WO2022166140A1 - FXR靶向的saRNA及其应用 - Google Patents

FXR靶向的saRNA及其应用 Download PDF

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WO2022166140A1
WO2022166140A1 PCT/CN2021/111137 CN2021111137W WO2022166140A1 WO 2022166140 A1 WO2022166140 A1 WO 2022166140A1 CN 2021111137 W CN2021111137 W CN 2021111137W WO 2022166140 A1 WO2022166140 A1 WO 2022166140A1
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sarna
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fxr
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郝海平
王洪
潘晓洁
王广基
于洋
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China Pharmaceutical University
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    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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  • the present invention relates to a type of saRNA targeted by FXR and its application, in particular to a type of saRNA that can up-regulate the expression level of FXR and its application.
  • Liver diseases including viral hepatitis (HBV/HCV, etc.), non-alcoholic fatty liver, alcoholic fatty liver, liver fibrosis, liver cirrhosis and liver cancer, etc.
  • HCV viral hepatitis
  • NASH non-alcoholic steatohepatitis
  • Apoptosis is a basic biological phenomenon that plays an important role in the evolution of organisms, homeostasis and development of the body. Disorders of the apoptosis process are closely related to the occurrence and development of various diseases, such as hepatocyte apoptosis. is a variety of liver diseases (including NALFD/NASH, alcoholic hepatitis, drug-induced liver injury, viral hepatitis, cholestatic liver injury, ischemia-reperfusion liver injury, hepatolenticular degeneration, liver fibrosis/cirrhosis, common features and pathological mechanisms of liver cancer, etc.).
  • liver diseases including NALFD/NASH, alcoholic hepatitis, drug-induced liver injury, viral hepatitis, cholestatic liver injury, ischemia-reperfusion liver injury, hepatolenticular degeneration, liver fibrosis/cirrhosis, common features and pathological mechanisms of liver cancer, etc.
  • FXR Farnesoid X Receptor
  • NR1H4 Farnesoid X Receptor
  • Inflammation is closely related to immunity, liver fibrosis and liver regeneration. Therefore, FXR is widely regarded as an important hepatoprotective drug development target.
  • drug development strategies based on FXR as a target mainly focus on the development of FXR agonists or antagonists, which aim to regulate the transcriptional activity state of FXR.
  • the research and development of FXR agonists as hepatoprotective drugs is a current research focus.
  • obeticholic acid a strong FXR agonist
  • FXR FXR
  • obeticholic acid is a drug for primary biliary cirrhosis, which was approved by the US FDA in May 2016.
  • 23% of patients taking high-dose obeticholic acid found improvements in liver fibrosis indicators, suggesting that the pharmacological effects of transcriptional activation of FXR by FXR agonists such as obeticholic acid are limited.
  • FXR in the liver is down-regulated in the state of NASH and liver fibrosis.
  • the lack of the target protein makes its agonists or antagonists unable to exert their effects by regulating the target, which is the main reason for the poor clinical efficacy of FXR agonists; Accelerated and aggravated hepatocyte apoptosis; and increased the expression of FXR has anti-apoptotic effect; in addition, with the increase of age, the expression of FXR in the liver was significantly down-regulated, which is an important factor causing fatty liver.
  • the first objective of the present invention is to provide a type of saRNA targeted by FXR, and the second objective is to provide the application of the saRNA.
  • the FXR-targeted saRNA of the present invention includes a nucleotide sequence complementary to the -1000 to -1 site region of the NR1H4 gene promoter region.
  • the saRNA includes a nucleotide sequence complementary to the region from the -102 site to the -84 site in the promoter region of the NR1H4 gene.
  • the saRNA includes a nucleotide sequence complementary to the region from the -103 site to the -85 site in the promoter region of the NR1H4 gene.
  • the saRNA includes a nucleotide sequence complementary to the region from the -105 site to the -87 site in the promoter region of the NR1H4 gene.
  • the saRNA includes a nucleotide sequence complementary to the region from the -318 site to the -300 site in the promoter region of the NR1H4 gene.
  • the saRNA includes a nucleotide sequence complementary to the region from the -319 site to the -301 site in the promoter region of the NR1H4 gene.
  • the saRNA consists of a sense strand whose sequence is shown in SEQ ID NO: 2 and an antisense strand whose sequence is shown in SEQ ID NO: 3, or the sense strand and sequence whose sequence is shown in SEQ ID NO: 4.
  • the shown sense strand and sequence are composed of the antisense strand shown in SEQ ID NO: 9, or the sense strand shown in SEQ ID NO: 10 and the antisense strand whose sequence is shown in SEQ ID NO: 11.
  • the present invention designs and synthesizes a series of saRNA specifically targeting FXR (NR1H4) for the first time, which has the characteristics of high specificity and strong specificity, and can significantly regulate the transcription and expression of FXR, thereby exerting anti-hepatocyte resistance.
  • Apoptotic activity can be used to prepare drugs against liver diseases with hepatocyte apoptotic phenotype.
  • the invention also discloses the application of the saRNA in the preparation of medicines for related diseases caused by the down-regulation of FXR expression.
  • the invention further discloses the application of the saRNA in the preparation of a drug against hepatocyte apoptosis.
  • liver diseases are drugs for the treatment of liver diseases, wherein liver diseases have hepatocyte apoptosis phenotype, and the specific treatment is not limited to the following diseases: viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, liver fibrosis, liver cirrhosis, Liver cancer, cholestatic liver disease, drug-induced liver damage, etc.
  • This series of saRNAs can promote the transcription of FXR gene, and can restore the down-regulated FXR expression level caused by various incentives, which is significantly different from the control group, and the optimal value is ***P ⁇ 0.001;
  • the series of saRNAs have precise targets, high specificity, strong specificity, and a wide range of indications, and can be prepared as drugs for the treatment of various liver diseases with hepatocyte apoptosis phenotype.
  • Figure 1 shows the expression level of FXR in the state of apoptosis, in which: *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001;
  • Figure 2 shows the regulatory effect of the saRNA of the application on the expression of NR1H4 gene in healthy cells, wherein: *P ⁇ 0.05, ***P ⁇ 0.001;
  • Figure 3 shows the regulatory effect of the saRNA of the present application on the expression of NR1H4 gene in apoptotic cells, wherein: *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001;
  • Figure 4 is the alleviation effect of the saRNA of the application on apoptosis, wherein: **P ⁇ 0.01, ***P ⁇ 0.001.
  • DMEM medium was purchased from GIBCO (Grand Island, New York, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, Utah, USA). Trypsin Trypsin was purchased from Amersco (Solon, Ohio, USA). Cell culture consumables were purchased from Costar (USA). Actinomycin D (Actinomycin, ActD) and cycloheximide (cycloheximide, CHX) were purchased from MedChem Express Company (NJ, USA).
  • Recombinant human tumor necrosis factor alpha (Tumor Necrosis Factor alpha, TNF ⁇ ) was purchased from Peprotech Company (Rocky Hill, USA), recombinant human FasL protein and recombinant human TRAIL protein were purchased from Sino Biological Company (Wayne, PA, USA).
  • RNAiso Plus was purchased from RNAiso Plus from TaKaRa Biotechnology (Dalian, China).
  • IIIRT SuperMix for qPCR and ChamQ SYBR qPCR Master Mix were purchased from Vazyme Biotechnology (Nanjing, China). All primers were designed and synthesized by Invitrogen (Shanghai, China).
  • DEPC water was purchased from Biyuntian Institute of Biotechnology (Shanghai, China).
  • Human hepatocyte cell line L02 was cultured in DMEM complete medium containing 10% fetal bovine serum, placed in a cell incubator at 37°C, 5% CO 2 , and the cells in the logarithmic growth phase were taken as 2 ⁇ 10 5 cells/well seeded in 12-well plates.
  • the apoptosis model was established by TRAIL, ActD/TNF ⁇ , CHX/FasL method respectively.
  • the administration concentration of TRAIL was 50ng/ml, and the administration time was 0, 2, 4, 8, 12, and 24 hours.
  • ActD/TNF ⁇ administration concentrations were 0.2 ⁇ M and 20 ng/ml, where ActD was pre-administered for 30 min, and the administration time was 0, 1, 2, 4, 8, and 12 hours.
  • the administration concentrations of CHX/FasL were 50 ⁇ M and 50 ng/ml, in which CHX was pre-administered for 30 minutes, and the administration time was 0, 2, 4, and 8 hours.
  • RNAiso Plus reagent was used to extract total cellular RNA, reverse transcribed it into cDNA, and analyze the transcriptional expression changes of FXR by fluorescence quantitative PCR.
  • the primers used in fluorescence quantitative PCR are shown in Table 1 below:
  • RNAiMAX Lipofectamine RNAiMAX was purchased from Thermo Fisher (CA, USA). Opti-MEM medium was purchased from GIBCO (Grand Island, New York, USA). All saRNAs were synthesized by Jiman Biotechnology Co., Ltd. (Shanghai, China). Recombinant human TRAIL protein is the same as Example 1.
  • the FXR promoter region sequence was obtained at the website NCBI (https://www.ncbi.nlm.nih.gov/).
  • sequence (SEQ ID NO: 1) of site -1000 to -1 in the FXR promoter region is as follows:
  • Human hepatocyte cell line L02 was cultured in DMEM complete medium containing 10% fetal bovine serum, placed in a cell incubator at 37°C, 5% CO 2 , and the cells in the logarithmic growth phase were taken as 2 ⁇ 10 5 cells/well seeded in 12-well plates. Cells were transfected at 50 nM after 24 h and harvested 48 h after transfection. RNAiso Plus reagent was used to extract total cellular RNA, reverse transcribed it into cDNA, and analyze the transcriptional expression changes of FXR by fluorescence quantitative PCR.
  • Antisense Strand 5'-UAAACAUAGAAAGGAACCU[dT][dT]-3' (SEQ ID NO:7)
  • Antisense Strand 5'-AAACAUAGAAAGGAACCUU[dT][dT]-3' (SEQ ID NO:3)
  • Antisense Strand 5'-ACAUAGAAAGGAACCUUGG[dT][dT]-3' (SEQ ID NO:9)
  • Antisense strand 5'-GAAUAGCUCUAUCACUUCC[dT][dT]-3' (SEQ ID NO:11)
  • Antisense Strand 5'-AAUAGCUCUAUCACUUCCC[dT][dT]-3' (SEQ ID NO:5)
  • the regulation effect of the above saRNA on FXR gene expression was investigated and verified in normal L02 cells.
  • the five pairs of saRNAs all have activation effects, and can significantly promote the transcription of the FXR gene and up-regulate its mRNA level; and hdsFXR-103 and hdsFXR-319 are better than hdsFXR-102, hdsFXR-105 and hdsFXR-318 .
  • the expression level of FXR gene was down-regulated in a time-dependent manner in apoptotic state.
  • Figure 3 under TRAIL-induced L02 apoptotic state, the expression of FXR gene was down-regulated in a time-dependent manner; the above saRNA had a significant reversal effect on this, and down-regulated FXR at various time points and to varying degrees in the apoptosis model All have significant reversal effects.
  • Thiazolyl blue (Methylthiazolyldiphenyl-tetrazolium bromide, MTT) was purchased from MedChem Express (NJ, USA).
  • the human hepatocyte cell line L02 was cultured in DMEM complete medium containing 10% fetal bovine serum, placed in a cell incubator at 37°C, 5% CO 2 , and the cells in the logarithmic growth phase were taken as 5 ⁇ 10 3 cells/well seeded in 96-well plates. Cells were transfected at 50 nM after 24 h, and apoptosis modeling was performed 48 h after transfection.
  • TRAIL was administered at a concentration of 50ng/ml for 24h. ActD/TNF ⁇ administration concentration was 0.2 ⁇ M and 20ng/ml, where ActD was pre-administered for 30min and administration time was 8h.
  • the administration concentration of CHX/FasL was 50 ⁇ M and 50 ng/ml, in which CHX was pre-administered for 30 minutes and the administration time was 8 hours.
  • hdsFXR-102, hdsFXR-103, hdsFXR-105, hdsFXR-318 and hdsFXR-319 were significantly different in TRAIL (Figure 4A and 4D), ActD/TNF ⁇ ( Figure 4B and 4E) and CHX/FasL ( Figure 4C and Figure 4F) all have significant anti-apoptotic effects under the three classical apoptosis models.

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Abstract

提供了一种FXR靶向的saRNA及其在制备对抗肝细胞凋亡的药物中的应用。该saRNA由序列如SEQ ID NO：2所示的正义链和序列如SEQ ID NO：3所示的反义链组成，或者由SEQ ID NO：4所示的正义链和序列如SEQ ID NO：5所示的反义链组成，或者由SEQ ID NO：6所示的正义链和序列如SEQ ID NO：7所示的反义链组成，或者由SEQ ID NO：8所示的正义链和序列如SEQ ID NO：9所示的反义链组成，或者由SEQ ID NO：10所示的正义链和序列如SEQ ID NO：11所示的反义链组成。该saRNA通过促进FXR的转录并上调该基因的表达水平，使疾病状态下下调的肝脏FXR表达水平恢复正常，并改善细胞凋亡。

Description

FXR靶向的saRNA及其应用 技术领域
本发明涉及一类FXR靶向的saRNA及其应用，尤其涉及一类可上调FXR表达水平的saRNA及其应用。
背景技术
肝脏疾病，包括病毒性肝炎(HBV/HCV等)、非酒精性脂肪肝、酒精性脂肪肝、肝纤维化、肝硬化和肝癌等，在全球范围的发病率居高不下，肝病已经成为威胁人类健康的重要杀手。目前全球约有1.85亿人感染HCV，其中慢性感染者约1.3-1.7亿人；另外，全球一般人群的非酒精性脂肪性肝病(Nonalcoholic fatty liver disease,NAFLD)患病率超过25％，且已有超过1亿人口的进展为非酒精性脂肪性肝炎(Nonalcoholic steatohepatitis,NASH)，NASH已发展成为肝移植的主要原因，然而目前治疗各类肝脏疾病的特异性药物并不多。
细胞凋亡是一种基本的生物学现象，对生物体进化、内环境稳态及机体发育等具有重要作用；而凋亡过程紊乱则与多种疾病的发生发展密切相关，如肝细胞凋亡是各种肝病(包括NALFD/NASH，酒精性肝炎、药物性肝损伤、病毒性肝炎、胆汁淤积型肝损伤、缺血-再灌注性肝损伤、肝豆状核变性、肝纤维化/硬化、肝癌等)的共同特征和病理机制。
法尼醇X受体(Farnesoid X Receptor，FXR，又称NR1H4)是核受体超家族的一员，它在调节胆汁酸代谢、脂质代谢和糖代谢中发挥重要功能，同时它与能量平衡、炎症与免疫、肝纤维化以及肝脏再生密切相关。因此，FXR被广泛认为是重要的保肝药物研发靶标。目前基于FXR为靶点的药物研发策略主要集中于FXR激动剂或拮抗剂的研发，即旨在调控FXR的转录活性状态。FXR激动剂作为保肝药物的研发是当前的研究热点，其中，奥贝胆酸作为一种强FXR激动剂，为抗原发性胆汁性肝硬化药物，已于2016年5月被美国FDA批准上市，是以FXR为靶点成功上市的第一例药物；其抗NASH药效已完成临床III期研究，然而其临床药效欠佳：服用安慰剂组的患者中有12％发现肝纤维化指标有改善，而服用高剂量奥贝胆酸的患者中有23％发现肝纤维化指标有改善，提示通过奥贝胆酸等FXR激动剂转录激活FXR后的药理效应比较局限。
研究表明，NASH以及肝纤维化状态下，肝脏中FXR表达下调。靶标蛋白的缺失使得其激动剂或者拮抗剂无法通过调控该靶标来发挥作用，这是导致FXR激动剂临床药效不佳的主要原因；此外，肝细胞凋亡进程中伴随着FXR的表达 下调，加速加重肝细胞凋亡；并且提高FXR表达具有抗凋亡作用；另外，随着年龄的增大，肝脏中FXR的表达显著下调，这是引起脂肪肝的重要因素。因此，需重视FXR自身表达对疾病发生发展以及其配体药理活性的作用；基于此，调控FXR的自身表达是新的靶向FXR的重要策略。然而如何调控FXR蛋白的基因表达尚无成熟的技术手段。
发明内容
发明目的：本发明的第一目的是提供一类FXR靶向的saRNA，第二目的是提供所述saRNA的应用。
技术方案：本发明的FXR靶向的saRNA包括与NR1H4基因启动子区-1000至-1位点区域互补的核苷酸序列。
进一步地，所述saRNA包括与NR1H4基因启动子区-102位点至-84位点区域互补的核苷酸序列。
进一步地，所述saRNA包括与NR1H4基因启动子区-103位点至-85位点区域互补的核苷酸序列。
进一步地，所述saRNA包括与NR1H4基因启动子区-105位点至-87位点区域互补的核苷酸序列。
进一步地，所述saRNA包括与NR1H4基因启动子区-318位点至-300位点区域互补的核苷酸序列。
进一步地，所述saRNA包括与NR1H4基因启动子区-319位点至-301位点区域互补的核苷酸序列。
更具体地，所述saRNA由序列如SEQ ID NO：2所示的正义链和序列如SEQ ID NO：3所示的反义链组成，或者由SEQ ID NO：4所示的正义链和序列如SEQ ID NO：5所示的反义链组成，或者由SEQ ID NO：6所示的正义链和序列如SEQ ID NO：7所示的反义链组成，或者由SEQ ID NO：8所示的正义链和序列如SEQ ID NO：9所示的反义链组成，或者由SEQ ID NO：10所示的正义链和序列如SEQ ID NO：11所示的反义链组成。
本发明从saRNA的角度，首次设计合成了特异性靶向FXR(NR1H4)的系列saRNA，其具有特异性高、专一性强的特点，可显著调控FXR的转录和表达，从而发挥抗肝细胞凋亡的活性，可用于制备对抗具有肝细胞凋亡表型的肝脏疾病的药物。
本发明还公开了所述的saRNA在制备由于FXR表达下调引起的相关疾病的药物中的应用。
本发明又公开了所述的saRNA在制备对抗肝细胞凋亡的药物中的应用。
上述药物为治疗肝脏疾病的药物，其中肝脏疾病具有肝细胞凋亡表型，可具体治疗不限于以下疾病：病毒性肝炎、酒精性肝病、非酒精性脂肪性肝病、肝纤维化、肝硬化、肝癌、胆汁淤积性肝病、药物性肝损害等。
有益效果：与现有技术相比，本发明具有如下显著优点：
(1)该系列saRNA均能够促进FXR基因的转录，均能够恢复多种诱因导致下调的FXR表达水平，与对照组具有显著差异，最优达到***P<0.001；
(2)该系列saRNA均能够对抗各种诱因导致的肝细胞凋亡，与对照组具有显著差异，最优达到***P<0.001；
(3)该系列saRNA作用靶点精准，特异性高、专一性强，适应症广泛，可制备为治疗具有肝细胞凋亡表型的多种肝脏疾病的药物。
附图说明
图1为细胞凋亡状态下FXR的表达水平，其中：*P<0.05，**P<0.01，***P<0.001；
图2为本申请的saRNA对健康细胞中NR1H4基因表达的调控作用，其中：*P<0.05，***P<0.001；
图3为本申请的saRNA对凋亡细胞中NR1H4基因表达的调控作用，其中：*P<0.05，**P<0.01，***P<0.001；
图4为本申请的saRNA对细胞凋亡的缓解作用，其中：**P<0.01，***P<0.001。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1：细胞凋亡状态下FXR的表达变化
1、实验材料
DMEM培养基购自GIBCO(Grand Island,New York,USA)。胎牛血清(Fetal bovine serum,FBS)购自Hyclone(Logan,Utah,USA)。胰蛋白酶Trypsin购自Amersco(Solon,Ohio,USA)。细胞培养耗材购于Costar(USA)。放线菌素D(Actinomycin,ActD)和放线菌酮(cycloheximide,CHX)购于MedChem Express公司(NJ,USA)。重组人肿瘤坏死因子α(Tumor Necrosis Factor alpha,TNFα)购于Peprotech公司(Rocky Hill,USA)，重组人FasL蛋白和重组人TRAIL蛋白购于Sino Biological公司(Wayne,PA,USA)。RNAiso Plus购于RNAiso Plus购于TaKaRa Biotechnology(大连，中国)。
Figure PCTCN2021111137-appb-000001
ⅢRT SuperMix for qPCR及 ChamQ SYBR qPCR Master Mix均购于Vazyme Biotechnology(南京，中国)。所有引物均由Invitrogen公司(上海，中国)设计合成。DEPC水购于碧云天生物技术研究所(上海，中国)。
2、实验方法
2.1细胞培养及给药处理
人肝细胞系L02培养于含10％胎牛血清的DMEM完全培养基，置于37℃，5％CO 2的细胞培养箱中培养，取对数生长期的细胞按2×10 5个/孔接种于12孔板中。24h后分别用TRAIL，ActD/TNFα，CHX/FasL法造凋亡模型。TRAIL给药浓度为50ng/ml，给药时间为0、2、4、8、12、24h。ActD/TNFα给药浓度为0.2μM和20ng/ml，其中ActD预给30min，给药时间为0、1、2、4、8、12h。CHX/FasL给药浓度为50μM和50ng/ml，其中CHX预给30min，给药时间为0、2、4、8h。
2.2 PCR
RNAiso Plus试剂提取细胞总RNA，将其反转录为cDNA然后通过荧光定量PCR法分析FXR的转录表达变化。荧光定量PCR所使用的引物见下表1：
表1 荧光定量PCR所使用的引物
Figure PCTCN2021111137-appb-000002
3、实验结果
如图1所示，在TRAIL、ActD/TNFα、CHX/FasL 3种凋亡模型下，FXR的mRNA水平均表现出时间依赖性的下降。
实施例2：靶向FXR的saRNA设计及验证
1、实验材料
Lipofectamine RNAiMAX购于Thermo Fisher(CA,USA)。Opti-MEM培养基购于GIBCO(Grand Island,New York,USA)。所有saRNA均由吉满生物科技有限公司(上海，中国)合成。重组人TRAIL蛋白同实施例1。
其余试剂同实施例1。
2、实验方法
2.1 FXR启动子序列查询
在网站NCBI(https://www.ncbi.nlm.nih.gov/)获得FXR启动子区序列。
根据saRNA序列的设计原则，设计获得FXR的5对saRNA及其对应FXR启动子区位点。FXR启动子区位点-1000位点至-1位点的序列(SEQ ID NO:1) 如下所示：
Figure PCTCN2021111137-appb-000003
2.2细胞培养及saRNA转染
人肝细胞系L02培养于含10％胎牛血清的DMEM完全培养基，置于37℃，5％CO 2的细胞培养箱中培养，取对数生长期的细胞按2×10 5个/孔接种于12孔板中。24h后以50nM转染细胞，转染48h后收获细胞。RNAiso Plus试剂提取细胞总RNA，将其反转录为cDNA然后通过荧光定量PCR法分析FXR的转录表达变化。
2.3 TRAIL诱导的细胞凋亡模型
同实施例1。
2.4 PCR
同实施例1。
3、实验结果
3.1 saRNA序列
hdsFXR-102：
正义链：5’-AGGUUCCUUUCUAUGUUUA[dT][dT]-3’(SEQ ID NO:6)
反义链：5’-UAAACAUAGAAAGGAACCU[dT][dT]-3’(SEQ ID NO:7)
(FXR启动子区-102位点至-84位点)。
hdsFXR-103：
正义链：5’-AAGGUUCCUUUCUAUGUUU[dT][dT]-3’(SEQ ID NO:2)
反义链：5’-AAACAUAGAAAGGAACCUU[dT][dT]-3’(SEQ ID NO:3)
(FXR启动子区-103位点至-85位点)。
hdsFXR-105：
正义链：5’-CCAAGGUUCCUUUCUAUGU[dT][dT]-3’(SEQ ID NO:8)
反义链：5’-ACAUAGAAAGGAACCUUGG[dT][dT]-3’(SEQ ID NO:9)
(FXR启动子区-105位点至-87位点)。
hdsFXR-318：
正义链：5’-GGAAGUGAUAGAGCUAUUC[dT][dT]-3’(SEQ ID NO:10)
反义链：5’-GAAUAGCUCUAUCACUUCC[dT][dT]-3’(SEQ ID NO:11)
(FXR启动子区-318位点至-300位点)。
hdsFXR-319：
正义链：5’-GGGAAGUGAUAGAGCUAUU[dT][dT]-3’(SEQ ID NO:4)
反义链：5’-AAUAGCUCUAUCACUUCCC[dT][dT]-3’(SEQ ID NO:5)
(FXR启动子区-319位点至-301位点)。
3.2 saRNA验证
首先在正常L02细胞中考察验证了上述saRNA对FXR基因表达的调控作用。如图2所示，5对saRNA均具有激活效果，均可以显著促进FXR基因的转录，使其mRNA水平上调；且hdsFXR-103和hdsFXR-319优于hdsFXR-102、hdsFXR-105和hdsFXR-318。
3.3 saRNA对凋亡细胞中FXR表达的调控作用
同实施例1所述，凋亡状态下FXR基因表达水平呈时间依赖性的下调。如图3所示，TRAIL诱导的L02凋亡状态下，FXR基因表达呈时间依赖性的下调；而上述saRNA对此具有显著的逆转作用，对凋亡模型中各个时间点和不同程度的FXR下调均具有显著的逆转作用。
实施例3：系列saRNA对细胞凋亡的药理活性
1、实验材料
噻唑蓝(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)购于MedChem Express公司(NJ,USA)。
其余试剂同实施例2。
2、实验方法
2.1细胞培养、转染和给药处理
人肝细胞系L02培养于含10％胎牛血清的DMEM完全培养基，置于37℃，5％CO 2的细胞培养箱中培养，取对数生长期的细胞按5×10 3个/孔接种于96孔板中。24h后以50nM转染细胞，转染48h后进行凋亡造模。TRAIL给药浓度为50ng/ml，给药24h。ActD/TNFα给药浓度为0.2μM和20ng/ml，其中ActD预给30min，给药时间为8h。CHX/FasL给药浓度为50μM和50ng/ml，其中CHX预给30min，给药时间为8h。
2.2细胞活力检测
给药时间结束后，每孔加入20μl MTT(5mg/ml)，37℃继续孵育4h，吸出弃去培养基，每孔加入150μl DMSO，37℃孵育10min，490nm波长测定吸光度。
3、实验结果
如图4所示，hdsFXR-102、hdsFXR-103、hdsFXR-105、hdsFXR-318和hdsFXR-319在TRAIL(图4A和图4D)、ActD/TNFα(图4B和图4E)和CHX/FasL(图4C和图4F)3种经典凋亡模型下均具有显著的抗细胞凋亡效果。

Claims (10)

  1. 一种FXR靶向的saRNA，其特征在于，所述saRNA包括与NR1H4基因启动子区-1000至-1位点区域互补的核苷酸序列。
  2. 根据权利要求1所述的saRNA，其特征在于，所述saRNA包括与NR1H4基因启动子区-102位点至-84位点区域互补的核苷酸序列。
  3. 根据权利要求1所述的saRNA，其特征在于，所述saRNA包括与NR1H4基因启动子区-103位点至-85位点区域互补的核苷酸序列。
  4. 根据权利要求1所述的saRNA，其特征在于，所述saRNA包括与NR1H4基因启动子区-105位点至-87位点区域互补的核苷酸序列。
  5. 根据权利要求1所述的saRNA，其特征在于，所述saRNA包括与NR1H4基因启动子区-318位点至-300位点区域互补的核苷酸序列。
  6. 根据权利要求1所述的saRNA，其特征在于，所述saRNA包括与NR1H4基因启动子区-319位点至-301位点区域互补的核苷酸序列。
  7. 根据权利要求1所述的saRNA，其特征在于，所述saRNA由序列如SEQ ID NO：2所示的正义链和序列如SEQ ID NO：3所示的反义链组成，或者由SEQ ID NO：4所示的正义链和序列如SEQ ID NO：5所示的反义链组成，或者由SEQ ID NO：6所示的正义链和序列如SEQ ID NO：7所示的反义链组成，或者由SEQ ID NO：8所示的正义链和序列如SEQ ID NO：9所示的反义链组成，或者由SEQ ID NO：10所示的正义链和序列如SEQ ID NO：11所示的反义链组成。
  8. 一种权利要求1-7任一所述的saRNA在制备由于FXR表达下调引起的相关疾病的药物中的应用。
  9. 一种权利要求1-7任一所述的saRNA在制备对抗肝细胞凋亡的药物中的应用。
  10. 根据权利要求8或9所述的应用，其特征在于，所述药物为治疗肝脏疾病的药物。
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