CN116617391A - tRF-Gln-TTG靶向抑制剂在制备治疗肝癌的药物中的应用 - Google Patents
tRF-Gln-TTG靶向抑制剂在制备治疗肝癌的药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药领域，具体涉及tRF‑Gln‑TTG靶向抑制剂在制备治疗肝癌的药物中的应用。所述tRF‑Gln‑TTG的核苷酸序列如SEQ ID NO.1所示，所述靶向抑制剂具有如SEQ ID NO.2所示的核苷酸序列。本发明实验结果显示，tRF‑Gln‑TTG具有促进肝癌细胞增殖和抑制凋亡的能力，敲低tRF‑Gln‑TTG能够抑制肝癌细胞增殖，促进肝癌细胞凋亡，该结果为阐明肝癌发生发展分子机理提供新的理论依据，为肝癌临床治疗提供新型策略，为新药开发提供潜在靶点。
Description
技术领域
本发明属于生物医药领域，具体涉及tRF-Gln-TTG靶向抑制剂在制备治疗肝癌的药物中的应用。
背景技术
肝细胞癌(hepatocellular carcinoma，HCC，以下简称肝癌)是一种常见的恶性肿瘤，严重危害着人类生命健康。据统计数据显示，在所有癌症的新发病例和死亡人数中，肝癌发病率占比4.7％，但其死亡率却高达8.3％，是癌症相关死亡的第三大原因。
肿瘤发生发展的分子机制复杂多样，新的机制不断被阐明；近年来有关转移RNA(transfer RNA,tRNA)衍生的RNA片段(tRNA-derived fragment,tRF)参与肿瘤调节的作用逐渐受到人们关注。tRF是一类新型非编码小RNA，与半tRNA(tRNA halve,又名tiRNA)类似，均由tRNA酶切而来。tRF作为一组高度保守的tRNA切割产物，大致可以分为五类：tRF-1、tRF-2、tRF-3、tRF-5和i-tRF(图1)。tRF-1是由RnaseZ/ELAC2切割前体tRNA的3′端产生的小片段，是一个含有poly-U序列的3’末端序列。tRF-2是衍生于tRNA-Glu、tRNA-Asp、tRNA-Gly和tRNA-Tyr的小片段，由tRNA上的反密码子环分解而来。tRF-3包括tRF-3a和tRF-3b，是ANG、Dicer或核糖核酸酶家族的成员在成熟tRNA的T-环中切割产生的包含CCA序列的3’末端部分。tRF-5是Dicer酶在成熟tRNA的D-环处切割产生的衍生片段。根据切割位置的不同，tRF-5可分为tRF-5a(14-16nt)、tRF-5b(22-24nt)和tRF-5c(28-30nt)三种亚型。i-tRF是从成熟的tRNA区域切割出来的，并含有反密码子环、D-环和T-环的片段。
tRFs在体内广泛存在，它可以通过不同的作用机制调控基因的表达，进而在疾病的发生发展中发挥重要的作用。tRFs作用机制主要可以分为三类：①tRFs表现出类似microRNA的作用。tRFs可以与Argonaute(AGO)蛋白形成复合物，与靶基因mRNA的3'非翻译区域(3'UTR)相互作用并抑制靶基因的表达；②tRFs通过与RNA结合蛋白(RBP)相互作用间接调控基因的表达；③tRFs具有调控翻译的作用；tRFs通过作用于翻译起始复合物，将翻译相关起始因子eIF4G/A置换出翻译起始复合物，进而抑制翻译进程。迄今为止，已有多项研究揭示，tRFs在恶性肿瘤中表达失调，并且在癌症的发生发展过程中扮演了重要的角色。但是，截至目前，肝癌中关于tRFs的报道仍然较少，tRFs在肝癌中的角色及作用仍然未知。
发明内容
有鉴于上述技术问题，本发明提供了tRF-Gln-TTG靶向抑制剂在制备治疗肝癌的药物中的应用，所述tRF-Gln-TTG的核苷酸序列如SEQ ID NO.1所示。
进一步的，所述靶向抑制剂具有如SEQ ID NO.2所示的核苷酸序列。
基于同一个发明构思，本发明还提供了一种治疗肝癌的药物，所述药物包括所述tRF-Gln-TTG靶向抑制剂。
进一步的，所述药物为任何药物治疗学上可接受的剂型。
更进一步的，所述药物为任何药物治疗学上可接受的剂量。
本发明具有如下有益效果：
本发明的tRF-Gln-TTG是来源于tRNA-Gln-TTG-3-3的tRF-1，长度为14个核苷酸，实验结果显示，tRF-Gln-TTG具有促进肝癌细胞增殖和抑制凋亡的能力，敲低tRF-Gln-TTG能够抑制肝癌细胞增殖，促进肝癌细胞凋亡，该结果为阐明肝癌发生发展分子机理提供新的理论依据，为肝癌临床治疗提供新型策略，为新药开发提供潜在靶点。
附图说明
图1为tRF-Gln-mimic的过表达效率(A)和tRF-Gln-inhibitor的敲低效率(B)。
图2为免疫印迹评估转染tRF-Gln-mimic的SMMC-7721细胞中增殖及凋亡标记物表达水平。
图3为为过表达tRF-Gln-TTG对SMMC-7721细胞(A)和HepG2细胞(B)增殖的影响，以及敲低tRF-Gln-TTG对SMMC-7721细胞(C)和HepG2细胞(D)增殖的影响。
图4为克隆形成实验，A为过表达tRF-Gln-TTG的SMMC-7721细胞克隆形成情况，B为敲低tRF-Gln-TTG的HepG2细胞克隆形成情况，C为过表达tRF-Gln-TTG的SMMC-7721细胞克隆形成的定量统计，D为敲低tRF-Gln-TTG的HepG2细胞克隆定量统计。
图5为过表达或敲低tRF-Gln-TTG对肝癌细胞凋亡的影响，A为过表达tRF-Gln-TTG对SMMC-7721细胞凋亡的影响，B为过表达tRF-Gln-TTG对HepG2细胞凋亡的影响，C为敲低tRF-Gln-TTG对SMMC-7721细胞凋亡的影响，D为敲低tRF-Gln-TTG对HepG2细胞凋亡的影响，其中early代表早期凋亡比率、late代表晚期凋亡比率，total代表总凋亡比率。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：tRF-Gln-TTG mimic的过表达效率及inhibitor的敲低效率检测
1.人工合成tRF-Gln-TTG mimic(记作tRF-Gln-mimic)和tRF-Gln-TTG inhibitor(记作tRF-Gln-inhibitor)，以及tRF-Gln-TTG过表达及敲低专用的对照tRF-mimic-Control和tRF-inhibitor-Control。
tRF-Gln-mimic的序列(SEQ ID NO.1)为：5'-UCUUGGCUCUUUUU-3'。
tRF-Gln-inhibitor的序列(SEQ ID NO.2)为：5'-AAAAAGAGCCAAGA-3'。
tRF-mimic-Control的序列(SEQ ID NO.3)为：5'-UUUGUACUACACAA AAGUACUG-3'。
tRF-inhibitor-Control的序列(SEQ ID NO.4)为：5'-CAGUACUUUUGUGUAGUACAAA-3'。
2.细胞培养
SMMC-7721细胞：SMMC-7721细胞购买自ATCC。SMMC-7721细胞在添加10％胎牛血清(上海双洳生物,中国)的DMEM(武汉塞维尔生物,中国)中培养，常氧，37℃，5％ CO2环境下培养。
HepG2细胞：HepG2细胞购买自ATCC。HepG2细胞在添加10％胎牛血清(上海双洳生物,中国)的DMEM(武汉塞维尔生物,中国)中培养，常氧，37℃，5％CO2环境下培养。
2.转染
采用Lipofectamine 2000(Invitrogen，美国)用于mimic或inhibtor瞬时转染。
3.qRT-PCR验证过表达及敲低效率
根据制造商说明使用Trizol试剂(Takara,中国)从细胞中提取总RNA。随后，根据rtStar tRF&tiRNA Pretreatment Kit和rtStar First-Strand cDNA Synthesis Kit(Arraystar,Inc，美国)制造商说明制备tRF&tiRNA第一链cDNA样品。用SYBR体系在QuantStudioTM 3(Applied Biosystems，美国)上进行荧光定量PCR分析。U6作为样品的对照。相关引物序列如下：
U6-F(SEQ ID NO.5):5'-GCTTCGGCAGCACATATACTAAAAT-3'。
U6-R(SEQ ID NO.6):5'-CGCTTCACGAATTTGCGTGTCAT-3'。
tRF-Gln-TTG-F(SEQ ID NO.7):5'-TACAGTCCGACGATCTCTTGG-3'。
tRF-Gln-TTG-R(SEQ ID NO.8):5'-GTGTGCTCTTCCGATCTAAAAA-3'。
实验结果如图1所示，tRF-Gln-mimic可以显著提高tRF-Gln-TTG的表达，tRF-Gln-inhibitor可以显著抑制tRF-Gln-TTG的表达。
实施例2：免疫印迹实验
用含有1nmol/L PMSF(碧云天生物，中国)的RIPA缓冲液(碧云天生物，中国)从细胞样品中提取总蛋白。蛋白质经SDS-PAGE凝胶电泳150V，50分钟，随后转移到聚偏二氟乙烯膜(PVDF)膜上(Millipore,美国)。用5％脱脂牛奶在室温下孵育2小时后，TBST缓冲液清洗3次，每次5分钟。之后采用一抗在4℃下杂交过夜，TBST缓冲液清洗3次，每次5分钟，然后用二抗在室温下孵育1小时，TBST缓冲液清洗3次，每次5分钟。使用ECL化学发光试剂盒(白鲨,中国)对蛋白条带进行可视化。本研究中使用的一抗为β-Actin(上海泊湾生物，中国，AB0035)、claved-caspase-3(Cell Signaling，美国，#9664)、p53(武汉三鹰生物，中国，10442-1-AP)、Bax(Cell Signaling，美国，#5023)、Bcl-2(Abcam，ab182858)、Ki67(亲科生物，中国，#AF0198)。
结果如图2所示，tRF-Gln-TTG上调了增殖标记物Ki-67和抗凋亡基因Bcl-2的表达水平，同时抑制了促凋亡基因p53、Bax和激活型凋亡激酶caspase3的表达水平。
实施例3：CCK-8和克隆形成实验
1.CCK-8实验
细胞以2000个/孔的密度铺设在96孔板中。待细胞稳定贴壁后，采用Lipofectamine 2000进行相应的转染处理。在检测细胞增殖能力之前，每孔加CCK-8试剂10μL,在常氧，37℃，5％ CO2的培养箱中孵育2小时。随后，采用多波长酶标仪在450nm处读取细胞样品的吸光光度值。细胞增殖试验在0、24、48、72小时进行，每个样品在5个重复孔中进行，实验独立进行3次。
2.克隆形成实验
将细胞铺设至24孔板，用含10％胎牛血清的DMEM在常氧、5％ CO2的37℃培养箱中培养12小时。然后，用相应的mimic或inhibtor转染细胞。转染48小时后，将细胞消化重悬，铺设至6孔板内，并在无血清的DMEM液体中培养15天。培养基每两天更换一次。菌落形成后，用预冷PBS冲洗2次，用4％多聚甲醛固定30分钟。随后用1％结晶紫在室温下染色15分钟，双蒸水冲洗3次。自然风干后，观察克隆形成情况并分析。
结果如图3-4所示，过表达tRF-Gln-TTG提升了肝癌细胞的增殖能力，相反，敲低tRF-Gln-TTG抑制了细胞的增殖能力。
实施例4：流式细胞术检测肝癌细胞凋亡
将细胞铺设至24孔板，转染48小时后收取细胞。用预冷的PBS洗涤后，加入结合缓冲液重悬细胞。向每个Ep管中加入500μL裂解缓冲液(凯基生物，中国)然后用5μL AnnexinV-FITC和5μL PI(凯基生物，中国)冰上孵育5分钟染色细胞。1小时内采用流式细胞仪检测凋亡细胞。每组设3个重复。
结果如图5所示，在肝癌细胞中过表达tRF-Gln-TTG能够抑制细胞凋亡，而敲低tRF-Gln-TTG可使肝癌细胞凋亡水平升高。以上数据提示，tRF-Gln-TTG在肝癌细胞中抑制凋亡。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (5)
1.tRF-Gln-TTG靶向抑制剂在制备治疗肝癌的药物中的应用，其特征在于，所述tRF-Gln-TTG的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于，所述靶向抑制剂具有如SEQ ID NO.2所示的核苷酸序列。
3.一种治疗肝癌的药物，其特征在于，所述药物包括权利要求1中所述tRF-Gln-TTG靶向抑制剂。
4.根据权利要求3所述的药物，其特征在于，所述药物为任何药物治疗学上可接受的剂型。
5.根据权利要求4所述的药物，其特征在于，所述药物为任何药物治疗学上可接受的剂量。
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