CN113862269A - tsRNA分子及其用途 - Google Patents
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Abstract
本发明公开了一种tsRNA分子及其用途。该新型分子与多发性骨髓瘤有关,命名为tRF‑1:29‑Thr‑TGT‑2,二代测序及qPCR验证确定了其在复发难治多发性骨髓瘤中表达下调,该分子在以往对于多发性骨髓瘤、其他类型肿瘤及非肿瘤的研究中均尚未被发现,因此具有较好的创新性。另一方面,ROC曲线显示了该分子对于复发难治患者的预测价值,生存分析提示tRF‑1:29‑Thr‑TGT‑2低表达的多发性骨髓患者预后不良,初步的细胞实验也验证了tRF‑1:29‑Thr‑TGT‑2在骨髓瘤细胞株中的作用机制,这一发现为临床诊断及预后分层提供了新的标记,并可能成为将来精准化治疗的新靶点,具有较好的临床应用价值。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及一种多发性骨髓瘤相关的tsRNA分子及其用途。
背景技术
多发性骨髓瘤(MM)是一种无法治愈的具有复发倾向的浆细胞肿瘤,是第二常见的血液系统恶性肿瘤,常表现为贫血、骨痛、感染、神经症状、淀粉样变等。尽管近年来如蛋白酶体抑制剂、CD38单克隆抗体等新药的出现显着改善了预后,但临床上我们仍经常面临多发性骨髓瘤的复发和耐药。因此,探索复发耐药的机制的是改善多发性骨髓瘤患者临床疗效和延长生存期的关键。
细胞遗传学异常、染色体易位、致癌基因的激活及骨髓微环境等多种因素与多发性骨髓瘤的复发难治相关。非编码RNA(ncRNA)是一类不编码蛋白质的RNA,过去的研究已经发现了非编码RNA在各种生物过程中的重要作用,它们在人类疾病中的基本作用已经得到了很好的证实。转移RNA(tRNAs)是在蛋白质生物合成过程中传递氨基酸到核糖体的管家ncRNA。tRNA片段(tRNA derived fragments)是一种通过酶切tRNAs产生的小非编码RNA,并已被证明在多种生物进程中发挥类似microRNAs的关键调控作用。
tRNAs在其生命周期中经历了广泛的加工和一系列的化学修饰,在tRNA成熟过程中,根据前体、成熟tRNAs的裂解位置,这种tRNA衍生片段(tRNA derived fragments)可以大致分为tRF(tRNA related Fragments)和tiRNA(tRNA halves),又可以进一步细分为tRF-5、tRF-3、tRF-1、tRF-2和tiRNA。tRFs/tiRNAs广泛存在于各种组织细胞,表达水平具有高度组织、疾病和时序特异性,可参与基因沉默、蛋白质翻译、细胞应激和细胞分化等多种分子过程。虽然tRFs/tiRNAs的生物学功能及其复杂,有待进一步阐明,但目前对其功能的认识可归纳为:RNA沉默、翻译调控和表观遗传调控。
发明内容
本发明的首要目的是提供一种多发性骨髓瘤相关的tsRNA分子。tsRNA在多发性骨髓瘤病程中的相关研究少有报道。本发明利用高通量测序鉴定出一种在多发性骨髓瘤中异常表达的新型tsRNA分子,命名为tRF-1:29-Thr-TGT-2。
本发明的多发性骨髓瘤相关的tsRNA分子的cDNA序列为GGCTCCATAGCTCAGTGGTTAGAGCACTG。见SEQ ID NO.1所示。
本发明的第二个目的是提供检测上述的tsRNA分子的试剂的用途,用于制备复发难治多发性骨髓瘤诊断和/或预后制剂。主要是区分初诊多发性骨髓瘤患者和复发难治多发性骨髓瘤患者。
本发明后续再通过实时定量PCR(Real-time quantitative polymerase chainreaction,qRT-PCR)扩大样本验证,并通过生存分析探索了该分子的临床意义。
进一步地,所述的检测上述的tsRNA分子的试剂包括PCR试剂;引物序列优选如下:
F:5’-TCCATAGCTCAGTGGTTAGAG-3’,见SEQ ID NO.2所示。
R:5’-GTCCAGTTTTTTTTTTTTTTTCAGTG-3’,见SEQ ID NO.3所示。。
但本发明检测tsRNA分子的PCR试剂中的引物包括但不限于上述序列。
本发明的第三个目的是提供一种复发难治多发性骨髓瘤诊断和/或预后试剂盒,包括检测上述的tsRNA分子的试剂。
进一步地,所述的检测上述的tsRNA分子的试剂包括PCR试剂;
引物序列优选如下:
F:5’-TCCATAGCTCAGTGGTTAGAG-3’,
R:5’-GTCCAGTTTTTTTTTTTTTTTCAGTG-3’。
但本发明检测tsRNA分子的PCR试剂中的引物包括但不限于上述序列。
本发明通过过表达上述的tsRNA分子表达,多发性骨髓瘤细胞增殖速率、细胞侵袭性明显下降,对药物敏感性增加。
因此,本发明的第四个目的是提供过表达上述的tsRNA分子的试剂用于制备多发性骨髓瘤治疗、降低多发性骨髓瘤细胞增殖速率、降低多发性骨髓瘤细胞侵袭性或者增加对多发性骨髓瘤治疗药物敏感性制剂的用途。
进一步地,所述的过表达上述的tsRNA分子表达的试剂包括转染试剂。
转染试剂包括:RNA mimic;
RNA mimic序列优选:
sense 5’-GGCUCCAUAGCUCAGUGGUUAGAGCACUG-3’,见SEQ ID NO.4所示。
anti-sense 5’-GUGCUCUAACCACUGAGCUAUGGAGCCUU-3’,见SEQ ID NO.5所示。
采用GP-transfect-Mate转染试剂(吉玛基因)转染。
本发明的第五个目的是提供一种治疗多发性骨髓瘤(尤其是复发难治多发性骨髓瘤)、降低多发性骨髓瘤细胞增殖速率、降低多发性骨髓瘤细胞侵袭性或者增加对多发性骨髓瘤治疗药物敏感性的制剂,也就包括过表达上述的tsRNA分子的试剂。
过表达上述的tsRNA分子的试剂包括过表达转染试剂。
尽管多发性骨髓瘤疗效近年来得到了显著改善,但其仍不可治愈,肿瘤的耐药/复发极大的威胁着公众健康并带来长期的经济负担。在本发明中,发现了一个全新的分子tRF-1:29-Thr-TGT-2,并明确了该分子用于临床的预后意义。
新的tRFs/tiRNAs命名采用来自加州大学圣克鲁兹分校Lowe实验室的标准化tDR命名系统(tDRnamer),该命名规则分5个部分,依次表示前缀、位置、来源的tRNA、匹配的tRNA转录本和变异。二代测序及qPCR验证确定了tRF-1:29-Thr-TGT-2在复发难治多发性骨髓瘤中表达下调,该分子在以往对于多发性骨髓瘤、其他类型肿瘤及非肿瘤的研究中均尚未被报道,本发明首次发现了其用于复发难治性多发性骨髓瘤的诊断价值,具有较好的创新性。另一方面,生存分析提示tRF-1:29-Thr-TGT-2低表达的多发性骨髓患者预后不良,初步的机制探索也验证了tRF-1:29-Thr-TGT-2在多发性骨髓瘤发生发展中的作用。这一发现为临床疗效预测及预后分层提供了新的标记,并可能成为将来新的治疗靶点,因此也具有较好的应用价值。
总之,本发明发现了一个新的tsRNA分子,该分子在复发难治多发性骨髓瘤患者中表达下调,且与预后不良相关,还可能成为潜在的治疗靶点,以及为以后开发多发性骨髓瘤的治疗药物打下基础。这一发现具有良好的创新性及应用性。
以下结合附图和具体实施方式进一步详细说明本发明,而不会形成对本发明的限制。
附图说明
图1A为检测出的复发难治和初治多发性骨髓瘤患者之间的20多条差异表达tsRNA分子结果;图1B为对所有24位初诊患者(NDMM)、19位复发难治多发性骨髓瘤患者(RRMM)采用qPCR验证tRF-1:29-Thr-TGT-2的表达水平结果。
图2A为ROC曲线评价tRF-1:29-Thr-TGT-2用于区分复发难治多发性骨髓瘤与初治多发性骨髓瘤的敏感性和特异性结果;图2B为新诊断多发性骨髓瘤患者的生存曲线分析结果。
图3为转染RNA mimic后对tRF-1:29-Thr-TGT-2表达量的影响。
图4为上调tRF-1:29-Thr-TGT-2表达后U266细胞增殖速率变化结果。
图5为上调tRF-1:29-Thr-TGT-2G1/S转化结果。
图6为transwell迁移实验上调tRF-1:29-Thr-TGT-2表达后U266细胞侵袭性结果。
图7为采用5nM硼替佐米处理实验组(+RNA mimic)和对照组U266细胞24h,AnnexinV-FITC/PI双染法检测细胞凋亡结果。
具体实施方式
方法
1.临床样本
本发明共选取2016年3月-2018年3月期间来自中南大学湘雅三医院的24位初治以及19位复发难治多发性骨髓瘤患者。多发性骨髓瘤的诊断参考美国国家综合癌症网络(NCCN)定义的症状性多发性骨髓瘤的诊断标准,复发难治的评价标准也依据NCCN指南定义,在知情同意后采集患者骨髓,通过磁珠分选CD138阳性浆细胞,并搜集相关临床信息。
2.磁珠分选
(1)骨髓标本室温环境下1500转离心5分钟,移液枪收集上清到一个1.5ml无菌无酶的EP管中,加入与离心后采血管内剩余细胞同等量的PBS,吹打混匀;
(2)离心管中加入3.5ml红细胞裂解液,吹打混匀后,放入4℃冰箱避光10分钟后1500转离心5分钟;弃上清,加入3.5ml PBS吹打混匀,随后1500转离心5分钟,弃上清后,500μl磁珠分选缓冲液重悬细胞显微镜下计数单个核细胞;
(3)根据分选试剂盒说明书,每107细胞加入80μL MACS Buffer,25μL CD1388+细胞磁珠抗体,吹打混匀,放入4℃冰箱中避光孵育15min;
(4)细胞分选前,首先500μL Buffer两次润洗分选柱,之后将待分选细胞沿壁缓慢加入MS分选柱中,当分选柱中最后一滴细胞液留下去后,再加入500μL MACS Buffer冲洗两次;
(5)从分选器上取下分选柱,将分选柱置15ml离心管中,之后迅速用移液器吸取1mL MACS Buffer加到分选柱中,快速推动活塞,从分选柱流出的细胞即为分选出的CD138+浆细胞;
(6)将分选出的CD138+浆细胞放入离心机中,1500转离心5分钟,之后加入700μLTrizol,吹打混匀,置于无菌1.5ml冻存管内,最后冻存-80℃冰箱中,以备送检。
3.RNA提取
按照说明书提供流程,使用TRIzol(Invitrogen,美国)提取浆细胞RNA,具体流程为:
(1)将标本置于冰上,加入Trizol裂解5-10分钟,用枪头轻轻吹打后吸取液体放入EP管中;
(2)加入1/5体积的氯仿,上下混匀液体,4℃静置10-15分钟;
(3)4℃离心15分钟,离心后分成三层,从离心机轻轻拿出EP管,吸取上清,避免吸到下层沉淀,将液体置于新的EP管中;
(4)按0.5ml异丙醇/ml Trizol加入异丙醇,静置10min,然后4℃12000rpm离心10min,弃上清;
(5)按1ml 75%乙醇/ml Trizol加入75%乙醇,4℃离心5min,吸弃上清;
(6)将EP管再次放于离心机中瞬时离心,弃掉管壁残余液体。然后将EP管置于超净台中干燥5-10min。加入50ul DEPC处理水,震荡充分溶解沉淀
(7)采用NanoDrop ND-1000(NanoDrop,美国)测定RNA浓度和活性,然后采用甲醛变性琼脂糖凝胶电泳方法检测RNA的纯度及完整性。提取后的RNA保存在-80℃。
4.tsRNA测序
使用Agilent BioAnalyzer 2100对测序文库进行鉴定和绝对定量,对于Illumina二代测序仪器上的标准小RNA测序流程,测序类型为50bp单端。细胞质的tRNA序列从GtRNAdb下载,线粒体的tRNA序列用tRNAscan-SE软件预测。通过删除了预测内含子序列(如果存在)并向每个tRNA添加一个额外的3’末端“CCA”构建成熟的tRNA文库,在原始tRNA序列的两侧加入了40个核苷酸的侧翼基因组序列构建前体tRNA文库。
首先,对总RNA样品进行预处理以去除干扰小RNA-seq文库构建的RNA修饰。然后将文库变性为单链DNA分子,在Illumina流动池上捕获,原位扩增为测序簇,并按照制造商的说明在Illumina NextSeq 500系统上测序50个循环。图像分析和碱基调用由Solexapipeline v1.8(Off-Line Base Caller软件,v1.8)进行。FastQC检测测序质量。通过测序计数评估tsRNA的丰度,并将其标准化为每百万总比对读数(CPM)的计数。使用R软件limma包对数据进行归一化和后续分析,通过倍数变化以及p值定义差异表达的tRFs/tiRNAs,为显著上调和下调基因设定的阈值是倍数变化(FC,fold change)>1.5且p值<0.05。
5.定量RT-PCR验证
按照SuperScript III Reverse Transcriptase(Invitrogen,Grand Island,NY,USA)试剂盒说明将样品的RNA逆转录cDNA,采用ViiA 7Real-time PCR System(AppliedBiosystems)及2×PCR Master Mix来执行定量实时qRT-PCR(Arraystar)。具体为:每个反应混合物(10μL)包含2×Master Mix 5ul,PCR特异引物F 0.5μL,PCR特异引物R 0.5μL,2μLcDNA。将混合液加到384-PCR板对应的每个孔中,随后将384-PCR板置于Realtime PCR仪上进行PCR反应。反应条件设置为95℃温育10分钟,然后进行40个循环的95℃10秒和60℃1分钟。U6小核RNA作为内源参考转录物并将其标准化,ΔCt值反映tRFs/tiRNAs表达水平。各样品目的表达量以内参校正的计算结果。
tRF-1:29-Thr-TGT-2引物序列为:
F:5’-TCCATAGCTCAGTGGTTAGAG-3’,
R:5’-GTCCAGTTTTTTTTTTTTTTTCAGTG-3’。
U6引物序列为:
F:5’-GCTTCGGCAGCACATATACTAAAAT-3’见SEQ ID NO.6所示。,
R:5’-CGCTTCACGAATTTGCGTGTCAT-3’,见SEQ ID NO.7所示。
6.转染RNA mimic的具体步骤,
(1)细胞铺板。转染时细胞密度一般设置在60-80%。
(2)复合物的制备及转染。1)使用前将GP-transfect-Mate转染试剂(吉玛基因)放置于室温中,使用前轻轻混匀;2)在1.5ml无菌离心管中加入50μl无血清培养基或OPTI-MEM,并添加适量的转染试剂,用移液器轻轻混匀,室温静置5min;3)同时在另一1.5ml无菌离心管中加入50μl无血清培养基或OPTI-MEM,并添加适量的RNA oligo/DNA,用移液器轻轻混匀,室温静置5min。4)将GP-transfect-Mate-培养基混合物滴加至RNA oligo/DNA-培养基混合物中,用移液器轻轻混匀,室温静置15-20min后,立即转染。注:复合物尽量在60min内使用,并且GP-transfect-Mate-培养基混合物和RNA oligo/DNA-培养基混合物的混合次序非常重要,切勿颠倒。
RNA mimic序列:
sense 5’-GGCUCCAUAGCUCAGUGGUUAGAGCACUG-3’,
anti-sense 5’-GUGCUCUAACCACUGAGCUAUGGAGCCUU-3’。
(3)转染过程1)趁静置时,给24孔板换液,每孔换上400μl预热的新鲜培养基;2)将100μl转染混合物加入孔中,终体系为500μl。加完后将板轻轻晃动以使复合物均匀分布;3)37℃静置培养细胞,4-6h换成完全培养基。24-72h后qPCR检测转染效率。
7.细胞计数
(1)将计数板及盖片擦拭干净,并将盖片盖在计数板。
(2)将细胞悬液吹打均匀后吸出10ul,与0.4%台盼蓝染液1:1混合均匀后滴加10ul在盖片边缘,使悬液充满盖片和计数板之间,注意盖片下不要有气泡,也不能让悬液流入旁边槽中。
(3)计算板四大格细胞总数,压线细胞只计左侧和上方的。然后按公式计算细胞数目:细胞数/mL=四大格细胞总数/2×104。
8.Annexin V-FITC/PI染色细胞流式
(1)细胞收集。离心5分钟,用预冷1×PBS(4℃)重悬细胞一次,2000rpm离心5~10分钟,洗涤细胞;
(2)加入300μL的1×Binding Buffer悬浮细胞;
(3)Annexin V-FITC标记:加入5μL的Annexin V-FITC混匀后,避光,室温孵育15分钟;(注意:若仅进行PI染色检测细胞周期则无需进行此步)
(4)PI标记:上机前5分钟再加入5μL的PI染色。
(5)上机前,补加200μL的1×Binding Buffer。
9.transwell细胞迁移侵袭实验
(1)制备细胞悬液:细胞提前饥饿12h以去除血清影响,重悬,调整细胞密度至5×105/mL;
(2)取细胞悬液100ul加入transwell小室上室,24孔板下室加入600ul含20%FBS的培养基,避免产生气泡;
(3)培养24h后,取出transwell小室,结晶紫染色,显微镜下拍照。
10.统计分析
使用SPSS 23.0软件进行统计分析,数据表示为平均值±标准偏差,对于正态分布样本,通过双尾非配对Student t检验进行数据分析,否则采用非参数检验。结合临床资料数据进行分析时,采用Kaplan-Meier生存曲线,通过Log-rank检验作显著性检验。所有统计检验显著性水平定义为p<0.05认为有统计学意义。
结果
新诊断多发性骨髓瘤患者中位年龄59岁(42-78岁),其中男性15例(62.5%),复发难治多发性骨髓瘤中位年龄62岁(43-77岁),其中男性13例(65%)。为鉴定MM患者差异表达的特异性tsRNA,对5例复发难治及5例初治多发性骨髓瘤患者骨髓来源浆细胞标本进行了二代测序,检测到900多条tsRNAs的表达,其中包括20多条差异表达的tsRNA分子(p<0.05,FC(fold change)≥1.5认为有差异)(图1A)。
其中tRF-1:29-Thr-TGT-2分子在既往研究中未被报道,该分子的cDNA碱基序列为GGCTCCATAGCTCAGTGGTTAGAGCACTG,长度29个碱基,属于tRF-5c,在复发难治患者中表达显著下调(FC=0.041,p=0.0002)。对所有24位初治及19位复发难治多发性骨髓瘤患者采用qPCR验证tRF-1:29-Thr-TGT-2的表达水平,结果同样提示tRF-1:29-Thr-TGT-2在复发难治患者中显著下调(p=0.004)(图1B)。
通过比较复发难治及初治多发性骨髓瘤患者tRF-1:29-Thr-TGT-2的表达水平,并采用ROC曲线评估tRF-1:29-Thr-TGT-2的评价价值,结果显示AUC(曲线下面积)为0.948,95%CI 0.886-1.000(图2A),说明tRF-1:29-Thr-TGT-2用于多发性骨髓瘤复发难治的评估具有非常好的准确性。
对于初治多发性骨髓瘤患者,按tRF-1:29-Thr-TGT-2表达水平从低到高排序,前50%定义为低表达组,后50%定义为高表达组。随访4年,24例MM患者随访期内无失访。生存分析显示,高表达组无进展生存期(PFS)优于低表达组,差异具有统计学意义(p=0.038)(图2B)。
为了进一步探索tRF-1:29-Thr-TGT-2的作用机制,通过过表达技术在骨髓瘤细胞株U266中上调tRF-1:29-Thr-TGT-2的表达,进一步检测细胞周期、药物敏感性及细胞侵袭性的变化。首先通过qPCR验证转染效率,转染RNA mimic后U266细胞tRF-1:29-Thr-TGT-2表达水平明显上调(图3)。上调tRF-1:29-Thr-TGT-2表达后,分别在24h、48h、72h镜下计数,结果显示U266细胞增殖速率较对照组下降(图4)。流式PI染色显示,上调tRF-1:29-Thr-TGT-2表达后,G1/S转化受到抑制,被阻滞在G1期(图5)。transwell迁移实验显示上调tRF-1:29-Thr-TGT-2表达后U266细胞侵袭性下降(图6)。采用5nM硼替佐米处理实验组(U266+RNAmimic)和对照组(U266)24h,Annexin V-FITC/PI双染法检测细胞凋亡,结果显示上调tRF-1:29-Thr-TGT-2表达后骨髓瘤细胞对药物敏感性增加(图7)。
序列表
<110> 中南大学湘雅三医院
<120> tsRNA分子及其用途
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> DNA
<213> 智人(Homo sapiens)
<400> 1
ggctccatag ctcagtggtt agagcactg 29
<210> 2
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tccatagctc agtggttaga g 21
<210> 3
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gtccagtttt tttttttttt tcagtg 26
<210> 4
<211> 29
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggcuccauag cucagugguu agagcacug 29
<210> 5
<211> 29
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 5
gugcucuaac cacugagcua uggagccuu 29
<210> 6
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gcttcggcag cacatatact aaaat 25
<210> 7
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cgcttcacga atttgcgtgt cat 23
Claims (10)
1.一种tsRNA分子,其特征在于,其cDNA序列为GGCTCCATAGCTCAGTGGTTAGAGCACTG。
2.检测权利要求1所述的tsRNA分子的试剂的用途,其特征在于,用于制备复发难治多发性骨髓瘤诊断和/或预后制剂。
3.根据权利要求2所述的用途,其特征在于,所述的检测权利要求1所述的tsRNA分子的试剂包括PCR试剂;引物序列优选如下:
F:5’-TCCATAGCTCAGTGGTTAGAG-3’,
R:5’-GTCCAGTTTTTTTTTTTTTTTCAGTG-3’。
4.一种复发难治多发性骨髓瘤诊断和/或预后试剂盒,其特征在于,包括检测权利要求1所述的tsRNA分子的试剂。
5.根据权利要求4所述的试剂盒,其特征在于,所述的检测权利要求1所述的tsRNA分子的试剂包括PCR试剂;
引物序列优选如下:
F:5’-TCCATAGCTCAGTGGTTAGAG-3’,
R:5’-GTCCAGTTTTTTTTTTTTTTTCAGTG-3’。
6.过表达权利要求1所述的tsRNA分子的试剂的用途,其特征在于,用于制备多发性骨髓瘤治疗、降低多发性骨髓瘤细胞增殖速率、降低多发性骨髓瘤细胞侵袭性或者增加对多发性骨髓瘤治疗药物敏感性制剂。
7.根据权利要求6所述的用途,其特征在于,所述的过表达权利要求1所述的tsRNA分子的试剂包括过表达转染试剂。
8.根据权利要求7所述的用途,其特征在于,转染试剂包括:RNA mimic;
RNA mimic序列优选:
sense 5’-GGCUCCAUAGCUCAGUGGUUAGAGCACUG-3’,
anti-sense 5’-GUGCUCUAACCACUGAGCUAUGGAGCCUU-3’。
9.一种治疗多发性骨髓瘤、降低多发性骨髓瘤细胞增殖速率、降低多发性骨髓瘤细胞侵袭性或者增加对多发性骨髓瘤治疗药物敏感性的制剂,其特征在于,包括过表达权利要求1所述的tsRNA分子的试剂。
10.根据权利要求9所述的制剂,其特征在于,过表达权利要求1所述的tsRNA分子的试剂包括过表达转染试剂。
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