CN117024555A - circLRRFIP2-60aa及其在肺鳞癌治疗中的应用 - Google Patents
circLRRFIP2-60aa及其在肺鳞癌治疗中的应用 Download PDFInfo
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Abstract
本发明公开了circLRRFIP2‑60aa及其在肺鳞癌治疗中的应用；所述circLRRFIP2‑60aa的氨基酸序列如SEQ ID NO.1所述，还提供了编码circLRRFIP2‑60aa的核酸分子以及包含上述核酸分子的表达载体或重组细胞；本发明还发现circLRRFIP2‑60aa在肺鳞癌中充当肿瘤抑制基因，能有效抑制肺鳞癌细胞的增殖、迁移和侵袭；可以用于防治肺鳞癌，具有很好的应用前景。
Description
技术领域
本发明属于生物医药技术领域，具体涉及circLRRFIP2-60aa及其在肺鳞癌治疗中的应用。
背景技术
肺癌(lung cancer)是全球肿瘤相关病死率第一的恶性肿瘤，其中85％以上为非小细胞肺癌(NSCLC)，而NSCLC中30％为肺鳞状细胞癌(lung squamous cell carcinoma，LUSC，即：肺鳞癌)。LUSC早期诊断率不足15％，基因突变率低，靶向治疗机会少，对放化疗不敏感，5年生存率仅为17.8％，严重威胁人类的生命健康。因此，寻找肺鳞癌新的诊断生物标志物和治疗靶点具有重要的临床意义。期待通过揭示肺鳞癌的发生发展过程为临床治疗提供新的思路，为开发有效治疗方案奠定基础。
近年来，随着高通量测序技术和生物信息学技术的发展，大量的环状RNA(circular RNA，circRNA)在人体内被发现。circRNA是一类以共价闭环结构为特征的单链RNA分子，是由前体mRNA中的外显子或内含子反向剪接产生。其耐受核酸外切酶，组织特异性高且稳定表达，具有成为独特诊断标志物和治疗靶点的潜力。circRNA被认为是人类癌症的重要调节因子，它们与特定miRNA、mRNA和蛋白质的相互作用，参与肿瘤的发生、恶化进展、复发及耐药等病理过程。然而，大多数circRNA的功能仍然是未知的。最近的研究表明，circRNA具有翻译的功能，内部核糖体进入位点(IRES)和N 6-甲基腺苷(m6A)介导的帽独立翻译被认为是circRNA翻译的机制。circRNA编码的蛋白质或肽在疾病的发生发展中发挥生物学作用。
发明内容
本发明的目的提供一种由circLRRFIP2编码的功能性蛋白circLRRFIP2-60aa，其在肺鳞癌中充当肿瘤抑制基因，能有效抑制肺鳞癌细胞的增殖、迁移和侵袭，可用于制备抗肺鳞癌的药物。
本发明所采取的技术方案是：
本发明的第一方面，提供一种蛋白质，所述蛋白质的氨基酸序列如SEQ ID NO.1所示。
本发明的第二方面，提供一种核酸分子，所述核酸分子编码本发明第一方面所述的蛋白质。
优选地，所述核酸分子的序列如SEQ ID NO.2或SEQ ID NO.3或所示。
本发明的第三方面，提供一种重组载体，所述重组载体包含本发明第二方面所述的核酸分子。
优选地，所述载体包括质粒、噬菌体或病毒载体。
优选地，所述病毒性载体包括慢病毒载体、腺病毒载体、杆状病毒载体、反转录病毒载体、痘病毒载体、仙台病毒载体、单纯疱疹病毒载体中的至少一种。
优选地，所述病毒载体包括pCD5-ciR、pCD25-ciR、pLO5-ciR、pLC5-ciR、pK5ssAAV-ciR和pK25ssAAV-ciR等常用的circRNA表达载体。
本发明的第四方面，提供一种重组细胞，所述细胞包括本发明第三方面所述的重组载体。所述细胞非植物或动物新品种。
优选地，所述细胞包括原核细胞或真核细胞。
优选地，所述原核细胞包括大肠杆菌、链霉菌、枯草芽孢杆菌等本领域熟知的能够用于表达目的蛋白的原核细胞。
优选地，所述真核细胞包括酵母细胞、哺乳动物细胞、植物细胞和昆虫细胞等本领域熟知的能够用于表达目的蛋白的真核细胞。
优选地，所述哺乳动物细胞包括HEK293T、HEK293、BHK、MDCK、Hela、CHO、Vero、COS-7等细胞系中的至少一种。
本发明的第五方面，提供本发明第一方面所述的蛋白质和/或本发明第二方面所述的核酸分子和/或本发明第三方面所述的重组载体和/或本发明第四方面所述的重组细胞在制备预防和/或治疗肺鳞癌的药物中的应用。
优选地，所述治疗肺鳞癌包括抑制肺鳞癌细胞的增殖和/或迁移和/或侵袭能力。
本发明的第六方面，提供一种药物，所述药物包含本发明第一方面所述的蛋白质和/或本发明第二方面所述的核酸分子和/或本发明第三方面所述的重组载体和/或本发明第四方面所述的重组细胞。
优选地，所述药物还包括至少一种其他预防和/或治疗肺鳞癌的活性成分。
优选地，所述药物还包括药学上可接受的辅料。
优选地，所述药学上可接受的辅料包括：稀释剂、黏合剂、润湿剂、润滑剂、崩解剂、溶剂、乳化剂、助溶剂、防腐剂、pH调节剂、渗透压调节剂、表面活性剂、包衣材料、抗氧剂或缓冲剂中的至少一种。
优选地，所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、滴丸剂、注射剂、栓剂、气雾剂或滴剂中的至少一种。
优选地，所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药或雾化给药中的至少一种。
本发明的有益效果是：
本发明发现了一种由circLRRFIP2编码的新的功能性蛋白circLRRFIP2-60aa，其氨基酸序列如SEQ ID NO.1所述，还提供了编码circLRRFIP2-60aa的核酸分子以及包含上述核酸分子的表达载体或重组细胞；本发明还发现circLRRFIP2-60aa在肺鳞癌中充当肿瘤抑制基因，能有效抑制肺鳞癌细胞的增殖、迁移和侵袭；可以用于防治肺鳞癌，具有很好的应用前景。
附图说明
图1为环状RNA circLRRFIP2形成的示意图。
图2为circLRRFIP2编码蛋白质潜力的预测。A：circPrimer2.0预测circLRRFIP2具有一个ORF和M6A位点；B：SRAMP数据库预测circLRRFIP2具有很高的m6A修饰潜力。
图3为circLRRFIP2-60aa验证实验。A：circRNA慢病毒表达载体；B：构建的Lv-vector control、Lv-flag-circLRRFIP2、Lv-flag-circLRRFIP2-mut、Lv-circLRRFIP2-60aa重组载体；C：qRT-PCR验证过表达效率；D：western检测到Flag标签蛋白的表达；E：SDS-PAGE电泳及银染。
图4为液相色谱-串联质谱(LC-MS/MS)分析。
图5为细胞平板克隆形成实验。
图6为HCC1588细胞Transwell迁移侵袭实验。
图7为H1703细胞Transwell迁移侵袭实验。
图8为细胞划痕实验。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。
实施例1circLRRFIP2-60aa序列预测
1、通过UCSC(http://genome.ucsc.edu/)在线数据库软件分析发现circLRRFIP2基因定位于人的第3号染色体短臂chr3(p22.2)区域，基因组跨越19977bp，来源于人的LRRFIP2基因的3-4号外显子(图1)；根据环状RNA权威数据库circBase(http://circrna.org/)收录的LRRFIP2产生的环状RNA信息，circLRRFIP2的circRNA ID为hsa_circ_0003264，成熟环状RNA序列为232nt，命名为circLRRFIP2，其序列如下：GGCTGAAAACACAGTTAAATCACTTTCAAAGTTAAAAGACTATTAAGAACACAAGATGGGGACTCCTGCTTCTGGAAGGAAAAGAACACCTGTGAAAGACCGATTTTCTGCAGAAGATGAAGCTTTGAGTAACATTGCCAGAGAGGCAGAGGCAAGGCTGGCAGCAAAACGGGCTGCCCGGGCAGAAGCAAGAGATATACGCATGAGAGAACTGGAACGACAACAAAAAGAG(SEQ IDNO.2)。
2、通过circPrimer2.0预测circLRRFIP2可能具有编码蛋白质的潜力，预测发现circLRRFIP2成熟RNA序列232nt，包含一个开放阅读框(ORF)和M6A修饰位点(图2A)；SRAMP数据库分析表明，circLRRFIP2具有很高的m6A修饰潜力(图2B)；因此，理论上circLRRFIP2可以翻译出由60个氨基酸组成的小分子蛋白质。通过蛋白质分子量预测软件(http://www.bio-soft.net/sms/prot_mw.html)预测circLRRFIP2翻译的新蛋白的分子量约10kDa，将其命名为circLRRFIP2-60aa，如图2A所示，其序列如下所示：MGTPASGRKRTPVKDRFSAEDEALSNIAREAEARLAAKRAARAEARDIRMRELERQQKEG(SEQ ID NO.1)。
实施例2circLRRFIP2-60aa验证实验
为了进一步验证circLRRFIP2-60aa的存在，在circRNA慢病毒表达载体(图3A)上构建带flag标签的载体：Lv-vector control(对照载体)、Lv-flag-circLRRFIP2(将flag标记的circLRRFIP2序列克隆到CMV诱导的表达载体中)、Lv-flag-circLRRFIP2-mut(将flag标记且起始密码子ATG突变为ACG的circLRRFIP2序列克隆到CMV诱导的表达载体中)；Lv-circLRRFIP2-60aa是将flag标记的circLRRFIP2-60aa序列克隆到CMV诱导的表达载体pCDH-CMV-MCS-EF1-CopGFP-Puro中(图3B)，其中circLRRFIP2-60aa的核苷酸序列是circLRRFIP2开放阅读框(ORF)的序列，即ATGGGGACTCCTGCTTCTGGAAGGAAAAGAACACCTGTGAAAGACCGATTTTCTGCAGAAGATGAAGCTTTGAGTAACATTGCCAGAGAGGCAGAGGCAAGGCTGGCAGCAAAACGGGCTGCCCGGGCAGAAGCAAGAGATATACGCATGAGAGAACTGGAACGACAACAAAAAGAGGGCTGA(SEQ IDNO.3)，该序列是克隆到pCDH-CMV-MCS-EF1-CopGFP-Puro这个慢病毒载体中，因为该序列呈线性，不是环状，所以克隆到线性基因的载体中；根据转染试剂说明书制作慢病毒液转染HCC1588、H1703细胞，经嘌呤霉素筛选出稳定表达上述载体的稳转株细胞，qRT-PCR验证过表达效率(图3C)。
用RIPA裂解液分别提取转染上述四种载体的293T细胞总蛋白，BCA蛋白定量法对提取的蛋白进行定量；配置5％SDS-PAGE浓缩胶、15％SDS-PAGE分离胶，上样总蛋白为40微克；采用80V 30分钟、120V 1h跑蛋白电泳；转膜采用360A转膜30min；封闭液常温封闭30min；Flag鼠单克隆抗体(AffinityBiosciences Cat#T0053,RRID:AB_2843447)(1:1000)、GAPDH抗体(Affinity BiosciencesCat#AF7021,RRID:AB_2839421)(1:3000)；4度孵育过夜；第二天采用小鼠二抗(1:10000)常温孵育90min，TBST洗涤5遍每次5min，然后发光，显影，定影。
western检测到Flag标签蛋白的表达(图3D)。另外，针对Lv-flag-circLRRFIP2-60aa转染293T细胞的裂解物进行免疫共沉淀(Co-IP)，拉下来的蛋白液进行SDS-PAGE电泳及银染(图3E)，手动切10-25kDa的蛋白质条带进行液相色谱-串联质谱(LC-MS/MS)分析(图4)，结果证实Lv-flag-circLRRFIP2和Lv-flag-circLRRFIP2-60aa都表达60aa蛋白，证明circLRRFIP2-60aa是由circLRRFIP2翻译来的，并且鉴定出circLRRFIP2-60aa含有一个独特aa序列(Gly)。综上所述，circLRRFIP2具有利用其ORF编码蛋白质的潜力，circLRRFIP2编码一种新的未表征的蛋白质circLRRFIP2-60aa。
实施例3 circLRRFIP2-60aa蛋白提取
用6×105个HEK 293T细胞接种于10cm培养皿中，24h后将上述4种质粒载体进行转染；按照转染试剂操作说明书，转染前，将500微升无血清培养基DMEM和质粒制备成混合液；将500微升无血清培养基DMEM和60ul均匀混合，做成转染试剂混合液；将上述两种混合液等混合，室温放置15min后转入培养皿中，最终10cm培养皿中的液体终体积为10毫升，细胞培养条件37℃，5％CO2，培养48h后提取蛋白质。
实施例4细胞平板克隆形成实验
克隆形成实验评估肺鳞癌细胞的增殖能力。对数生长期的HCC1588 Lv-vectorcontrol、Lv-flag-circLRRFIP2、Lv-flag-circLRRFIP2-mut、Lv-circLRRFIP2-60aa经胰酶消化后加入1ml贴壁细胞培养基重悬为单细胞悬液，细胞计数板计数并适当调整细胞密度。六孔板中每孔中加入1000个上述四组细胞，再加入2ml含10％ FBS的1640培养基，放于37℃、5％ CO2的孵箱培养，隔日换液，培养14d后取出培养板使用2ml PBS清洗2遍，4％多聚甲醛室温固定30min，吸净多聚甲醛，等待水分吹干，每孔加结晶紫染液1ml染色15min,弃去结晶紫染液，2ml PBS洗涤3次，待水分吹干，扫描拍照并计数；结果见图5，可以看出，相比于Lv-vector control、Lv-flag-circLRRFIP2-mut组，Lv-flag-circLRRFIP2和Lv-flag-circLRRFIP2-60aa两组的细胞增殖能力显著降低。
实施例5细胞Transwell迁移侵袭实验
Transwell实验评估肺鳞癌细胞的迁移侵袭能力。用10000个上述四组HCC1588、H1703肺鳞癌细胞接种于Transwell小室中，细胞培养条件37℃，5％CO2，培养24h，4％多聚甲醛固定滤膜30min，结晶紫染色20min，拍照观察统计穿过滤膜及Martrigel胶的细胞数。结果如图6和图7所示，可以看出Lv-flag-circLRRFIP2及Lv-circLRRFIP2-60aa组抑制肺鳞癌细胞的迁移及侵袭能力。
实施例6细胞划痕实验
细胞划痕测定评估肺鳞癌细胞的迁移能力。用5×106个上述四组HCC1588、H1703肺鳞癌细胞接种于六孔板中，培养至细胞汇合度达到95％以上，使用中枪头在六孔板中进行垂直划痕后，在其中加入无血清培养基。随后在0h和24h进行图像采集和迁移距离测量。结果如图8所示，Lv-flag-circLRRFIP2及Lv-circLRRFIP2-60aa组抑制肺鳞癌细胞的迁移能力，与Transwell迁移实验结果一致。
上述具体实施方式对本发明作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。
Claims (10)
1.一种蛋白质，所述蛋白质的氨基酸序列如SEQ ID NO.1所示。
2.一种核酸分子，所述核酸分子编码权利要求1所述的蛋白质。
3.根据权利要求2所述的核酸分子，其特征在于，所述核酸分子的序列如SEQ ID NO.2或SEQ ID NO.3所示。
4.一种重组载体，所述重组载体包含权利要求2或3所述的核酸分子。
5.一种重组细胞，所述细胞包括权利要求4所述的重组载体。
6.权利要求1所述的蛋白质和/或权利要求2～3任一项所述的核酸分子和/或权利要求4所述的重组载体和/或权利要求5所述的重组细胞在制备预防和/或治疗肺鳞癌的药物中的应用。
7.根据权利要求6所述的应用，其特征在于，所述预防和/或治疗肺鳞癌包括抑制肺鳞癌细胞的增殖和/或迁移和/或侵袭能力。
8.一种药物，所述药物包含权利要求1所述的蛋白质和/或权利要求2～3任一项所述的核酸分子和/或权利要求4所述的重组载体和/或权利要求5所述的重组细胞。
9.根据权利要求8所述的药物，其特征在于，所述药物还包括至少一种其他预防和/或治疗肺鳞癌的活性成分。
10.根据权利要求8或9所述的药物，其特征在于，所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、滴丸剂、注射剂、栓剂、气雾剂或滴剂中的至少一种。
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