CN108409851B - 肾细胞癌中高水平表达的蛋白及其应用 - Google Patents

肾细胞癌中高水平表达的蛋白及其应用 Download PDF

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CN108409851B
CN108409851B CN201810084391.8A CN201810084391A CN108409851B CN 108409851 B CN108409851 B CN 108409851B CN 201810084391 A CN201810084391 A CN 201810084391A CN 108409851 B CN108409851 B CN 108409851B
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高维强
李力
饶汉钰
刘敏
蔡彪
刘璟
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Abstract

一种肾细胞癌中高水平表达的蛋白及其应用，该Di‑Ras2蛋白氨基酸序列如Seq ID No.1所示。本发明Di‑Ras2蛋白在肾细胞癌肿瘤中的过表达能够促进肾细胞癌的增殖和浸润，因此检测Di‑Ras2的表达水平来进行早期诊断，通过抑制Di‑Ras2的表达而由此将Di‑Ras2作为药物靶标来辅助临床手术治疗提高肾细胞癌病人的治愈率，改善病人的生活质量。

Description

肾细胞癌中高水平表达的蛋白及其应用
技术领域
本发明涉及的是一种生物医疗领域的技术，具体是一种在肾细胞癌中高水平表达的蛋白Di-Ras2及其在肾细胞癌诊断标志物或治疗靶标的上的应用。
背景技术
肾细胞癌的临床表现多样，从典型的三联症，血尿、疼痛和可能触及的肾脏肿块，到较隐匿的肿瘤周围综合征，有时肿瘤体积很大，甚至出现肺、骨等转移征象，但依然无任何症状。虽然以手术为主的肾细胞癌后期治疗可以在很大程度上缓解病情，但对发生侵袭转移的肾细胞癌的疗效并不十分理想。早期肾细胞癌的手术疗效虽较好，但对某些病例也可采取非手术的方法治愈，因此除了定期体检外，还应积极探索其他治疗方法，或者以手术治疗为主的综合治疗方案，以提高疗效。到目前为止，很多生物标志物都不能很好的用来作为临床诊断标识，仅有助于判别肿瘤的预后及化疗疗效。所以还没有很好的分子诊断标识来辅助常规诊断检查，以及作为治疗靶标辅助外科手术治疗。
寻找肿瘤组织特异性高水平表达的蛋白标志物，并以之作为诊断治疗的靶点，是近来被认为是手术治疗的积极有效的辅助手段。肿瘤的发生发展与某些特意的信号通路激活、基因活化有关。某些膜蛋白的激活导致信号通路的活化，进一步导致某些基因的激活，维持肿瘤的发生发展。寻找这些差异性表达的(膜)蛋白，以此作为诊断肿瘤的标志物。如果是有具体功能的蛋白标志物，则可以结合开发特异性的封闭/激活抗体(药物)，定向的干扰/活化肿瘤组织中标志物的活性，结合临床手术治疗达到更有效的治疗肿瘤。
发明内容
本发明针对现有技术存在的上述不足，提出一种肾细胞癌中高水平表达的蛋白及其应用。
本发明是通过以下技术方案实现的：
本发明通过基因芯片筛选得到Di-Ras2等在肾细胞癌病人组织中高表达的基因，其中Di-Ras2的表达变化最为显著，其氨基酸序列如Seq ID No.1所示。
本发明涉及一种Di-Ras2蛋白的应用，将其用于制备肾细胞癌药物或治疗靶标。
技术效果
与现有技术相比，本发明Di-Ras2蛋白在肾细胞癌肿瘤中的过表达能够促进肾细胞癌的增殖和浸润，因此检测Di-Ras2的表达水平来进行早期诊断，通过抑制Di-Ras2的表达而由此将Di-Ras2作为药物靶标来辅助临床手术治疗提高肾细胞癌病人的治愈率，改善病人的生活质量。
附图说明
图1A为通过免疫组化检测组织样品中Di-Ras2的表达情况，比较成对的肿瘤组织与相邻的癌旁组织，Di-Ras2的表达水平；图1B为TCGA数据库中Di-Ras2表达强度分析，Di-Ras2的表达水平与临床分级的相关性，n＝623；图1C为Di-Ras2表达水平与病人预后生存情况的相关性分析；图1D和E为通过western blot，RT-PCR实验检测14对新鲜组织样本中，成对的样本(正常组织，肿瘤组织)中Di-Ras2蛋白水平的表达量；实验结果表明Di-Ras2在部分肾细胞癌中表达升高，并且与临床症状有紧密的相关性；
图2A为在786O和A498转染Di-Ras2过表达病毒建立的稳定细胞系中，通过RT-PC和Rwestern blot实验检测Di-Ras2的表达情况；图2B为两组细胞系通过细胞数目和MTS方法检测细胞的增值情况；图2C为两组细胞系通过细胞划痕方法检测细胞的迁移情况；图2D为两组细胞系通过metri-gel trans-well方法检测细胞的侵袭情况；图2E为两组细胞系通过核酸PI染色方法检测细胞的周期变化情况；其中*表示p<0.05,**表示p<0.01,***表示p<0.001；
图3A为在786O和A498中转染干扰Di-Ras2表达的慢病毒，通过RT-PCR和westernblot实验检测Di-Ras2的表达情况；图3B为两组细胞系通过细胞数目和MTS方法检测细胞的增值情况；图3C为两组细胞系通过细胞划痕方法检测细胞的迁移情况；图3D为两组细胞系通过metri-gel trans-well方法检测细胞的侵袭情况；图3E为两组细胞系通过核酸PI染色方法检测细胞的周期变化情况；其中*表示p<0.05,**表示p<0.01,***表示p<0.001。
具体实施方式
首先检测新的肾细胞癌肿瘤的诊断标志物和治疗靶标-Di-Ras2。Di-Ras2在肾细胞癌组织中比配对的正常肾组织中的表达水平明显升高，前期实验证明Di-Ras2与肾细胞癌的发生发展临床上存在紧密相关性，可以作为肾细胞癌诊断、辅助治疗的靶标分子。
本结果证实：肾细胞癌组织中Di-Ras2的表达量高于配对的癌旁组织，(图1A，B)。并且Di-Ras2的表达水平与病人的预后(图1C)都有一定的负相关性。通过western blot，RT-PCR对新鲜组织样本，从蛋白，RNA水平上对Di-Ras2表达量进行检测(图1D、E)，发现Di-Ras2在癌组织中的表达量高于在正常组织中的表达。
在此基础上通过干扰/过表达Di-Ras2的表达，研究缺失/激活Di-Ras2对肾细胞癌细胞增殖、迁移和克隆形成的影响。通过增强肾透明细胞癌细胞786O和A498中Di-Ras2的表达，可以促进786O和A498的增殖速率(图2B、2E)，增强迁移(图2C)和侵袭能力(图2D)，反之在786O和A498中降低Di-Ras2的表达，可以减弱细胞的增值速率(图3B、3E)，抑制迁移(图3C)和侵袭能力(图3D)。
实施例1
免疫组化实验
材料：10例新鲜组织样本(配对正常肾组织、肾细胞癌组织)；一抗为兔抗人Di-Ras2抗体(Proteintech，货号ag7926)，后续染色使用GTVision III免疫组化试剂盒(上海基因科技)。
方法：
1、四度冰箱中放置的组织切片，取出室温放置恢复室温，置于56℃孵箱中烘烤20min。将石蜡切片浸于二甲苯中5min，三次。取出切片置于100％无水乙醇中3min两次；依次置入90％-70％各级酒精各3min。用PBS冲洗3次，每次3min。
2、将组织用多聚甲醛固定液固定15min，PBS洗5min3次。
3、抗原修复：用0.01M柠檬酸盐溶液暴露抗原决定簇。微波炉高火3min加热至修复液沸腾(4min)，放入芯片再低火微波1min两次(及时补充修复液防止修复液沸腾溢出)。冷却至室温，PBS洗5min。用含0.5％Triton的PBS破膜15min，PBS洗5min3次。
4、封闭非特异性蛋白
1)3％H2O2-甲醇(30％H2O2 10mL+甲醇90mL)室温浸泡30min，消除内源性氧化还原酶。
2)自来水冲洗10min，PBS浸泡3min3次，将玻片甩干或用无尘纸吸去多余液体(勿碰到组织)。
3)PBS配制10％山羊血清。
4)向玻片组织上滴加10％山羊血清(用PBS配制)，放置于湿盒中封闭非特异性抗原(200μL/玻片)，室温1h。
5、一抗孵育:甩去切片上的10％山羊血清封闭液，用无尘纸擦干组织周围，直接加入已稀释的兔抗人的Di-Ras2抗体(约100μL，用PBS配置的1％山羊血清1:400配好一抗反应液)，置于湿盒中4℃过夜。第二天从冰箱中取出需37℃复温1h。
6、二抗孵育:
1)将一抗洗掉，将玻片插入塑料玻片架，然后整个放入塑料盒中，加PBS浸泡洗15min3次。
2)用无尘纸将玻片周围的PBS吸去，加入二抗(GTVisionTM III型聚合物)于，室温30min。孵育完毕，将切片置入PBS缓冲液中，冲洗3次，每次3min，取出切片，甩掉并擦干组织周围的液体(组织切勿干燥)，平放于湿盒中。
3)滴加预备好的显色剂DAB工作液50-100μL，室温孵育5-10min，或光镜下控制显色，自来水冲洗终止显色。
7、用苏木精复染细胞核，室温30秒，用自来水冲洗1h复染。
8、封片:各级酒精(70％-100％)脱水，每级3min。取出切片置入二甲苯5min，三次。在玻片上滴管滴加中性树脂，然后盖上盖玻片，用镊子轻轻挤压，并赶走气泡，通风橱中静置吹干，显微镜观察。
实施例2
实时PCR实验
材料：肾细胞癌组织样本，肾细胞癌细胞系。
1，利用传统的Trizol-氯仿-异丙醇抽提RNA的方法，通过组织冰冻碾磨，细胞裂解等手段，抽提组织和细胞中的RNA。运用PrimeScript RT reagent Kit with gDNA Eraser(Takara)试剂盒反转录RNA合成cDNA。进一步通过SYBR Green实时定量RT-PCR技术(TakaraRR420A)，检测组织，细胞水平上Di-Ras2基因的表达水平。在ABI 7900HT实时定量检测PCR结果。
2，用ΔΔct方法以内参GAPDH的表达水平标准化Di-Ras2基因的表达水平。用正常的肾脏组织作为对照，量化Di-Ras2的表达水平。
实施例3
细胞增殖实验
材料：细胞增殖检测试剂盒CellTiter 96AQueous(MTS)(Promega，G358A)；Matrigel(BD，货号：356234)。
1、体外细胞增值实验：取96孔板，每孔3,000个细胞，体积100μL，待到细胞贴壁后(大约四个h)加入20μL MTS反应液(作为起始时间0hr，此后选取24、48、72、96hrs时间点)，在37℃孵育2-4h，然后在酶标仪(BioTek)检测490nm处的吸光度。由于吸光度与细胞数目和活力是成正比的，所以能够线性
直观反映细胞数目大小。
2、体内成瘤实验：将786O和CAKI以及相应的干扰/过表达细胞系细胞悬浮于不含血清的基本培养基中，按照50μL基本培养基的含有1,000,000个肿瘤细胞的悬浮液与等体积的基质胶(货号356234)混合均匀，然后注射入小鼠的皮下100μL/只。每十天测量肿瘤的大小，计算的方法是V＝a2*b(其中V是体积，a是最短边的长度，b是最长边的长度)，称量裸鼠的体重。
实施例4
肿瘤迁移实验
材料：RMPI 1640基本培养液，PBS，六孔板。
步骤1)在六孔板中加入约5X105个细胞，具体数量因细胞不同而不同，掌握为过夜能铺满。
步骤2)第二天用枪头比着直尺，尽量垂至于背后的横线划痕，枪头要垂直，不能倾斜。
步骤3)用PBS洗细胞3次，去处划下的细胞，加入无血清培养基。
步骤4)放入37度5％CO2培养箱，培养。按0、6、12、24h取样，拍照。
步骤5)将786O和CAKI以及相应的干扰/过表达细胞系种于六孔板中。
步骤6)在六孔板中加入786O和CAKI以及相应的干扰/过表达细胞系，具体数量因细胞不同而不同，掌握为过夜能铺满。
步骤7)第二天细胞长满后用枪头比着直尺，尽量垂至于背后的横线划痕，枪头要垂直，不能倾斜。
步骤8)用PBS洗细胞3次，去处划下的细胞，加入无血清培养基。
步骤9)放入37度5％CO2培养箱，培养。按0、6、12、24h取样，拍照。
实施例5
肿瘤侵袭实验
材料：8μm的transwell小室(Corning，3422)，按照说明用matrigel包被。
步骤：做transwell实验时，先将细胞在无血清培养基中饥饿24h，然后加入tanswell小室上面1％BSA活化小室上面的基质胶半h，在transwell小室的上面加入100μL细胞悬液(无血清)，含有细胞10,000个。下面加入500μL的10％血清的完全培养液。在37度细胞培养箱中过夜。第二天，用4％多聚甲醛固定10min，置于PBS中放置10min，然后用0.1％的结晶紫染色10min，PBS清洗干净，用棉签将上面的细胞轻轻擦掉(不要影响小室下层细胞)，将小室放于置有500μLPBS的24孔板中，于倒置显微镜下观察、拍照。
实施例6
细胞周期实验
材料：无水乙醇20X PI(1mg/mL)100X RNase(10mg/mL)，细胞染液：5％PI(约50μg/mL)，100μg/mL RNAse，0.2％triton X-100。
步骤：
细胞消化后，600μLPBS重悬，加1400无水乙醇(终浓度为70％)，-20℃放置过夜；离心后用200μL染液重悬，37℃孵育40min；转移至1.5mL EP管，加PBS至400μL。流式细胞仪检测PI通道，分析DNA含量高的分裂期细胞数目。
综上，本发明Di-Ras2蛋白可以用来作为新型的肾细胞癌临床诊断标志物，并且可以用来作为新的药物靶标分子，通过抑制Di-Ras2的活性，结合临床手术来辅助治疗肾细胞癌等肿瘤。
上述具体实施可由本领域技术人员在不背离本发明原理和宗旨的前提下以不同的方式对其进行局部调整，本发明的保护范围以权利要求书为准且不由上述具体实施所限，在其范围内的各个实现方案均受本发明之约束。
序列表
<110> 上海交通大学
<120> 肾细胞癌中高水平表达的蛋白及其应用
<130> f-b111e
<141> 2018-01-29
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 199
<212> PRT
<213> Di-Ras2
<400> 1
Met Pro Glu Gln Ser Asn Asp Tyr Arg Val Ala Val Phe Gly Ala Gly
1 5 10 15
Gly Val Gly Lys Ser Ser Leu Val Leu Arg Phe Val Lys Gly Thr Phe
20 25 30
Arg Glu Ser Tyr Ile Pro Thr Val Glu Asp Thr Tyr Arg Gln Val Ile
35 40 45
Ser Cys Asp Lys Ser Ile Cys Thr Leu Gln Ile Thr Asp Thr Thr Gly
50 55 60
Ser His Gln Phe Pro Ala Met Gln Arg Leu Ser Ile Ser Lys Gly His
65 70 75 80
Ala Phe Ile Leu Val Tyr Ser Ile Thr Ser Arg Gln Ser Leu Glu Glu
85 90 95
Leu Lys Pro Ile Tyr Glu Gln Ile Cys Glu Ile Lys Gly Asp Val Glu
100 105 110
Ser Ile Pro Ile Met Leu Val Gly Asn Lys Cys Asp Glu Ser Pro Ser
115 120 125
Arg Glu Val Gln Ser Ser Glu Ala Glu Ala Leu Ala Arg Thr Trp Lys
130 135 140
Cys Ala Phe Met Glu Thr Ser Ala Lys Leu Asn His Asn Val Lys Glu
145 150 155 160
Leu Phe Gln Glu Leu Leu Asn Leu Glu Lys Arg Arg Thr Val Ser Leu
165 170 175
Gln Ile Asp Gly Lys Lys Ser Lys Gln Gln Lys Arg Lys Glu Lys Leu
180 185 190
Lys Gly Lys Cys Val Ile Met
195

Claims (1)

1.一种Di-Ras2蛋白的应用，其特征在于，将其用于制备调节肾透明细胞癌细胞786O和A498的增殖和迁移以及侵袭的药物；所述Di-Ras2蛋白的氨基酸序列如Seq ID No.1所示。
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