CN112961857A - 一种介导肺腺癌细胞焦亡的非编码长链rna基因 - Google Patents
一种介导肺腺癌细胞焦亡的非编码长链rna基因 Download PDFInfo
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Abstract
本发明属于生物技术领域,涉及一种未见报道的新发现非编码长链RNA基因TCONS‑14036介导肺腺癌NCI‑H1299细胞焦亡的机理及其实施方法。本发明首次公开了TCONS‑14036基因的序列,并根据此序列构建TCONS‑14036过表达质粒,填补了新发现非编码长链RNA基因TCONS‑14036在肺癌治疗领域的空白,明确其转染NCI‑H1299细胞后具有介导细胞焦亡的能力;明确了TCONS‑14036对肺腺癌NCI‑H1299细胞焦亡的作用机理,为推动TCONS‑14036进入抗肺腺癌临床应用提供依据。
Description
技术领域
本发明属于生物技术领域,涉及一种未见报道的新发现非编码长链RNA基因TCONS-14036介导肺腺癌NCI-H1299细胞焦亡的机理及实施方法。
背景技术
细胞焦亡是一种伴随炎症反应,并参与机体免疫应答的细胞程序性死亡方式。2005年,Susan L. Fink等人首次提出细胞焦亡的定义,并指出,由于炎症反应伴随,细胞焦亡形态学上同时具有细胞坏死和凋亡的特征。细胞焦亡与肿瘤相关既往研究中,NLRP3、AIM2等炎性小体诱发肿瘤焦亡抑制效应已被证实,P53等典型抑癌基因诱导细胞焦亡抑制肿瘤生长已被报道。近期,Zhang Z等人对GSDME蛋白通过激活细胞焦亡增强抗肿瘤免疫,达到肿瘤抑制作用的报道,则将研究氛围推向高潮。
长链非编码RNA是一类具有多种大量未明确生物学功能的转录本。已知的调控方式中,lncRNA通过染色质重塑、转录及转录后调控,行使对下游DNA、RNA和蛋白质的调节功能。
肺癌是目前世界范围内发病率和死亡率增长最快,对人类健康和生命威胁最大的恶性肿瘤之一。由于肺癌高度恶性的生物行为学表现,新疗法的开发与应用迫在眉睫。在既有研究中,lncRNA致癌与抑癌功能已被证实,但是,利用新发现非编码长链RNA基因用于肺癌治疗依然是空白。
发明内容
为了解决现有技术存在的问题,本发明的目的是填补新发现非编码长链RNA基因TCONS-14036在肺癌治疗领域的空白,明确TCONS-14036对肺腺癌NCI-H1299细胞焦亡的作用机理,提供理论依据推动TCONS-14036进入抗肺腺癌临床应用。
为了达到上述技术目的,本发明提供的技术方案如下:一种介导肺腺癌细胞焦亡的非编码长链RNA基因TCONS-14036,具体基因序列如SEQ ID NO:1所示。
所提供非编码长链RNA基因TCONS-14036过表达质粒通过PGMLV-6395载体构建。TCONS-14036 过表达质粒的过表达效率在938-6525倍。
进一步的,所述TCONS-14036过表达质粒转染NCI-H1299细胞后,诱导NCI-H1299细胞死亡。
进一步的,所述TCONS-14036过表达质粒转染NCI-H1299细胞后,诱导NCI-H1299细胞炎症因子IL-1β和IL-18释放。
进一步的,所述TCONS-14036过表达质粒转染NCI-H1299细胞后,诱导NCI-H1299细胞ASC蛋白寡聚化,并形成ASC瘢痕。
进一步的,所述TCONS-14036过表达质粒转染NCI-H1299细胞后,促进NCI-H1299细胞caspase1、IL-1β和GSDMD蛋白剪切。
进一步的,所述TCONS-14036过表达质粒转染NCI-H1299细胞后,诱导NCI-H1299细胞中NLRP3炎症小体释放至胞浆。
进一步的,运用RT-qPCR检测TCONS-14036表达水平的特异性扩增引物序列为:
TCONS-14036-F: 5’-CCGTGGACCCCGCCCTTC-3’;
TCONS-14036-R: 5’-CCTCACCTCAGCCATTGAACTCAC-3’。
TCONS-14036过表达质粒所包被的慢病毒抑制NCI-H1299细胞造肺腺癌模型小鼠原位肿瘤生长。
TCONS-14036在制备肺腺癌预防或治疗药物中的应用。
本发明的有益技术效果是:首次公开了TCONS-14036基因的序列,并根据此序列构建TCONS-14036过表达质粒,填补了新发现非编码长链RNA基因TCONS-14036在肺癌治疗领域的空白,明确其转染NCI-H1299细胞后具有介导细胞焦亡的能力;明确了TCONS-14036对肺腺癌NCI-H1299细胞焦亡的作用机理,为推动TCONS-14036进入抗肺腺癌临床应用提供依据。
附图说明
图1为Annexin V/PI流式细胞术证实TCONS-14036过表达质粒诱导NCI-H1299细胞死亡。
图2为ELISA法证实TCONS-14036过表达质粒诱导炎症因子IL-1β和IL-18释放。
图3为免疫荧光染色证实TCONS-14036过表达质粒诱导ASC蛋白寡聚化,并形成ASC瘢痕。
图4为western blot实验证实TCONS-14036过表达质粒促进caspase1、IL-1β和GSDMD蛋白剪切。
图5为免疫荧光染色证实TCONS-14036过表达质粒诱导NLRP3炎症小体释放。
图6为HE染色证实TCONS-14036过表达质粒所包被的慢病毒抑制NCI-H1299细胞造肺腺癌模型小鼠原位肿瘤生长。
图7、8为活体荧光成像证实新TCONS-14036过表达质粒所包被的慢病毒抑制NCI-H1299细胞造肺腺癌模型小鼠原位肿瘤生长及远端转移。
具体实施方式
下面结合附图和实施例对本发明作进一步的解释说明。
实施例1
TCONS-14036的RT-qPCR引物设计。
实验方法:依据RT-qPCR引物设计原则,使用Primer5.0软件设计实时定量荧光PCR引物。
实验结果:TCONS-14036的RT-qPCR引物,引物序列如SEQ ID NO:2/3所示。
TCONS-14036过表达质粒构建。
实验方法:依据PCR引物设计原则,设计PCR扩增片段引物,并在引物5’端引入线性化克隆载体末端的同源序列,引物序列如SEQ ID NO:4/5所示。利用稀释的引物及模板进行PCR扩增。将载体质粒(PGMLV-6395)进行扩增、双酶切后,加入目标片段,无缝克隆,转化,并通过NCI-H1299细胞转染及RT-qPCR对所得质粒进行检测。
实验结果:如图1所示,所得质粒转染后,TCONS-14036基因较正常细胞及空载质粒转染组显著上升,过表达效率在938-6525倍,过表达质粒构建成功。
实施例2
TCONS-14036过表达质粒转染NCI-H1299细胞后,Annexin V/PI流式细胞术细胞死亡检测。
实验方法:将正常培养的NCI-H1299细胞以5×105 个/ml密度,2ml/孔种入6孔板。24 H贴壁培养后,运用Lipofectamine 2000及Opti MEM将TCONS-14036过表达质粒或空载质粒转染至NCI-H1299细胞中。转染24 H后收集细胞,运用FITC-Annexin V流式细胞检测试剂盒,检测细胞死亡情况。
实验结果:如图2所示,肺腺癌NCI-H1299细胞在TCONS-14036过表达质粒转染的死亡数量较正常培养及空载质粒转染有显著上升,表明TCONS-14036过表达质粒对肺腺癌NCI-H1299细胞具有显著的杀伤作用。
实施例3
TCONS-14036过表达质粒转染NCI-H1299细胞后,细胞炎症因子IL-1β和IL-18检测。
实验方法:将正常培养的NCI-H1299细胞以5×105 个/ml密度,2ml/孔种入6孔板。24 H贴壁培养后,运用Lipofectamine 2000及Opti MEM将TCONS-14036过表达质粒或空载质粒转染至NCI-H1299细胞中。24 H干预后收集细胞,运用IL-1β和IL-18的ELISA检测试剂盒,检测细胞炎症因子IL-1β和IL-18释放情况。
实验结果:如图3所示,肺腺癌NCI-H1299细胞炎症因子IL-1β和IL-18释放在TCONS-14036过表达质粒转染组显著上升,表明TCONS-14036过表达质粒对肺腺癌NCI-H1299细胞具有显著的促炎症因子释放作用,而IL-1β和IL-18释放现象为细胞焦亡的特征炎症因子。
实施例4
TCONS-14036过表达质粒转染NCI-H1299细胞后,ASC蛋白免疫荧光检测。
实验方法:将正常培养的NCI-H1299细胞以5×105 个/ml密度,2ml/孔种入激光共聚焦皿。24 H贴壁培养后,运用Lipofectamine 2000及Opti MEM将TCONS-14036过表达质粒或空载质粒转染至NCI-H1299细胞中。24 H干预后固定,并孵育ASC蛋白一抗,水平摇床过夜。孵育荧光二抗,激光共聚焦显微镜观察拍照。
实验结果:如图4所示,TCONS-14036过表达质粒转染组肺腺癌NCI-H1299细胞形成ASC瘢痕,表明TCONS-14036过表达质粒转染组对肺腺癌NCI-H1299细胞中ASC蛋白寡聚化具有促进作用,为细胞焦亡的典型特征。
实施例5
TCONS-14036过表达质粒转染NCI-H1299细胞后,caspase1、IL-1β和GSDMD蛋白剪切检测。
实验方法:将正常培养的NCI-H1299细胞以5×105 个/ml密度,2ml/孔种入激光共聚焦皿。24 H贴壁培养后,运用Lipofectamine 2000及Opti MEM将TCONS-14036过表达质粒或空载质粒转染至NCI-H1299细胞中。24 H干预后提取细胞总蛋白,以β-actin为内参,western blot检测caspase1、IL-1β和GSDMD前体蛋白及剪切蛋白量。
实验结果:如图5所示,TCONS-14036过表达质粒转染组肺腺癌NCI-H1299细胞cleaved-caspase1、cleaved-IL-1β和cleaved-GSDMD蛋白量显著上升,表明TCONS-14036促进肺腺癌NCI-H1299细胞中caspase1、IL-1β和GSDMD蛋白剪切,细胞焦亡经典通路被激活。
实施例6
TCONS-14036过表达质粒转染NCI-H1299细胞后,NLRP3炎症小体免疫荧光检测。
实验方法:将正常培养的NCI-H1299细胞以5×105 个/ml密度,2ml/孔种入激光共聚焦皿。24 H贴壁培养后,运用Lipofectamine 2000及Opti MEM将TCONS-14036过表达质粒或空载质粒转染至NCI-H1299细胞中。24 H干预后固定,并孵育NLRP3炎症小体一抗,水平摇床过夜。孵育荧光二抗,激光共聚焦显微镜观察拍照。
实验结果:如图6所示,TCONS-14036过表达质粒组肺腺癌NCI-H1299细胞使NLRP3表达上调,表明TCONS-14036促进肺腺癌NCI-H1299细胞NLRP3炎症小体释放,而炎症小体释放为细胞焦亡必经之路。
实施例7
TCONS-14036过表达质粒所包被的慢病毒干预NCI-H1299细胞造肺腺癌模型小鼠后,肿瘤生长状况检测。
实验方法:将Luciferase基因标记的NCI-H1299细胞以3×108 个/只接种于裸鼠左肺。以1×106/只小鼠原位注射TCONS-14036过表达质粒所包被的慢病毒。运用活体成像技术连续3周检测肿瘤荧光强度。三周后处死,HE染色检测肿瘤组织。
实验结果:如图7和图8所示,TCONS-14036过表达质粒所包被的慢病毒干预NCI-H1299细胞造肺腺癌模型小鼠后,小鼠体内肿瘤细胞生长被显著抑制,肿瘤取出后,较模型组显著缩小,HE染色表明肺实质化减轻,肿瘤被抑制。
综上实验可见:TCONS-14036促进肺腺癌NCI-H1299细胞释放炎症因子IL-1β和IL-18,以炎症小体NLRP3为介导,通过ASC蛋白寡聚化、caspase1、IL-1β和GSDMD蛋白剪切,激活细胞焦亡经典通路,导致NCI-H1299细胞焦亡发生,且体内研究证实TCONS-14036抑制NCI-H1299细胞造肺腺癌模型小鼠肿瘤生长。因此,本发明所述TCONS-14036能作为治疗靶点,或作为药物作用靶点,在临床抗肺腺癌治疗中应用发挥价值。
上述内容为本发明的示例及说明,但不意味着本发明可取得的优点受此限制,凡是本发明实践过程中可能对机制发生的简单变换、和/或一些实施方式中实现的优点的其中一个或多个均在本申请的保护范围内。
序列表
<110> 上海中医药大学
<120> 一种介导肺腺癌细胞焦亡的非编码长链RNA基因
<141> 2021-04-13
<160> 5
<170> SIPOSequenceListing 1.0
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<211> 191
<212> DNA/RNA
<213> Homo sapiens
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ggcccggcgg atgcctcctt tgccggagct tggaacagac tcacggccag cgaagtgagt 120
tcaatggctg aggtgaggta ccccgcaggg gacctcataa cccaattcag actactctcc 180
tccgcccatt t 191
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<212> DNA/RNA
<213> Artificial Sequence
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<212> DNA/RNA
<213> Artificial Sequence
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cctcacctca gccattgaac tcac 24
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Claims (10)
1.一种介导肺腺癌细胞焦亡的非编码长链RNA基因,其特征在于:非编码长链RNA基因TCONS-14036介导肺腺癌NCI-H1299细胞焦亡。
2.根据权利要求1所述的介导肺腺癌细胞焦亡的非编码长链RNA基因,其特征在于:所述非编码长链RNA基因TCONS-14036序列为SEQ ID NO:1序列表。
3.根据权利要求1所述的介导肺腺癌细胞焦亡的非编码长链RNA基因,其特征在于:所述非编码长链RNA基因TCONS-14036过表达质粒通过PGMLV-6395载体构建。
4.根据权利要求1所述的介导肺腺癌细胞焦亡的非编码长链RNA基因,其特征在于:所述非编码长链RNA基因TCONS-14036表达水平的特异性扩增引物序列为:
TCONS-14036-F: 5’-CCGTGGACCCCGCCCTTC-3’;
TCONS-14036-R: 5’-CCTCACCTCAGCCATTGAACTCAC-3’。
5.根据权利要求1所述的介导肺腺癌细胞焦亡的非编码长链RNA基因,其特征在于:所述非编码长链RNA基因TCONS-14036基因介导的细胞焦亡是发生于肺腺癌NCI-H1299细胞中的,TCONS-14036过表达质粒转染NCI-H1299细胞后,诱导NCI-H1299细胞死亡。
6.根据权利要求1所述的介导肺腺癌细胞焦亡的非编码长链RNA基因,其特征在于:所述非编码长链RNA基因TCONS-14036过表达质粒转染NCI-H1299细胞后,诱导NCI-H1299细胞炎症因子IL-1β和IL-18释放。
7.根据权利要求1所述的介导肺腺癌细胞焦亡的非编码长链RNA基因,其特征在于:所述非编码长链RNA基因TCONS-14036过表达质粒转染NCI-H1299细胞后,诱导NCI-H1299细胞ASC蛋白寡聚化,并形成ASC瘢痕。
8.根据权利要求1所述的介导肺腺癌细胞焦亡的非编码长链RNA基因,其特征在于:所述非编码长链RNA基因TCONS-14036过表达质粒转染NCI-H1299细胞后,诱导促进NCI-H1299细胞caspase1、IL-1β和GSDMD蛋白剪切量上升。
9.根据权利要求1所述的介导肺腺癌细胞焦亡的非编码长链RNA基因,其特征在于:所述非编码长链RNA基因TCONS-14036过表达质粒转染NCI-H1299细胞后,诱导NCI-H1299细胞中NLRP3炎症小体释放至胞浆。
10.如权利要求1所述的介导肺腺癌细胞焦亡的非编码长链RNA基因TCONS-14036在制备肺腺癌预防或治疗药物中的应用。
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