CN109355389A - B4galnt2基因作为肝癌检测的生物标志物及应用 - Google Patents
B4galnt2基因作为肝癌检测的生物标志物及应用 Download PDFInfo
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Abstract
本发明确立了B4GALNT2基因可作为检测肝癌的标志物，进而得出：B4GALNT2基因的一种用途，用于制备肝癌分子检测的试剂或试剂盒，所述试剂是对应于B4GALNT2基因的特异性试剂；而且，适于靶向敲减RIMS3基因的表达的某些免疫细胞，可用于抑制肝肿瘤，例如CAR‑T细胞、CIK细胞和/或NK细胞；同理，特异性抑制B4GALNT2基因的化合物或抗体，可作为抑制剂用于抑制肝肿瘤。应用本发明进行肝癌检测，灵敏度高、特异性强、快速简便，将成为肝癌早期诊断和预后预测的重要手段；并且，可通过靶向敲减B4GALNT2基因的表达实现精准医疗。
Description
技术领域
本发明属于生物医学领域，涉及一种特定基因作为检测肝癌的标志物的应用。
背景技术
肝癌(Hepatocellular Carcinoma,HCC)是一种严重危害人类健康的常肿瘤，在全球癌症相关死亡率中排名第三。HCC肿瘤发生相对沉默，患者出现症状表现通常意味着已发展为晚期疾病。临床上有效治疗HCC多选择进行早期干预，中晚期潜在的治疗选择非常有限。如何针对肝癌的发生发展，阐明其分子调控机制，进行有效的治疗干预，是治疗肝癌的重要问题之一。
细胞遗传学、分子生物学及治疗靶点识别技术的发展，推动了靶向治疗及分层治疗水平的进步，明显提高了HCC临床疗效及总体生存率。但临床仍有部分难治性病例无法得到根治，同时对有些预后良好者又存在不同程度的过度治疗问题。由肿瘤组织所产生的能反映肿瘤存在的生化物质叫肿瘤标志物。
随着研究的不断深入，细胞遗传学和分子生物学技术在临床肝癌患者的诊断及治疗中的应用越来越广泛，已经成为肝癌患者的常规检测项目之一。在疾病的诊断、治疗、微小残留病(Mininal residual disease，MRD)的监测及预后判断等多个方面均发挥了重要作用。
近年来，蛋白组学技术的发展使得筛选新的肿瘤标志物成为可能，各种有希望的新的肿瘤标志物被相继发现，其中高尔基体蛋白73(GP73)最有可能成为更好的诊断肝癌，尤其是早期肝癌的血清标志物。在仅有的少数报道中，其敏感性可达69％，特异性可达90％。而其异构体敏感性和特异性可达90％和100％。除此之外，新发现的肝癌患者血清中高表达的标志物有甲胎蛋白异质体(AFP-L3)、异常凝血酶原(DCP)、α-L-岩藻糖苷酶(AFU)、磷脂酰基醇蛋白聚糖-3(GPC-3)、肝细胞生长因子(HGF)、转化生长因子β1(TGFβ1)、血管内皮生长因子(VEGF)、粘液素1(MUC-1，KL-1)等。这些新的肝癌标志物，正在被各种临床研究进行检测，有希望改变肝癌早期诊断的现状。
发明内容
在人体中，β-1，4-N-乙酰氨基半乳糖转移酶(Beta-1,4N-acetylgalactosaminyltransferase2，B4GALNT2)由B4GALNT2基因编码。申请人对B4GALNT2基因的功能进行了深入研究，具体如下：
以GAPDH为内参，qRT-PCR法检测B4GALNT2基因在肝癌模式细胞中的表达，结果表明B4GALNT2基因在模式细胞BEL-7404中表达显著增加。B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲低可以抑制人肝癌BEL-7404细胞增殖，诱导细胞凋亡，并增加Caspase3的活性。体内试验显示，接种B4GALNT2基因敲减的肝癌BEL-7404细胞裸鼠肝癌模型，肿瘤的生长受到显著抑制，动物的存活时间显著延长。
研究结论：B4GALNT2基因在肝癌组织中表达异常升高，敲减B4GALNT2的表达，可以诱导肝癌细胞凋亡，抑制细胞迁移和肿瘤的发生发展。
在以上研究结论的基础上，本发明确立了B4GALNT2基因可作为检测肝癌的标志物，进而得出以下方案：
第一方面，提供了B4GALNT2基因的一种用途，用于制备肝癌分子检测的试剂或含有该试剂的试剂盒，所述试剂是对应于B4GALNT2基因的特异性试剂。
进一步的，所述的检测为组织学检测或血液学检测。
进一步的，所述的试剂包括引物、探针、核酸芯片等。
第二方面，提供了一种用于检测肝癌的试剂或试剂盒，所述的试剂或试剂盒中含有检测B4GALNT2基因的相应试剂。
进一步的，所述的检测B4GALNT2基因的相应试剂选自下组：PCR检测试剂、分子杂交检测试剂等。
可在试剂盒中添加标签或说明书，所述标签或说明书注明所述试剂盒用于检测肝癌。
相应的，适于靶向敲减B4GALNT2基因的表达的某些免疫细胞，可用于抑制肝肿瘤，例如CAR-T细胞、CIK细胞和/或NK细胞。
相应的，特异性抑制B4GALNT2基因的化合物或抗体，可作为抑制剂用于抑制肝肿瘤。
本发明的效果如下：
应用本发明进行肝癌检测，灵敏度高、特异性强、快速简便，将成为肝癌早期诊断和预后预测的重要手段；并且，可通过靶向敲减B4GALNT2基因的表达实现精准医疗。
附图说明
图1.B4GALNT2基因在BEL-7402，BEL-7404，BEL-7404和SMMC-7721四种肝癌模式细胞中表达。
图2.Celigo法检测B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲低对人肝癌BEL-7404细胞的增殖的影响。
图3.CCK-8法检测B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲低对肝癌BEL-7404细胞增殖速率的影响。
图4.B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲除对肝癌BEL-7404凋亡率的影响。
图5.B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲低对人肝癌BEL-7404细胞Caspase3活性的影响。
图6.B4GALNT2(PSC38671)敲低对肝癌动物模型的抗肿瘤效果；其中，图6A为对照组和RIMS3敲低组动物中的肿瘤组织，图6B为对照组和RIMS3敲低组动物中的肿瘤组织重量的柱状图。
具体实施方式
本领域技术人员可以通过常规方法获得B4GALNT2基因的核苷酸序列，例如从NCBI、Genebank等数据库获取。
1.B4GALNT2基因在BEL-7402，BEL-7404，BEL-7404和SMMC-7721四种肝癌模式细胞中表达
细胞培养：培养人肝癌细胞的培养基为RPM I-640(含10％小牛血清，抗生素浓度为100μg/ml链霉素和l00U/L青霉素)。细胞培养在CO2浓度为5％，37℃的细胞培养箱中。收集对数生长期的肝癌细胞到一个离心管中，然后加入1-2倍吸取量的PBS充分混匀，室温1500rpm离心5min。离心结束后弃上清，重新加入1ml PBS重悬离心管中的细胞团块，并移至1.5ml的EP管中，室温1500rpm离心5min。去除残留的PBS，根据离心管中团块的量进行分装。分别用于mRNA和蛋白质的检测。
总RNA提取：将需要提取总RNA的细胞室温静置5分钟，用12,000g转速4℃离心5分钟，而后将上清转移至新的1.5ml RNase-free的EP管中。在新的EP管中加入200μl的氯仿，振荡混匀，室温静置5分钟，用12,000g转速4℃离心15分钟。吸取上清液转移至新的1.5mlRNase-free的EP管中，加入600μl的异丙醇。上下颠倒数次后，室温静置10分钟，用12,000g转速4℃离心15分钟。小心弃去上清，加入1ml 75％乙醇清洗沉淀，用7500g转速4℃离心5分钟。轻轻弃上清，保留沉淀，然后打开EP管盖，室温干燥10分钟左右。最后加入适量的RNase-free水溶解沉淀。用Nanodrop微量核酸蛋白定量仪进行RNA纯度和浓度的测定。样本保存于-80℃冰箱中以供后续实验检测。总RNA的提取：急性髓系白血病患者白细胞，收集到1.5mL EP管中，采用Trizol试剂盒提取总RNA。提取完毕后取1μL加入79μL DEPC水测OD260/OD280，计算浓度与纯度，-70℃保存。
cDNA第一链的合成：应用GIBICOL公司SuperScript TM PreamplificationSystem for First Strand cDNA Synthesis试剂盒合成cDNA。
PCR扩增：取0.5ml PCR管，加入cDNA、上游引物(10pM)、下游引物(10pM)、dNTP(2mM)、10×PCR buffer、Taq酶(2u/μL)、适量的ddH2O，使总体积达50μL。轻轻混匀，离心，设定PCR程序扩增条件：先94℃变性模板5min，然后分别进行94℃1min，60℃1min，72℃将产物延伸3min，此循环进行30次，最终产物72℃5min。
结果：以GAPDH为内参，QPCR结果提示B4GALNT2基因在模式细胞BEL-7404中表达较高(图1)。图中纵坐标为ΔCt，ΔCt＝Ct目的基因-Ct内参基因，ΔCt相对较大的细胞，目的基因表达丰度相对较低。
2.B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲低对人肝癌BEL-7404细胞的增殖的影响
参与肝癌细胞凋亡的分子机制研究需要设计合成特异性shRNA，转染方法参照HiPerFect transfection reagent试剂盒说明书进行。本研究所用的目的基因shRNA以及阴性对照shRNA由美国Invitrogen公司设计和合成。进行shRNA转染时，参照转染试剂HiPerFect transfection reagent使用说明书，将50nM的shRNA与12μl的转染试剂轻轻混匀，室温静置5-10分钟后，将转染试剂与shRNA混合物均匀滴加在6孔细胞培养板相应孔中，轻轻摇晃孔板混匀，置于细胞培养箱中继续培养。3天后经Real-time PCR和Westernblotting检测干扰效率。从第四天开始，每天观察细胞形态，并用荧光显微镜观察细胞增殖状态。
结果：B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲低的人肝癌BEL-7404细胞的数量显著少于shRNA组细胞(图2)。
3.CCK-8法检测B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲低对肝癌BEL-7404细胞增殖率的影响
将180μl(含细胞0.5×104)对数生长期shB4GALNT2干扰的肝癌BEL-7404细胞悬液加入到96孔培养板中，培养24h后，每孔加入CCK-8溶液20μl。继续培养6h，观察细胞形态后，小心地吸去培养液，再向每孔加二甲基亚砜150μl后，在微孔板震荡器上震荡10min，再在490nm波长的条件下，用RF-6100酶标分析仪，测定吸光值(OD值)。
结果：经shRNA慢病毒感染3天后，连续5天用CCK-8连续检测细胞增殖，发现B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲低的肝癌BEL-7404细胞增殖速率受到显著抑制，表明B4GALNT2基因与BEL-7404细胞的增殖能力显著相关(图3)
4.B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲减对肝癌BEL-7404凋亡的影响
将BEL-7404细胞及B4GALNT2基因敲除的肝癌BEL-7404细胞培养24小时后，胰酶消化，然后1000rpm离心5min，弃去培养液，收集细胞。再用PBS清洗两次，调整各组细胞数为2×106/ml。然后取400μl细胞悬液，加入200μl PBS洗涤，弃上清，加入400μl bindingbuffer和10μl AnnexinV-FITC(20μg/ml)，混匀，4℃避光孵育15分钟，再加5μl碘化丙啶(PI)
(20μg/ml)于细胞悬液中，混匀，4℃避光孵育5分钟，室温放置10分钟，采用流式细胞仪检测细胞凋亡率。该实验重复三次，最后数据做统计学分析。
结果：shB4GALNT2慢病毒感染4天后，与shRNA组比较，shB4GALNT2组凋亡细胞数量显著增多，表明B4GALNT2(PSC38671)和B4GALNT2(PSC38672)敲减可以诱导肝癌BEL-7404凋亡(图4)。
5.B4GALNT2(PSC38671)、B4GALNT2(PSC38672)敲低对人肝癌BEL-7404细胞Caspase3活性的影响
将shRNA干扰BEL-7404细胞及B4GALNT2基因敲除的肝癌BEL-7404细胞培养24小时后，胰酶消化，然后1000rpm离心5min，弃去培养液，收集细胞。再用PBS清洗两次，用Caspase3/7检测试剂盒，检测Caspase3/7的活性。该实验重复三次，最后数据做统计学分析。
结果：shRNA慢病毒感染4天后检测Caspase3/7活性，结果显示：实验组Caspase3/7活性增加，提示B4GALNT2基因与BEL-7404细胞的凋亡显著相关(图5)。
6.B4GALNT2(PSC38671)敲低对肝癌动物模型的肿瘤抑制作用的影响
将BEL-7404细胞及B4GALNT2基因敲除的肝癌BEL-7404细胞培养24小时后，胰酶消化，然后1000rpm离心5min，弃去培养液，收集细胞。
将BALB/C-nu裸鼠在SPF条件下饲养，收集EDTA消化的对数生长期的shRNA干扰的人肝癌BEL-7404(NC组)细胞及B4GALNT2基因敲减的肝癌BEL-7404细胞(KD组)，将其配制成浓度为1×107个/ml的单细胞悬液，按照0.3ml/只接种到裸鼠皮下，接种后观察。对于各组裸鼠的精神、饮食、活动等情况每日详细观察并且进行记录，用药后处死动物，称重肿瘤组织。
结果：接种B4GALNT2基因敲减的肝癌BEL-7404细胞的裸鼠肝癌模型，肿瘤的生长受到显著抑制(图6A为对照组和RIMS3敲低组动物中的肿瘤组织)，动物的存活时间显著延迟(图6B为对照组和RIMS3敲低组动物中的肿瘤组织重量的柱状图)。
以上实验表明，B4GALNT2基因可作为肝癌检测的生物标志物，用于肝癌的临床分子诊断标志物及药物开发、体外诊断试剂及相关试剂盒的开发。其中涉及的特异性检测B4GALNT2基因的引物、探针以及核酸芯片的设计方法属于常规技术手段。
Claims (9)
1.B4GALNT2基因作为肝癌检测的生物标志物的应用，用于制备肝癌早期检测的试剂或含有该试剂的试剂盒，所述试剂是对应于B4GALNT2基因的特异性试剂。
2.根据权利要求1所述的应用，其特征在于：所述的检测为组织检测或血液学检测。
3.根据权利要求1所述的应用，其特征在于：所述的试剂为引物、探针或核酸芯片。
4.一种用于检测肝癌的试剂，其特征在于：所述的试剂中含有检测B4GALNT2基因的相应试剂。
5.根据权利要求4所述的用于检测肝癌的试剂，其特征在于：所述的检测B4GALNT2基因的相应试剂为PCR检测试剂或分子杂交检测试剂。
6.包含有权利要求4所述用于检测肝癌的试剂的试剂盒。
7.特定免疫细胞在抑制肝肿瘤方面的应用，其特征在于：所述特定免疫细胞适于靶向敲减B4GALNT2基因的表达。
8.根据权利要求7所述的应用，其特征在于：所述特定免疫细胞为CAR-T细胞、CIK细胞和/或NK细胞。
9.一种抑制剂在抑制肝肿瘤方面的应用，其特征在于：所述抑制剂为特异性抑制B4GALNT2基因的化合物或抗体。
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