WO2021022888A1 - 靶向长链非编码rna ddx11-as1的aso、试剂盒及在肝癌治疗中的应用 - Google Patents

靶向长链非编码rna ddx11-as1的aso、试剂盒及在肝癌治疗中的应用 Download PDF

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WO2021022888A1
WO2021022888A1 PCT/CN2020/094281 CN2020094281W WO2021022888A1 WO 2021022888 A1 WO2021022888 A1 WO 2021022888A1 CN 2020094281 W CN2020094281 W CN 2020094281W WO 2021022888 A1 WO2021022888 A1 WO 2021022888A1
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ddx11
liver cancer
aso
cells
kit
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杨忠丽
赵忻艺
徐梦详
李明定
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浙江大学
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  • the invention belongs to the field of biomedicine, and specifically relates to an ASO targeting long-chain non-coding RNA DDX11-AS1, a kit, and application in the treatment of liver cancer.
  • lncRNA Long-chain non-coding RNA
  • lncRNAs may participate in the regulation of gene expression at the apparent, transcriptional and post-transcriptional levels. lncRNAs regulate pathophysiological processes through genetic imprinting, histone modification, chromatin remodeling, transcription activation, transcription interference, nuclear transport, and cell cycle regulation.
  • lncRNAs may be used as cavernous bodies to competitively bind to microRNAs (miRNAs), thereby inhibiting their function (ie, blocking the interaction with target mRNAs).
  • miRNAs microRNAs
  • Many lncRNAs are abnormally expressed in different disease types, especially in refractory tumors with unclear pathogenesis.
  • Abnormal lncRNA regulation usually promotes tumor progression by promoting malignant biological behaviors (such as proliferation, invasion and metastasis) in tumor cells.
  • lncRNAs have high tissue specificity, high efficiency and high stability, and are expected to become potential therapeutic targets and biomarkers for diagnosis and prognosis.
  • Hepatocellular Carcinoma ranks sixth and third in the world's cancer incidence and mortality respectively, and the incidence is increasing year by year.
  • China is an area with a high incidence of liver cancer, and the number of deaths from liver cancer each year accounts for about half of the world's deaths from liver cancer.
  • liver cancers in my country have a background of hepatitis B infection and are often associated with cirrhosis, liver insufficiency, and early spread and metastasis, the resection rate of surgery is low, the recurrence rate is high, and only 20%-30% of patients can get the chance of surgical resection.
  • liver cancer Although some genomic and epigenetic changes have been found in liver cancer, mainly focusing on apoptosis, autophagy and hepatitis virus, the molecular mechanism of liver cancer is still unclear, and the treatment options are extremely limited. In recent years, studies have shown that lncRNAs are closely related to the occurrence and development of liver cancer, mainly including tumor cell hyperproliferation, tumor suppressor gene inactivation, tumor cell immortalization, tumor metastasis, invasion and apoptosis inhibition.
  • lncRNA and DDX11-AS1 of liver cancer can provide a reference for the clinical diagnosis of liver cancer.
  • lncRNA can not only become a marker related to the early diagnosis and development of new liver cancer, but it is also expected to treat liver cancer and other diseases by changing the expression of lncRNA or its target genes. Find and identify lncRNA and its target genes related to the occurrence of liver cancer to provide evidence for the clinical treatment of liver cancer.
  • the purpose of the present invention is to provide the application and detection kit of ASO targeting long-chain non-coding RNA DDX11-AS1 in the treatment of liver cancer.
  • the first objective of the present invention is to provide an ASO, which is characterized in that the ASO can inhibit the expression of long non-coding RNA DDX11-AS1.
  • the ASO can down-regulate the expression level of long-chain non-coding RNA DDX11-AS1 in liver cancer cells by more than 60%.
  • the ASO is selected from one or more of SEQ ID NO. 1-3.
  • the present invention firstly adopts the method of fluorescent quantitative PCR to detect the expression of lncRNA and DDX11-AS1 in clinical liver cancer tissues/paracancerous tissues, liver cancer cell lines/normal liver cell lines.
  • the experiment of the present invention proves that the expression level of DDX11-AS1 in liver cancer tissue is significantly higher than the expression level of DDX11-AS1 in tissue adjacent to liver cancer.
  • the second object of the present invention is to provide a liver cancer detection kit, which includes reagents for detecting the expression level of DDX11-AS1 in liver cancer tissues of subjects. Compared with the expression level of DDX11-AS1 in the tissue adjacent to liver cancer, if the expression level of DDX11-AS1 in the liver cancer tissue of the subject is significantly increased by the kit, it is judged that the risk of liver cancer of the subject is high.
  • the reagent includes a primer for DDX11-AS1.
  • the forward primer of DDX11-AS1 is shown in SEQ ID NO. 4, and the reverse primer is shown in SEQ ID NO. 5, as shown in Table 1.
  • the reagent also includes primers for lncRNA that have been reported in the prior art and can be used to detect liver cancer.
  • the detection primers for multiple lncRNAs are placed in the same kit, and the combined detection of liver cancer by detecting multiple lncRNA indicators is also protected by the present invention.
  • kit also includes PCR Mix.
  • the components of the PCR Mix include PCR reaction buffer, Taq DNA polymerase, Mg 2+ and dNTPs.
  • the concentration of each specific primer in the reaction system is preferably 1 uM.
  • reaction system of the kit for performing fluorescent quantitative PCR is: primers with a concentration of 1 uM and a volume of 2.5 ul; 5 ⁇ l of 2 ⁇ SYBR Mix, supplemented with ddH 2 O until the total volume of the reaction is 10 ul.
  • the procedure for performing the fluorescent quantitative PCR reaction with the kit is: 50°C 2 min, 95°C 10 min, (95°C 15s, 60°C 1 min) ⁇ 40 cycles, 95°C 15s, 60°C 15s.
  • the third objective of the present invention is to provide an application of ASO targeting long non-coding RNA DDX11-AS1 in the preparation of drugs for the treatment of liver cancer, which can effectively inhibit the expression of DDX11-AS1 and chemical modification of its nucleic acid sequence and A pharmaceutically acceptable carrier, the carrier includes a lentiviral vector, cholesterol or liposome.
  • the present invention finds that the lncRNA DDX11-AS1 is expressed very low in normal liver tissues or adjacent tissues, but is significantly high in liver cancer tissues.
  • the present invention designs and synthesizes ASO by targeting long-chain non-coding RNA DDX11-AS1, which can reduce the expression level of DDX11-AS1, and significantly inhibit the proliferation, cell cycle progress, migration and invasion of liver cancer cells, which is useful for the development of new anti- Gene drugs for liver cancer are of great significance, with broad application prospects and great economic value.
  • Figure 1 shows the use of qRT-PCR to detect the expression of DDX11-AS1 in liver cancer tissues
  • Figure 2 shows the use of qRT-PCR to detect the expression of DDX11-AS1 in liver cancer cell lines
  • Figure 3 shows the use of qRT-PCR to detect the level of inhibition of DDX11-AS1 by ASO;
  • Figure 4 shows that knocking down DDX11-AS1 leads to a decrease in the proliferation of liver cancer cells
  • Figure 5 shows that knocking down DDX11-AS1 leads to hepatocellular carcinoma cell division arrest in G0/G1 phase
  • Figure 6 shows that knocking down DDX11-AS1 leads to a decrease in the migration ability of liver cancer cells
  • Figure 7 shows that knocking down DDX11-AS1 leads to a decrease in the invasion ability of liver cancer cells.
  • Example 1 Screening of lncRNA related to liver cancer
  • Sample collection 38 cases of clinically confirmed liver cancer and matched tissues adjacent to liver cancer were collected. These samples were surgical resection samples of liver cancer patients. The samples were all from the First affiliated Hospital of Zhejiang University School of Medicine. All the above specimens were obtained with the approval of the ethics committee of the hospital. Clinical data of tissue samples, including clinical features such as tumor size, capsule, lung metastasis, TNM staging, distant metastasis, histopathological grading, postoperative tumor-free survival time and survival time.
  • RNA-seq2 and edgeR RNA-seq difference analysis algorithms
  • Example 2 Expression of DDX11-AS1 in liver cancer cell lines
  • the conventional culture of SMMC-7721 cell line uses 1640 cell culture medium containing 10% inactivated fetal bovine serum and 1% double antibody (penicillin-streptomycin); the conventional culture of Huh7 cell line uses 10% inactivated fetal bovine serum and 1 % Double antibody (penicillin-streptomycin) in DMEM high glucose medium. And put it in a CO 2 incubator at 37°C, 5% CO 2 , and saturated humidity. When the cells are fused to about 80% of the culture dish, they are passaged, the original culture medium is aspirated, and an appropriate amount of trypsin (containing phenol red) solution is added to digest the cells. Observe the cell status under an inverted microscope.
  • RNA template 500ng-1ug of total RNA template with 4ul of 5 ⁇ iScript Reaction Mix, 1ul of iScript Reverse Transcriptase and RNase free water, the final volume is 20ul, the reverse transcription program is: 25 °C 5min, 42°C 30min, 85°C 5min.
  • qRT-PCR reaction A 10ul reaction system is used, with 3 parallel tubes for each sample, and all amplification reactions are repeated more than three times to ensure the reliability of the results.
  • the amplification procedure is: 50°C 2min, 95°C 10min, (95°C 15s, 60°C 1min) ⁇ 40 cycles, 95°C 15s, 60°C 15s.
  • SYBR Green as the fluorescent marker
  • the PCR reaction was performed on the Applied Biosystems real-time quantitative PCR instrument.
  • the forward primer sequence for amplifying DDX11-AS1 is shown in SEQ ID NO. 4, and the reverse primer is shown in SEQ ID NO. 5.
  • the target band was determined by melting curve analysis and electrophoresis, and relative quantification was performed by ⁇ CT method.
  • Example 3 Effect of ASO on the function of liver cancer cells after inhibiting the expression of DDX11-AS1
  • Cells Human liver cancer cell lines SMMC-7721 and Huh7, purchased from Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences.
  • transfection reagent Lipofectamine 2000 was purchased from Thermo Fisher Scientific, and CCK8 and PI cell cycle detection kits were purchased from China MULTISCIENCES.
  • DDX11-AS1 gene sequence (NR-) and mfold software from the NCBI database.
  • NR- non-coding RNA DDX11-AS1
  • design a specifically targeted ASO and select 3 of them to synthesize and make an ASO mixture (Equimolar ratio)
  • the specific sequence of ASO is shown in SEQ ID NO.1-3.
  • the above ASO was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., China.
  • the control ASO was provided by Guangzhou Ruibo Biotechnology Co., Ltd., China.
  • Transiently transfected cells are set as 0h at the time of transfection (refer to cell transfection steps, 96-well plate system for transfection method), and other time points are counted from the time of transfection, and each time point is equipped with 10% CCK8 medium , 100ul per hole is added to the detection hole in the form of changing the liquid, and the incubator is directly tested after 2h incubation.
  • Microplate reader detects absorbance: dual-wavelength detection, CCK8 detection wavelength is 490nm, reference wavelength 650nm. Result statistics: Calculate the average absorbance of each group of repeated wells at each time point for statistical analysis
  • SMMC-7721 cells and Huh7 cells were respectively planted in 6-well plates, and ASO and control ASO were transfected for 48 hours and then collected.
  • Cell sample preparation Wash cells with PBS 2-3 times to wash away dead cells, trypsinize and centrifuge to discard the supernatant, add 1ml PBS to resuspend, so that each sample cell is 2 ⁇ 10 5 -1 ⁇ 10 6 . Discard PBS by centrifugation. Add 1ml DNA Staining Solution and 10 ⁇ l Permeabilization Solution for staining, vortex for 5-10 seconds to mix. Incubate in the dark at room temperature for 30 minutes. Filter with a 300 mesh screen, and autoclave the filter in advance. The flow cytometer is tested on the machine, and the off-machine data is analyzed by Flowjo7.6 software.
  • SMMC-7721 cells and Huh7 cells were planted in 12-well plates, and ASO and control ASO were transfected for 48 hours and then collected.
  • Prepare cell suspension and count wash the cells with PBS for 1-2 times, then digest the cells, terminate the digestion and discard the culture solution by centrifugation, resuspend in serum-free medium, and adjust the cell density to 5 ⁇ 10 5 /ml.
  • SMMC-7721 cells and Huh7 cells were planted in 12-well plates, and ASO and control ASO were transfected for 48 hours and then collected. Dilute 1:8 with Matrigel from BD Company, coat the upper chamber surface of the bottom membrane of Transwell chamber, and place it at 37°C for 30 minutes to polymerize Matrigel into a gel.
  • the next step is to prepare and count the cell suspension: wash the cells with PBS for 1-2 times and then digest the cells, terminate the digestion and discard the culture solution by centrifugation, resuspend in serum-free medium, and adjust the cell density to 1 ⁇ 10 6 /ml. Place the 8um Transwell cell on a 24-well plate (24-well cell culture plate should be matched with the purchased Transwell cell).
  • the ASO mixture can significantly inhibit the expression of DDX11-AS1, and the expression of DDX11-AS1 in SMMC-7721 cells was down-regulated by 81%. See Figure 3(a); DDX11-AS1 expression in Huh7 cells was down-regulated by 66%, see Figure 3(b).
  • the knockdown DDX11-AS1 experiment group reduced the number of cells passing through the basement membrane of the transwell chamber that had been covered with Matrigel by 85%.
  • ASO that specifically targets the non-coding RNA DDX11-AS1 provided by the present invention has a good inhibitory effect, and knockdown of DDX11-AS1 can significantly inhibit the proliferation, cycle, migration and migration of SMMC-7721 cells and Huh7 cells.
  • ASO targeting the long-chain non-coding RNA DDX11-AS1 can be used to develop new anti-liver cancer gene drugs and prepare liver cancer detection kits, which has important significance, broad application prospects and huge economic value.

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Abstract

提供了一种靶向长链非编码RNA DDX11-AS1的反义寡核苷酸及其在制备用于治疗肝癌的药物中的应用，以及一种肝癌检测试剂盒。该反义寡核苷酸能够通过抑制DDX11-AS1的表达来显著地影响肝癌的发生及发展。

Description

靶向长链非编码RNA DDX11-AS1的ASO、试剂盒及在肝癌治疗中的应用 技术领域
本发明属于生物医药领域，具体涉及靶向长链非编码RNA DDX11-AS1的ASO、试剂盒及在肝癌治疗中的应用。
背景技术
长链非编码RNA(lncRNA)是长度大于200bp的非编码RNA，主要由RNA聚合酶II转录，缺乏明显的开放阅读框。lncRNAs可能在表观、转录和转录后水平上参与调控基因的表达。lncRNAs调节病理生理过程通过基因印迹、组蛋白修饰、染色质重塑、转录激活、转录干扰、核转运和细胞周期调控等机制。最近提出的竞争性内源性RNA(ceRNA)调控网络也表明，lncRNAs可能作为海绵体竞争性地与microRNAs(miRNAs)结合，从而抑制其功能(即阻断与目标mRNAs的相互作用)。多种lncRNAs在不同的疾病类型中异常表达，尤其是在发病机制不明确的难治性肿瘤中。lncRNA调节异常通常通过促进肿瘤细胞中的恶性生物学行为(例如增殖，侵袭和转移)而促进肿瘤的进展。另外，lncRNAs具有较高的组织特异性、高效率和高稳定性，有望成为诊断和预后的潜在治疗靶点和生物标志物。
肝癌(Hepatocellular Carcinoma，HCC)在全世界癌症发病率和死亡率中分别位居第六和第三，而且发病率呈逐年上升的趋势。中国是肝癌的高发区，每年死于肝癌的人数约占全世界肝癌死亡人数的一半。由于我国肝癌多有乙肝感染背景且常合并肝硬化、肝功能不全以及早期播散和转移，因此手术的切除率低，复发率高，能获得手术切除机会的患者仅占20％-30％。虽然在肝癌中已经发现了一些基因组和表观遗传学的改变，主要集中在细胞凋亡、自噬和肝炎病毒，但肝癌的分子机制仍不清楚，且治疗手段极其有限。近年来，有研究表明lncRNAs与肝癌的发生发展密切相关，主要包括肿瘤细胞过度增殖、抑癌基因失活、肿瘤细胞永生化、肿瘤转移、浸润和凋亡抑制等。
肿瘤分子的靶向治疗作为一种新型肿瘤疗法，正在逐渐成为临床肿瘤治疗的重要手段。分子靶向治疗的原理是通过抑制或干扰特异性分子(肿瘤驱动基因或信号传导通路关键基因)的表达，从而抑制肿瘤细胞的生长、转移或者诱导其凋亡，到达治疗肿瘤的目的。与传统的治疗手段相比，具有更好的准确性，能选择性的杀死肿瘤细胞，对正常组织或细胞的损伤较低或无损伤，副作用小，不易产生耐药性。但目前市场上靶向分子治疗的药物非常有限，其主要原因还是在于已知的特异靶点较奇缺，迫切需要寻找到新的有效的药物靶点。
检测肝癌lncRNA DDX11-AS1的表达水平可以为肝癌的临床诊断提供参考。在未来临床 治疗中，lncRNA不仅可以成为新的肝癌早期诊断和发展进程相关的标记物，而且也有望通过改变lncRNA或其靶基因的表达来治疗肝癌等疾病。寻找和鉴定与肝癌发生相关的lncRNA及其靶基因，为肝癌的临床治疗提供证据。
发明内容
本发明的目的在于提供靶向长链非编码RNA DDX11-AS1的ASO在肝癌治疗中的应用和检测试剂盒。
为了实现上述目的，本发明采用了如下技术方案：
本发明的第一目的是提供一种ASO，其特征在于：该ASO能够抑制长链非编码RNA DDX11-AS1的表达。
进一步的，该ASO能够使肝癌细胞中长链非编码RNA DDX11-AS1的表达水平下调60％以上。
进一步的，所述长链非编码RNA上的ASO作用靶点在1-3个。
进一步的，所述的ASO选自SEQ ID NO.1-3中的一种或多种。
本发明首先采用荧光定量PCR的方法对临床肝癌组织/癌旁组织、肝癌细胞系/正常肝细胞系的lncRNA DDX11-AS1的表达进行检测。本发明的实验证明肝癌组织中DDX11-AS1的表达水平显著高于肝癌旁组织中DDX11-AS1的表达水平。针对DDX11-AS1基因序列设计并合成多条特异性靶向DDX11-AS1的ASO，采用脂质体介导的方法转染肝癌细胞后，通过荧光定量PCR的方法检测ASO沉默DDX11-AS1的效率，同时通过CCK8、PI染色流式分析、Transwell、Invasion等实验方法检测ASO对细胞增殖、周期、迁移和侵袭的影响，结果显示多条ASO联合转染对DDX11-AS1具有很高的沉默效率，可显著抑制肝癌细胞的增殖、周期、迁移和侵袭能力，抑制肝癌细胞的进程。
本发明的第二目的是提供一种肝癌检测试剂盒，所述试剂盒包括用于检测受试者肝癌组织中DDX11-AS1的表达水平的试剂。与肝癌旁组织中的DDX11-AS1的表达水平相比，如果通过试剂盒检测受试者肝癌组织中DDX11-AS1的表达水平显著升高，则判断该受试者肝癌发生的风险很高。
进一步的，所述试剂包括针对DDX11-AS1的引物。所述DDX11-AS1的正向引物如SEQ ID NO.4所示，反向引物如SEQ ID NO.5所示，见表1.
Figure PCTCN2020094281-appb-000001
Figure PCTCN2020094281-appb-000002
所述试剂还包括对现有技术中已经报道的可用于检测肝癌的lncRNA的引物。将多种lncRNA的检测引物放置在同一试剂盒中，通过检测多种lncRNA指标联合检测肝癌也在本发明的保护之内。
进一步的，所述试剂盒还包括PCR Mix。
进一步的，所述PCR Mix的成分包括PCR反应缓冲液、Taq DNA聚合酶、Mg 2+和dNTPs。
进一步的，反应体系中优选各条特异性引物的浓度为1uM。
进一步的，所述试剂盒进行荧光定量PCR反应体系为：浓度为1uM、体积为2.5ul的引物；5μl 2×SYBR Mix，补加ddH 2O至反应的总体积为10ul。
进一步的，所述试剂盒进行荧光定量PCR反应程序为：50℃2min，95℃10min，(95℃15s，60℃1min)×40个循环，95℃15s，60℃15s。
本发明第三目的是提供一种靶向长链非编码RNA DDX11-AS1的ASO在制备用于治疗肝癌的药物中的应用，能高效抑制DDX11-AS1表达的ASO及其核酸序列的化学修饰以及药学上接受的载体，所述载体包括慢病毒载体，胆固醇或者脂质体。
相对于现有技术，本发明的有益效果如下：本发明发现该lncRNA DDX11-AS1在肝正常组织或癌旁组织中表达很低，但在肝癌组织中却显著高表达。本发明通过靶向长链非编码RNA DDX11-AS1设计并合成ASO，其能够降低DDX11-AS1的表达水平，并且显著抑制肝癌细胞的增殖、细胞周期进程、迁移和侵袭能力，对于开发新的抗肝癌基因药物具有重要意义，具有广阔的应用前景和巨大的经济价值。
附图说明
图1显示利用qRT-PCR检测DDX11-AS1在肝癌组织中的表达情况；
图2显示利用qRT-PCR检测DDX11-AS1在肝癌细胞系中的表达情况；
图3显示利用qRT-PCR检测ASO对DDX11-AS1的抑制水平；
图4显示敲低DDX11-AS1导致肝癌细胞增殖能力的减弱；
图5显示敲低DDX11-AS1导致肝癌细胞分裂阻滞在G0/G1期；
图6显示敲低DDX11-AS1导致肝癌细胞迁移能力的减弱；
图7显示敲低DDX11-AS1导致肝癌细胞的侵袭能力减弱。
具体实施方式
下面结合具体的实施例进一步说明本发明。本发明的实施例仅用于解释本发明，并不意味着限制本发明的保护范围。
下述实施例中所使用的实验方法如无特殊说明，均为常规试验方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从现有的商业途径得到。
实施例1：与肝癌相关lncRNA的筛选
1、样本收集：取临床确诊的肝癌及配对肝癌旁组织标本38例，这些样本为肝癌患者的手术切除标本，样本均来自于浙江大学医学院附属第一医院。上述所有标本的取得均通过该院组织伦理委员会的同意。组织样本临床数据，包括肿瘤大小、包膜、肺转移、TNM分期、远处转移、组织病理分级、术后无瘤生存时间及生存时间等临床特征。
2、肝癌及配对肝癌旁组织总RNA的提取
取100mg冰冻组织并在液氮中快速充分研磨，加入1mL Trizol和约1/5体积的氯仿，上下颠倒2min，室温下静置5min；然后4℃，12,000rpm离心15min；取出离心管并转移上清液到新的1.5ml离心管中，加入等体积的异丙醇，上下颠倒混匀，室温静置10min。4℃，12000rpm离心10min，去上清；向沉淀中加入1：1体积的70％乙醇，4℃，12000rpm离心5min，去上清；沉淀在室温下放置10min后加入适量无RNA酶的水充分溶解，离心管放置在冰上，测定其浓度。琼脂糖胶电泳评价总RNA质量，28S:18S≥2被认为总RNA质量较好。
3、使用Bio-Rad逆转录试剂盒进行逆转录，并用Illumina HiSeq2500测序仪进行RNA深度测序
使用DESeq2和edgeR两种主流的RNA-seq差异分析算法计算，且FDR<0.05和改变量在2倍及以上的基因被认为具有显著性差异。基于TCGA的lncRNA表达数据和肝癌临床预后数据，对潜在具有凋亡功能的lncRNA进行生存分析，根据log-rank p<0.05，同时结合通路富集程度或共表达相关性，筛选出新的具有细胞凋亡功能的lncRNA分子标志物。
4、结果
如图1所示，与癌旁组织相比，肿瘤组织中的DDX11-AS1表达明显升高(P<0.05)。
实施例2：DDX11-AS1在肝癌细胞系中的表达
1、细胞培养
SMMC-7721细胞株常规培养采用含10％灭活胎牛血清和1％双抗(青霉素-链霉素)的1640细胞培养基；Huh7细胞株常规培养采用含10％灭活胎牛血清和1％双抗(青霉素-链霉素)的DMEM高糖培养基。并静置于37℃、5％CO 2、饱和湿度条件的CO 2培养箱中培养。当细胞融合到培养皿的80％左右时予以传代，吸出原培养液，加入适量的胰酶(含酚红)溶液消化细胞。倒置显微镜下观察细胞状态，待细胞呈圆形(胞质回缩)，吸出胰酶，加入RPMI1640或DMEM高糖完全培养基1ml终止消化，反复吹打以分散细胞，移入1ml离心管，1200rpm离心5min，弃上清后加入1ml新培养液，轻轻吹打混匀，按1：2的量接种于RPMI1640或高糖完全培养基，置于培养箱中继续培养。
2、qRT-PCR
2.1细胞总RNA提取：用Trizol提取细胞总RNA。
2.2逆转录：将500ng-1ug的总RNA模板与4ul的5×iScript Reaction Mix、1ul的iScript Reverse Transcriptase和无核糖核酸酶水(RNase free water)混合，体积最后为20ul，逆转录程序为：25℃5min，42℃30min，85℃5min。
2.3 qRT-PCR反应：采用10ul反应体系，每个样本设置3个平行管，所有扩增反应均重复三次以上以保证结果的可靠性。配制以下反应体系：SYBR Green聚合酶链式反应体系5ul，引物混合物(1uM)2.5ul，模板cDNA 1ul，无酶水1.5ul，各项操作均于冰上进行。扩增程序为：50℃2min，95℃10min，(95℃15s，60℃1min)×40个循环，95℃15s，60℃15s。以SYBR Green作为荧光标记物，在Applied Biosystems荧光实时定量PCR仪上进行PCR反应。扩增DDX11-AS1的正向引物序列如SEQ ID NO.4所示，反向引物如SEQ ID NO.5所示。以GAPDH作为参照基因。通过融解曲线分析和电泳确定目的条带，ΔΔCT法进行相对定量。
3、结果
如图2所示，与正常细胞相比，肝癌细胞株SMMC-7721和Huh7中DDX11-AS1的表达明显升高(P<0.05)。
实施例3：ASO抑制DDX11-AS1表达后对肝癌细胞功能的影响
1、材料
细胞：人肝癌细胞细胞系SMMC-7721和Huh7，购自中国科学院上海生命科学研究 所细胞资源中心。
试剂：转染试剂Lipofectamine 2000购自Thermo Fisher Scientific公司，CCK8和PI细胞周期检测试剂盒购自中国MULTISCIENCES公司。
2、细胞培养同实施例2
3、靶向DDX11-AS1的ASO序列的设计和合成
通过NCBI数据库获得DDX11-AS1基因序列(NR-)以及mfold软件，根据长链非编码RNA DDX11-AS1的二级结构，设计特异靶向的ASO，从中选出3条进行合成并制成ASO混合物(等摩尔比)，ASO具体序列为SEQ ID NO.1-3所示。上述ASO由中国广州锐博生物科技有限公司合成。对照ASO由中国广州锐博生物科技有限公司提供。
4、细胞转染：分别将人肝癌细胞SMMC-7721和Huh7接种于6孔板，在CO2培养箱中培养过夜。生长至汇合度为60％-70％，参考Lipofectamine 2000转染试剂说明书进行转染，操作步骤如下：
(1)将10μl的ASO混合物与5μl的Lipofectamine 2000分别与250μl的培养基进行混匀，室温静置5min。
(2)将250μl混合好的Lipofectamine 2000混合物加入到含有ASO混合物的培养基中，轻轻混匀，室温放置20min。
(3)取出6孔板，吸取500μl的混合体系，逐滴加在孔的四周，轻拍板的四边摇匀，放回CO2培养箱中，培养48h。
(4)转染后收集细胞，采用qRT-PCR方法检测ASO对DDX11-AS1的干扰效率，以及进行后续的细胞增殖、周期、迁移和侵袭等相关细胞功能学实验。
2、细胞增殖
制备细胞悬液：用0.25％的胰酶消化处于对数生长期的细胞，1500r/min低速离心5min，弃去上清液加培养基制成细胞悬液并进行细胞计数，充分混匀铺板：按照每孔5000个细胞的100ul细胞悬液接种到96孔板中，分别设有Case和Control两组，每组至少设置3个重复孔并设置0，24h，48h,72h,96h四个检测时间点，在37℃的5％CO 2培养箱中培养使细胞贴壁。瞬时转染的细胞在转染时定为0h(转染方法参照细胞转染步骤，96孔板体系)，其他时间点从转染时算起，每个时间点配置含10％CCK8的培养基，每孔100ul以换液的形式加入检测孔中，培养箱培养2h后直接检测。酶标仪检测吸光度：采用双波长检测，CCK8检测波长为490nm，参比波长650nm。结果统计：计算每个时间点每组重复孔的吸光度均值进行统计分析
3、细胞周期
将SMMC-7721细胞和Huh7细胞分别种于6孔板中，ASO和对照ASO转染48h后收集细胞。细胞样本准备：用PBS清洗细胞2-3次洗去死细胞，胰酶消化离心弃上清，加入1ml PBS重悬，使每个样细胞在2×10 5-1×10 6个。离心弃去PBS。加入1ml DNA Staining Solution和10μl Permeabilization Solution进行染色，涡旋振荡5-10秒混匀。室温避光孵育30分钟。用300目筛网过滤，滤筛提前高压灭菌。流式细胞仪上机检测，下机数据采用Flowjo7.6软件分析。
4、迁移实验
将SMMC-7721细胞和Huh7细胞分别种于12孔板中，ASO和对照ASO转染48h后收集细胞。制备细胞悬液并计数：用PBS清洗细胞1-2遍后消化细胞，终止消化后离心弃去培养液，用无血清的培养基重悬，调整细胞密度至5×10 5/ml。将孔径8μm的Transwell小室置于24孔板上(24孔细胞培养板应当与购买的Transwell小室相配套)。向每孔Transwell上室中加入200ul制备好的转染细胞悬液，下室中加入700ul含10％FBS的培养基，调整小室的位置注意避免产生气泡。在37℃的5％CO 2培养箱中培养24小时。取出Chamber，吸干上室的液体用棉签小心擦干上室残留的细胞，再转移到已加入500ul甲醇的孔中，室温固定30min。吸干上室的甲醇再转移到加入0.1％的结晶紫染液中染色20-30min。用干棉签从上室底部擦干膜表面残留的染液。镜下观察，每孔随机拍五个视野，计数穿过的细胞个数并做统计。
3、侵袭实验
将SMMC-7721细胞和Huh7细胞分别种于12孔板中，ASO和对照ASO转染48h后收集细胞。用BD公司的Matrigel 1：8稀释，包被Transwell小室底部膜的上室面，置37℃30min使Matrigel聚合成凝胶。下一步制备细胞悬液并计数：用PBS清洗细胞1-2遍后消化细胞，终止消化后离心弃去培养液，用无血清的培养基重悬，调整细胞密度至1×10 6/ml。将孔径8um的Transwell小室置于24孔板上(24孔细胞培养板应当与购买的Transwell小室相配套)。向每孔Transwell上室中加入200ul制备好的细胞悬液，下室中加入700ul含10％FBS的培养基，调整小室的位置注意避免产生气泡。在37℃的5％CO 2培养箱中培养24小时。取出chamber，吸干上室的液体用棉签小心擦干上室残留的细胞，再转移到已加入500ul甲醇的孔中，室温固定30min。吸干上室的甲醇再转移到加入0.1％的结晶紫染液中染色20-30min。用干棉签从上室底部擦干膜表面残留的染液。镜下观察，每孔随机拍五个视野，计数穿过的细胞个数并做统计。
4、结果
如图3所示，在干扰实验中，与抑制阴性对照组(ASO-Ctrl)相比，ASO混合物可明显 抑制DDX11-AS1的表达，SMMC-7721细胞中DDX11-AS1的表达下调了81％，见图3中的(a)；Huh7细胞中的DDX11-AS1表达下调了66％，见图3中的(b)。
如图4所示，在增殖实验中：与抑制阴性对照组(ASO-Ctrl)相比，两个敲低DDX11-AS1实验组的细胞增殖速率均减弱了约60％，见图4中(a)的SMMC-7721细胞和图4中(b)的Huh7细胞。
如图5所示，在周期实验中：与抑制阴性对照组(ASO-Ctrl)相比，两个敲低DDX11-AS1实验组的细胞周期明显阻滞在G0/G1期，见图5中(a)的SMMC-7721细胞和图4中(b)的Huh7细胞。
如图6所示，在迁移实验中：与抑制阴性对照组(ASO-Ctrl)相比，敲低DDX11-AS1实验组穿过Transwell小室基底膜的细胞减少了约60％。
如图7所示，在侵袭实验中：与抑制阴性对照组(ASO-Ctrl)相比，敲低DDX11-AS1实验组穿过已经铺过基质胶的transwell小室基底膜的细胞减少了85％。
上述实验结果表明，本发明提供的特异靶向非编码RNA DDX11-AS1的ASO具有良好的抑制效果，并且敲低DDX11-AS1后能够显著抑制SMMC-7721细胞和Huh7细胞的增殖、周期、迁移和侵袭能力，靶向长链非编码RNA DDX11-AS1的ASO可用于开发新的抗肝癌基因药物以及制备肝癌检测试剂盒，具有重要意义和广阔的应用前景及巨大的经济价值。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。

Claims (10)

  1. 一种ASO，其特征在于：该ASO能够抑制长链非编码RNA DDX11-AS1的表达。
  2. 根据权利要求1所述的ASO，其特征在于：该ASO能够使肝癌细胞中长链非编码RNADDX11-AS1的表达水平下调60％以上。
  3. 根据权利要求1所述的ASO，其特征在于：所述长链非编码RNA上的ASO作用靶点在1-3个。
  4. 一种靶向长链非编码RNA DDX11-AS1的ASO在制备用于治疗肝癌的药物中的应用。
  5. 根据权利要求4所述的应用，其特征在于：所述的ASO选自SEQ ID NO.1-3中的一种或多种。
  6. 一种肝癌检测试剂盒，其特征在于：所述试剂盒包括用于检测受试者肝癌组织中DDX11-AS1的表达水平的试剂，所述试剂包括针对DDX11-AS1的引物。
  7. 根据权利要求6所述的肝癌检测试剂盒，其特征在于：所述DDX11-AS1的正向引物如SEQ ID NO.4所示，反向引物如SEQ ID NO.5所示。
  8. 根据权利要求6所述的试剂盒，其特征在于：所述试剂盒还包括PCR Mix。
  9. 根据权利要求8所述的试剂盒，其特征在于：所述PCR Mix的成分包括PCR反应缓冲液、Taq DNA聚合酶、Mg 2+和dNTPs。
  10. 根据权利要求6所述的试剂盒，其特征在于：反应体系中优选各条特异性引物的浓度为1uM。
PCT/CN2020/094281 2019-08-07 2020-06-04 靶向长链非编码rna ddx11-as1的aso、试剂盒及在肝癌治疗中的应用 WO2021022888A1 (zh)

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