作用的敏感性。
6、应用miRanda、TargetScan等软件,在hWNK4第3775-3795nt位置预测到一个miR-296的结合位点,其与小鼠、大鼠、狗和马的同源性高达95%。
7、在HEK293细胞中抑制miR-296表达可使带有hWNK4 3'-UTR野生型序列的报告载体荧光素酶活性上升；过表达miR-296,使荧光素酶活性下降,而且不论是SV40启动子还是WNK4启动子都有同样的作用。对带有hWNK4 3'-UTR突变型的报告载体给予同样的处理因素,荧光素酶活性基本没有变化。证实了miR-296靶向作用于hWNK4 3'-UTR,而且miR-296对基因的调节作用并不依赖于启动子。
8、将pSV40-luc-UTR和pmiR-296共转染到HEK293,BGC823和Hela细胞中,发现在HEK293和Hela细胞中过表达miR-296使荧光素酶活性下降；而在BGC823细胞中没有抑制作用,提示miR-296对基因表达的抑制作用具有细胞特异性。
9、将pmiR-296转染到HEK293细胞中,应用Real-time PCR检测内源性hWNK4 mRNA水平没有减低。将pHA-WNK4与pmiR-296共转染到HEK293细胞中,应用Western Blot检测hWNK4蛋白水平有下降,并呈剂量依赖性,提示miR-296在转录后水平抑制hWNK4的表达。
结论
1、hWNK4 3'-UTR具有增强子作用,并且与启动子之间存在远隔交互作用,某些转录因子及其结合位点可能是3'-UTR和启动子彼此连接的桥梁。
2、miR-296与hWNK4 3'-UTR存在靶向作用关系,且miR-296对基因表达的抑制作用存在细胞特异性。miR-296在转录后水平抑制WNK4的表达。
Introduction
Human with-no-lysine kinase-4 (hWNK4) is a recently discovered member of the serine-threonine protein kinase family with unusual protein kinase domains. hWNK4 mutations in exon 7 and exon 17 result in pseudohypoaldosteronism typeⅡ(PHA-Ⅱ) which is an autosomal dominant disease featuring hypertension with hyperkalemia. hWNK4 expresses mainly in kidney and partly in polarized epithelia. It acts as a multifunctional regulator of diverse ion transporters including NaCl cotransporter (NCCT) and renal outer medullary K+ channel (ROMK) in the distal convoluted tubule, and can vary the balance between NaCi reabsorption and K+ secretion to maintain integrated homeostasis. So, it may be involved in pathophysiological processes of fluid and electrolyte perturbations and hypertension. So far, most of studies focus on the function of hWNK4 and the molecular regulation mechanisms of hWNK4 gene expression are still in question.
The genome basically comprises genes, coding sequences that instruct the synthesis of RNA or proteins, and non-coding sequences. In humans, coding regions represent<5% of the genome, the information contained in the remaining 95% is still unknown for us. Within these non-coding sequences, which for a long time were considered junk DNA, is located the regulatory DNA that performs the essential role of tightly controlling the levels of transcription of genes in time and space and plays essential roles in organizing the structure of the chromatin, which is also essential for the control of gene expression. The 5'and 3'flanking sequences of genes are the most interesting regions among these non-coding sequences.
Our research previously focused on the 5'promoter region of hWNK4 and found that the hWNK4 promoter contained numerous putative transcription factor binding sites, such as Spl, AP1, AP2, NF-κB, and C/EBPa, but not TATA-box. Li et al have identified two negative glucocorticoid response elements (GREs) at position-285 and -337 of hWNK4 promoter, through which dexamethasone (Dex) inhibited the WNK4 transcriptional activity in glucocorticoid receptor (GR)-dependent pattern. Li et al have clarified there was a GATA-1 responsive element at position-426 of hWNK4 promoter. Its binding to GATA-1 transcriptional factor was augmented by trichostatin A (TSA), a histone deacetylase inhibitor, resulting in upregulation of hWNK4 expression. These studies on the 5'promoter of hWNK4 for gene regulation are of important significance for illustrating the pathogenesis of hypertension. So far, however, less is known about the 3'-untranslated region (3'-UTR) of hWNK4 for gene regulation.
MicroRNAs (miRNAs) is a family of small non-coding, single-stranded RNAs (19～25 nucleotides), which has been a hotspot of biological fields recently. MiRNAs represent a novel large class of gene regulatory molecules that control diverse biological processes, such as early development, differentiation, proliferation, apoptosis and death. Most animal miRNAs exert their regulatory effects by binding to imperfect complementary sites within the 3'-UTRs of their mRNA targets, and they repress target-gene expression post-transcriptionally, apparently at the level of translation. Therefore, the purpose of the current study was to determine how the 3'-UTR of hWNK4 was involved in the regulation of gene expression and validate the potential binding site of miRNA in the 3'-UTR of hWNK4, applying the methods of bioioformatics, luciferase reporter assay, chromosome conformation capture (3C) assay, Real-time PCR and Western Blot. Our data will further unravel the molecular mechanism of hWNK4 expression regulating by the hWNK4 3'-UTR.
Materials and Methods
Based on the lucferase reporter vector pGL3-Basic, several constructs were generated using gene cloning and recombination technology, including pSV40-luc, pSV40-luc-UTR, p484-luc, p484-luc-UTR, p337-luc, p337-luc-UTR, p216-luc and p216-luc-UTR. Transfection was done with Lipofectamine 2000 in HEK293, BGC823 and Hela cell lines, which were derived from human embryonic kidney, gastric adenocarcinoma and cervical adenocarcinoma, respectively. Luciferase activity was measured using the dual luciferase assay system and a luminometer to observe the effect of hWNK4 3'-UTR on the promoter.3C assay was done to determine the distant interaction between hWNK4 promoter and 3'-UTR.
Potential miRNAs targeting on hWNK4 3'-UTR were predicted via bioioformatics software. The expression vector of miR-296 was constructed based on the plasmid pcDNA3.1(+) and the miR-296 inhibitor was purchased from Ambion. The predicted information was verified under the conditions of overexpression or knock-down of miR-296 using luciferase reporter assay, real-time PCR and western blot.
Results
1. Alignment (http://genome.ucsc.edu) of WNK4 sequences in several mammals showed there was high conservation among them, especially in 3'-UTR, the similarity reached 95%.
2. Transfecting pSV40-luc or pSV40-luc-UTR into HEK293, BGC823 and Hela cells, the luciferase activity of pSV40-luc-UTR was siginificantly higher than that of pSV40-luc. Drived by the homologous hWNK4 484-promoter, the hWNK4 3'-UTR still enhanced the transcriptional activity of the reporter vectors, but the enhancing intensity were different with the different cell types. These results suggested that the hWNK4 3'-UTR acted as an enhancer.
3.3C assay showed that the 3' enhancer and 5'promoter of the hWNK4 gene interact physically, with the intervening DNA looped out in WNK4-expressing HEK293 cells, but not in non-WNK4-expressing Hela cells.
4. A series of 5'-deleted hWNK4 promoter-luciferase constructs were successfully generated, with or without the hWNK4 3'-UTR, which were transfected into HEK293, BGC823 and Hela cells, respectively. The results showed that 484-promoter activity was increased,337-promoter not changed, and 216-promoter markedly dropped off by the hWNK4 3'-UTR. It indicated that 3'-UTR may interact with the different cis-elements in the hWNK4 promoter and cooperatively control the hWNK4 gene expression. The region from-484 to-337 in the hWNK4 promoter could be the key site for the 3'-UTR enhancer.
5. Transfecting with p484-luc or p484-luc-UTR, HEK293 cells were administrated with positive stimuli of hWNK4 promoter (TSA) or negative stimuli (Dex). These stimuli did not change the enhancing trend of hWNK4 3'-UTR for 484-promoter. And the transcriptional activity of 484-promoter with 3'-UTR was further improved 40% in response to TSA, compared with 484-promoter without 3'-UTR, that implied 3'-UTR may increase the sensitivity of 484-promoter to TSA.
6. In silico analysis was performed using miRanda and TargetScan, and showed a putative miR-296 binding site at nt 3775-3795 of hWNK4 relative to the first nucleotide defined in AF390018 (GenBank), with 95% homology compared with mouse, rat, dog and horse.
7. In HEK293 cells, inhibiting of miR-296 leaded to the luciferase activity of the reporter vectors carrying the wild type of h.WNK4 3'-UTR increasing and overexpression of miR-296 resulted in decreasing, no matter of under SV40 promoter or 484-promoter. While for the vector with the mutant type of hWNK4 3'-UTR, p484-luc-UTRmut, overexpression or inhibition of miR-296 did not change the luciferase activity. These indicated that miR-296 regulated gene expression through binding to the specific sequence of the hWNK4 3'-UTR, not depending on the promoter.
8. Cotransfecting pSV40-luc-UTR with pmiR-296 into HEK293, BGC823 and Hela cells, the luciferase activity of pSV40-luc-UTR were suppressed in HEK293 and Hela, compared with pmiR-control, but not in BGC823. This result suggested the function of miR-296 was characterized with cell-specific.
9. Overexpression of miR-296 did not repress the level of endogenous h.WNK4 mRNA detecting by real-time PCR. While cotransfecting pHA-WNK4 with pmiR-296 into HEK293 cells, the level of HA-tagged hWNK4 protein decreased in dose-dependence pattern though Western Blot. That demonstrated miR-296 downregulated hWNK4 expression at the posttranscriptional level.
Conclusion 1. The hWNK4 3'-UTR acted as an enhancer and crosstalked with the promoter in
cell-specific manner. Some transcriptional factor and their binding motif might be a part of the bridge between the promoter and 3'-UTR of hWNK4.
2. A miR-296 binding site was validated in the hWNK4 3'-UTR, through which miR-296 downregulated the hWNK4 expression at the posttranscriptional level in cell-specific manner.
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