WO2020000466A1 - Lncrna afap1-as1 overexpression vector and preparation method thereof - Google Patents

Lncrna afap1-as1 overexpression vector and preparation method thereof Download PDF

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WO2020000466A1
WO2020000466A1 PCT/CN2018/093876 CN2018093876W WO2020000466A1 WO 2020000466 A1 WO2020000466 A1 WO 2020000466A1 CN 2018093876 W CN2018093876 W CN 2018093876W WO 2020000466 A1 WO2020000466 A1 WO 2020000466A1
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毛吉炎
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深圳市博奥康生物科技有限公司
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  • the invention belongs to the field of biotechnology, and particularly relates to an lncRNA AFAP1-AS1 overexpression vector and a preparation method thereof.
  • Lung cancer is one of the most common malignant tumors in the world. Worldwide, the incidence of lung cancer has increased significantly, and its incidence has ranked first among various tumors in men, while the incidence of lung cancer in women has also increased significantly. To date, lung cancer has become the leading cause of cancer-related deaths worldwide. In China, the incidence of lung cancer has increased rapidly year by year. In the past few decades, lung cancer mortality has increased by 465%, and its mortality rate ranks first in urban areas and second in rural areas. Among all lung cancers, non-small cell lung cancer accounts for 75-80%. Despite many new advances in both clinical and experimental oncology, the prognosis for lung cancer is still unsatisfactory, with a 5-year survival rate of only about 11%. The main reason for the high mortality rate of lung cancer is that tumor cells usually have invasion and metastasis when patients are diagnosed, and lack of effective treatment measures.
  • lncRNA Long-chain non-coding RNA
  • lncRNA is a type of large-molecule non-coding RNA that can play a regulatory role in a variety of biological processes. It is widely distributed, typically more than 200 bases in length, and lacks or has little ability to encode proteins due to the lack of effective open reading frames.
  • lncRNA is a form of RNA that plays a role in regulating gene expression at multiple levels, mainly from three levels: epigenetic regulation, transcriptional regulation, and post-transcriptional regulation.
  • epigenetic regulation As an important part of mammalian transcriptome, the function of lncRNA needs to be further studied. At first lncRNA was considered as a by-product of RNA polymerase II transcription.
  • LncRNAs are found to be dysregulated in many types of cancer, such as breast, prostate, lung, colon, and liver cancer.
  • lncRNA AFAP1-AS1 is a lncRNA first studied in Barrett's esophagus and esophageal adenocarcinoma. Its low methylation and high expression indicate that lncRNA AFAP1-AS1 is closely related to Barrett's esophagus and esophageal adenocarcinoma. However, the role and mechanism of lncRNA AFAP1-AS1 in the pathogenesis of Barrett's esophagus and esophageal adenocarcinoma are unclear, and there is also no existing overexpression vector that can be used to study the function of lncRNA AFAP1-AS1 in the prior art.
  • An lncRNA AFAP1-AS1 overexpression vector includes a promoter-lncRNA AFAP1-AS1 fragment-terminator, and a sequence of the lncRNA AFAP1-AS1 fragment is shown in SEQ ID NO. 1
  • the promoter is pCMV and the terminator is SV40 pA.
  • the lncRNA AFAP1-AS1 overexpression vector and preparation method thereof include the following steps:
  • the lncRNA AFAP1-AS1 overexpression vector and the preparation method thereof provided by the present invention can greatly increase the expression level of lncRNA AFAP1-AS1 in tumor cells, and provide a new technical means for studying the role of lncRNA AFAP1-AS1 in tumorigenesis and development.
  • Figure 1 is a pCMV-C-Flag vector map
  • Figure 2 shows the lncRNA AFAP1-AS1 expression levels of SKGT-4 cells in the control and experimental groups.
  • a 5 'end of SEQ ID NO. 1 was added with a Bam HI digestion site, and a 3' end was added with an EcoR I digestion site. Shanghai Biotech was entrusted to directly synthesize the sequence by gene synthesis.
  • Bam HI and EcoR I enzymes were used to double digest the plasmid containing the synthetic sequence and the pCMV-C-Flag vector, respectively, and then recovered and purified.
  • the recovered synthetic sequence was mixed with the pCMV-C-Flag vector 1: 4, and then ligated with NEB T4 DNA ligase.
  • the ligation product was transformed into competent E. coli Top 10, followed by expansion and sequencing, and screening of bacteria that completely matched the expected results. Then expand the culture and use the endotoxin-free plasmid extraction kit to extract the recombinant plasmid in E. coli and name it pCMV-lncRNA-AFAP1-AS1.
  • Embodiment two pCMV-lncRNA-AFAP1-AS1 Transfection SKGT-4 cell
  • SKGT-4 cells were seeded in a six-well plate with 1,000,000 cells per well, and the cell density was about 50% after 12 h. Lipofectamine 2000 was used to transfer 1 ⁇ g The pCMV-lncRNA-AFAP1-AS1 plasmid was transfected into SKGT-4 cells, and the cells were further cultured.
  • SKGT-4 cells control group
  • transfected SKGT-4 cells experimental group
  • Example 2 Normal SKGT-4 cells (control group) and the transfected SKGT-4 cells (experimental group) obtained in Example 2 were respectively inoculated into a six-well plate.
  • the cell density reached 80% -90%
  • total RNA of each group of cells was extracted with RNeasy Mini Kit, and mRNA was reverse transcribed into cDNA using PrimeScrip RT reagent Kit, and stored at -20 ° C.
  • the lncRNA AFAP1-AS1 overexpression vector and the preparation method thereof provided by the present invention can greatly increase the expression level of lncRNA AFAP1-AS1 in tumor cells, and provide a new technical means for studying the role of lncRNA AFAP1-AS1 in tumorigenesis and development.

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Abstract

Provided are a lncRNA AFAP1-AS1 overexpression vector and a preparation method thereof. The lncRNA AFAP1-AS1 overexpression vector contains complete lncRNA AFAP1-AS1 expression sequence as shown in SEQ ID NO.1. The constructed lncRNA AFAP1-AS1 overexpression vector can efficiently express lncRNA AFAP1-AS1, thereby improving the efficiency of functional studies thereof.

Description

一种lncRNA AFAP1-AS1过表达载体及其制备方法LncRNA AFAP1-AS1 overexpression vector and preparation method thereof 技术领域Technical field
本发明属于生物技术领域，尤其涉及一种lncRNA AFAP1-AS1过表达载体及其制备方法。 The invention belongs to the field of biotechnology, and particularly relates to an lncRNA AFAP1-AS1 overexpression vector and a preparation method thereof.
背景技术Background technique
肺癌是全球最常见的恶性肿瘤之一。 在世界范围内，肺癌的发病率明显升高，其发病率已经位于男性各种肿瘤的首位，而女性肺癌的发病率也明显增高，至今，肺癌已成为全世界癌相关性死亡的主要原因。在中国，肺癌的发病率逐年快速上升，在过去的几十年里，肺癌死亡率增加了465%，其死亡率在城市中居首位，在农村中位于第二位。在所有的肺癌中，非小细胞肺癌占75-80%。尽管临床和实验肿瘤学均取得了许多新的进展，但是肺癌的预后仍不如人意，5 年生存率只有约11%。造成肺癌高死亡率的主要原因是患者确诊时肿瘤细胞通常已发生了侵袭和转移，缺乏有效的治疗措施。Lung cancer is one of the most common malignant tumors in the world. Worldwide, the incidence of lung cancer has increased significantly, and its incidence has ranked first among various tumors in men, while the incidence of lung cancer in women has also increased significantly. To date, lung cancer has become the leading cause of cancer-related deaths worldwide. In China, the incidence of lung cancer has increased rapidly year by year. In the past few decades, lung cancer mortality has increased by 465%, and its mortality rate ranks first in urban areas and second in rural areas. Among all lung cancers, non-small cell lung cancer accounts for 75-80%. Despite many new advances in both clinical and experimental oncology, the prognosis for lung cancer is still unsatisfactory, with a 5-year survival rate of only about 11%. The main reason for the high mortality rate of lung cancer is that tumor cells usually have invasion and metastasis when patients are diagnosed, and lack of effective treatment measures.
长链非编码RNA（lncRNA）是一类能在多种生物学过程中发挥调控作用的大分子非编码RNA。其分布广泛，长度一般多于200个碱基，由于缺少有效开放阅读框而无或很少有编码蛋白的能力。作为分子生物学中的一个全新领域，lncRNA是以RNA的形式在多个层面上发挥调控基因表达的作用，主要是从三个层面，分别是表观遗传调控、转录调控以及转录后调控。作为哺乳动物转录组的重要组成部分，lncRNA的功能目前还有待于深入研究。起初lncRNA被认为是RNA聚合酶Ⅱ转录的副产物，是基因组转录的“噪音”，不具有生物功能。但近年来的研究结果显示，实际上lncRNAs 在癌症的发生发展中发挥重要作用，与癌症的生物学过程有关，比如癌症的形成、浸润和转移过程[19]。 lncRNAs 表达失调在许多类型的癌症中被发现，比如乳腺癌、前列腺癌、肺癌、结肠癌、肝癌。Long-chain non-coding RNA (lncRNA) is a type of large-molecule non-coding RNA that can play a regulatory role in a variety of biological processes. It is widely distributed, typically more than 200 bases in length, and lacks or has little ability to encode proteins due to the lack of effective open reading frames. As a new field in molecular biology, lncRNA is a form of RNA that plays a role in regulating gene expression at multiple levels, mainly from three levels: epigenetic regulation, transcriptional regulation, and post-transcriptional regulation. As an important part of mammalian transcriptome, the function of lncRNA needs to be further studied. At first lncRNA was considered as a by-product of RNA polymerase II transcription. It was the "noise" of genome transcription and had no biological function. However, recent research results show that lncRNAs actually play an important role in the occurrence and development of cancer, and are related to the biological processes of cancer, such as the formation, invasion and metastasis of cancer [19]. LncRNAs are found to be dysregulated in many types of cancer, such as breast, prostate, lung, colon, and liver cancer.
技术问题technical problem
lncRNA AFAP1-AS1是一个首先在 Barrett食管和食管腺癌中被研究的 lncRNA，它在其中低甲基化、高表达，说明lncRNA AFAP1-AS1与Barrett食管和食管腺癌有密切联系。但lncRNA AFAP1-AS1在Barrett食管和食管腺癌发病过程中所起的作用及其机制尚不清楚，现有技术中也缺乏可用于lncRNA AFAP1-AS1功能研究的过表达载体。lncRNA AFAP1-AS1 is a lncRNA first studied in Barrett's esophagus and esophageal adenocarcinoma. Its low methylation and high expression indicate that lncRNA AFAP1-AS1 is closely related to Barrett's esophagus and esophageal adenocarcinoma. However, the role and mechanism of lncRNA AFAP1-AS1 in the pathogenesis of Barrett's esophagus and esophageal adenocarcinoma are unclear, and there is also no existing overexpression vector that can be used to study the function of lncRNA AFAP1-AS1 in the prior art.
技术解决方案Technical solutions
有鉴于此，有必要针对上述的问题，提供一种lncRNA AFAP1-AS1过表达载体及其制备方法。In view of this, it is necessary to provide an lncRNA AFAP1-AS1 overexpression vector and a method for preparing the same in view of the above problems.
一种lncRNA AFAP1-AS1过表达载体，包含启动子-lncRNA AFAP1-AS1片段-终止子，所述lncRNA AFAP1-AS1片段的序列如SEQ ID NO. 1所示An lncRNA AFAP1-AS1 overexpression vector includes a promoter-lncRNA AFAP1-AS1 fragment-terminator, and a sequence of the lncRNA AFAP1-AS1 fragment is shown in SEQ ID NO. 1
优选地，所述启动子为pCMV，所述终止子为SV40 pA。Preferably, the promoter is pCMV and the terminator is SV40 pA.
所述lncRNA AFAP1-AS1过表达载体及其制备方法，包含如下步骤：The lncRNA AFAP1-AS1 overexpression vector and preparation method thereof include the following steps:
S1、在SEQ ID NO. 1序列的5’端加上Bam HI酶切位点，3’端加上EcoR I酶切位点，然后采用基因合成的方式直接合成；S1. Add a Bam HI digestion site to the 5 ′ end of SEQ ID NO. 1 sequence, add an EcoR I digestion site to the 3 ′ end, and then directly synthesize it by gene synthesis;
S2、双酶切pCMV-C-Flag载体和上述合成的序列，用连接酶将该序列连接至pCMV-C-Flag载体中CMV启动子下游，测序验证正确得到lncRNA AFAP1-AS1过表达载体。S2. Double-digest the pCMV-C-Flag vector and the above-synthesized sequence, and ligase this sequence to the downstream of the CMV promoter in the pCMV-C-Flag vector. Sequencing verified that the lncRNA AFAP1-AS1 overexpression vector was correctly obtained.
有益效果Beneficial effect
本发明提供的lncRNA AFAP1-AS1过表达载体及其制备方法可大幅提升lncRNA AFAP1-AS1在肿瘤细胞中的表达水平，为研究lncRNA AFAP1-AS1在肿瘤发生发展中的作用提供了新的技术手段。The lncRNA AFAP1-AS1 overexpression vector and the preparation method thereof provided by the present invention can greatly increase the expression level of lncRNA AFAP1-AS1 in tumor cells, and provide a new technical means for studying the role of lncRNA AFAP1-AS1 in tumorigenesis and development.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为pCMV-C-Flag载体图谱；Figure 1 is a pCMV-C-Flag vector map;
图2为对照组和实验组SKGT-4细胞的lncRNA AFAP1-AS1表达水平。Figure 2 shows the lncRNA AFAP1-AS1 expression levels of SKGT-4 cells in the control and experimental groups.
本发明的实施方式Embodiments of the invention
下面结合附图与具体实施例对本发明做进一步的说明。The invention is further described below with reference to the drawings and specific embodiments.
实施例一Example one ：: lncRNAlncRNA AFAP1-AS1 AFAP1-AS1 过表达载体的构建Construction of overexpression vectors
在SEQ ID NO. 1序列的5’端加上Bam HI酶切位点，3’端加上EcoR I酶切位点，委托上海生工采用基因合成的方式直接合成该序列。A 5 'end of SEQ ID NO. 1 was added with a Bam HI digestion site, and a 3' end was added with an EcoR I digestion site. Shanghai Biotech was entrusted to directly synthesize the sequence by gene synthesis.
使用Bam HI和EcoR I酶分别对含有合成序列的质粒和pCMV-C-Flag载体进行双酶切，然后进行回收纯化。回收后的合成序列与pCMV-C-Flag载体按1:4混匀后，用NEB T4 DNA连接酶进行连接。Bam HI and EcoR I enzymes were used to double digest the plasmid containing the synthetic sequence and the pCMV-C-Flag vector, respectively, and then recovered and purified. The recovered synthetic sequence was mixed with the pCMV-C-Flag vector 1: 4, and then ligated with NEB T4 DNA ligase.
连接产物转化感受态大肠杆菌Top 10，扩大培养后测序，筛选出测序结果与预期完全相符的菌。再扩大培养，并应用无内毒素质粒提取试剂盒提取大肠杆菌中的重组质粒，命名为pCMV-lncRNA-AFAP1-AS1。The ligation product was transformed into competent E. coli Top 10, followed by expansion and sequencing, and screening of bacteria that completely matched the expected results. Then expand the culture and use the endotoxin-free plasmid extraction kit to extract the recombinant plasmid in E. coli and name it pCMV-lncRNA-AFAP1-AS1.
实施例二：Embodiment two: pCMV-lncRNA-AFAP1-AS1pCMV-lncRNA-AFAP1-AS1 转染Transfection SKGT-4SKGT-4 细胞cell
接种SKGT-4细胞于六孔板中，每孔1000000个细胞，12h后细胞密度约为50%，，用Lipofectamine 2000将1 μg pCMV-lncRNA-AFAP1-AS1质粒分别转染至SKGT-4细胞，并对细胞进行继续培养。SKGT-4 cells were seeded in a six-well plate with 1,000,000 cells per well, and the cell density was about 50% after 12 h. Lipofectamine 2000 was used to transfer 1 μg The pCMV-lncRNA-AFAP1-AS1 plasmid was transfected into SKGT-4 cells, and the cells were further cultured.
实施例三：荧光定量Example 3: Fluorescence Quantification PCRPCR 检测Detection lncRNAlncRNA AFAP1-AS1 AFAP1-AS1 表达水平The expression level
分别接种正常SKGT-4细胞（对照组）、实施例二中获得经转染的SKGT-4细胞（实验组）至六孔板。细胞密度达到80%-90%时，用RNeasy Mini Kit提取各组细胞的总RNA，利用PrimeScrip RT reagent Kit将mRNA逆转录为cDNA，-20℃保存。Normal SKGT-4 cells (control group) and the transfected SKGT-4 cells (experimental group) obtained in Example 2 were respectively inoculated into a six-well plate. When the cell density reached 80% -90%, total RNA of each group of cells was extracted with RNeasy Mini Kit, and mRNA was reverse transcribed into cDNA using PrimeScrip RT reagent Kit, and stored at -20 ° C.
取各组细胞的cDNA 1 μL为模板，以β-actin为内参，实时荧光定量PCR检测lncRNA AFAP1-AS1的相对表达水平，设置反应条件：95℃ 30s，1循环；60℃ 30s 40循环；95℃ 5s，60℃ 1min，95℃ 15s。结果如图1所示，可以看到，实验组细胞的lncRNA AFAP1-AS1表达水平较对照组细胞有21倍以上的升高，说明本发明提供的lncRNA AFAP1-AS1过表达载体能特异、高效地促进lncRNA AFAP1-AS1过表达。Take 1 μL of cDNA from each group of cells as the template and use β-actin as the internal reference to detect the relative expression level of lncRNA AFAP1-AS1 by real-time quantitative PCR. Set the reaction conditions: 95 ° C 30s, 1 cycle; 60 ° C 30s 40 cycles; 95 5 ° C, 1min at 60 ° C, 15s at 95 ° C. The results are shown in FIG. 1. It can be seen that the expression level of lncRNA AFAP1-AS1 in the experimental group cells is 21 times higher than that in the control group, indicating that the lncRNA AFAP1-AS1 overexpression vector provided by the present invention can be specifically and efficiently Promote lncRNA AFAP1-AS1 overexpression.
工业实用性Industrial applicability
本发明提供的lncRNA AFAP1-AS1过表达载体及其制备方法可大幅提升lncRNA AFAP1-AS1在肿瘤细胞中的表达水平，为研究lncRNA AFAP1-AS1在肿瘤发生发展中的作用提供了新的技术手段。The lncRNA AFAP1-AS1 overexpression vector and the preparation method thereof provided by the present invention can greatly increase the expression level of lncRNA AFAP1-AS1 in tumor cells, and provide a new technical means for studying the role of lncRNA AFAP1-AS1 in tumorigenesis and development.

Claims (3)

  1. 一种lncRNA AFAP1-AS1过表达载体，其特征在于，包含启动子-lncRNA AFAP1-AS1片段-终止子，所述lncRNA AFAP1-AS1片段的序列如SEQ ID NO. 1所示。An lncRNA AFAP1-AS1 overexpression vector, comprising a promoter-lncRNA AFAP1-AS1 fragment-terminator, and a sequence of the lncRNA AFAP1-AS1 fragment is shown in SEQ ID NO. 1.
  2. 根据权利要求1所述的一种lncRNA AFAP1-AS1过表达载体，其特征在于，所述启动子为pCMV，所述终止子为SV40 pA。The lncRNA AFAP1-AS1 overexpression vector according to claim 1, wherein the promoter is pCMV and the terminator is SV40 pA.
  3. 根据权利要求1所述的一种lncRNA AFAP1-AS1过表达载体，其特征在于，该载体制备过程包含如下步骤：The lncRNA AFAP1-AS1 overexpression vector according to claim 1, wherein the vector preparation process comprises the following steps:
    S1、在SEQ ID NO. 1序列的5’端加上Bam HI酶切位点，3’端加上EcoR I酶切位点，然后采用基因合成的方式直接合成；S1. Add a Bam HI digestion site to the 5 ′ end of SEQ ID NO. 1 sequence, add an EcoR I digestion site to the 3 ′ end, and then directly synthesize it by gene synthesis;
    S2、双酶切pCMV-C-Flag载体和上述合成的序列，用连接酶将该序列连接至pCMV-C-Flag载体中CMV启动子下游，测序验证正确得到lncRNA AFAP1-AS1过表达载体。S2. Double-digest the pCMV-C-Flag vector and the above-synthesized sequence, and ligase this sequence to the downstream of the CMV promoter in the pCMV-C-Flag vector. Sequencing verified that the lncRNA AFAP1-AS1 overexpression vector was correctly obtained.
PCT/CN2018/093876 2018-06-29 2018-06-29 Lncrna afap1-as1 overexpression vector and preparation method thereof WO2020000466A1 (en)

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