CN113373151B - 环状RNAhsa_circ_0008399的应用 - Google Patents
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Abstract
本发明属于环状RNA技术研究领域,具体公开了环状RNA hsa_circ_0008399的应用。环状RNA hsa_circ_0008399,其核苷酸序列如SEQ ID NO.1所示。膀胱癌分子标志物,所述分子标志物为所述的环状RNA hsa_circ_0008399。顺铂抗性抑制剂,包括能敲低或敲除所述环状RNA hsa_circ_0008399基因表达的制剂。肿瘤疾病顺铂化疗敏感性提高药物,包括顺铂抗性抑制剂。有益效果是:针对肿瘤疾病提供了一种辅助治疗药物;尤其能够针对顺铂化疗中的抗药性起到较好的缓解作用。
Description
技术领域
本发明属于环状RNA技术研究领域,尤其涉及环状RNAhsa_circ_0008399的应用。
背景技术
环状RNA(circRNA)属于以共价闭合连续环为特征的非编码RNA转录物家族,circRNA是区别于线性RNA的一类新型RNA,它的3’端和5’端是链接在一起的,与线性转录物相比,它具有高度稳定性、物种保守性,对外界因素的变化更具抵抗力。1979年,通过电子显微镜首次在真核细胞中观察到circRNA,认为其是前体mRNA反向剪接的副产物,功能潜力很小。随着第二代RNA测序和生物信息学技术的发展,circRNA被国内外很多知名研究者证明其具有强大的生物学功能,包括促进癌症的增殖、血管生成、转移、抑制癌症的发生发展等。
膀胱癌是泌尿***最常见的恶性肿瘤之一,在全球肿瘤发病率中排名第9,在全球肿瘤相关死亡率中排名第13。在美国,2021年有83,730例患者被诊断出患有膀胱癌,17,200例膀胱癌患者死亡。临床上,以顺铂为基础的化疗是肌肉浸润性和转移性膀胱癌的一线治疗方法。尽管60-70%的患者初始治疗时对顺铂有反应,但大多数患者会在短时间内产生耐药性。由于顺铂化疗耐药,膀胱癌患者的无进展生存期和总生存期并不令人满意。因此,确定介导顺铂化疗敏感性的关键靶点对于准确提供膀胱癌诊断和治疗至关重要。
以RNA干扰技术(RNAi)为基础的治疗方法对开发展新型有效的肿瘤治疗药物、减少药物不良反应有重要意义。RNAi分子通过不同细胞途径特异性靶向治疗各种肿瘤,沉默致癌基因,可有效抑制恶性肿瘤的增长。
发明内容
针对上述问题,本发明提供环状RNAhsa_circ_0008399的应用,为肿瘤治疗提供了一种新的药物,同时针对顺铂治疗也提供一种降低抗性的方法和药物。
为了解决上述问题,本发明采用如下技术方案:
环状RNA hsa_circ_0008399,其核苷酸序列如SEQ ID NO.1所示;和/或,其结构是由SEQ ID NO.1所示核苷酸序列转录后经剪接形成的3’端和5’端相连的环状RNA结构。
膀胱癌分子标志物,所述分子标志物为所述的环状RNA hsa_circ_0008399;和/或包括能特异性识别环状RNA hsa_circ_0008399的物质。
所述环状RNA hsa_circ_0008399在制备***药物中的应用。
能敲低或敲除所述环状RNA hsa_circ_0008399基因表达的制剂在制备***疾病药物中的应用。
***疾病的药物,包括能敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂,在一些情况中,所述制剂为siRNA或shRNA,更优选的,所述肿瘤疾病包括膀胱癌。
抑制肿瘤细胞增殖或促进肿瘤细胞凋亡的药物,包括能敲低或敲除所述环状RNAhsa_circ_0008399基因表达的制剂,优选的,所述制剂为siRNA或shRNA,更优选的,所述肿瘤细胞包括膀胱癌细胞。
敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂在制备膀胱癌顺铂抗性抑制剂中的应用。
敲低或敲除环状RNA hsa_circ_0008399基因表达制剂在制备提高膀胱癌顺铂化疗敏感性药物中的应用。
顺铂抗性抑制剂,包括能敲低或敲除所述环状RNA hsa_circ_0008399基因表达的制剂,在一些情况中,所述制剂为siRNA或shRNA。
肿瘤疾病顺铂化疗敏感性提高药物,包括顺铂抗性抑制剂,在一些情况中,所述肿瘤疾病包括膀胱癌。
能敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂作为顺铂抗性抑制剂的应用,在一些情况中,所述制剂为siRNA或shRNA。
顺铂化疗耐药性检测试剂盒,包括能扩增所述环状RNA hsa_circ_0008399的引物;在一些情况中,所述引物序列为:
前引物:GGCTATGGGAGTGGCAGGTATT,
后引物:TCGTCGGTGTTAAAGTTGAGCC。
所述环状RNA hsa_circ_0008399过表达载体制备方法,包括下述步骤:
A.以cDNA为模版进行PCR合成带有载体环化位点的circ0008399的序列;
B.重组,将带有载体环化位点的circ0008399的序列、线性载体、2X SoSoo Mix混合,***序列与线性载体摩尔比在1:1-10:1之间;
C.转化,
取100ul冰浴上融化的感受态细胞,加入上述步骤A产物,混匀,冷却,升温,转移至冰浴中,
向离心管中加入无菌液体LB培养基,混匀,复苏,
吸取复苏液体涂布在含有相应抗生素的LB固体培养基上,将平板倒置在培养箱过夜培养。
环状RNA hsa_circ_0008399过表达载体制备方法,
步骤B重组反映的条件为45-54℃,10-20min;
PCR合成带有载体环化位点的circ0008399的序列步骤为:体积比为22:1:1:1:25的cDNA、前引物GGCTATGGGAGTGGCAGGTATT、后引物TCGTCGGTGTTAAAGTTGAGCC、ROX Dye1、SYBR Color qPCR Master Mix酶混合,
a.95-96℃反应3-5mins,
b.94-95℃反应10-15s,
c.55-56℃反应30s,
将b、c步骤进行30-50个循环;
环状RNA hsa_circ_0008399过表达载体制备方法,
模版cDNA由原料RNA经过逆转录制备而得;
原料RNA提取步骤为:
细胞中加入胰蛋白酶进行消化,将细胞溶液离心,收集细胞沉淀,加入TRIzonReagent,混匀,
振荡使样品裂解,
加入氯仿,震荡,
离心,取上层水相,
加入等体积的70%乙醇,混匀,
将上述液体转移至装有收集管的吸附柱中,离心,去除滤液,
向吸附柱中加入Buffer RW1,离心,去除滤液,
向吸附柱中加入Buffer RW2,离心,去除滤液,
向吸附柱中加入无RNase水,离心,收集RNA溶液。
本发明的有益效果是:
针对肿瘤疾病提供了一种辅助治疗药物;尤其能够针对顺铂化疗中的抗药性起到较好的缓解作用。
附图说明
图1为制备环状RNA hsa_circ_0008399结果测试图;
图2为实施例1结果图;
图3为实施例2结果图;
图4为实施例3结果图;
图5为实施例4结果图;
图6为实施例5结果图。
具体实施方式
下面对本发明做进一步说明:
环状RNA hsa_circ_0008399,其核苷酸序列如SEQ ID NO.1所示;和/或,其结构是由SEQ ID NO.1所示核苷酸序列转录后经剪接形成的3’端和5’端相连的环状RNA结构。
环状RNA hsa_circ_0008399的一种具体来源为:将RBM3基因的2~6号外显子环化形成所述环状RNA。实验室可以通过基因工程技术构建cirRNA过表达载体进行合成circRNA,但这个载体中涵盖的序列不是纯的circRNA序列,其中还包括载体必要的基本原件序列。该载体可以通过转染细胞进行cirRNA的细胞水平过表达。
膀胱癌分子标志物,所述分子标志物为环状RNA hsa_circ_0008399;和/或包括能特异性识别环状RNA hsa_circ_0008399的物质,该物质能够使得环状RNA hsa_circ_0008399的存在(或基因较高表达)得到可测量性显示,从而实现对相关分子的标识。
环状RNA hsa_circ_0008399在制备***药物中的应用。
***疾病的药物,包括能敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂,优选的,所述制剂为siRNA或shRNA,更优选的,所述肿瘤疾病包括膀胱癌。
抑制肿瘤细胞增殖或促进肿瘤细胞凋亡的药物,包括能敲低或敲除所述环状RNAhsa_circ_0008399基因表达的制剂,优选的,所述制剂为siRNA,更优选的,所述肿瘤细胞包括膀胱癌细胞。
顺铂抗性抑制剂,包括能敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂,优选的,所述制剂为siRNA或shRNA。其中顺铂抗性抑制剂为降低人体对顺铂药物抗药性的制剂。
敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂在制备膀胱癌顺铂抗性抑制剂中的应用。
敲低或敲除环状RNA hsa_circ_0008399基因表达制剂在制备提高膀胱癌顺铂化疗敏感性药物中的应用。
肿瘤疾病顺铂化疗敏感性提高药物,包括顺铂抗性抑制剂,优选的,所述肿瘤疾病包括膀胱癌。顺铂化疗敏感性主要表现为人体对顺铂药物的敏感性,也一定程度表现为顺铂药物的作用效果。
能敲低或敲除所述环状RNA hsa_circ_0008399基因表达的制剂作为顺铂抗性抑制剂的应用,优选的,所述制剂为siRNA或shRNA。
顺铂化疗耐药性检测试剂盒,包括能扩增所述环状RNA hsa_circ_0008399的引物;本发明中,该引物主要能够较好的对过表达环状RNA hsa_circ_0008399进行标志,使得过表达环状RNA得到更好的标记,提高其可检测性;所述引物序列为:
前引物:GGCTATGGGAGTGGCAGGTATT,
后引物:TCGTCGGTGTTAAAGTTGAGCC。
膀胱癌患者在顺铂化疗过程中容易形成化疗抵抗,导致患者对顺铂化疗不敏感,治疗效果不理想。发现一种新型环状RNA,hsa_circ_0008399, 它在膀胱癌患者中高表达,且与患者的生存率呈负相关。降低膀胱癌中hsa_circ_0008399的表达水平后可以增加顺铂化疗敏感性,为临床膀胱癌顺铂化疗耐药患者提供新的治疗策略。
本发明中的siRNA、shRNA均以能够实现敲低和敲除RNA hsa_circ_0008399基因表达为基础,满足前序条件的落入本发明的范围内;其他不能满足敲低和敲除RNA hsa_circ_0008399基因表达的siRNA、shRNA,但经过简单常规改造满足前序条件的,也应当在本发明范围内。
siRNA:即小干扰RNA,是一种双端片段双链RNA分子,能够以同源互补序列的RNA为靶标降低特定的RNA。
shRNA:即短发卡RNA,可以利用基因工程技术克隆到载体上,进行细胞转染后,其可以表达短的干扰RNA(siRNA)发挥作用,我们可以根据具体RNA靶标设计shRNA序列将其克隆到载体上。
将膀胱癌细胞系种植于6孔板,次日其密度达60-80%作用时进行提取RNA步骤,使用Ultraure RNA Kit试剂盒(CWBIO,China)。将提取得到的RNA进行逆转录得到cDNA,使用HiScript III RT SuperMix试剂盒(Vazyme,China),再用SYBR Green Master Mix试剂盒(Vazyme,China)进行实时定量PCR(qRT-PCR),qRT-PCR分析使用Step OnePlus Real TimePCR System (Applied Biosystems,USA)进行,将其产物进行sanger一代测序得到此图。长箭头表示circ0008399的环化位点。图1说明circ0008399由RBM3基因的2号到6号外显子环化形成。
列举一些具体的步骤作为补充说明,其对现有的制备技术也作出了补充完善:
(一)Ultraure RNA Kit试剂盒(CWBIO,China)进行RNA提取方法:
细胞培养液弃去,PBS清洗一遍,加入1ml胰蛋白酶进行消化,将细胞溶液转移至无RNase的离心管中,300g离心5min,收集细胞沉淀,仔细吸除所有上清,加入TRIzon Reagent1ml混匀;
将样品用涡旋振荡器振荡10s,促使样品充***解。室温放置5 mins,使蛋白核酸复合物完全分离;
样品中加入200ul氯仿,盖好管盖,剧烈震荡15s,室温放置2mins
4度,12000rpm 离心10 mins,将上层水相移至一个新的无RNase的离心管中;
向样品中加入等体积的70%乙醇,颠倒混匀;
将上述液体完全加入至装有收集管的吸附柱中(RM),12000rpm 离心20s,倒掉滤液;
再向吸附柱中加入700ul Buffer RW1,12000rpm 离心20s,倒掉滤液;
向吸附柱中加入500ul Buffer RW2,12000rpm离心20s,倒掉滤液;
重复步骤8;
12000rpm离心2mins,倒掉滤液,将吸附柱置于室温数分钟,彻底晾干;
将吸附柱置于一个新的无RNase的离心管,向吸附柱中间位置加入30ul的无RNase水,室温放置1min,12000rpm离心1min,收集RNA溶液,-80度保存RNA,防止降解。
(二)逆转录步骤:
逆转录体系10ul:500ngRNA+2ul逆转录酶+补无RNase水至10ul;
逆转录条件:37度,15mins;85度,5s;4度,保存样品。
(三)实时定量PCR(qRT-PCR)步骤:
实时定量PCR体系10ul:4.4ul的cDNA+0.2ul前引物+0.2ul后引物+0.2ulROX Dye1+5ul SYBR Color qPCR Master Mix酶;
条件:①95度3mins;②95度10s;③55度30s;将②③步骤进行40个循环。
(四)构建circ0008399过表达载体序列:
1.合成带有载体环化位点的circ0008399的引物如下:
前引物:CTAATGACTTTTTTTTTATACTTCAGGACTTGAACTGCCATGTCCT
后引物:GCCTAATTCTTTTCCTTGCTTCTTACCTATATTATGTGCATGTCTCAT。
2.以cDNA为模板进行普通PCR合成带有载体环化位点的circ0008399的序列;
3.将上述序列利用同源重组的基因工程构建技术合成至pcDNA3.1(+) CircRNAMini Vector载体上,具体步骤入下:
①10ul体系:***circ0008399的带头载体环化位点的序列n ul+线性载体 n ul+2X SoSoo Mix 5ul+补ddH2O至10ul。***序列:线性载体摩尔比在1:1至10:1之间;
②重组反应条件:50度反应15mins;
③转化:取100ul冰浴上融化的感受态细胞,加入上述产物,轻轻混匀,冰上静止25mins;42度水浴热激45s,迅速转移至冰浴中,静止2mins;向离心管中加入500ul的不含抗生素的无菌液体LB培养基,混匀后37度,200rpm复苏1h;吸取不同体积的复苏液体均匀涂布在含有相应抗生素的LB固体培养基上,将平板倒置在37度培养箱过夜培养;
④次日挑单个菌进行扩大培养,再进行质粒提取进行测序,观察circ0008399序列是否成功***载体上。
实施例1
将90对膀胱癌标本及其对应的邻近的膀胱黏膜正常上皮标本各自提取RNA,再进行逆转录后实施实时定量PCR(同上),检测膀胱癌标本相对于癌旁正常膀胱黏膜中的circ0008399的表达量,可以看出circ0008399在膀胱癌标本中高表达。图2说明circ0008399在膀胱癌临床组织样本中相对于癌旁的正常组织的表达水平是升高的。
实施例2
将实施例2进行的90例膀胱癌标本检测出的circ0008399表达量分为两组(高、低表达),根据临床患者术后随访相关资料,根据每例患者的生存时间(定义为从手术日期到死亡日期或幸存者最后一次随访日期)作图得到此生存曲线,可以看出circ0008399越高,生存时间越短。图3说明circ0008399的表达量与患者的生存期呈负相关。
实施例3
将两种膀胱癌细胞EJ和T24T种于6孔板,次日其密度约60-80%时进行转染小干扰RNA进行敲低circ0008399分子,转染试剂使用RNAiMAX(Invitrogen,USA),步骤按照说明书进行(无血清培养基:siRNA:转染试剂比例约100ul:2ug:10ul),24h后收取细胞进行RNA提取,逆转录,实时定量PCR检测circ0008399敲低效率。图4验证小干扰RNA敲低circ0008399的效率。
实施例4
将两种膀胱癌细胞EJ和T24T种于6孔板,次日其密度约60-80%时进行转染小干扰RNA进行敲低circ0008399分子,转染试剂使用同上,24h后收取细胞用流式细胞仪分别检测两种细胞凋亡程度,使用的凋亡试剂盒为FITC Annexin V Apoptosis Detection Kit(BD,USA),流式分析仪型号为BD LSRFortessaTMX-20 Special Order Product(BD,USA),可以观察到敲低circ0008399可以促进膀胱癌细胞发生凋亡。图5说明敲低circ0008399可以促进膀胱癌细胞发生凋亡。
实施例5
将两种膀胱癌细胞EJ和T24T种于6孔板,次日其密度约60-80%时进行转染小干扰RNA进行敲低circ0008399分子,再分别向对照组细胞和处理组细胞中加入DMSO和顺铂(浓度为30μM),24h后进行流式细胞凋亡检测(试剂步骤同上),可以观察到敲低circ0008399可以促进膀胱癌细胞对顺铂治疗的敏感性。图6说明敲低circ0008399可以提高膀胱癌细胞的顺铂化疗敏感性,降低了顺铂抗性。
本领域的技术人员可以明确,在不脱离本发明的总体精神以及构思的情形下,可以做出对于以上实施例的各种变型。其均落入本发明的保护范围之内。本发明的保护方案以本发明所附的权利要求书为准。
序列表
<110> 华中科技大学同济医学院附属协和医院
<120> 环状RNAhascirc0008399及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 510
<212> DNA
<213> 人(Homo)
<400> 2
gacttgaact gccatgtcct ctgaagaagg aaagctcttc gtgggagggc tcaactttaa 60
caccgacgag caggcactgg aagaccactt cagcagtttc ggacctatct ctgaggtggt 120
cgttgtcaag gaccgggaga ctcagcggtc caggggtttt ggtttcatca ccttcaccaa 180
cccagagcat gcttcagttg ccatgagagc catgaacgga gagtctctgg atggtcgtca 240
gatccgtgtg gatcatgcag gcaagtctgc tcggggaacc agaggaggtg gctttggggc 300
ccatgggcgt ggtcgcagct actctagagg tggtggggac cagggctatg ggagtggcag 360
gtattatgac agtcgacctg gagggtatgg atatggatat ggacgttcca gagactataa 420
tggcagaaac cagggtggtt atgaccgcta ctcaggagga aattacagag acaattatga 480
caactgaaat gagacatgca cataatatag 510
Claims (6)
1.敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂在制备治疗膀胱癌药物中的应用;其中,所述敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂为siRNA或shRNA。
2.敲低或敲除环状RNA hsa_circ_0008399基因表达制剂在制备促进膀胱癌细胞凋亡的药物中的应用;其中,所述敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂为siRNA或shRNA。
3.敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂在制备膀胱癌顺铂抗性抑制剂中的应用;其中,所述敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂为siRNA或shRNA。
4.敲低或敲除环状RNA hsa_circ_0008399基因表达制剂在制备提高膀胱癌顺铂化疗敏感性药物中的应用;其中,所述敲低或敲除环状RNA hsa_circ_0008399基因表达的制剂为siRNA或shRNA。
5.能扩增环状RNA hsa_circ_0008399的引物在制备膀胱癌顺铂化疗耐药性检测试剂中的应用。
6.根据权利要求5所述的应用,其中,所述能扩增环状RNA hsa_circ_0008399的引物的序列为:
前引物:GGCTATGGGAGTGGCAGGTATT,
后引物:TCGTCGGTGTTAAAGTTGAGCC。
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