CN106834292A - 一种与卵巢癌相关的环状非编码RNA‑PUM1的shRNA和应用 - Google Patents
一种与卵巢癌相关的环状非编码RNA‑PUM1的shRNA和应用 Download PDFInfo
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Abstract
本发明属于肿瘤分子生物学技术领域,具体涉及一种与卵巢癌相关的环状RNA PUM1的shRNA和应用。针对circPUM1基因设计的shRNA,具体为shRNA1或shRNA2;所述的环状RNA PUM1可作为卵巢癌诊断和治疗的重要靶点。所述的与卵巢癌相关的环状RNA PUM1的shRNA可以用于制备抗卵巢癌药物。
Description
技术领域
本发明属于肿瘤分子生物学技术领域,具体涉及一种与卵巢癌相关的环状RNAPUM1的shRNA和应用。
技术背景
卵巢癌是女性生殖系统死亡率最高的恶性疾病,发病率居妇科肿瘤第三位,其较高的死亡率由于大多数患者确诊时已为晚期,手术治疗后常发生复发和转移,远期预后差。因此,探索卵巢癌发生发展的分子生物学机制对于卵巢癌的早期诊断、预后判断及个体化靶向治疗具有重要意义。
近年来随着高通量测序等技术的不断进展,环状RNA(circRNA)越来越多地进入研究者的视野,环状RNA是一种高度保守的闭合的非编码RNA,富含微小RNA(miRNA)结合位点,能够吸附并解除miRNA对靶基因的抑制作用,调控肿瘤发生、发展通路中癌基因或抑癌基因的表达,影响肿瘤细胞的增殖、凋亡、侵袭及转移能力。PUM(Homo sapiens pumilio)基因已被证实能够影响细胞周期、增殖与分化、调控癌症相关基因的表达,在肿瘤发生和进展中发挥着重要作用,但目前并没有关于PUM1基因转录形成的circPUM1结构在卵巢癌组织中表达情况及作用机制的相关报道。
发明内容
针对上述问题,本发明提供PUM1基因在卵巢癌的诊断、治疗及预后评估的应用,尤其是一种与卵巢癌相关的环状RNA PUM1的shRNA和应用。该环状RNA与癌细胞的增殖能力相关,针对其设计的shRNA可应用于抗肿瘤的药物的制备。
为了实现上述目的,本发明提供一种与卵巢癌相关的circPUM1基因,其DNA序列如SEQ.ID. NO.1所示;针对circPUM1基因设计的shRNA,具体为shRNA1或shRNA2;针对circPUM1设计shRNA1,所述的shRNA1的top strand和bottom strand的核苷酸序列分别SEQ.ID.NO.2和SEQ.ID.NO.3所示;针对circPUM1设计的 shRNA2,所述的shRNA的topstrand和bottom strand的核苷酸序列分别SEQ.ID.NO.4和SEQ.ID.NO.5所示。
SEQ.ID.NO.2:GATTCTCAACAACAGGGCCCAAGGGATTCAAGAGATCCCTTGG
GCCCTGTTGTTGAGAATTTTTTT。
SEQ.ID.NO.3:AAAAAAATTCTCAACAACAGGGCCCAAGGGATCTCTTGAATCCCT
TGGGCCCTGTTGTTGAGAATC。
SEQ.ID.NO.4:GAACAACAGGGCCCAAGGGATGCAGATTCAAGAGATCTGCAT
CCCTTGGGCCCTGTTGTTTTTTTT。
SEQ.ID.NO.5:AAAAAAAACAACAGGGCCCAAGGGATGCAGATCTCTTGAATC
TGCATCCCTTGGGCCCTGTTGTTC。
所述的环状RNA PUM1可作为卵巢癌诊断和治疗的重要靶点。
所述的与卵巢癌相关的环状RNA PUM1的shRNA可以用于制备抗卵巢癌药物。
本发明的有益效果。
本发明首次发现circPUM1在卵巢癌组织中高表达,通过转染shRNA能够下调circPUM1的表达,显著抑制癌细胞活性,为卵巢癌相关circRNA的临床治疗和科学研究提供了基础。
附图说明
图1利用qRT-PCR检测circPUM1在卵巢癌及正常组织中的表达情况。
图2 circPUM1 shRNA转染后,卵巢癌细胞circPUM1表达情况的QRT-PCR。
图3 利用MTT法检测干扰circPUM1对卵巢癌细胞活性的影响。
图4检测干扰circPUM1对卵巢癌细胞凋亡的影响。
具体实施方式
下面结合具体的实施例对本发明作进一步的说明,以下实施例将有助于对本发明的了解,但这些实施例仅为了对本发明加以说明,本发明并不限于这些内容。在实施例中未作特殊说明的操作方法均为本技术领域常规操作方法。
实施例1。
一、 circPUM1在卵巢癌组织、正常卵巢组织中的表达情况。
1.标本采集。
在患者知情的情况下,于术中采集卵巢癌、正常卵巢组织标本,生理盐水清洗后,保存于液氮或-80℃超低温冰箱中,备用;组织标本于2014年6月-2016年6月,在中国医科大学附属第一医院手术室,由陈医生采集。
2.引物设计。
circPUM1 RT-PCR引物序列如SEQ.ID.NO.6-7所示。
上游引物(SEQ.ID.NO.6):AGTGTACTGGGAGGAGG。
下游引物(SEQ.ID.NO.7):ATAAGTCCGTGCGTCC。
3.应用qRT-PCR方法分别检测circPUM1在卵巢癌、正常卵巢组织的表达量。
3.1总RNA抽提。
使用RNA抽提试剂RNAiso Plus试剂(宝生物工程有限公司)从正常卵巢组织、卵巢癌组织分离总RNA。将匀浆液转移至离心管中,室温静置5分钟;4℃条件下12,000r离心处理5分钟;小心吸取上清液,移入新的离心管中;加入RNAiso Plus的1/5体积量的氯仿,剧烈振荡15秒,待溶液充分乳化后,室温静置5分钟;4℃条件下,12,000r离心处理15分钟,吸取上清液转移至另一个新的离心管中,向上清液中加入等体积的异丙醇,上下颠倒离心管充分混匀后,在30℃下静置10分钟;4℃条件下12,000r离心处理10分钟,弃去上清,沿管壁加入lml的75%的乙醇,上下颠倒洗涤离心管管壁,4℃条件下12,000r离心处理5分钟后,弃去乙醇,室温干燥沉淀2-5分钟,加入适量的RNase-free水溶解沉淀后,用UV-2800A型紫外可见分光光度计测定RNA浓度和纯度(定量RNA浓度1ug/ul, OD260/OD280 1.8-2.0之间表示RNA纯度较高)。
3.2逆转录合成cDNA。
逆转录试剂盒High Capacity cDNA Reverse Transcription Kits购自Invitrogen公司,定量PCR试剂盒Power SYBR Green PCR Master Mix购自Invitrogen公司。
采用 GoScript 反转录系统(A5000、A5001),按照以下操作步骤进行反转录得到的 cDNA。
第一步:取一定量模板 RNA 加入引物。
RNA ( 1µg/ ul) 5μl。
Random Primers (0.5 µg / ul) 1 μl。
Oligo(dT)15 Primer (0.5 µg/ ul) 1 μl。
Nuclease-Free Water (加至 10 μl) 3μl。
第二步:将模板 RNA 与反转录引物( Random Primers和Oligo(dT)15 Primer)的混合物进行 70℃、5 min 预变性,完成后取出置于冰上。
第三步:配制 RT -Mix,向每个样品管加入 10 μl。
组分 反转录混合液 终浓度。
Nuclease-Free Water 1.6 μl。
GoScript™ 5X Reaction Buffer 4 μl 1X。
MgCl2 (25 mM) 2 μl 2.5mM。
PCR Nucleotide Mix 1 μl 0.5mM。
Recombinant RNasin® Ribonuclease Inhibitor 0.4 μl 20units。
GoScript™ Reverse Transcriptase 1 μl。
第四步:设置反转录程序,包括退火、延伸、逆转录酶失活三步(退火25℃ 5min,延伸42℃ 60 min,失活70℃ 15 min,4℃ + ∞。
程序完成后得到 cDNA。
3.3 Real-time PCR。
(1)按下列配置PCR反应混合液(反应液配置可在室温进行),并分至各反应管,然后加入2ul模板。
组分 体积(20ul反应体系) 终浓度。
Nuclease-Free Water 7 μl。
上游引物(10 uM) 0.4ul 0.2uM。
下游引物(10 uM) 0.4ul 0.2uM。
GoTaq®qPCR Master Mix,2X 10ul 1X。
CXR 100X 0.2ul 1X。
(2)采用ABI PRISM®7500 Real-Time PCR System,两步法进行PCR标准扩增程序。
(3)导出数据,Realtime PCR结果用2-△△CT法进行分析。用Graphad Prism软件分别绘制出卵巢癌和正常卵巢组织癌的图表,结果如图1表示相比正常组织,circPUM1(circ_100133)在卵巢癌组织中高表达(p<0.05)。
二、体外细胞功能实验。
1.细胞培养与转染。
在含10%胎牛血清(FBS)DMEM(购自HyClone公司)培养液中,加入100 U/mL青霉素和链霉素在饱和湿度,5%二氧化碳,37℃孵箱中,按照常规培养方法进行细胞传代培养卵巢癌细胞系 A2780(购自中科院细胞库)。根据制造商的说明书使用Lipofectamine 2000将circPUM1 shRNA转入细胞中,进行培养。shRNA干扰片段序列由上海汉恒生物科技有限公司合成,circPUM1基因和管家基因18S引物由上海生工生物工程技术服务有限公司合成。
2.干扰效率检测:转染效率结果采用QRT-PCR检测。
QRT-PCR结果见图2,结果显示卵巢癌细胞系circPUM1 shRNA转染降低了circPUM1表达水平(P <0.05)。
3.MTT细胞增殖活性测定。
将细胞以3,000个细胞/孔的密度接种在96孔板中。瞬时转染后分别孵育0h、24h、48h、和72h;然后加入20ul MTT(5毫克/毫升)在37℃ 孵育4小时;弃掉上清液,再加入150ulDMSO,使用分光光度计在490nm下测OD值;每个实验设置三个复孔。
结果见图3,结果显示,与对照组和空载体转染组相比,转染后48h、72h的卵巢癌细胞活性明显受到抑制(P < 0.05);即shRNA1、2处理后,circPUM1表达下调,抑制了卵巢癌细胞的生长增殖活性。
4.细胞凋亡实验。
实验前将细胞在6cm盘培养,加入处理因素(circPUM1 RNA干扰质粒)转染48小时后,进行实验。细胞用不含EDTA胰蛋白酶消化,1500r离心处理5分钟后收集细胞,预冷的PBS洗涤两次,离心后,收集细胞约5×10 5个,加入1×Binding Buffer,重悬细胞100ul,加入5μL Annexin V-FITC和5uL PI Staining Solution,轻轻混匀,室温避光孵育10分钟,再加入400μL 1×Binding Buffer,轻轻混匀;样品在1小时内流式细胞仪检测。
结果显示circPUM1干扰后的卵巢癌细胞系凋亡实验结果见图4;其中图4-1表示利用shRNA1沉默circPUM1后,卵巢癌细胞A2780凋亡率显著增加(P <0.05);图4-2表示利用shRNA2沉默circPUM1后,卵巢癌细胞A2780凋亡率显著增加(P <0.05)。
因此,circPUM1可以作为卵巢癌的治疗靶点,采用特异性的shRNA、PUM1特异性的抗体或者小分子药物,沉默或者降低肿瘤组织中的circPUM1的表达,可用于卵巢癌的临床治疗。
序列表
<110>中国医科大学附属第一医院
<120>一种与卵巢癌相关的环状非编码RNA-PUM1的shRNA和应用
<160>7
<210>1
<211>438
<212>DNA
<213>环状RNA PUM1的核苷酸序列
<400>1
ggcccaaggg atgcagacag tgatgaaaac gacaaaggtg aaaagaagaa caagggtacg 60
tttgatggag ataagctagg agatttgaag gaggagggtg atgtgatgga caagaccaat 120
ggtttaccag tgcagaatgg gattgatgca gacgtcaaag attttagccg tacccctggt 180
aattgccaga actctgctaa tgaagtggat cttctgggtc caaaccagaa tggttctgag 240
ggcttagccc agctgaccag caccaatggt gccaagcctg tggaggattt ctccaacatg 300
gagtcccaga gtgtcccctt ggaccccatg gaacatgtgg gcatggagcc tcttcagttt 360
gattattcag gcacgcaggt acctgtggac tcagcagcag caactgtggg actttttgac 420
tacaattctc aacaacag 438
<210>2
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<212>DNA
<213>人工序列
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gattctcaac aacagggccc aagggattca agagatccct tgggccctgt tgttgagaat tttttt 66
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aaaaaaattc tcaacaacag ggcccaaggg atctcttgaa tcccttgggc cctgttgttg agaatc 66
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gaacaacagg gcccaaggga tgcagattca agagatctgc atcccttggg ccctgttgtt tttttt 66
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Claims (4)
1.一种与卵巢癌相关的环状非编码RNA-PUM1的shRNA,其特征在于,所述的shRNA为shRNA1或shRNA2;针对circPUM1设计shRNA1,所述的shRNA的top strand和bottom strand的核苷酸序列分别SEQ.ID.NO.2和SEQ.ID.NO.3所示;针对circPUM1设计的 siRNA2,所述的shRNA的top strand和bottom strand的核苷酸序列分别SEQ.ID.NO.4和SEQ.ID.NO.5所示。
2.如权利要求1所述的环状RNA PUM1的shRNA,其特征在于,应用qRT-PCR方法分别检测RNA PUM1在卵巢癌、正常卵巢组织的表达量。
3.如权利要求2所述的环状RNA PUM1的shRNA,其特征在于,所述的qRT-PCR方法检测所用的引物核苷酸序列分别为:上游引物:AGTGTACTGGGAGGAGG;下游引物:ATAAGTCCGTGCGTCC。
4.如权利要求1所述的与卵巢癌相关的环状RNA PUM1的shRNA用于制备抗卵巢癌药物。
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CN112921086A (zh) * | 2019-12-06 | 2021-06-08 | 广州医科大学附属第三医院(广州重症孕产妇救治中心、广州柔济医院) | Circ-CRIM1作为卵巢癌诊断标志物及其应用 |
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CN113893261A (zh) * | 2021-06-22 | 2022-01-07 | 中山大学肿瘤防治中心(中山大学附属肿瘤医院、中山大学肿瘤研究所) | circITGB6-ASO与铂类化疗药物联合使用在制备治疗卵巢癌的药物中的应用 |
CN113893261B (zh) * | 2021-06-22 | 2023-01-13 | 中山大学肿瘤防治中心(中山大学附属肿瘤医院、中山大学肿瘤研究所) | circITGB6-ASO与铂类化疗药物联合使用在制备治疗卵巢癌的药物中的应用 |
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