CN111647597B - An siRNA for inhibiting the expression of hsa_circ_0027479 and its application - Google Patents

Abstract

The invention discloses a nucleotide for inhibiting the expression of human hsa _ circ _0027479 circular RNA and application thereof, which is characterized in that: the nucleotide specifically binds to human hsa _ circ _0027479 circular RNA, the sequence of the nucleotide consisting of 18-22 consecutive nucleotides of the AAAUUCUUGGGACCCUGAUGCUC sequence or the complementary sequence, in particular it comprises the sequence: AAAUUCUUGGGACCCUGAU, and UCUUGGGACCCUGAUGCUC. The invention can inhibit the human hsa _ circ _0027479 circular RNA, can effectively inhibit the expression of hsa _ circ _0027479 circular RNA in A549 cells and XWLC-05 cells, can increase the inhibition of the growth and the proliferation of the human hsa _ circ _0027479 circular RNA by combining with the antitumor drug, and thus can effectively treat various tumors.

Description

Translated fromChinese

一种抑制hsa_circ_0027479表达的siRNA及其应用An siRNA for inhibiting the expression of hsa_circ_0027479 and its application

技术领域technical field

本发明属于医学材料技术和药物领域 ，具体地本发明涉及一种环状RNAs核苷酸，尤其是涉及人环状RNAs核苷酸。该核苷酸可与hsa_circ_0027479互补，从而抑制人hsa_circ_0027479的表达，与其他抗肿瘤药物其起到抗肿瘤的作用。The present invention belongs to the field of medical material technology and medicine, and in particular the present invention relates to a circular RNAs nucleotide, in particular to a human circular RNAs nucleotide. The nucleotide can be complementary to hsa_circ_0027479, thereby inhibiting the expression of human hsa_circ_0027479, and it plays an anti-tumor effect with other anti-tumor drugs.

背景技术Background technique

CircRNA是由外显子或内含子反向剪接形成的闭合环结构的非编码RNA。CircRNA主要存在于细胞质中，根据其基因组的来源及构成序列的不同，可分为外显子来源的环状RNA、内含子来源的环状RNA、由内含子及外显子共同形成的环状RNA 。CircRNA不仅可调控基因表达，也可作为原癌基因和抑癌基因在肿瘤发生发展过程中发挥特殊作用。CircRNAs are non-coding RNAs with closed loop structures formed by back-splicing of exons or introns. CircRNAs mainly exist in the cytoplasm and can be divided into exon-derived circular RNAs, intron-derived circular RNAs, and circRNAs formed by introns and exons according to their genome origins and constituent sequences. circular RNA. CircRNAs can not only regulate gene expression, but also play special roles as proto-oncogenes and tumor suppressor genes in the process of tumorigenesis and development.

CircRNA在肿瘤组织及癌旁正常组织中的表达差异显著，可作为微小RNAs海绵、调节亲本基因的表达、与 RNA 结合蛋白相互作用等在肿瘤细胞周期、 细胞凋亡、 血管形成、侵袭等方面发挥作用。 CircRNA最经典的作用是海绵样作用，通过竞争性结合数个 miRNA，从而抑制了该 miRNA 对靶基因的负性调节作用，形成CircRNA-miRNA-mRNA调控网络来调节肿瘤的发生发展。此外CircRNA不仅可通过结合和调控致癌蛋白促进肿瘤进展，而且其生成过程中与线性剪接竞争 ，影响其线性基因表达，为肿瘤发生创造有利条件。CircRNAs have significant differences in expression between tumor tissues and adjacent normal tissues, and can act as microRNAs sponges, regulate the expression of parental genes, and interact with RNA-binding proteins in tumor cell cycle, apoptosis, angiogenesis, and invasion. effect. The most classic role of circRNA is sponge-like action, which inhibits the negative regulation of the miRNA on target genes by competitively binding to several miRNAs, forming a circRNA-miRNA-mRNA regulatory network to regulate the occurrence and development of tumors. In addition, circRNAs can not only promote tumor progression by binding and regulating oncogenic proteins, but also compete with linear splicing during their generation, affecting their linear gene expression and creating favorable conditions for tumorigenesis.

circRNAs 是肿瘤防御系统的重要组成部分，其异常表达可能是肿瘤启动的早期关键性事件。其可作为肿瘤的分子标志物或潜在治疗靶点，为肿瘤的早期诊断、疗效评价、预后预测和肿瘤基因治疗提供一个新的靶点。。CircRNAs are an important part of the tumor defense system, and their abnormal expression may be an early key event in tumor initiation. It can be used as a molecular marker or potential therapeutic target of tumor, providing a new target for early diagnosis, efficacy evaluation, prognosis prediction and tumor gene therapy of tumor. .

RNA干扰(RNA interference，RNAi)技术利用双链小RNA高效、特异性降解细胞内同源RNA从而沉默靶基因，从而实现干扰靶基因的功能。RNA interference (RNAi) technology utilizes double-stranded small RNAs to efficiently and specifically degrade homologous RNAs in cells to silence target genes, thereby interfering with the function of target genes.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供一种能够特异高效地抑制hsa_circ_0027479表达的siRNA及其应用。The purpose of the present invention is to provide an siRNA capable of specifically and efficiently inhibiting the expression of hsa_circ_0027479 and its application.

本发明的目的是这样实现的，包括合成核苷酸的序列由AAAUUCUUGGGACCCUGAUGCUC(SEQ ID NO.1)序列中的18-22个连续核苷酸序列组成或者互补序列中的18-22个连续核苷酸序列组成。特别是其包含序列：AAAUUCUUGGGACCCUGAU(SEQ IDNO.2)和UCUUGGGACCCUGAUGCUC(SEQ ID NO.3)或者互补序列。核苷酸为核糖核苷酸、脱氧核糖核苷酸或者核糖核苷酸与脱氧核糖核苷酸的嵌合体。核苷酸进一步被核糖修饰、碱基修饰、磷酸骨架修饰、氟代修饰、硫代修饰、甲氧基修饰、胆固醇修饰的一种或几种。核苷酸和其他抗肿瘤治疗药物特别是三价无机砷。制备治疗癌症药物中的应用。癌症包括 ：肝癌、贲门癌、结胃癌、、鼻咽癌、卵巢癌、前列腺癌症、慢性或急性白血病、脑瘤、食道 癌、口腔癌、尿道癌、皮肤癌、直肠癌、中耳癌、骨癌、肠癌、胆囊癌、喉癌、牙龈癌、肺癌、胰腺癌、乳腺癌、宫颈癌、、大肠癌、 睾丸癌、内分泌系统的癌症、淋巴细胞性淋巴瘤。本研究受国家自然科学基金的支持（81860572）。The object of the present invention is achieved by the sequence comprising synthetic nucleotides consisting of 18-22 consecutive nucleotide sequences in the AAAUUCUUGGGACCCUGAUGCUC (SEQ ID NO. 1) sequence or 18-22 consecutive nucleosides in the complementary sequence acid sequence composition. In particular it comprises the sequences: AAAUUCUUGGGACCCUGAU (SEQ ID NO. 2) and UCUUGGGACCCUGAUGCUC (SEQ ID NO. 3) or complementary sequences. Nucleotides are ribonucleotides, deoxyribonucleotides, or chimeras of ribonucleotides and deoxyribonucleotides. The nucleotides are further modified by one or more of ribose modification, base modification, phosphate backbone modification, fluorine modification, thio modification, methoxy modification and cholesterol modification. Nucleotides and other antineoplastic therapeutics especially trivalent inorganic arsenic. Application in the preparation of medicines for the treatment of cancer. Cancers include: liver cancer, cardia cancer, gastric cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, chronic or acute leukemia, brain tumor, esophageal cancer, oral cancer, urethral cancer, skin cancer, rectal cancer, middle ear cancer, bone cancer Cancer, colon cancer, gallbladder cancer, throat cancer, gum cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, colorectal cancer, testicular cancer, cancer of the endocrine system, lymphocytic lymphoma. This research was supported by the National Natural Science Foundation of China (81860572).

本发明（优点）：发明的核苷酸具有很好的hsa_circ_0027479抑制效果，作用于特异性的靶位点，特异性强、毒性低、副作用小和修饰半衰期长，可以与多种抗肿瘤药物合用。The present invention (advantage): The nucleotides of the invention have good hsa_circ_0027479 inhibitory effect, act on specific target sites, have strong specificity, low toxicity, small side effects and long modification half-life, and can be used in combination with a variety of antitumor drugs .

具体实施方式Detailed ways

下面对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换或替换，均属于本发明的保护范围。The present invention is further described below, but the present invention is not limited in any way, and any transformation or replacement based on the teaching of the present invention belongs to the protection scope of the present invention.

具体实施方式。Detailed ways.

实施例Example

本实施例中的沉默RNA序列为：AAAUUCUUGGGACCCUGAU(SEQ ID NO.2)和UCUUGGGACCCUGAUGCUC(SEQ ID NO.3)以及互补序列,所有的序列都是从5端到3端,所有序列都在末端加上dTdT。hsa_circ_0027479的RNA序列信息存在于UCSC数据库中。本课题受本研究受国家自然科学基金的支持（81860572）。The silencing RNA sequences in this example are: AAAUUCUUGGGACCCUGAU (SEQ ID NO.2) and UCUUGGGACCCUGAUGCUC (SEQ ID NO.3) and their complementary sequences, all sequences are from the 5th end to the 3rd end, and all sequences are added at the end dTdT. The RNA sequence information of hsa_circ_0027479 exists in the UCSC database. This subject is supported by the National Natural Science Foundation of China (81860572).

对照合成control synthesis

首先，由上海吉玛制药技术有限公司合成核苷酸，合成包含其互补序列的双链RNA序列，对照组使用吉玛制药技术有限公司的阴性对照siRNA货号为A06001序列为对照组的阴性对照。First, nucleotides were synthesized by Shanghai Gema Pharmaceutical Technology Co., Ltd., and a double-stranded RNA sequence containing its complementary sequence was synthesized. The negative control siRNA product number A06001 of Gema Pharmaceutical Technology Co., Ltd. was used as the negative control for the control group.

细胞培养：将A549或者XWLC-05细胞在含有10％胎牛血清的1640培养基于37℃，5％CO2条件下培养。以2500个每孔铺于96孔板内。RNA转染采用百代公司Rfect转染试剂进行转染，通过荧光定量PCR来经常RNA干扰的效率。以下分别为对照组、沉默组1、沉默组的沉默效率，A549细胞分别为： 1.001±0.041，0.199±0.021，0.243±0.038；XWLC-05细胞分别为0.998±0.071,0.181±0.014, 0.254±0.020,数据使用2^^-ΔΔCt表示。Cell culture: A549 or XWLC-05 cells were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. 2500 cells were plated in a 96-well plate. The RNA transfection was carried out using the EMI company's Rfect transfection reagent, and the efficiency of RNA interference was determined by fluorescence quantitative PCR. The following are the silencing efficiencies of the control group, silence group 1, and silence group, respectively, A549 cells are: 1.001±0.041, 0.199±0.021, 0.243±0.038; XWLC-05 cells are 0.998±0.071, 0.181±0.014, 0.254±0.020 , the data are represented by 2^^-ΔΔCt .

荧光定量使用的引物：hsa_circ_0027479上游：ATGAGAAAATTCTTGGGACCCTG (SEQID NO.4)下游：GCTCTCCATGCTTGACCACA(SEQ ID NO.5)，内参采用“陈江容,张若冰,张媛,胡娟,周梅,何越峰.砷及其代谢产物对P21基因表达的影响[J].职业与健康,2018,34(23):3213-3216.”文章中的ACTB基因。Primer used for fluorescence quantification: hsa_circ_0027479 Upstream: ATGAGAAAATTCTTGGGACCCTG (SEQ ID NO.4) Downstream: GCTCTCCATGCTTGACCACA (SEQ ID NO.5), internal reference used "Chen Jiangrong, Zhang Ruobing, Zhang Yuan, Hu Juan, Zhou Mei, He Yuefeng. Arsenic and its metabolites Effects on P21 gene expression [J]. Occupation and Health, 2018, 34(23): 3213-3216.” ACTB gene in the article.

两个细胞使用96孔板转染转染后48小时加入砷，亚砷酸钠溶于培养基中，不加砷的加入等量培养基，亚砷酸钠的终浓度为A549为60微摩尔每升，XWLC-05细胞砷的终浓度为40微摩尔每升，再培养48小时后MTS检测细胞活力。用Promega公司MTS检测，采用490nm吸光度，计算细胞活力。Two cells were transfected using a 96-well plate. Arsenic was added 48 hours after transfection, and sodium arsenite was dissolved in the medium. Without arsenic, an equal amount of medium was added. The final concentration of sodium arsenite was 60 micromolar for A549. The final concentration of arsenic in XWLC-05 cells was 40 μmol per liter per liter, and cell viability was detected by MTS after another 48 hours of culture. The cell viability was calculated by MTS detection of Promega company and the absorbance at 490nm.

未加砷对照组：为使用阴性对照片段后不加入亚砷酸钠，本组的活力定为100.0%。未加砷实验组1：核苷酸片段1沉默的结果。未加砷实验组2：沉默核苷酸片段2沉默的结果。加砷对照组：为使用阴性对照片段后加入亚砷酸钠的结果。加砷实验组1：沉默核苷酸片段1沉默后加入亚砷酸钠的结果。加砷实验组2：沉默核苷酸片段2沉默后加入亚砷酸钠的结果。以下为各组的细胞活力均为百分比（%）：Control group without arsenic: In order to use the negative control fragment without adding sodium arsenite, the activity of this group was set as 100.0%. Arsenic-free experimental group 1: Results of silencing of nucleotide fragment 1. Arsenic-free experimental group 2: Silencing results of silencing nucleotide fragment 2. Arsenic-added control group: It is the result of adding sodium arsenite after using the negative control fragment. Arsenic-added experimental group 1: the result of adding sodium arsenite after silencing the silencing nucleotide fragment 1. Arsenic-added experimental group 2: the result of adding sodium arsenite after silencing the silencing nucleotide fragment 2. The following are the percentages (%) of cell viability in each group:

未加砷对照组：A549为101.4±2.9，XWLC-05为99.9±4.1，Arsenic-free control group: A549 was 101.4±2.9, XWLC-05 was 99.9±4.1,

未加砷实验组1： A549为93.9±5.0， XWLC-05为94.7±4.0，Arsenic-free experimental group 1: A549 was 93.9±5.0, XWLC-05 was 94.7±4.0,

未加砷实验组2: A549为94.1±4.2，XWLC-05为93.9±2.9，Experimental group 2 without arsenic: A549 was 94.1±4.2, XWLC-05 was 93.9±2.9,

加砷对照组： A549为87.1±4.2，XWLC-05为86.4±3.3，Arsenic-added control group: A549 was 87.1±4.2, XWLC-05 was 86.4±3.3,

加砷实验组1： A549为64.3±2.8，XWLC-05为57.4±3.1，Arsenic-added experimental group 1: A549 was 64.3±2.8, XWLC-05 was 57.4±3.1,

加砷实验组2： A549为65.1±2.9，XWLC-05为58.1±3.7。Arsenic-added experimental group 2: A549 was 65.1±2.9, XWLC-05 was 58.1±3.7.

序列表sequence listing

<110> 昆明医科大学<110> Kunming Medical University

<120> 一种抑制hsa_circ_0027479表达的siRNA及其应用<120> An siRNA for inhibiting the expression of hsa_circ_0027479 and its application

<141> 2019-12-24<141> 2019-12-24

<160> 5<160> 5

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 23<211> 23

<212> RNA<212> RNA

<213> 人工序列(rengongxulie)<213> Artificial sequence (rengongxulie)

<400> 1<400> 1

aaauucuugg gacccugaug cuc 23aaauucuugg gacccugaug cuc 23

<210> 2<210> 2

<211> 19<211> 19

<212> RNA<212> RNA

<213> 人工序列(rengongxulie)<213> Artificial sequence (rengongxulie)

<400> 2<400> 2

aaauucuugg gacccugau 19aaauucuugg gacccugau 19

<210> 3<210> 3

<211> 19<211> 19

<212> RNA<212> RNA

<213> 人工序列(rengongxulie)<213> Artificial sequence (rengongxulie)

<400> 3<400> 3

ucuugggacc cugaugcuc 19ucuugggacc cugaugcuc 19

<210> 4<210> 4

<211> 23<211> 23

<212> DNA<212> DNA

<213> 人工序列(rengongxulie)<213> Artificial sequence (rengongxulie)

<400> 4<400> 4

atgagaaaat tcttgggacc ctg 23atgagaaaat tcttgggacc ctg 23

<210> 5<210> 5

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人工序列(rengongxulie)<213> Artificial sequence (rengongxulie)

<400> 5<400> 5

gctctccatg cttgaccaca 20gctctccatg cttgaccaca 20

Claims (3)

1. The application of nucleotide for inhibiting expression of human hsa _ circ _0027479 circular RNA in preparing medicine for treating lung cancer is characterized in that the sequence of the nucleotide is AAAUUCUUGGGACCCUGAU and complementary sequence thereof or UCUUGGGACCCUGAUGCUC and complementary sequence thereof.

2. Use of a pharmaceutical composition comprising the nucleotide of claim 1 and sodium arsenite in the preparation of a medicament for the treatment of lung cancer.

3. The use of the pharmaceutical composition of claim 2 in the manufacture of a medicament for the treatment of lung cancer, wherein the concentration of sodium arsenite in the composition is 40 to 60 uM for the treatment of lung cancer.