CN117310167B - 一种蛋白质amotl2在制备子宫内膜癌诊断标志物中的应用 - Google Patents
一种蛋白质amotl2在制备子宫内膜癌诊断标志物中的应用 Download PDFInfo
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Abstract
本发明公开了一种AMOTL2蛋白质作为子宫内膜癌诊断标志物的应用,该蛋白质是AMOTL2,由779个氨基酸组成,蛋白大小约为85.8kDa,其特点是蛋白质AMOTL2在子宫内膜癌诊断中的应用。主要通过免疫组织化学技术进行分析,比较AMOTL2蛋白质在子宫内膜癌患者样本和正常样本中的表达,以用于辅助诊断子宫内膜癌,具有灵敏性高、特异性强、周期短等特点,有利于子宫内膜癌的早期诊断和治疗,并且在子宫内膜癌中具有成为新型生物标志物及有效治疗靶点的潜力。
Description
技术领域
本发明属于分子生物学领域,具体涉及蛋白质AMOTL2作为子宫内膜癌诊断标志物的应用。
背景技术
子宫内膜癌(Endometrial cancer,EC)是女性中第六大常见癌症,2020年全球有41.7万例新诊断。女性患子宫内膜癌的终生风险约为3%,而在过去30年中,总发病率上升了132%。其中2型包括3级子宫内膜样癌和非子宫内膜样癌,它们由萎缩性子宫内膜癌发展而来,预后较差。手术是治疗子宫内膜癌较为有效且常用的治疗手段,虽然该疾病的普遍预后较好,但低分化的子宫内膜癌存在极高的复发转移的倾向,晚期子宫内膜癌患者复发率约为52%,且复发性子宫内膜癌预后较差,成为困扰许多学者的问题。虽然临床已有应用术后辅助放化疗来减少子宫内膜癌患者的复发率,但大量研究报道显示,放化疗并不能改善子宫内膜癌患者的5年生存率和无瘤生存时间。而随着精准医疗的发展,根据患者基因谱特征实施的靶向治疗逐步成为研究热点,精准医疗也成为子宫内膜癌治疗的具有指导意义和广阔应用前景的新疗法因此,寻找子宫内膜癌治疗的新靶点十分必要。
目前子宫内膜癌的治疗首选手术切除,并根据手术的病理分期和组织学分级,辅以放疗和化疗。此外,雌激素受体和孕激素受体阳性的患者也可以考虑进行内分泌治疗。研究表明,抗雌激素药物他莫昔芬(Tamoxifen,TAM)对雌激素受体阳性乳腺癌患者的治疗效果显著,但对子宫内膜癌的治疗效果甚微,甚至可能导致子宫内膜癌发病率的增加。这些治疗对提高子宫内膜癌患者的生存率固然有效,但对于III、IV期子宫内膜腺癌患者,传统的手术治疗和辅助放化疗并不能提高患者的生存率,反而会导致预后不良。而随着精准医疗的发展,根据患者基因谱特征实施的靶向治疗逐步成为研究热点,精准医疗也成为子宫内膜癌治疗方法中具有指导意义和广阔应用前景的新疗法。因此,寻找在子宫内膜癌复发转移中起重要作用的关键信号分子,阐明其参与的相关信号通路,并设计靶向的诊疗手段或许是未来解决子宫内膜癌复发转移问题的可行思路。
AMOTL2的表达具有细胞类型和组织特异性,AMOTL2主要在心脏和骨骼肌中表达,AMOTL2调节细胞骨架组织和尖端紧密连接。在培养的人脐静脉内皮细胞和MAE细胞中,AMOTL2基因敲除抑制细胞的增殖和迁移,并破坏细胞的极性。此外,在乳腺上皮细胞中,AMOTL2的下调促进了细胞形态和上皮-间充质转化(epithelial-mesenchymaltransition,EMT)的变化,暗示了AMOTL2潜在的抑癌作用。目前国内外还没有公开任何关于在子宫内膜癌中将AMOTL2蛋白作为诊治标志物的相关研究报道。
发明内容
本发明所要解决的技术问题是提供一种灵敏度和特异性高且与子宫内膜癌的患病率呈负相关的蛋白质AMOTL2在制备子宫内膜癌诊断标志物或子宫内膜癌分子靶向药物中的应用。
本发明解决上述技术问题所采用的技术方案为:一种蛋白质AMOTL2在制备子宫内膜癌诊断标志物的应用。
进一步,所述的蛋白AMOTL2由779个氨基酸组成,蛋白大小约为85.8kDa。
一种蛋白质AMOTL2在制备子宫内膜癌分子靶向药物中的应用。
进一步,所述的蛋白质AMOTL2的基因片段或基因的剪切体表达产物在制备子宫内膜癌分子靶向药物中的应用。
进一步,所述的蛋白质AMOTL2的基因编码的蛋白质抗体在制备子宫内膜癌分子靶向药物中的应用。
进一步,所述的蛋白质AMOTL2的基因编码的蛋白质抗体在制备子宫内膜癌分子靶向药物中的应用。
与现有技术相比,本发明的优点在于:本发明首次公开了可用于检测与子宫内膜癌相关的蛋白质AMOTL2及其在制备检测子宫内膜癌试剂或者辅助诊断子宫内膜癌试剂中的应用,所述的检测主要通过免疫组织化学技术完成,利用对免疫组织化学染色图片进行分析,通过Image J软件计算比较AMOTL2蛋白质在子宫内膜癌患者样本和正常样本中的表达,以用于辅助诊断子宫内膜癌。相对传统子宫内膜癌检测技术具有灵敏性高、特异性强、周期短等特点,有利于子宫内膜癌的早期诊断和治疗,并且AMOTL2在子宫内膜中具有成为新型生物标志物及有效治疗靶点的潜力。
附图说明
图1为免疫组化验证AMOTL2在人子宫内膜癌组织及配对癌旁组织中的表达情况。(A)69对人子宫内膜癌组织及配对癌旁组织中,AMOTL2免疫组织化学染色的三个代表性图像(n=69);(B)69对人子宫内膜癌组织及配对癌旁组织免疫组化学染色结果的堆积柱状图(n=69,****:p<0.0001)。(其中“Negative”表示AMOTL2不表达,“Low Positive”表示AMOTL2低表达,“High Positive”表示AMOTL2高表达);
图2为蛋白质免疫印迹技术和qRT-PCR筛选AMOTL2敲除的siRNA,证实si-AMOTL2#4为敲除的siRNA,作为后续实验所描述的si-AMOTL2。(A、B)蛋白质免疫印迹技术和qRT-PCR筛选AMOTL2敲除的AN3CA。(C、D)蛋白质免疫印迹技术和qRT-PCR筛选AMOTL2敲除的HEC-1-A(***:p<0.001);
图3为CCK8实验验证AMOTL2表达水平子宫内膜癌细胞生长的影响。(A)CCK8实验检测AMOTL2表达水平对AN3CA细胞生长的影响;(B)CCK8实验检测AMOTL2表达水平对HEC-1-A细胞生长的影响(**:p<0.01,***:p<0.001,****:p<0.0001);
图4为Transwell迁移实验验证AMOTL2表达水平对子宫内膜癌细胞迁移的影响(A)过表达或敲除AMOTL2后AN3CA和HEC-1-A细胞的迁移实验;(B)细胞迁移实验结果量化(**:p<0.01,***:p<0.001);
图5为克隆形成验证AMOTL2表达水平对子宫内膜癌细胞生长的影响。(A)克隆形成实验检测过表达或敲除AMOTL2后AN3CA和HEC-1-A集落形成能力;(B)克隆形成试验结果的量化(**:p<0.01,***:p<0.001)。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
一、实验方法
1、子宫内膜癌组织标本收集
纳入研究的69对人子宫内膜癌组织白片源自2019年01月01日至2022年12月31日宁波市临床病理诊断中心和宁波大学附属人民医院确诊为子宫内膜癌。其中21对人子宫内膜癌组织白片来源于宁波市临床病理诊断中心,48对人子宫内膜癌组织白片来源于宁波大学附属人民医院。
本实验涉及的所有人体组织标本,均经课题组申请,在宁波大学医学院人体伦理委员会同意的条件下,只限实验室研究。
2、标本免疫组织化学染色
a.烤片:将附有组织的载玻片置于已预热65℃的烤片机上进行烤片,至少烘烤2h,或放于37℃烘箱过夜;
b.脱蜡与复水:将满载切片的玻片架依次放入二甲苯中浸泡20min;无水乙醇溶液中浸泡5min×2次;95%乙醇溶液中浸泡5min×1次;75%乙醇溶液中浸泡5min×1次;最后将玻片架置于去离子水中洗涤5min;
c.抗原修复:将50×的pH9.0 EDTA抗原修复液用去离子水稀释到1×并摇晃均匀,将1×EDTA抗原修复液倒进高压锅中,合上锅盖,待锅内液体沸腾,将装有切片的玻片架置于高压锅中,使柠檬酸钠抗原修复液完全浸没切片,旋紧锅盖,待高压锅气阀开始均匀冒气后开始计时,10min后关闭电磁炉电源,结束加热。将高压锅置于流动自来水下降温,打开锅盖,使石蜡切片冷却至室温;
d.洗涤:首先使用去离子水充分洗涤切片5min,接着用PBST缓冲液洗涤切片3min,重复洗涤3次;
e.封闭内源性过氧化物酶:将30%过氧化氢水溶液用去离子水稀释至3%浓度,每次现配现用,将石蜡切片放于湿盒内,在载玻片上组织所在位置滴加适量3%过氧化氢水溶液,合上湿盒的盖子,37℃封闭20min;
f.洗涤:用PBST缓冲液洗涤切片3min×3次,甩掉载玻片上的液体;
g.血清封闭:洗涤后的切片摆放于湿盒内,每张切片上滴加适量10%驴血清封闭液,室温下封闭15min后,甩掉切片上的封闭液;
h.一抗孵育:擦净组织周围的封闭液,用免疫组织化学染色专用疏水笔围绕组织画圈,将切片置于湿盒内,滴加适当浓度的AMOTL2抗体稀释液,使抗体稀释液浸没组织,合上湿盒盖子,置于4℃冰箱过夜;
i.洗涤:用PBST缓冲液洗涤切片3min×3次;
j.二抗孵育:擦干组织周围的液体,在组织上滴加适当浓度的HRP标记驴二抗,使抗体完全覆盖组织,合上湿盒盖子,37℃孵育1h;
k.洗涤:用PBST缓冲液洗涤切片3min×3次;
l.DAB显色:将DAB显色液滴加到载玻片上组织所在位置,将载玻片置于倒置显微镜上观察组织染色情况,待染色强度达到最佳时甩掉显色液,使用去离子水洗涤3min×3次;
m.苏木素复染:在载玻片上组织所在位置滴加适量改良Lillie-Mayer苏木素染液,染色10s,甩掉染色液,将装有载玻片的玻片架置于流动自来水下洗涤1min以返蓝;
n.脱水:将装有切片的玻片架依次浸泡于75%乙醇溶液5min×1次;95%乙醇溶液中浸泡5min×1次;无水乙醇溶液中浸泡5min×3次;
o.透明:将切片置于二甲苯溶液中浸泡5min×2次;
p.封片:在载玻片上组织所在位置滴加适量中性树胶,用镊子夹取一张盖玻片轻轻覆盖在载玻片上,置于通风橱中晾干成片;
q.评分:所有样本均由两名在IHC评估方面经验丰富的独立病理学家审查,他们不了解这些患者的临床结果。我们评估了阳性染色细胞的百分比和染色强度,以半定量地确定AMOTL2表达。阳性染色细胞百分比评分如下:0,<10%;1,10%-50%;2,>50%。染色强度分级如下:0(无或弱染色=浅黄色)、1(中度染色=黄褐色)和2(强染色=棕色)。AMOTL2表达的总分是对阳性染色评分的细胞百分比和染色强度评分的总和,总分从0到4。对于统计分析,最终分数是本研究中报告的两位病理学家分配的独立分数的组合。通过两位病理学家之间的讨论解决了分数上的任何差异。
2、蛋白质免疫印迹技术以及qRT-PCR筛选si-AMOTL2
表1si-AMOTL2序列
a.蛋白质免疫印迹技术
(1)取于12孔板分别瞬时转染Negative Control,si-AMOTL2#1-4,48h后、生长良好的AN3CA和HEC-1-A子宫内膜癌细胞,吸尽培养液,每孔加入100ul细胞裂解液RIPA弱,4℃摇床20分钟后转入-80℃冰冻过夜;
(2)将样品从-80℃取出,4℃融化后转入1.5mlEp管中,4℃离心12000r 20min后,取上清加入4XSDS后,95℃金属浴煮5min;
(3)提前制备10%的WB胶备用,跑电泳恒压80V、转膜恒流220mA 120min、TBST洗膜5min,用TBST配置的5%奶粉封闭40min后洗膜,敷对应抗体过夜;
(4)提前于暗室准备显影液、水和定影液,将滴完ECL发光液的膜甩干后平铺在保鲜膜上,然后固定在暗盒中,于暗室进行压片曝光,曝光结束后打开暗盒,取出胶片,浸于显影液中,红灯下观察至条带出现,水中浸洗后放入定影液中,后开灯观察曝光结果。最后进行扫片分析结果。
b.qRT-PCR技术
(1)取分别瞬时转染Negative Control,si-AMOTL2#1-4,48h后、生长良好的AN3CA和HEC-1-A子宫内膜癌细胞,吸尽培养液,每孔加入1ml TRIGene,用加样器吹打数次,以确保细胞完全裂解,然后转移至离心管中;
(2)裂解产物于室温放置5min,使核酸-蛋白复合物完全分离;
(3)每1ml TRIGene加入0.2ml氯仿,盖紧管盖,剧烈振荡15s,室温放置2-3min;
(4)4℃12000xg离心15min,样品会分成三层:桔黄色的下层有机相,中间层和无色的上层水相;
(5)吸取含总RNA的上层水相至一新的离心管中,吸取水相的体积为所用TRIGene试剂的60%;
(6)按照每1ml TRIGene的最初使用量加入0.5ml异丙醇,颠倒数次混匀,室温放置10min;
(7)4℃12000xg离心10min,弃除上清,可见胶状的RNA沉淀;
(8)按照每1ml TRIGene的最初使用量加入1ml 75%乙醇,颠倒数次混匀,洗涤沉淀;
(9)4℃12000xg离心5min,弃除上清;
(10)室温倒置5-10min晾干;
(11)加入25μl DEPC-ddH2O,吹打数次溶解RNA;
(12)通过RNA电泳以及紫外分光光度计检测RNA的浓度、纯度和完整性;
(13)所得的RNA应立即使用或适量分装后-80℃保存,避免反复冻融;
(14)逆转录反应体系:
反应程序:
50℃ | 15min |
85℃ | 5sec |
(15)qRT-PCR检测
qRT-PCR引物:
AMOTL2-RTF | GCTCGTTGAGTGAACGGCT |
AMOTL2-RTR | CATGAGCTAGTACAACATGAGGG |
GAPDH-RTF | CATGGCCTTCCGTGTTCCTA |
GAPDH-RTR | CCCTCAGATGCCTGCTTCA |
qPCR管中配制如下混合液:
2×Taq Pro Universal SYBR qPCR Master Mix | 10.0μl |
Primer1(10μM) | 0.4μl |
Primer2(10μM) | 0.4μl |
Template DNA/Cdna | xμl |
ddH2O | To 20.0μl |
反应步骤:
3、CCK8细胞增殖试验
(1)取分别瞬时转染Negative Control,si-AMOTL2,FLAG-AMOTL224 h后、生长良好的AN3CA和HEC-1-A子宫内膜癌细胞,将贴壁细胞消化成均匀细胞悬液;
(2)吸取20μL上述均匀细胞悬液加入细胞计数板中,利用细胞计数仪测量其浓度,按照相应的比例稀释细胞悬液,用移液枪吹打均匀;
(3)取6个96孔细胞板,每孔加入2×103个细胞,定量0.1mL,每个处理组设置5个复孔,利用“8”字法摇匀,置于恒温培养箱;
(4)6块96孔细胞培养板分别培养至0,1,2,3,4,5天时,避光条件下每孔加入10μLCCK8试剂,放入恒温培养箱培养2h,见孔板变橙黄色。设置酶标仪波长为450nm,测定每个孔的吸光度,重读3次,记录并整理相应数据。
4、Transwell迁移实验
(1)取分别瞬时转染Negative Control,si-AMOTL2,FLAG-AMOTL224 h后、生长良好的AN3CA和HEC-1-A子宫内膜癌细胞,将贴壁细胞消化成均匀细胞悬液;
(2)将细胞悬液收集至1.5mL离心管中,以1,000rpm转速离心4min;
(3)弃上清液,用1mLPBS溶液重悬细胞沉淀,按上述方法再次离心,用1mLDMEM重悬细胞,取细胞计数板对其进行计数,用DMEM按需稀释;
(4)无菌镊子夹取3个Transwell小室,放进新的24孔细胞板,向上室中均加入150μL上述细胞悬液,定量5×104个细胞,在下室中加入500μL完全培养基,静置30min后将孔板放入恒温培养箱;
(5)48h后弃去小室内外培养基,用PBS缓冲液润洗小室和孔板底部2次;
(6)向小室内加入500μL多聚甲醛固定液,放置30min;
(7)弃固定液,PBS溶液润洗2次,加入500μL 0.1%结晶紫染料,将孔板放于摇床上染色20min;
(8)回收染色剂,用PBS溶液润洗3次,自然晾干小室,拍照并统计数据。
5、克隆形成
(1)取分别瞬时转染Negative Control,si-AMOTL2,FLAG-AMOTL224 h后、生长良好的AN3CA和HEC-1-A子宫内膜癌细胞,将贴壁细胞消化成均匀细胞悬液;
(2)吸取20μL上述均匀细胞悬液加入细胞计数板中,利用细胞计数仪测量其浓度,按照相应的比例稀释细胞悬液,用移液枪吹打均匀;
(3)取数个6孔细胞板,每孔加入1×103个细胞,定量2mL,每个处理组设置3个复孔,利用“8”字法摇匀,置于恒温培养箱,每5天换液一次,共培养10-15天。
(4)当六孔板内肉眼可见细胞集落时,弃去孔内培养基,用PBS缓冲液润洗2次,每孔加入适量4%多聚甲醛固定细胞,放置30min;
(5)弃去固定液,用PBS缓冲液润洗2次,向每孔加入800μL 0.1%结晶紫染料,放于摇床上染色10min,回收染料,用PBS缓冲液润洗3次,置于37℃烘箱过夜,待六孔板晾干后拍照并统计数据。
6、结果分析
本实验确保了实验数据及收集的原始临床资料进行全面检查、校对、整理,确保资料尽可能的完整、准确、无误。然后利用Excel软件分别建立组织、细胞数据库,对涉及的临床原始资料进行分组、归纳后将其录入表格中。最后利用SPSS26.0统计软件对最终的实验数据进行整理、分析。所有数据统计检验均取双侧概率,按照α=0.05的检验水准进行统计检验,若p<0.05,即认为两组间的差异有统计学意义,反之则认为差异无统计学意义。柱状图、折线图等实验结果图的绘制,利用GraphPad Prism 9.0、SPSS26.0软件共同完成。
二、实验结果
为了检验AMOTL2在子宫内膜癌组织中的表达情况,我们收集了69对人子宫内膜癌组织以及临近癌旁组织的石蜡切片,利用免疫组织化学染色检测临床样本中AMOTL2的表达水平。免疫组织化学染色结果显示,AMOTL 2染色表现为深浅不同的棕黄色且主要定位在癌旁组织及少量子宫内膜癌组织的细胞质内,与子宫内膜癌组织相比,AMOTL2在相应癌旁组织中表达较强。利用Image J软件对免疫组织化学染色图片进行分析,结果显示子宫内膜癌组织以及临近癌旁组织AMOTL2表达水平集中在negative(1分)、low positive(2分)、Positive(3分)这三个等级(图1A)。同时在子宫内膜癌组织中,AMOTL2的阳性表达率为33.9%(20/69),而癌旁组高达为69.6%(43/69),差异有统计学意义(X2=23.59,p<0.05)。AMOTL2在子宫内膜癌组织中的表达水平明显低于相应的癌旁组织(p<0.0001)(图1B)。
为了后续我们进一步验证AMOTL2蛋白功能的细胞功能实验,我们首先筛选了si-AMOTL2。我们使用si-AMOTL2敲低子宫内膜癌细胞株AN3 CA以及HEC-1-A中的AMOTL2,选择有效的si-AMOTL2,利用蛋白质免疫印迹技术及qRT-PCR技术进行进一步的确认(图2A-D)。最终结果显示si-AMOTL2#4为有效的si-AMOTL2。
在进一步验证AMOTL2对于子宫内膜癌细胞增殖以及迁移能力的影响的实验中,我们采用了CCK8实验、Transwell迁移实验、以及克隆形成实验。CCK8实验显示,与NC组子宫内膜癌细胞相比,AMOTL2高表达组细胞增殖的速度明显变慢,而si-AMOTL2组较NC组则表现出了增殖加快(图3A、B)。表明了AMOTL2具有可以抑制子宫内膜癌细胞的增殖的能力。
Transwell迁移实验显示,在子宫内膜癌细胞中,AMOTL2高表达组较NC组穿透Transwell小室的细胞数量明显减少,而si-AMOTL2组较NC组则表现出了穿透Transwell小室的细胞数量明显增多(图4A、B)。表明了AMOTL2具有可以抑制子宫内膜癌细胞迁移的能力。
克隆形成实验显示,NC组子宫内膜癌细胞相比,AMOTL2高表达组细胞集落的数量明显减少,而si-AMOTL2组较NC组则表现出了细胞集落的数量明显增加(图5A、B)。表明了AMOTL2具有可以促进子宫内膜癌细胞的增殖以及集落形成的能力。
这些结果提示检测AMOTL2蛋白质的相对用于的诊断具有较高价值,特别是免疫组化,AMOTL2在子宫内膜癌组织中的表达水平明显低于相应的癌旁组织(p<0.0001),因此检测AMOTL2蛋白质在子宫内膜组织的表达情况,可以做到灵敏度和特异性高的子宫内膜癌诊断。
该发明通过检测AMOTL2在子宫内膜癌组织的表达以及AMOTL2高表达以及低表达对子宫内膜癌细胞增殖迁移的影响,结合统计学原理和现代生物学技术,提供、灵敏度高、特异性强、周期短和结果稳定的检测方法,为子宫内膜癌患者的诊断和治疗提供科学依据,为分子靶向治疗提供可能。
上述说明并非对本发明的限制,本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内,做出的变化、改型、添加或替换,也应属于本发明的保护范围。
Claims (1)
1.一种非治疗目的的AMOTL2蛋白质在调控子宫内膜癌细胞增殖和/或迁移中的应用,其特征在于:敲低AMOTL2的表达,从而促进子宫内膜癌细胞的增殖和/或迁移,所述敲低AMOTL2的表达所采用的试剂为siRNA,所述siRNA的正义链为CCUGCGACUGUCAGAACAA,反义链为UUGUUCUGACAGUCGCAGG。
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