转录因子诱饵策略中由载体表达的双链寡核苷酸的功能研究

发布时间：2018-01-17 18:01

本文关键词：转录因子诱饵策略中由载体表达的双链寡核苷酸的功能研究　出处：《第四军医大学》2009年硕士论文　论文类型：学位论文

【摘要】： 转录因子诱饵策略(TFD strategy,Transcription Factor Decoy strategy)是近年来研究和应用较广的一种用来探索转录因子功能的策略。利用人工合成的含有某转录因子结合位点序列的小片段脱氧寡核苷酸双链能与该转录因子正常的结合位点竞争结合转录因子的特性,达到封闭该转录因子活性的目的。近年来已有众多利用TFD策略研究转录因子的报道,如NFκB、Sp1、NFAT等等。转录因子诱饵策略以其对转录因子封闭效果切实、功能分子为小分子等优点被越来越广泛地应用到分子生物学领域,并于近期初步用于临床,取得了较好效果。虽然转录因子诱饵策略有众多优点,但其缺陷同样突出,其中以功能分子脱氧寡核苷酸ODN(oligodeoxynucleotide)的细胞核转移效率过低最为明显。TFD策略发挥作用需要ODN进入细胞核内与转录因子结合,但未经修饰的ODN入核后有大约90%的分子被溶酶体摄取降解,仅有10%左右的ODN能进入细胞核发挥作用。这对TFD策略的实际应用产生很大的影响。目前有数种方案用以解决这项难题,如引入核定位信号肽(NLS)交联ODN以提高核转移效率、对ODN进行修饰以提高其对溶酶体降解的抵抗力以及利用新型转染试剂提高小分子(ODN)的转染效率等。但这些方案或者成本过高,或者工艺复杂,未能被广泛应用。因此尚需探索新的、更方便经济的方法。我们在对茎-环小干扰RNA即shRNA的研究中发现,其在细胞内表达过程会出现一个阶段,表达出的单链RNA分子会形成暂时性的局部双链。我们设想,既然含有转录因子结合位点序列的ODN(系双链DNA)具有相应的转录因子封闭功能,那么与某转录因子结合位点序列相似的双链RNA是否也具有类似的封闭效应呢?如果猜想是正确的话,那我们就可以利用shRNA来表达这种双链RNA,将载体包装病毒,这样既可以达到封闭的目的,又极大地提高了功能分子的核转移效率。因此本课题将对此进行探讨。【目的】 1.明确能够表达与某转录因子结合位点序列相似的双链RNA的载体是否能够封闭该转录因子的活性;2.探索这种载体发挥封闭功能的机制【方法】 1.选择在HEK293细胞中有表达的转录因子NFκB,构建其相关载体,包括目的载体即表达上述双链RNA的载体(vector-based ON, V-B ON)及其错序对照scrambled V-B ON,同时构建NFκB的shRNA作为阳性对照1,合成未经修饰的NFκB结合位点序列ODN作为阳性对照2及其错序对照scrambled ODN;2.将各种载体或小分子分别转染HEK293细胞,MTT试验检测细胞生长状态变化;流式细胞术检测转染后细胞受药物作用后的凋亡情况;实时定量PCR及荧光素酶报告基因实验检测转染后细胞内NFκB下游基因IL-8及MCP-1转录活性变化;3.随机选择在HEK293细胞中有表达的3种转录因子(FOS、AP-2α及RXRA),实时定量PCR检测转染后细胞内这三种转录因子下游基因转录活性的变化以明确这种封闭效应是否广泛存在;4.实时定量PCR检测转染后细胞内NFκB、FOS、AP-2α及RXRA各转录因子本身转录活性的变化,以明确封闭效应是否系载体的RNAi作用所致,探索载体V-B ON发挥作用的机制。【结果】 1.目的载体V-B ON能够抑制HEK293细胞的生长,促进转染后细胞凋亡我们在成功构建各种载体的基础上,将各载体及小分子ODN分别转染HEK293细胞,分别在24小时、48小时及72小时做MTT试验。结果证明,三个时段均出现相似的趋势,即V-B ON与shRNA对细胞生长的相对抑制率相似(V-B ON:42.3±0.152%、57.2±0.612%、29.1±0.441%; shRNA:54.2±0.231%、64.3±0.246%、31.6±0.225%),而合成的小片段ODN对细胞生长的相对抑制率则较低(ODN:18.2±0.294%、21.1±0.546%、11.6±0.331%)。按MTT试验得出的结论,选择48小时作为以下其他实验的处理时间。各载体及小片段转染细胞24小时后,向各组加依托伯苷(VP-16)至终浓度50umol/L。药物处理24小时后收集细胞做流式细胞术。结果显示,V-B ON与shRNA对转染后细胞凋亡的促进程度即相对抑制率相似(V-B ON:42.2±0.437% vs shRNA:50.2±0.512%),而小片段ODN促进程度则较低(22.3±0.601%)。以上相对抑制率为各载体或分子对各自阴性对照的相对抑制率(V-B ON vs scrambled V-B ON;shRNA vs空白载体;ODN vs scrambled ODN)。 MTT试验及流式细胞术均证明V-B ON能够抑制HEK293细胞生长,促进其凋亡,效果与shRNA相似。 2. V-B ON能够影响转染后细胞内NFκB、FOS、AP-2α及RXRA各因子下游基因转录成功构建了NFκB下游基因IL-8及MCP-1的荧光素酶报告基因载体。报告基因分析证明,转染V-B ON后细胞内IL-8及MCP-1的转录活性明显较阴性对照组降低。成功构建FOS、AP-2α及RXRA等因子的相关载体(V-B ON、scrambledV-B ON及shRNA)。将NFκB、FOS、AP-2α及RXRA等因子的各载体或小分子分别转染细胞,提取RNA,反转录并做实时定量PCR(SYBR Green)。结果显示,各因子下游基因mRNA表达出现程度不一的抑制(转录因子抑制其表达者则是活性升高)。V-B ON与shRNA的抑制率相似,而小片段ODN效果较差。 3. V-B ON并不会影响其相应转录因子在转染后细胞内的转录活性,说明V-B ON至少并非通过对相应转录因子的RNA干扰来发挥作用 NFκB、FOS、AP-2α及RXRA等因子的各载体或小分子分别转染细胞,提取RNA,反转录并做实时定量PCR,检测四种转录因子本身转录活性的变化。结果显示,与错序对照相比,V-B ON未表现出明显的抑制效果,而shRNA对各分子活性的抑制则很明显。小片段ODN对各分子活性亦无明显影响。【结论】本研究首次提出能够表达与某转录因子结合位点序列相似的双链RNA的载体可能具有与转录因子诱饵策略中ODN相似的转录因子封闭功能的假说,提供了该类载体封闭相应转录因子活性的功能性证据,并初步探讨了其可能机制。这种载体很可能是通过其表达的序列上与某转录因子结合位点相似的双链RNA发挥ODN样作用的。这些发现有助于提高对RNAi及TFD策略的进一步认识,并可能为分子生物学提供新的有力工具。
[Abstract]:Transcription factor decoy (TFD strategy, Transcription Factor Decoy strategy) is a transcription factor used to explore the function of strategy research in recent years and is widely used. The use of synthetic containing a transcription factor binding site sequence of small fragments of double stranded oligonucleotides can binding sites of transcription factors and competitive binding characteristics of the normal transcription factor the reach to close the transcription factor activity. In recent years there have been numerous transcription factors using TFD strategy research reports, such as Sp1, NF kappa B, NFAT and so on. Transcription factor decoy on transcription factors and blocking effect of functional molecules into small molecules, etc. are increasingly widely used in molecular biology the field, and preliminarily used in the recent clinical, and achieved good results.
Although the transcription factor decoy strategy has many advantages, but its defect is also prominent, with the function of molecular ODN oligodeoxynucleotide (oligodeoxynucleotide) of the nuclear transfer of low efficiency is the most obvious.TFD strategies play a role in combination with transcription factor ODN into the nucleus, but without modification of the ODN into the nucleus after about 90% of the uptake of lysosomal molecules degradation, only about 10% of the ODN can translocate into nucleus. This greatly affect the practical application of the TFD strategy. Several schemes to solve this problem, such as the introduction of nuclear localization signal peptide (NLS) in order to improve the efficiency of nuclear transfer ODN crosslinking of ODN, modified to improve the lysosomal degradation the resistance and improve the utilization of new small molecule transfection reagent (ODN) transfection efficiency. But these solutions or the cost is too high, or complex technology, has not been widely used. It is still necessary to explore new, more convenient and economical ways.
We are on the stem loop of small interfering RNA shRNA study found that the expression of the emergence of a stage in the cell, single stranded RNA molecules expressed will form a temporary local double chain. We assume that since it contains transcription factor binding sites of ODN (double stranded DNA) is a transcription factor the corresponding sealing function, then combined with a transcription factor of double stranded RNA sequences similar whether it has closed to similar effects? If the conjecture is correct, then we can use shRNA to express the double stranded RNA, the carrier for viral packaging, this can achieve the purpose of sealing, and greatly improve the efficiency of nuclear transfer function molecules. So this topic will be discussed.
[Objective]
1., it is clear whether a double stranded RNA that is similar to a transcription factor binding site can block the activity of the transcription factor. 2., explore the mechanism of this vector to play a closed function.
[method]
The 1. is the expression of the transcription factor NF kappa B in HEK293 cells, construct the relevant carrier carrier including objective vector that is the expression of double stranded RNA (vector-based ON, V-B ON) and scrambled V-B ON wrong sequence control, and construction of NF kappa B shRNA as a positive control 1, the synthesis of unmodified NF B binding site sequence ODN as positive control and 2 wrong sequence control scrambled ODN; 2. kinds of carrier or small molecules were transfected into HEK293 cells, MTT cell growth state change test; flow cytometry to detect the apoptosis of cells after transfection after drug action; downstream changes in intracellular NF kappa B gene IL-8 and MCP-1 the transcriptional activity of real-time PCR and luciferase reporter assay after transfection; 3. randomly selected 3 transcription factor expression in HEK293 cells (FOS, AP-2 alpha and RXRA), real time quantitative PCR detection of transfected cells in the three turn Changes of downstream transcriptional activity of transcription factors to determine whether the sealing effect exists; 4. real time quantitative PCR detection after transfection of NF kappa B, FOS, AP-2 and RXRA all the changes of alpha transcription factor itself transcriptional activity, the effect of RNAi clear whether the sealing effect caused by the carrier, to explore the role of ON carrier V-B the mechanism.
[results]
1. destination vector V-B ON can inhibit the growth of HEK293 cells, promote apoptosis after transfection, we based on the successful construction of various vectors, each vector and small molecule ODN were transfected into HEK293 cells, respectively, in 24 hours, 48 hours and 72 hours to do the MTT test. The results prove that the three periods showed similar trends V-B, ON and shRNA on cell growth inhibition rate relatively similar (V-B ON:42.3 + 0.152%, 57.2 + 0.612%, 29.1 + 0.441%; shRNA:54.2 + 0.231%, 64.3 + 0.246%, 31.6 + 0.225%), while the small fragments of ODN on the synthesis of cell growth inhibition rate is relatively low (ODN:18.2 0.294%, 21.1 + 0.546%, 11.6 + 0.331%).
According to the MTT result, 48 hours of processing time following other experiments. After 24 hours of each carrier and small fragments of transfected cells, and relying on the primary glycoside to each of the groups (VP-16) to a final concentration of 50umol/L. drugs after 24 hours of treatment were collected as flow cytometry. The results showed that the relative inhibition rate, degree of enhancement similar to cell apoptosis after transfection with V-B ON shRNA (V-B ON:42.2 + 0.437% vs shRNA:50.2 + 0.512%), while the small fragments of ODN promote the degree is low (22.3 + 0.601%). The relative inhibition rate for each carrier or molecules on their negative control of ON vs scrambled V-B (the inhibition rate of V-B ON; shRNA vs ODN vs scrambled ODN blank vector;).
Both MTT test and flow cytometry showed that V-B ON could inhibit the growth of HEK293 cells and promote its apoptosis, and the effect was similar to that of shRNA.
2. V-B ON can affect the downstream gene transcription of NF kappa B, FOS, AP-2 alpha and RXRA factors after transfection
The luciferase reporter vector of NF and B downstream genes IL-8 and MCP-1 were successfully constructed. The analysis of the reporter gene showed that the transcriptional activity of IL-8 and MCP-1 decreased significantly compared with the negative control group after transfection of V-B ON.
The successful construction of FOS, AP-2 and RXRA related carrier alpha factor (V-B ON, scrambledV-B ON and shRNA). NF FOS, RNA K B, extraction of each carrier or small molecule AP-2 alpha and RXRA factor were transfected cells, reverse transcription and quantitative real-time PCR (SYBR Green). The results showed that the expression of mRNA downstream genes of each factor appears to inhibit the level of (transcription factor inhibiting its expression is increased the activity of inhibition of.V-B ON and shRNA) were similar, and the poor small fragments of ODN.
3. V-B ON does not affect the transcriptional activity of its corresponding transcription factors after transfection, indicating that V-B ON does not play a role in RNA interference at least.
NF FOS, RNA K B, extraction of each carrier or small molecule AP-2 alpha and RXRA factor were transfected cells, reverse transcription and quantitative real-time PCR, change detection of four kinds of transcription factor itself transcriptional activity. The results showed that, compared with the wrong sequence control, V-B ON did not show inhibitory effect is obvious. The inhibition of shRNA on the activity of a molecule is very obvious. There is no obvious effect on the activity of small fragments of ODN molecules.
[Conclusion]
This is the first study to propose expression combined with the vector of double stranded RNA sequences with a similar transcription factor and transcription factor ODN may have similar closed function in transcriptional factor decoy strategy hypothesis, provides the carrier closed functional evidence corresponding transcription factor activity, and to explore its possible mechanism. This vector is double stranded RNA binding sites may be similar to a transcription factor through the sequence of its expression play ODN like effect. These findings are helpful to improve the further understanding of the RNAi and TFD strategy, and may provide a new powerful tool for molecular biology.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

【引证文献】