CN111424082A - lncRNA-SNHG6基因在制备治疗骨肉瘤的药物中的用途 - Google Patents
lncRNA-SNHG6基因在制备治疗骨肉瘤的药物中的用途 Download PDFInfo
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Abstract
本发明公开了lncRNA‑SNHG6基因在制备治疗骨肉瘤的药物中的用途。本发明证实了lncRNA‑SNHG6在骨肉瘤患者组织、骨肉瘤细胞株、包括骨肉瘤在内的各种肿瘤患者血浆中表达水平均上调,通过lncRNA‑SNHG6基因敲除和基因敲降、细胞和裸鼠移植瘤模型都证明判断lncRNA‑SNHG6与骨肉瘤发病、转移、生存、预后直接相关,可用于骨肉瘤早期诊断、早期治疗、改善预后的基因靶标,同时对于手术后患者,可以通过监测其血液中lncRNA‑SNHG6表达水平,判断是否出现转移、疾病进展和预后好坏。本发明还公开了通过RNA干扰抑制lncRNA‑SNHG6基因表达的siRNA、以及其它任何形式的lncRNA‑SNHG6抑制剂在制备治疗骨肉瘤的药物中的用途。
Description
技术领域
本发明属于生物技术领域,具体涉及lncRNA-SNHG6基因在骨肉瘤早期诊断、治疗和预后中的用途。
背景技术
骨肉瘤是以类骨质生成为特点的高度恶性骨源性癌症,是儿童和青少年(15-19岁)最常见原发性恶性骨肿瘤和第二癌症相关死亡原因。骨肉瘤是骨骼中最常见的原发性恶性肿瘤。通过手术和化疗相结合的方案改善了无转移骨肉瘤患者的生存状况,5年存活率达到了75%至77%。但这些治疗策略对出现转移的骨肉瘤患者效果有限,而且在过去的30年中没得到任何改善,具体说,转移性骨肉瘤的临床预后仍然不佳,发生转移的病人初次诊断后5年存活率低于30%,目前预防骨肉瘤转移和治疗转移性骨肉瘤面临着严峻的挑战,迫切需要发展治疗骨肉瘤的新疗法,但现有的针对该肿瘤的可用于开发新疗法的关键生物学机制数据非常少。
长链非编码RNAs(lncRNAs)是一类不编码蛋白质、长度大于200bp的RNA。研究表明lncRNAs具有保守的二级结构,可以与蛋白质、DNA和RNA相互作用,参与多种生物过程的调控,尤其在肿瘤发生发展过程中发挥重要调控作用,如染色质修饰、转录激活和抑制、转录后调节,诱导miRNA干扰靶基因表达等,与蛋白质编码基因比较,其显著优点表现在作为效应分子,是标记肿瘤固有特征更好的指标。未来用lncRNA治疗癌症的基因疗法令人鼓舞,如用RNAi介导选择性沉默、甚至用CRISPR/CAS9选择性敲除(knock out)致癌lncRNA,或将抑癌lncRNA靶向递送到特定细胞。且针对lncRNAs治疗的副作用会较少,是***很好的分子靶标。因此,lncRNAs不仅能作为筛选肿瘤标记物、且可以开发新的抗肿瘤方法。
lncRNA-SNHG6(ENST00000502076)是一种长链非编码RNA,位于8号染色体上,与邻近编码基因之间的关系为natural antisense。
发明内容
为了弥补现有技术的不足,根据本发明的实施例,希望提供一种可用于骨肉瘤(osteosarcoma)早期诊断、治疗和预后的分子标志物,并提出其在制备治疗骨肉瘤的药物中的用途。
本发明技术方案的提出基于:
1.本发明通过RT-PCR实验验证了lncRNA-SNHG6在骨肉瘤组织和细胞中表达上调。
2.本发明收集了骨肉瘤患者与正常人、其他内脏肿瘤患者与非肿瘤患者EDTA抗凝血,分离血浆,提取总RNA,反转为cDNA后,同过RT-PCR检测了lncRNA-SNHG6的表达水平。
3.本发明通过细胞核细胞质RNA分离-RT-PCR检测,以及荧光原位杂交(FISH)实验确定了lncRNA-SNHG6在细胞中的定位。
4.本发明通过信息学发现lncRNA-SNHG6有四个转录本,转录本1-4,通过细胞克隆基因测序发现,确定了骨肉瘤细胞143B中的主要转录本。
5.本发明建立了lncRNA-SNHG6基因敲除骨肉瘤细胞株。
6.本发明分别用构建了lncRNA-SNHG6基因的3个特异性慢病毒shRNA,感染骨肉瘤细胞后,通过RT-PCR检测证实,慢病毒成功介导敲降了骨肉瘤细胞143B、MG63、U2OS中lncRNA-SNHG6表达。
7.本发明比较了骨肉瘤lncRNA-SNHG6敲除和敲降前后各种生物学功能,明确了lncRNA-SNHG6在骨肉瘤发病中的特异性功能活性。
8.本发明通过将143B SNHG6-/-和143B SNHG6+/+细胞分别经胫骨平台注射入裸鼠骨髓腔,建立原位骨肉瘤和肺转移模型,明确了lncRNA-SNHG6在体内对骨肉瘤生长的抑制作用。
相对于现有技术,本发明证实了lncRNA-SNHG6在骨肉瘤患者组织、骨肉瘤细胞株、包括骨肉瘤在内的各种肿瘤患者血浆中表达水平均上调,通过lncRNA-SNHG6基因敲除和基因敲降、细胞和裸鼠移植瘤模型都证明判断lncRNA-SNHG6与骨肉瘤发病、转移、生存、预后直接相关,可用于骨肉瘤早期诊断、早期治疗、改善预后的基因靶标,同时对于手术后患者,可以通过监测其血液中lncRNA-SNHG6表达水平,判断是否出现转移、疾病进展和预后好坏。
相比传统的骨肉瘤诊断、治疗和预后方法,使用lncRNAs基因标志物来早期诊断、治疗和预后骨肉瘤具有及时性、特异性和灵敏性,从而使患者在疾病早期就能发现疾病风险,从而做到早期诊断、早期治疗、改善预后的目的,同时对于手术后患者,可以通过监测其血液中lncRNA-SNHG6表达水平,判断是否出现转移、疾病进展和预后好坏。
附图说明
图1显示利用RT-PCR检测lncRNA-SNHG6在骨肉瘤患者癌旁和骨肉瘤组织中的表达情况,表明lncRNA-SNHG6在不同骨肉瘤组织中表达显著上调;
图2显示利用RT-PCR检测lncRNA-SNHG6在人成骨细胞hFOB.19和不同骨肉瘤细胞中的表达情况,表明lncRNA-SNHG6在不同骨肉瘤细胞中表达均显著上调;
图3显示利用RT-PCR检测lncRNA-SNHG6在各种内脏瘤患者和非肿瘤患者血浆中的表达情况,表明lncRNA-SNHG6在各种内脏瘤患者血浆中表达显著上调;
图4显示利用RT-PCR检测lncRNA-SNHG6在骨肉瘤患者和正常人血浆中的表达情况,表明lncRNA-SNHG6在骨肉瘤患者血浆中表达显著上调;
图5显示利用RT-PCR检测发现lncRNA-SNHG6主要存在于骨肉瘤细胞的细胞浆中;
图6显示利用FISH检测发现lncRNA-SNHG6主要存在于骨肉瘤细胞的细胞浆中;
图7显示通过细胞克隆基因测序发现,骨肉瘤细胞143B中以转录本1为主;
图8显示利用RT-PCR检测CRISPR/Cas9双载体慢病毒***成功特异性敲除lncRNA-SNHG6基因;
图9显示利用RT-PCR检测3个特异性慢病毒shRNA成功特异性敲降lncRNA-SNHG6基因;
图10显示利用实时细胞仪检测发现骨肉瘤细胞中lncRNA-SNHG6敲除和敲降后存活率显著降低;
图11显示利用流式细胞仪检测发现骨肉瘤细胞中lncRNA-SNHG6敲降后细胞凋亡率显著增高;
图12显示利用细胞克隆形成检测发现骨肉瘤细胞中lncRNA-SNHG6敲降后细胞克隆形成显著降低;
图13显示利用流式细胞仪检测发现lncRNA-SNHG6敲除后细胞周期阻滞在G2/M期;
图14通过western blot检测关键蛋白表达发现骨肉瘤细胞中lncRNA-SNHG6敲除和敲降后将细胞周期阻滞在G2期;
图15通过western blot检测关键蛋白表达发现骨肉瘤细胞中lncRNA-SNHG6敲除和敲降后细胞凋亡相关蛋白显著改变;
图16显示分别利用用Transwell、实时细胞仪、划痕实验细胞检测发现,骨肉瘤细胞中敲除和lncRNA-SNHG6敲降后细胞迁移均降低;
图17显示利用实时细胞仪检测发现,骨肉瘤细胞中敲除lncRNA-SNHG6后细胞浸润能力降低;
图18显示利用移植瘤模型检测发现,骨肉瘤细胞中敲除lncRNA-SNHG6后,肿瘤原位生长和肺转移能力均降低。
具体实施方式
下面结合附图和具体实施例,进一步阐述本发明。这些实施例应理解为仅用于说明本发明而不用于限制本发明的保护范围。在阅读了本发明记载的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等效变化和修改同样落入本发明权利要求所限定的范围。
1.用RT-PCR实验检测了lncRNA-SNHG6在病人标本(骨肉瘤9例+癌旁对照9例),以及正常成骨细胞hFOB1.19和3种不同骨肉瘤细胞(143B,MG-63,U2OS,MNNG/HOS,Saos-2)中的表达水平:
分别取大约10mg人体组织标本(癌旁组织+肿瘤组织),加入1mlTRIZOL,用自动组织研磨仪研磨,提取总RNA;同时将正常成骨细胞hFOB1.19和3种不同骨肉瘤细胞(143B,MG-63,MNNG/HOS)以1×105个/ml的密度接种于6孔板,每孔2ml,培养48h,每孔加1ml TRIZOL提取总RNA。微量分光光度计测定提取样品的RNA浓度及纯度,逆转录试剂盒(Qiagen,Valencia,CA)将RNA逆转为cDNA,按照20μl体系45个循环进行扩增,采用2-△△Ct的计算方法计算不同标本间lncRNA-SNHG6的含量变化。结果发现了lncRNA-SNHG6在骨肉瘤组织(如图1所示)和骨肉瘤细胞(如图2所示)中表达均上调。
2.用RT-PCR检测了lncRNA-SNHG6在骨肉瘤患者与正常人、其它内脏肿瘤患者与非肿瘤患者之间表达差异:
我们收集了骨肉瘤患者与正常人、其它内脏肿瘤患者与非肿瘤患者EDTA抗凝血,分离血浆,提取总RNA,反转录为cDNA后,同上方法通过RT-PCR检测,结果发现在癌症患者血浆中显著高表达lncRNA-SNHG6,包括其它内脏肿瘤患者与非肿瘤患者比较(如图3所示)和骨肉瘤患者与正常人比较(如图4所示)。
4.分别通过细胞核细胞质RNA分离-RT-PCR检测和荧光原位杂交(FISH)确定了lncRNA-SNHG6在细胞中的亚细胞定位
细胞核细胞质RNA分离-RT-PCR检测:将生长状态良好、处于对数生长期的野生型和lncRNA-SNHG6敲降后骨肉瘤细胞消化,计数后用核质分离试剂盒按照说明书分别提取细胞核和细胞质RNA,再用上述RT-PCR方法分别检测细胞核和细胞质RNA中lncRNA-SNHG6的表达量,计算各自所占百分比。结果发现了lncRNA-SNHG6主要位于骨肉瘤细胞浆中(约占70%),细胞核中约占30%(如图5所示)。
荧光原位杂交(FISH)实验,将杂交液提前在水浴锅孵育2h,用DEPC水溶解探针(100μg/ml),用杂交液按照终浓度为12.5μg/ml、25μg/ml、50μg/ml,200μl/孔稀释并加入探针,在73℃水浴中变性5min;将铺于24孔板,浓度为5×104/孔、生长状态良好、处于对数生长期的野生型和lncRNA-SNHG6敲降后骨肉瘤细胞的培养基吸弃后,用PBS洗2次,每次5min;吸弃PBS,每孔加入200μl无水乙醇,室温固定15min;吸弃无水乙醇,每孔加入200μl 0.1%tritonX-100(现用现配);吸弃0.1%tritonX-100,用PBS洗2次,每次5min;吸弃PBS,每孔加入200μl 2×SSC,37℃,30min;吸弃2×SSC,每孔加入200μl 70%乙醇,室温30min;吸弃70%乙醇,每孔加入200μl 85%乙醇,室温30min;吸弃85%乙醇,每孔加入200μl 100%乙醇,室温30min;吸弃100%乙醇,室温干燥。每孔加入200μl上述准备的变性探针混合液,37℃杂交过夜。杂交次日,将样本从37℃取出,吸弃探针混合液,每孔加入200μl在65℃预热的0.4×SSC/0.3%Tween20洗涤2min;吸弃0.4×SSC/0.3%Tween20,加入200μl/孔2×SSC/0.1%Tween,室温洗涤2min,室温干燥;加入100μl DAPI染液(1:1000稀释),避光染色20min,吸弃,加入100μl PBS,荧光显微镜观察。结果发现了lncRNA-SNHG6主要位于骨肉瘤细胞浆中(如图6所示)。
5.通过细胞克隆团基因测序确定lncRNA-SNHG6转录本序列
为明确骨肉瘤细胞系143B中LncRNA-SNHG6以哪些转录异构体形式存在,通过对NCBI上存在的四个转录异构体差异区域进行引物设计,并以143B的cDNA为模板,通过高保真酶PCR扩增目的片段,胶回收后将目的片段连接到T载体,转化DH5α感受态,挑取60个克隆团进行测序分析,统计结果发现,86%的143B骨肉瘤细胞克隆团以SNHG6转录本(viariant)1形式存在(如图7所示),其序列见SEQ ID No.1所示。
6.通过CRISPR/Cas9双载体慢病毒***建立了lncRNA-SNHG6基因敲除骨肉瘤细胞株
1)制备过表达SNHG6基因的sgRNA慢病毒质粒:
针对SNHG6基因序列,设计sgRNA干扰靶点序列,合成两端含酶切位点粘端的单链DNA oligo(具有PAGE纯化体系),退火处理后形成双链DNA,连入Lenti-sgRNA-tag载体,并使用TOP10感受态转化,菌落进行PCR得到阳性克隆后测序,得到含有正确序列的、表达含SNHG6基因的sgRNA过表达慢病毒质粒。
2)慢病毒感染骨肉瘤细胞制备稳定敲除SNHG6基因的细胞株:
培养生长状态良好的骨肉瘤细胞143B,在病毒感染前一天将143B细胞铺板,于感染当天按实验设计组别加入Lenti-CAS9慢病毒颗粒进行骨肉瘤143B目的细胞感染实验,3天后用Puromycine筛选,之后用sgRNA慢病毒感染,在荧光显微镜下观察GFP表达情况,荧光率达80%以上,收集细胞提取总RNA。
按照同上所述的RT-PCR方法检测lncRNA-SNHG6表达,结果证明了CRISPR/Cas9双载体慢病毒***成功介导敲除了骨肉瘤细胞中lncRNA-SNHG6表达,获得了SNHG6control和SNHG6KO细胞(如图8所示)。
7.通过lncRNA-SNHG6shRNA在骨肉瘤细胞中敲降lncRNA-SNHG6表达
针对SNHG6基因序列,分别设计特异性shRNA序列,构建了针对lncRNA-SNHG6基因的3个特异性慢病毒shRNA,分别感染骨肉瘤细胞、用Puromycine筛选后,通过上面所述RT-PCR方法检测lncRNA-SNHG6表达,结果证明了慢病毒成功介导敲降了骨肉瘤细胞中lncRNA-SNHG6表达(如图9所示)。
8.通过比较骨肉瘤细胞中lncRNA-SNHG6敲除和敲降前后各种生物学功能,明确了lncRNA-SNHG6在骨肉瘤发病中的特异性功能活性。
(1)通过实时细胞仪检测了lncRNA-SNHG6敲除和敲降后骨肉瘤细胞存活率变化:
用E-Plate板,每孔加100μl完全培养基,设置程序,测Background;将生长状态良好、处于对数生长期的野生型和lncRNA-SNHG6敲除或敲降后骨肉瘤细胞消化、电子计数仪计数,以20000个/孔的密度接种,每孔100μl;室温孵育30min,上机Start实时检测48h,结果发现骨肉瘤细胞中lncRNA-SNHG6敲除和敲降后存活率显著降低(如图10所示)。
(2)通过流式细胞仪检测了lncRNA-SNHG6敲降后骨肉瘤细胞凋亡率变化:
将生长状态良好、处于对数生长期的野生型和lncRNA-SNHG6敲降后骨肉瘤细胞消化、电子计数仪计数,6孔板铺板,调整细胞密度至20万个细胞/孔(2ml,10万个细胞/ml),细胞培养箱中培养至细胞贴壁、状态良好后,吸弃旧培养液,用灭菌后的PBS溶液洗涤2次,消化细胞,离心机2000r/min离心5min,吸弃废液,加入1ml预冷无菌PBS(提前置于4℃冰箱过夜)洗涤细胞沉淀1次,离心机2000r/min离心5min,吸弃上层废液,用100μl Annexin VBinding Buffer(1×,用过滤过的去离子水稀释)重悬细胞沉淀,转移至1.5mlEP管中,加入5μl Annexin V-FITC,轻轻混匀,室温避光孵育15min,之后加入5μl碘化丙啶(PropidiumIodide Staining Solution,PI)染色液(50ug/ml),轻轻混匀,冰浴(冰盒)避光放置5-10min,根据细胞悬液浓度加入300-800μl 1×Binding Buffer终止染色,随即使用流式细胞仪(BD Accuri C6)进行检测,Annexin V-FITC为绿色荧光,PI为红色荧光,导出细胞凋亡数据进行统计分析。结果发现敲降lncRNA-SNHG6后骨肉瘤细胞凋亡率显著增高(如图11所示)。
(3)通细胞克隆形成实验检测敲降lncRNA-SNHG6后骨肉瘤细胞中细胞克隆形成:
取生长状态良好、处于对数生长期的野生型和lncRNA-SNHG6敲降后骨肉瘤细胞消化、电子计数仪计数,12孔板铺板,调整细胞密度至300个细胞/孔(1ml/孔),37℃、5%CO2培养箱中培养,每天显微镜下观察,每两天换一次培养基,7-9天后,吸取旧的培养基,PBS清洗一遍,每孔加入1ml多聚甲醛,放摇床上固定10min后,加入1ml10%的结晶紫染色,摇床上放置10min后,倒置显微镜下拍照,计数。结果发现了敲降lncRNA-SNHG6后骨肉瘤细胞中细胞克隆形成显著降低(如图12所示)。
(4)通过流式细胞仪检测了lncRNA-SNHG6敲除后骨肉瘤细胞细胞周期改变:
将生长状态良好、处于对数生长期的野生型和lncRNA-SNHG6敲除后骨肉瘤细胞消化、电子计数仪计数,6孔板铺板,调整细胞密度至20万个细胞/孔(2ml,10万个细胞/ml),细胞培养箱中培养至细胞贴壁、状态良好后,吸弃旧培养液,用灭菌后的PBS溶液洗涤2次,消化细胞,离心机2000r/min离心5min,吸弃废液,加入1ml预冷无菌PBS(提前置于4℃冰箱过夜)洗涤细胞沉淀1次,离心机2000r/min离心5min,吸弃上层废液,加入200μl冰70%乙醇将细胞沉淀重悬,将细胞悬液加入200μl预冷70%酒精(-20℃保存),转移至1.5ml EP管中,4℃固定过夜,隔日使用离心机2000r/min离心5min,小心吸弃上清,剩余沉淀,再用200ul冷PBS(提前4℃预冷)重悬细胞,离心机2000r/min离心5min,弃废液,然后每管样品中加入40μl RNaseA 37℃温水浴30min,再加入160μl PI染色液,缓慢并充分的重悬细胞沉淀,4℃避光30min,使用流式细胞仪在激发波长488nm处检测红色荧光,导出数据使用Modfit LT软件分析细胞周期,并导出细胞周期数据进行统计分析。结果发现lncRNA-SNHG6敲除后骨肉瘤细胞周期阻滞在G2/M期(如图13所示)。
(5)通过western blot检测敲除或敲降lncRNA-SNHG6后骨肉瘤细胞周期关键蛋白、细胞凋亡相关蛋白表达判断其对细胞周期和细胞凋亡的影响:
将生长状态良好、处于对数生长期的野生型和lncRNA-SNHG6敲除或敲降后骨肉瘤细胞用灭菌后的PBS溶液洗涤2次,消化细胞,离心机2000r/min离心5min,吸弃废液,加入1ml预冷无菌PBS(提前置于4℃冰箱过夜)洗涤细胞沉淀1次,加入适量裂解液(RIPA强裂解液:蛋白酶与磷酸酶抑制剂=50:1),冰上充***解30min,收取细胞裂解液,细胞超声破碎仪超声破碎(3×10s,间隔10s;使裂解更加充分),低温离心机4℃、14000rpm离心15min,收取上清。用BCA法检测蛋白浓度,蛋白定量与变性后用SDS-PAGE电泳、转膜、封闭、一抗孵育以及免疫印迹显影。结果发现骨肉瘤细胞中lncRNA-SNHG6敲除和敲降后将细胞周期阻滞在G2期(如图14所示)、促进细胞凋亡相关蛋白显著改变(如图15所示)。
(6)分别用Transwell、实时细胞仪、划痕实验检测骨肉瘤细胞中lncRNA-SNHG6敲除或敲降后细胞迁移能力:
Transwell检测细胞迁移能力,平衡Matrigel transwell,在小室的上室中分别加500μl野生型和lncRNA-SNHG6敲降后骨肉瘤细胞,下室中加500μl细胞迁移趋化剂,即含10%胎牛血清的相应细胞培养液。在37℃,CO2培养箱中培养24h后,用棉签擦去上室里面的非迁移细胞,移去transwell,倒置,风干,固定,染色,显微镜下观察、记数、拍照,根据细胞数量判断细胞迁移能力。
实时细胞仪检测细胞迁移能力,CIM-plate下室加入160μl预先温育的下室用液体,将上、下室安装在一起,上室加入20-50μl无血清培养液37℃孵育1h。设置layout和schedule后将CIM-plate放入station。启动Autoscan,当显示Connection ok后点击start检测Background,显示ok后收集野生型和lncRNA-SNHG6敲除或敲降后骨肉瘤细胞,在上室加100μl(20000个/孔)相应细胞悬液。将CIM-plate放入station,Autoscan显示Connectionok后,开始检测。
划痕实验检测细胞迁移能力,将单层平铺、约95%融合的野生型和lncRNA-SNHG6敲降后骨肉瘤细胞进行划痕处理,每隔12h对同一位置的划痕进行观察、测量、拍照,通过伤口愈合速度,比较细胞之间迁移能力的变化。
结果发现,骨肉瘤细胞中lncRNA-SNHG6敲除和敲降后细胞迁移能力均降低(如图16所示)。
(7)xCELLigence实时细胞检测仪检测浸润能力
此方法检测浸润能力的过程与检测迁移能力类似,只是在CIM-plate板的上室中预先铺上一层Matrigel胶,用无血清培养基配制1:40的胶,配好后置于冰上备用;在上室中加50μl配好的Matrigel胶,随后吸出30μl(这样形成的胶中没有气泡);37℃孵育4-5h;后续操作同迁移实验。结果发现骨肉瘤细胞中敲除lncRNA-SNHG6后细胞浸润能力降低(如图17所示)。
9.明确了lncRNA-SNHG6在体内对骨肉瘤生长的抑制作用
选择4周龄BALB/c雄性SPF级裸鼠,体重18-20g,分为2组,将野生型(Control)和lncRNA-SNHG6敲除后(SNHG6KO)骨肉瘤细胞143B(1*107个/ml,10μl)分别经胫骨平台注射入裸鼠骨髓腔,建立原位骨肉瘤和肺转移模型,观察肿瘤生长情况,Micro-CT(μCT)扫描骨密度和骨小梁容积,评价肿瘤对正常骨组织的破坏程度、监测骨肉瘤对胫骨骨质破坏情况。结果发现,lncRNA-SNHG6敲除(143B KO)组小鼠的肿瘤生长明显被抑制、对胫骨骨质破坏明显减轻,且肺转移能力均降低(如图18所示)。
序列表
<110> 上海中医药大学附属龙华医院
<120> lncRNA-SNHG6基因在制备治疗骨肉瘤的药物中的用途
<130> CNF2019111
<141> 2019-01-09
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 519
<212> RNA
<213> Homo sapiens
<400> 1
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Claims (9)
1.lncRNA-SNHG6基因在制备治疗骨肉瘤的药物中的用途。
2.根据权利要求1所述的用途,其特征在于,所述的lncRNA-SNHG6基因是一种长链非编码RNA,位于8号染色体上,与邻近编码基因之间的关系为natural antisense。
3.根据权利要求1所述的用途,其特征在于,所述的lncRNA-SNHG6基因选自人的lncRNA-SNHG6基因或其重组体。
4.根据权利要求1所述的用途,其特征在于,治疗骨肉瘤的药物为化合物,该化合物为核酸或蛋白质。
5.一种通过RNA干扰抑制lncRNA-SNHG6基因表达的siRNA,其特征在于,所述siRNA序列见SEQ ID No.1所示。
6.权利要求5所述的通过RNA干扰抑制lncRNA-SNHG6基因表达的siRNA在制备治疗骨肉瘤的药物中的用途。
7.根据权利要求5所述的用途,其特征在于,所述的lncRNA-SNHG6基因是一种长链非编码RNA,位于8号染色体上,与邻近编码基因之间的关系为natural antisense。
8.根据权利要求5所述的用途,其特征在于,所述的lncRNA-SNHG6基因选自人的lncRNA-SNHG6基因或其重组体。
9.根据权利要求5所述的用途,其特征在于,治疗骨肉瘤的药物为化合物,该化合物为核酸或蛋白质、以及其它任何形式的lncRNA-SNHG6抑制剂。
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