CN117264023A - 一种由长链非编码rna linc01007编码的微肽及其应用 - Google Patents
一种由长链非编码rna linc01007编码的微肽及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，尤其涉及一种由长链非编码RNA LINC01007编码的微肽及其应用。本发明通过对154例肝癌患者癌与癌旁的蛋白质组学分析，筛选出在肝癌组织中显著高表达的微肽，其中微肽LINC01007‑aa的表达对肝癌患者的总生存具有独立的预后意义（HR，95%CI=1.0‑1.7；p=0.025），是肝癌总生存的独立危险因子。过表达微肽LINC01007‑aa的肝癌细胞株侵袭与迁移能力显著增强，由此微肽LINC01007‑aa可作为肝癌的预后标志物及潜在的治疗靶点，具有很好的临床应用前景。
Description
技术领域
本发明属于生物技术领域，尤其涉及一种由长链非编码RNALINC01007编码的微肽及其在作为肝癌预后标志物和潜在治疗靶点中的应用。
背景技术
原发性肝癌(Primarylivercancer，PLC)是人体内常见的消化系统原发性实体恶性肿瘤，肝细胞癌(Hepatocellularcarcinoma，HCC)为原发性肝癌中最主要的类型，占原发性肝癌所有病理类型的75％～85％。HCC由于发病率高、发现时间晚、治疗策略有限等原因导致患者总体预后不佳，因此探索新的治疗靶点是肝癌治疗的重要研究方向，新的预后标志物在肝癌患者的治疗和研究中具有重要的意义。
长链非编码RNA(LongnoncodingRNA，LncRNA)属于非编码RNA中重要的组成部分，被证明在多种生物过程中发挥关键的作用，并正在成为一类重要的调节分子。蛋白质是遗传信息的最终产物及功能的体现，与生物体的生理活动密切相关，在癌症等疾病的发生发展中发挥重要的调节作用。已经有研究表明，LncRNA分子中的小开放阅读框(smallopenreadingframe，sORF)能够编码出功能性多肽微肽(sORF-encodedpeptide)。这些微肽在作为癌症生物标志物以及阐明肝癌发病机制方面具有巨大潜力。微肽作为此前被忽视的蛋白质家族的一部分，对于HCC的生物标志物以及机制研究具有积极意义。
发明内容
本发明的目的在于提供一种由长链非编码RNALINC01007编码的微肽及其在作为肝癌预后标志物和潜在治疗靶点中的应用。
为实现上述目的，本发明采用的技术方案如下：
一种由长链非编码RNALINC01007编码的微肽LINC01007-aa，其氨基酸序列如SEQID NO.1所示。
一种编码权利要求1所述的微肽LINC01007-aa的核酸分子，其核苷酸序列如SEQID NO.2所示。
一种重组载体，其含有编码微肽LINC01007-aa的核酸分子。
一种转基因细胞系，其含有编码微肽LINC01007-aa的核酸分子。
微肽LINC01007-aa在制备用于诊断肝癌的产品中的应用，所述产品为能够特异性检测微肽LINC01007-aa表达水平的产品。
微肽LINC01007-aa在制备用于对肝癌患者的预后进行评估的产品中的应用，所述预后为患者的总生存期。
微肽LINC01007-aa在制备具有促进肿瘤发展功能的产品中的应用，所述肿瘤为肝癌，所述肝癌的癌细胞为SK-HEP-1细胞，所述肿瘤发展体现为肿瘤细胞侵袭和/或迁移。
微肽LINC01007-aa作为靶点在制备具有抑制肿瘤发展功能的产品中的应用，所述肿瘤为肝癌，所述肝癌的癌细胞为SK-HEP-1细胞，所述肿瘤发展体现为肿瘤细胞侵袭和/或迁移。
微肽LINC01007-aa活性或表达的物质在制备抑制肿瘤发展的产品中的应用，所述肿瘤为肝癌，所述肝癌的癌细胞为SK-HEP-1细胞，所述肿瘤发展体现为肿瘤细胞侵袭和/或迁移。
本发明的显著优点在于：
本发明筛选了HCC新型预后生物标志物，可以用于预测HCC患者的预后总体生存，为HCC的精准医疗提供了依据，能够使广大患者从中获益，其意义如下：
1)本发明的微肽LINC01007-aa可作为HCC患者预后总体生存的新指标，通过制备相关检测试剂盒，可以预测HCC患者预后的总体生存率；
2)本发明所提供的微肽LINC01007-aa上游基因还可作为HCC治疗的靶基因，还可将微肽作为药物作用的新靶点，研制以微肽蛋白为靶点的新药。
因此，微肽LINC01007-aa的开发有可能开拓抗HCC的新途径，这是一项将产生重大社会效益的工程。
附图说明
图1为在154例肝癌患者中检测到的微肽的差异表达火山图。
图2为多因素cox分析微肽LINC01007-aa与总生存风险比森林图。
图3为微肽LINC01007-aa对应LncRNA在肝癌组织中的表达情况。
图4为微肽LINC01007-aa与EGFPmut融合蛋白表达情况。
图5为微肽LINC01007-aa促进肝癌细胞侵袭。
图6为微肽LINC01007-aa促进肝癌细胞迁移。
具体实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是本发明不仅限于此。
本发明所述微肽LINC01007-aa的序列为(N端-C端)：
MSPPGQWDICYSTRYCLRTLPLGSLRAGSLGRQ(SEQ ID NO.1)。
编码该微肽LINC01007-aa的长链非编码RNA LINC01007的核苷酸序列如SEQ IDNO.2所示。
实施例1
长链非编码RNA编码微肽的鉴定
1.数据来源
(1)质谱数据来源：
本发明的临床研究队列包含154例肝癌病人的癌组织及其配对的癌旁组织，均来自福建医科大学孟超肝胆医院生物样本库，标本采集经福建医科大学孟超肝胆医院伦理委员会批准，告知患者本人及家属样本用于科研并签署知情同意书。
根据以下标准入组研究：(1)实行原发性肝癌根治性切除手术；(2)术中超声检测肝脏其他的部分未发现病灶；(3)术后经病理学检测确诊为肝细胞癌，病理检查切缘未见癌细胞。排除标准：(1)术前针对肿瘤病灶进行介入、B超引导射频治疗获其他抗肿瘤治疗；(2)患者有其他重大疾病；(3)术后病理检测为胆管细胞癌、混合型肝癌、肝转移等。
本发明使用福建医科大学孟超肝胆医院前期根据以上标准入组了154例肝癌病人，提取癌组织与癌旁组织蛋白，混合Pool肽段制好样品，采用DDA进行蛋白质谱数据采集，获得原始DDA数据文件；同时对每一个蛋白样品采用DIA进行采集，获得原始蛋白质质谱DIA数据文件。
(2)蛋白质数据库来源：
本研究使用整合蛋白质数据库通过The Universal Protein Resource(Uniprot)提供的人类蛋白数据库混合已发表的公共微肽序列数据库得到。
A.人类Uniprot蛋白质数据库：http://www.uniprot.org/uniprot。本发明使用发布于2020年11月份，共包含20,381个非冗余蛋白的人类Uniprot蛋白质数据库。
B.已发表的公共微肽数据库：https://doi.org/10.1016/j.mcpro.2021.100109。
2.非编码RNA编码的微肽的筛选
(1)本地谱图库建立与搜库分析：使用Spectronaut软件进行DDA与DIA本地谱图库构建，DIA谱图库与整合蛋白质数据库匹配，共鉴定到肝癌组织中共208种微肽。
(2)微肽的差异分析(图1)：选取在癌与癌旁组织间具有显著差异(P<0.05，FoldChange>1.5)的44种微肽作为候选肝癌预后标志物进行后续分析，其中在癌组织中显著上调的有26个，显著下调的有18个(Wilcoxon秩和检验，p<0.05)。
(3)微肽的谱图质量控制与同源LncRNA分析：质谱谱图筛选标准为：
a)基峰呈明显峰形；
b)无双峰；
c)信号强度高(e5-e6)；
d)有三个及以上子离子检出。
对在癌组织中具有差异表达的微肽进一步筛选，筛选质谱峰图质量良好且具有同源来源LncRNA的微肽进行后续研究，共筛选到7个候选微肽。
(4)单多因素cox分析及多因素cox分析(图2)：选取通过以上筛选的7个候选微肽进行单多因素cox预后分析发现，微肽LINC01007-aa的表达量在肝癌中与总生存期(OS)具有显著的相关性(HR，95％CI＝1.1(0.91-1.4)，p＝0.033)。进一步采用多因素cox分析发现，微肽LINC01007-aa是影响术后总生存的独立危险因素(似然比检验，p<0.05，HR(95％CI)＝1.3(1.00-1.7))。
(5)微肽LINC01007-aa在RNA水平表达分析(图3)：对微肽LINC01007-aa对应LncRN的RNA水平表达进行探究，利用特异性引物5'-CTAGTCTAGAATGTCGCCCCCAGGTCAATG-3'和5'-CCGGAATTCTTGTCTCCCCAGTGACCCAGC-3'进行PCR扩增并对扩增产物进行测序分析，在肝癌组织的中检测到微肽LINC01007-aa的对应LncRNALINC01007。
(6)临床病理因素相关性(表1)：通过HCC病人的临床病理因素相关性分析显示，微肽LINC01007-aa的表达量与AFP、微卫星子灶具有显著相关性(Kruskal Wallis检验，p<0.05)。
表1
(7)微肽LINC01007-aa在蛋白质水平表达情况(图4)：通过构建微肽LINC01007-aa与EGFP起始密码子突变的融合蛋白，根据荧光表达情况能够对微肽LINC01007-aa的蛋白质水平进行探究。将微肽LINC01007-aa与EGFPmut的融合蛋白质粒转染HEK-293T细胞24h，能够检测到转染细胞内的荧光表达，而微肽LINC01007-aa起始密码子突变则不能检测到荧光表达。结果表明，微肽LINC01007-aa在蛋白水平能够稳定表达，且其表达能力受微肽LINC01007-aa自身起始密码子调控。
实施例2
微肽LINC01007-aa可促进肝癌细胞的侵袭迁移
1.微肽LINC01007-aa稳定表达细胞株的构建
(1)将HEK-293T细胞提前接种于10cm细胞培养皿，待细胞长到60％～70％汇合度时，进行慢病毒包装。
(2)在微肽LINC01007-aa的N末端融合Flag标签序列后连接入pCDH质粒，得到目的质粒。根据待包病毒的培养皿个数，计算病毒包装质粒PLP1、PLP2、PLPVSVG以及目的质粒的需要量。按照一个10cm培养皿PLP13.3μg、PLP23.3μg、PLPVSVG 3.3μg以及目的质粒10μg的体系进行转染。
(3)准备两个1.5mL EP管，分别标记为A、B管。A、B管各加入500μL opti-MEM，然后A管再加入PLP13.3μg、PLP23.3μg、PLPVSVG 3.3μg、目的质粒10μg以及LipofectamineP300020μL，B管中再加入Lipofectamine300020μL，分别混匀两管，室温静置5min。
(4)将B管中的混合液加入A管中，混匀后静置20min。
(5)静置的等待过程中，将要进行慢病毒感染的细胞培养基更换为无血清DMEM培养基。
(6)将A、B混合液滴加入换液后的HEK-293T细胞中，十字法摇匀后，放入细胞培养箱，8h后更换为含10％FBS的完全DMEM培养基。
(7)分别在24h，48h，72h收集病毒液即培养基上清，并更换新鲜的培养基，每次收集的病毒液可暂存在4℃冰箱，直至第三次收集完毕。
(8)将三次收集得到的病毒液用Backman超速低温离心机浓缩，浓缩后的病毒用600μL无血清培养基重悬，200μL每管分装后冻存于-80℃冰箱。
(9)用于构建稳定细胞株的SK-Hep-1细胞事先接种于六孔板中，待长到60％～70％汇合度后，更换新鲜的培养基，准备好的病毒液，每孔200μL，根据需要可每孔加入1μL的Polybrene，以提高病毒的感染效率。
(10)24h后用荧光显微镜观察荧光强度，根据荧光强度及比例判断病毒感染效率，然后加入Puromycin(10μg/mL)对细胞进行筛选。
(11)筛选期间可对细胞进行换液和传代，根据细胞状态及时调整筛选浓度，筛选过程持续至细胞不再死亡。
(12)收集部分构建的稳定细胞株的RNA进行PCR，蛋白质进行质谱的PRM靶向蛋白定量，以检测目的基因是否成功过表达。
(13)检测到稳定细胞株构建成功后，将细胞株冻存于液氮保种，用于后续实验。
2.Transwell实验检测微肽LINC01007-aa对肝癌细胞侵袭能力的影响
(1)将实验所需的SK-Hep-1细胞用胰酶消化下来，终止消化后离心弃去培养基，PBS清洗1～2遍以彻底去除血清的干扰，最后用无血清培养基重悬细胞，调整细胞密度为106个/mL；
(2)准备好Transwell小室(一般肿瘤细胞选用8μm孔径大小的小室)，在普通Transwell小室中提前铺好稀释过的基质胶(基质胶用无血清培养基稀释，比例一般为1:9，根据细胞侵袭能力可适当调整)；
(3)向铺了基质胶的Transwell小室中加入制备好的细胞悬液200μL，即2×105个细胞；
(4)向24孔板中加入500μL含10％FBS的完全培养基，将Transwell小室放入24孔板中。需要注意的是，下层培养基和小室间经常会产生气泡，而气泡的产生会影响下层培养基对上层细胞的趋化作用，因此需要提防气泡的产生，一旦产生气泡，需要将其除去后再将小室放入24孔板中培养；
(5)将带小室的24孔板放置于细胞培养箱中培养，18～48h后(时间根据细胞的侵袭能力不同而定)取出，进行后续处理；
(6)将培养基吸出，用PBS洗涤小室一到两遍后，4℃预冷的多聚甲醛固定液500μL加入下室中，对小室底部的细胞进行固定20min；
(7)弃去固定液，用PBS清洗3遍，将残余的PBS吸干净；
(8)加入500μL结晶紫染色液，室温孵育过夜；
(9)吸出结晶紫染液，PBS洗涤3遍后用棉签轻轻刮出小室中上层的细胞；
(10)室温风干后，显微镜下拍照，在20×10倍的视野下，取小室十字轴上的五个视野进行拍摄；
(11)计数每个视野的细胞数，最后进行统计分析，比较对照组和实验组间细胞侵袭速率的差异，结果如图5所示。
3.划痕实验检测SEP对细胞迁移能力的影响
(1)胰酶消化SK-Hep-1细胞，终止消化后300g离心3min，弃去培养基；
(2)用含10％FBS的完全培养基重悬细胞，调整细胞数为106个/mL；
(3)向Culture-Inserts划痕插件每小格内加入细胞悬液70μL，缓慢滴加，避免产生气泡，影响细胞贴壁，将划痕插件放入细胞培养箱；
(4)待细胞长满小格，尤其是要待划痕边缘的细胞完全长满后才可拔除插件，用无菌镊子夹着划痕插件一角，轻柔而快速地移除插件，注意不要手抖，导致细胞脱落，边缘不整齐；
(5)用37℃预热的PBS轻轻润洗，去除未粘附的细胞，再加入1mL完全培养基继续培养观察；
(6)以拔除划痕插件的时间点记为0h，显微镜下进行拍照观察，之后每隔一段时间进行一次拍照观察，直至细胞划痕完全愈合；
(7)用ImageJ软件对划痕面积进行统计，数据分析后作图，比较对照组和实验组的划痕差异。结果如图6所示。
综上所述，本发明通过蛋白质谱筛选提供了一种肝癌组织中LncRNA编码的微肽--LINC01007-aa在肝癌组织中显著高表达。通过单多因素cox分析显示微肽LINC01007-aa的表达量与肝癌患者的总生存显著相关。由此说明，微肽LINC01007-aa能够作为肝癌的潜在预后标志物。通过Transwell实验检测微肽LINC01007-aa对肝癌细胞侵袭能力的影响，结果发现过表达微肽LINC01007-aa的SK-Hep-1肝癌细胞株侵袭能力明显增强。通过划痕实验检测微肽LINC01007-aa对肝癌细胞迁移能力的影响，结果发现过表达微肽LINC01007-aa的SK-Hep-1肝癌细胞株迁移能力明显增强。由此说明，微肽LINC01007-aa也能够作为潜在的肝癌治疗靶点。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
Claims (9)
1.一种由长链非编码RNA LINC01007编码的微肽LINC01007-aa，其特征在于：其氨基酸序列如SEQ ID NO.1所示。
2.一种编码权利要求1所述的微肽LINC01007-aa的核酸分子，其特征在于：其核苷酸序列如SEQ ID NO.2所示。
3.一种重组载体，其特征在于：其含有权利要求2所述的核酸分子。
4.一种转基因细胞系，其特征在于：其含有权利要求3所述的重组载体。
5.如权利要求1所述的微肽LINC01007-aa在制备用于诊断肝癌的产品中的应用，其特征在于：所述产品为能够特异性检测微肽LINC01007-aa表达水平的产品。
6.如权利要求1所述的微肽LINC01007-aa在制备用于对肝癌患者的预后进行评估的产品中的应用，其特征在于：所述预后为患者的总生存期。
7.如权利要求1所述的微肽LINC01007-aa在制备具有促进肿瘤发展功能的产品中的应用，其特征在于：所述肿瘤为肝癌，所述肝癌的癌细胞为SK-HEP-1细胞，所述肿瘤发展体现为肿瘤细胞侵袭和/或迁移。
8.如权利要求1所述的微肽LINC01007-aa作为靶点在制备具有抑制肿瘤发展功能的产品中的应用，其特征在于：所述肿瘤为肝癌，所述肝癌的癌细胞为SK-HEP-1细胞，所述肿瘤发展体现为肿瘤细胞侵袭和/或迁移。
9.抑制权利要求1所述的微肽LINC01007-aa活性或表达的物质在制备抑制肿瘤发展的产品中的应用，其特征在于：所述肿瘤为肝癌，所述肝癌的癌细胞为SK-HEP-1细胞，所述肿瘤发展体现为肿瘤细胞侵袭和/或迁移。
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