CN116334226A - lncRNA SNHG26在制备肿瘤防治药物中的应用 - Google Patents
lncRNA SNHG26在制备肿瘤防治药物中的应用 Download PDFInfo
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Abstract
本发明公开了lncRNASNHG26在制备肿瘤防治药物中的应用。本发明通过在线数据库LncExpDB和实时荧光定量PCR技术确定了SNHG26在脑胶质瘤中显著升高；进一步在U251细胞转染特异性针对SNHG26的siRNA，结果表明敲降SNHG26可以显著抑制脑胶质瘤细胞U251的增殖和迁移。
Description
技术领域
本发明属于生物医药技术领域，具体涉及lncRNA SNHG26在制备肿瘤防治药物中的应用。
背景技术
脑胶质瘤是由于大脑和脊髓胶质细胞癌变所产生的原发性颅脑肿瘤。在中枢神经系统原发恶性肿瘤中，脑胶质瘤最为常见。在全球范围内，每年有超过25.6万人被诊断为恶性脑肿瘤，其中有19万死于脑肿瘤，尽管诊疗条件不断提高，但近年来我国脑肿瘤的发病率仍无明显下降趋势。目前脑胶质瘤的标准治疗是指外科手术、放射治疗和化学治疗的综合性治疗。然而，临床上并不是所有脑胶质瘤患者都是适合手术切除，需根据肿瘤的位置、大小、涉及的重要结构及预后来决定治疗策略，而且脑胶质瘤术后常常行全脑放疗，放射损伤重，并发症多。因此，寻找新的治疗策略尤为必要。目前人们越来越关注脑胶质瘤新分子治疗方法的研究，其中基因治疗和免疫治疗等在早期临床研究中显示出极好的前景。
随着功能基因组学的快速发展，长链非编码RNA(long noncoding RNA,LncRNA)作为非编码RNA(noncoding RNA,ncRNA)家族的重要成员受到了人们越来越多的关注。它是一类超过200个核苷酸(nt)的非编码RNA转录本亚群，大部分的LncRNA由RNA聚合酶Ⅱ转录，含有5’甲基化帽子及3’多聚腺苷酸(polyA)尾。LncRNA对基因表达的调控主要通过表观遗传学、转录及转录后调控等层面实现，从而在多种生物学过程中发挥重要作用，并与人类疾病的发生发展有着密切的关系。SNHG26(small nucleolar RNA host gene 26，[Homosapiens(human)]，Gene ID:109729180)是其中的一个lncRNA分子，到目前为止，还未曾有关于SNHG26在脑胶质瘤方面的报道。
发明内容
本发明的是提供lncRNA SNHG26的新用途，特别是用于制备肿瘤诊断试剂和防治药物中的用途。
为了实现上述目的，本发明采用以下技术方案：
lncRNA SNHG26在制备肿瘤诊断试剂中的应用。具体地，所述肿瘤为脑胶质瘤。
lncRNA SNHG26在筛选肿瘤防治药物中的应用。具体地，所述肿瘤为脑胶质瘤。
lncRNA SNHG26抑制剂在制备肿瘤防治药物中的应用。具体地，所述肿瘤为脑胶质瘤。
进一步地，所述lncRNA SNHG26抑制剂为化合物、蛋白、多肽、多糖、糖蛋白、糖肽、核酸中的一种或几种。
更进一步地，所述核酸为siRNA，该siRNA的序列如下：
正义链：GGGAGGUUAUGCCACCAUTT(seq-1)
反义链：AUGGUGGCAUAAUCCUCCCTT(seq-2)。
所述SNHG26的核苷酸序列如下：
Homo sapiens small nucleolar RNA host gene 26(SNHG26),long non-codingRNA NCBI Reference Sequence:NR_146320.1
GenBank Graphics
>NR_146320.1Homo sapiens small nucleolar RNA host gene 26(SNHG26),long non-coding RNA
本发明通过在线数据库LncExpDB和实时荧光定量PCR技术确定了SNHG26在脑胶质瘤中显著升高；进一步在U251细胞转染特异性针对SNHG26的siRNA，结果表明敲降SNHG26可以显著抑制脑胶质瘤细胞U251的增殖和迁移。因此，lncRNASNHG26不仅可以用于作为设计诊断试剂的靶点，还可以用于制备肿瘤防治药物。
附图说明
图1为SNHG26在脑胶质瘤中的表达情况。
图2为SNHG26 siRNA的干扰效率结果。
图3为敲降SNHG26后抑制脑胶质瘤细胞U251的增殖和迁移结果。其中：A为EdU实验结果，B为Ibidi小室迁移实验结果。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
实施例1
SNHG26在脑胶质瘤中的表达
通过在线数据库LncExpDB(https://ngdc.cncb.ac.cn/lncexpdb/)查阅SNHG26在不同肿瘤中的表达情况，采用实时荧光定量PCR技术检测其在人脑胶质瘤细胞系U251中的表达，结果如图1所示，SNHG26在脑胶质瘤中显著升高。
实施例2
SNHG26的功能验证
一、人脑胶质瘤细胞系U251的培养
从-80℃冰箱取出冻存的U251细胞，在37℃水浴锅中快速融解，在超净台内将细胞悬液吸出，加入2mL DMEM高糖完全培养基，离心，用适量完全培养基清洗一遍后，将细胞均匀种在60mm培养皿中，置于37℃细胞培养箱常规培养，隔天更换一次培养基，待细胞长至80％-90％可用于后续实验。
二、细胞RNA的提取
将培养在35mm培养皿中的U251用PBS洗3遍，加入1mL 
Reagent(Thermofisher)，然后放入1.5mL RNase-free EP管，冰上裂解5min。加入三氯甲烷200μL，涡旋剧烈振荡20s，室温静置5min；13000rpm，4℃离心15min。小心仔细吸取上清，加入异丙醇500μL，上下颠倒轻柔混匀，室温静置10min，13000rpm，4℃离心15min，弃除上清。加入1mL 75％乙醇，轻轻洗涤沉淀，4℃离心13000rpm，5min；去除上清，吹干。加入适量RNase-free H2O，65℃促溶10min；检测RNA的OD值及浓度，-80℃保存备用。
三、RNA逆转录合成cDNA
使用逆转录试剂盒(诺唯赞公司，R312-01)，将500ng的RNA逆转录为cDNA。
冰上操作，每个反应体系20μL，如下：
反应程序为：37℃15min，85℃5sec，4℃∞。
四、实时荧光定量PCR(qRT-PCR)
根据引物设计原则设计SNHG26 qRT-PCR引物序列。
SNHG26 qRT-PCR引物：
SNHG26-F：GGGCCAGTTGTCTCAGAATC(seq-4)
SNHG26-R：CTTGTCCAGCTGCAAAATCA(seq-5)。
将逆转录反应得到的cDNA，按照1∶5稀释，进行如下qRT-PCR反应。
(1)按下列组份配制qRT-PCR反应液：
(2)反应液混匀，Real-time PCR仪反应程序如下：
(3)反应设置3个复孔，内参为GAPDH，程序结束后，查看溶解曲线与扩增曲线，舍去误差较大的实验数据，根据具体实验要求，对数据进行统计分析。
五、U251的siRNA电转染
将培养至P2代的U251计数，吸取一定量的细胞与siRNA混合，充分混匀，使其最终浓度达到每管100μL混合液中含有2.0×106细胞+200nM的siRNA，其中细胞体积为90μL，siRNA体积为10μL。然后参照NEPA21(NEPAGENE公司)内皮细胞系(275V，1ms)电转染操作步骤进行电转。
SNHG26 siRNA如下所示：
正义链:GGGAGGUUAUGCCACCAUTT
反义链:AUGGUGGCAUAAUCCUCCCTT。
六、siRNA干扰效率鉴定
U251细胞分别转染特异性针对SNHG26 siRNA及其对照Ctrl(Control)siRNA，24h后，收细胞，按
Reagent(Thermo fisher)说明书提取U251细胞RNA，逆转录，进行qRT-PCR。结果如图2所示，siRNA处理24h后，SNHG26 siRNA可以显著降低U251中SNHG26的表达，SNHG26 siRNAvs.Ctrl siRNA，P值标注在图中。
七、敲降SNHG26对脑胶质瘤细胞U251增殖和迁移的影响
U251细胞分别转染特异性针对SNHG26 siRNA及其对照Ctrl(Control)siRNA，24h后，消化计数后，接种8×103个细胞于96孔板中，继续培养22个小时，按照广州锐博公司生产的EdU试剂盒(Cell-Light EdU Apollo567 In Vitro Kit(100T)，C10310-1)操作说明进行实验。Ibidi每个小室接种1.5×103个细胞进行迁移实验，待细胞完全贴壁后的0h，12h，24h，36h，和48h进行拍照，然后对迁移指标进行统计。结果如图3所示，敲降SNHG26显著抑制了脑胶质瘤细胞U251的增殖和迁移。
Claims (8)
1.lncRNA SNHG26的检测引物在制备肿瘤诊断试剂中的应用。
2.根据权利要求1所述的应用，其特征在于：所述肿瘤为脑胶质瘤。
3.lncRNA SNHG26在筛选肿瘤防治药物中的应用。
4.根据权利要求3所述的应用，其特征在于：所述肿瘤为脑胶质瘤。
5.lncRNA SNHG26抑制剂在制备肿瘤防治药物中的应用。
6.根据权利要求5所述的应用，其特征在于：所述肿瘤为脑胶质瘤。
7.根据权利要求6所述的应用，其特征在于：所述lncRNA SNHG26抑制剂为化合物、蛋白、多肽、多糖、糖蛋白、糖肽、核酸中的一种或几种。
8.根据权利要求7所述的应用，其特征在于：所述核酸为siRNA，该siRNA的序列如下：
正义链：GGGAGGUUAUGCCACCAUTT
反义链：AUGGUGGCAUAAUCCUCCCTT。
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