CN116814642A - 一种预测肝癌患者预后的生物标志物及其应用 - Google Patents
一种预测肝癌患者预后的生物标志物及其应用 Download PDFInfo
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Abstract
本发明公开了一种预测肝癌患者预后的生物标志物及其应用，所述生物标志物为SLC4A11基因。本发明通过研究发现SLC4A11基因在HCC中显著高表达且其表达与HCC的预后、TNM分期、血管浸润、肿瘤大小具有显著相关性；荧光多种免疫组织化学显示其表达与M2型巨噬细胞及CD4+T细胞的表达呈现显著正相关；利用特异性siRNA靶向调控SLC4A11基因的表达，减少SLC4A11基因的表达水平使得肝癌细胞的增殖和迁移能力大大降低，这说明SLC4A11基因的表达对于肝癌细胞至关重要。本发明为预防、诊断、检测和研究HCC提供了一种新的靶点来源，为治疗HCC提供了巨大临床应用前景。
Description
技术领域
本发明属于生物技术和医学领域，具体涉及一种预测肝癌患者预后的生物标志物及其应用。
背景技术
肝细胞癌(hepatocellular carcinoma，HCC)是世界上第六大最常见的恶性肿瘤，死亡率位居第三。虽然在临床和实验癌症治疗方面取得了很大的进展，但由于手术后肿瘤复发和转移率高，HCC患者的整体预后较差。
癌症的发展是在是在一个复杂的组织环境中，而肿瘤微环境(TME)是一个强有吸引力的治疗靶点。而TME中的肿瘤浸润免疫细胞(TIIC)在肿瘤进展方面起着重要作用临床数据强有力的证明，HCC的免疫细胞组成显然与整体预后和治疗反应有关。肝癌的免疫疗法很有希望但具有极大的挑战，因此，寻找可靠的肝癌免疫疗法和有效的肝癌治疗是非常重要的。
已知各种类型的免疫细胞表达单个溶质载体(SLC)，且SLC在调节巨噬细胞中也起着重要作用。肿瘤部位对癌症有着代谢活动极高的T细胞反应。SLC4家族成员的失调与某些癌症的病理发展有关，作为该家族中争议最大的SLC4A11，迄今为止还未证明其调控机制。近年来，陆续有学者进行了相关研究，对其在角膜内皮功能障碍以及听觉功能、肾脏、颌下腺、卵巢癌等进行了初步观察，但SLC4A11在HCC中的进展及在TME中的调节尚未有研究报道。因此，SLC4A11有望在HCC中的免疫治疗和预后评估方面带来新的突破，成为重要的治疗靶标。
发明内容
针对现有技术的不足，本发明提供一种预测肝癌患者预后的生物标志物及其应用，为肝癌预后和免疫治疗反应预测提供了一种新的指标，并为治疗肝癌提供了一种新的药物来源。
本发明是通过以下技术方案实现的：
一种预测肝癌患者预后的生物标志物，所述生物标志物为SLC4A11基因。
检测上述的生物标志物的产品在制备用于预测肝癌患者预后的试剂盒中的应用。
优选地，所述试剂盒通过检测受试者样本组织中SLC4A11基因的表达水平进行诊断。
优选地，肝癌预后良好的患者的样本中SLC4A11基因的表达水平显著下调。
靶向上述的生物标志物的siRNA在制备治疗肝癌的药物中的应用。
优选地，所述siRNA的正义链核苷酸序列如SEQ ID NO.1所示，反义链核苷酸序列如SEQ ID NO.2所示。
一种治疗肝癌的药物组合物，包括上述的siRNA，以及药学上可接受的辅料。
优选地，所述辅料包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体和润滑剂中的一种或多种。
优选地，所述药物组合物为口服剂型或非口服剂型。
优选地，所述药物组合物为片剂、胶囊剂、散剂、丸剂、颗粒剂、溶液剂、混悬剂、糖浆剂、注射剂、栓剂、吸入剂或喷雾剂。
本发明的有益效果如下：
本发明提供了HCC组织中的SLC4A11基因作为肝癌诊断、预后及免疫治疗预测的生物标志物的应用，明确了高表达SLC4A11基因预示着HCC的更差的预后。SLC4A11基因在HCC中显著高表达且其表达与HCC的预后、TNM分期、血管浸润、肿瘤大小具有显著相关性；荧光多种免疫组织化学显示其表达与M2型巨噬细胞及CD4+T细胞的表达呈现显著正相关；利用特异性siRNA靶向调控SLC4A11基因的表达，减少SLC4A11基因的表达水平使得肝癌细胞的增殖和迁移能力大大降低，这说明SLC4A11基因的表达对于肝癌细胞至关重要，为肝癌的临床治疗提供了新的靶点，通过检测该生物标志物的表达水平可判断患者是否患有肝癌，具有显著的临床应用前景。本发明为预防、诊断、检测和研究HCC提供了一种新的靶点来源，为治疗HCC提供了巨大临床应用前景。
附图说明
图1为实施例1中SLC4A11高、低表达患者的生存分析曲线图；
图2为实施例1中SLC4A11表达与各种免疫细胞的相关性；
图3为实施例2中SLC4A11在各肝细胞及肝癌细胞中的Western Blot结果(A)及mRNA表达水平(B)；
图4为实施例3中在Huh-7细胞中敲降SLC4A11的Western Blot结果(A)及mRNA表达水平(B)；
图5为实施例3中SLC4A11对Huh-7细胞增殖的影响。
具体实施方式
下面结合附图与具体实施例对本发明做进一步详细说明。
若无特殊说明，以下实施例中所用的技术手段，均为本领域技术人员所熟知的常规手段，未注明具体条件的实验方法，均为本领域常规方法。
以下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。
以下实施例中涉及的定量实验，均设置至少三次重复实验，结果取平均值。
以下实施例中所用HUVEC、THP-1、Huh1、Huh7、HLF、HepG2、LO2、LX2均购自中国科学院干细胞库。
实施例1SLC4A11基因与HHC患者临床病理特征及预后的相关性
1、组织信息
所有肝组织样本均来自南通大学附属第一医院肿瘤免疫治疗的HCC患者。本实施例共检测了120份HCC组织样本。所有患者在手术或活检前均未接受化疗或放疗。本实施例的研究经南通大学伦理委员会批准，并由患者或其监护人提供知情同意书。
本实施例中使用的组织微阵列(TMA)由南通大学医院临床生物标本库制作。TMA上代表直径为2mm的样品。本实施例从南通大学附属医院临床生物库的165名患者(120个肝癌组织和45个正常肝组织)中进行了回顾性采集的组织样本。在手术前，没有放疗、化疗、免疫治疗或其他治疗。道德批准得到了南通大学附属医院人类研究伦理委员会(中国江苏)的批准。
2、荧光多重免疫组织化学(mIHC)染色
用二甲苯脱蜡后用梯度乙醇法再水化。所有组织微阵列经脱蜡复水后，置于沸腾的AR6(AR600，AKOYA)缓冲液中，微波热基于荧光的mIHC的TMA切片以修复抗原诱导表位提取15min，修复抗原。组织芯片被阻断缓冲液阻断10min，4℃下加入一抗过夜，第二天，将其拿出复温30min，并在室温下加入二抗聚合物避光孵化10min，细胞核用DAPI染色并闭合。用Vectra 3扫描玻片。自动定量病理成像系统，用于生物标志物的检测和测量。
3、数据分析
根据mIHC的染色结果鉴定SLC4A11的表达，采用IBM SPSS Statistics v26(Endicott，New York，NY)对mIHC实验数据进行统计分析。从X-tile软件中选取61.4作为分界点将患者分为高表达组和低表达组，使用Pearson’sχ2用于确定SLC4A11表达与临床病理参数之间的相关性。P＜0.05表示差异具有统计学意义。
4、实验结果
表1 SLC4A11基因表达与临床特征的相关性
如表1所示，SLC4A11基因的高表达与TNM分期、血管浸润、肿瘤大小具有显著相关性(*P＜0.05)。
生存分析结果如图1所示，SLC4A11基因高表达患者的生存期较短(P＜0.001)，说明高表达的SLC4A11基因与较差的预后相关。
如图2所示，SLC4A11基因的表达与CD68、CD206、CD4的表达呈显著正相关，说明其与M2型巨噬细胞及CD4+T细胞的丰度呈正相关。
实施例2SLC4A11基因在肝癌细胞中的表达
1、肝细胞及肝癌细胞培养
将HUVEC、THP-1、LX2、LO2、Huh-1、Huh-7、Hepg2细胞培养于DMEM培养基(#11965092，Gibco，Thermo，USA)中，加入10％胎牛(#10099141，Gibco)和1％青霉素-链霉素(#15140122，Gibco)。
2、Western Blot检测
用RIPA裂解液(#P0013B，Beyotime Biotechnology，Shanghai，China)提取细胞总蛋白，与Gel Loading Buffer II(#AM8547，Invitrogen，USA)混合，在100℃下加热10min。等量的蛋白样品加入SDS-PAGE凝胶电泳分离，然后转移到PVDF膜上。将膜与5％脱脂牛奶室温孵育1h，然后将一抗与膜一起在4℃孵育过夜。将膜冲洗干净，与二抗室温孵育1h。使用ECL化学发光检测系统(Biorad)可视化蛋白质表达。
3、实验结果
如图3所示，与LO2细胞相比，SLC4A11基因在肝癌细胞系Huh-1、Huh7、Hepg2中相对高表达，说明SLC4A11基因在肝癌中的表达较高，可以作为一种肝癌筛查标志物。
实施例3干扰SLC4A11基因的表达对肝癌细胞增殖的影响
1、SLC4A11干扰片段的验证
Huh-7细胞被播种到6孔培养板中，并使用LipofectamineTM 2000用小干扰RNA(siRNA)转染给人类SLC4A11基因(siSLC4A11，GenePharma)。
靶向SLC4A11基因的siRNA(siSLC4A11)，核苷酸序列如下：
正义链(SEQ ID NO.1)：5’-GGACUUCACUGAUGGCAUUTT-3’；
反义链(SEQ ID NO.2)：5’-AAUGCCAUCAGUGAAUGCCTT-3’。
非靶向对照siRNA(siNC，GenePharma)被用作阴性对照。使用NCFAM荧光标记siRNA转染控制(Negative control FAM，GenePharam)来优化siRNA转染的浓度。击倒效率由转染48h后的实时定量反向转录PCR(RT-PCR)及Western Blot确定。
2、CCK-8增殖实验
使用计数室或细胞计数Kit-8试剂(CCK8，Dojindo Laboratories，熊本，日本)计数来测量HCC细胞的增殖能力。细胞被播种在96孔板中以每孔2.5×103个细胞播种，培养0h、24h、48h、72h。对于在96孔板中培养的细胞，CCK8的解决方案是添加10μL和37℃孵化2h。450nm的光学密度读数由微板读卡器(日本东京Bio-Rad)确定。
3、统计学方法。
使用GraphPad Prism 6软件进行统计分析。对于定量数据，将组报告为mean±SEM，并使用未配对的双尾t检验或One-way ANOVA检验，比较各组间差异的显著性，P＜0.05表示差异具有统计学意义。除非另有说明，否则所有实验重复三次以上。
4、实验结果
如图4所示，SLC4A11基因被干扰表达后，SLC4A11的表达在Huh-7细胞中的明显降低，差异有统计学意义(***P＜0.001)。说明干扰SLC4A11的表达是成功的。
如图5所示，干扰SLC4A11基因表达后Huh-7细胞增殖能力受到显著影响(**P＜0.01，***P＜0.001)。
以上实施例仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明的保护范围。
Claims (10)
1.一种预测肝癌患者预后的生物标志物，其特征在于，所述生物标志物为SLC4A11基因。
2.检测权利要求1所述的生物标志物的产品在制备用于预测肝癌患者预后的试剂盒中的应用。
3.根据权利要求2所述的应用，其特征在于，所述试剂盒通过检测受试者样本组织中SLC4A11基因的表达水平进行诊断。
4.根据权利要求3所述的应用，其特征在于，肝癌预后良好的患者的样本中SLC4A11基因的表达水平显著下调。
5.靶向权利要求1所述的生物标志物的siRNA在制备治疗肝癌的药物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述siRNA的正义链核苷酸序列如SEQ IDNO.1所示，反义链核苷酸序列如SEQ ID NO.2所示。
7.一种治疗肝癌的药物组合物，其特征在于，包括权利要求6所述的siRNA，以及药学上可接受的辅料。
8.根据权利要求7所述的药物组合物，其特征在于，所述辅料包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体和润滑剂中的一种或多种。
9.根据权利要求7所述的药物组合物，其特征在于，所述药物组合物为口服剂型或非口服剂型。
10.根据权利要求7所述的药物组合物，其特征在于，所述药物组合物为片剂、胶囊剂、散剂、丸剂、颗粒剂、溶液剂、混悬剂、糖浆剂、注射剂、栓剂、吸入剂或喷雾剂。
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