CN104004831B - Kin17基因或蛋白在制备肺癌诊断及治疗药剂中的应用 - Google Patents

Kin17基因或蛋白在制备肺癌诊断及治疗药剂中的应用 Download PDF

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CN104004831B
CN104004831B CN201410188632.5A CN201410188632A CN104004831B CN 104004831 B CN104004831 B CN 104004831B CN 201410188632 A CN201410188632 A CN 201410188632A CN 104004831 B CN104004831 B CN 104004831B
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Abstract

本发明公开了Kin17基因或蛋白在制备肺癌诊断、预后评估及治疗药剂中的应用，本发明研究发现，Kin17基因或蛋白可作为肺癌新的诊断标志及药物靶标，Kin17蛋白在肺癌中的表达明显升高，维持了肿瘤细胞的快速增殖活性，在肺癌的发生发展中发挥重要作用。沉默肺癌中Kin17的表达能抑制肺癌细胞的增值活性，也能增强肺癌细胞对化疗药物的敏感性。Kin17基因或蛋白可用于肺癌的早期鉴别诊断、患者预后评估及临床治疗。

Description

Kin17基因或蛋白在制备肺癌诊断及治疗药剂中的应用
技术领域
本发明涉及肺癌新的诊断标志及药物靶标，具体涉及Kin17基因或蛋白在制备肺癌诊断、预后评估及治疗药剂中的应用。
背景技术
肺癌是目前全球范围内发病率与致死率最高的恶性肿瘤，每年的新发肺癌病例有160万，占所有恶性肿瘤的13%，每年因肺癌导致的死亡患者有140万，占所有恶性肿瘤死亡的18%，严重危害着人类的生命健康。肺癌的恶性程度较高，易复发、转移，绝大多数患者在确诊时已属相对晚期，失去了根治性手术的机会。早期诊断和及早治疗对肺癌患者的疗效与预后至关重要。对于肺癌高危人群的筛查，一般根据病人的胸部症状、影像学检查（如X线胸片、CT、MRI、PET-CT等）和血液中肿瘤标志物（如CEA、CA125、NSE和CYFRA2121等）等。目前这些筛查指标还不够理想。很多肺癌患者在手术后易复发，而且外科手术和放疗都是局部治疗方式，无法控制肺癌的晚期转移。化疗是对晚期转移性癌症患者的最主要治疗手段。但常规的细胞毒性化疗药缺乏肿瘤细胞特异性，在杀死肿瘤细胞的同时，也杀伤了骨髓及其他增殖旺盛的正常细胞，不良反应严重；而且多次化疗后产生的耐药性也让化疗的疗效已经到达了平台期。为了进一步提高晚期肺癌的治疗效果，近年来靶向治疗方法越来越受到推崇。分子靶向治疗方法特异性强，细胞毒性小，在治疗晚期肺癌上具有独有的优势。虽然目前很多靶向药物的出现给肺癌患者带来了希望，但也在临床应用中存在着药物适用对象不广、单药疗效不强、与化疗药协同作用不佳、耐药性与不良反应严重等诸多问题。
肺癌癌细胞具有旺盛的增殖力、持续的DNA复制潜能和蓄积的DNA损伤。Kin17蛋白是一个在物种进化过程中十分保守的蛋白质，它主要与细胞的DNA复制、DNA修复及细胞增殖功能有关，在人体各组织器官中表达普遍较低。目前，关于Kin17基因或蛋白肺癌新的诊断标志及药物靶标还末见报道。
发明内容
本发明的目的在于提供Kin17基因或蛋白在制备肺癌诊断、预后评估及治疗药物中的应用。
发明人研究发现，Kin17蛋白在肺癌中的表达明显升高，维持了肿瘤细胞的快速增殖活性，在肺癌的发生发展中发挥重要作用。因此，Kin17基因（核苷酸序列见SEQIDNO：1）或蛋白（氨基酸序列见SEQIDNO：2）可作为肺癌新的诊断标志及药物靶标，Kin17基因或蛋白可用于肺癌的早期鉴别诊断、肺癌患者的预后评估分析及临床治疗。
附图说明
图1：Kin17在肺癌患者标本与良性病变患者标本中的表达情况；
图2：Kin17在肺癌标本的癌巢组织与A549细胞系中的表达明显高于癌旁正常组织；
图3：重组质粒pET32a-KIN17中插入的KIN17基因的部分测序图；
图4：IPTG诱导Kin17蛋白在大肠杆菌中原核表达的SDS-PAGE电泳图；
图5：纯化后的His标签Kin17蛋白的SDS-PAGE电泳图（左）与Western-blot鉴定图（右）；
图6：用siRNA_Kin17下调Kin17表达（左），抑制了肺癌A549细胞的生长增殖活性（右）；
图7：用siRNA_Kin17剔降Kin17，能增强化疗药卡铂对肺癌A549细胞增殖的抑制作用。
具体实施方式
Kin17基因或蛋白的定名与序列
编码Kin17蛋白的基因定名为“KIN17基因”，KIN17基因编码的蛋白定名为“Kin17蛋白”，KIN17基因的登录号为：NM_012311。
KIN17基因的核苷酸序列如SEQIDNO：1所示。
Kin17蛋白的氨基酸序列如SEQIDNO：2所示。
下面结合具体的实验对本发明作进一步的说明，但并不局限于此。
Kin17基因或蛋白应用于肺癌诊断：
随机选取施行手术活检并经病理学诊断的肺癌患者90例（其中非小细胞患者65例、小细胞肺癌患者25例）和和良性病变（如肺炎与肺纤维化等）患者40例，用免疫组化方法检测Kin17在这些患者病理标本中的表达情况。
免疫组化检测的步骤：
1)组织切片脱蜡至水，置于0.01M柠檬酸盐缓冲液（pH6.0）中，100℃微波抗原修复处理20min，自然冷却至室温；
2)蒸馏水冲洗后，滴加3%H2O2孵育15min；然后用0.01M磷酸盐缓冲液（PBS，pH7.4）冲洗3次，每次3min；
3)滴加1∶150稀释的Kin17抗体（购自Santacruz），37℃孵育60min后，用PBS冲洗3次，每次3min；
4)接着滴加辣根过氧化物酶（HRP）标记的EnVisionTM二抗试剂（购自Dako公司），37℃孵育30min后，用PBS冲洗3次，每次3min。最后用3,3’-二氨基联苯胺（DAB）显色3min，Mayer苏木素复染3min；
5)梯度酒精脱水、透明、封固。
染色评分：分别就每张切片的染色强度（－,±,1＋,2＋,3＋,4＋）与阳性细胞的百分率(0-100)%进行打分。计算染色得分=染色强度（0，0.5，1，2，3，4）×（0-100）。比如，某张玻片的染色强度是2＋，阳性细胞的百分率是65%，那得分等于2×60=130。得分≤100定义为低表达，得分＞100定义为高表达。全部切片由两个病理科医生在不知道组织诊断性质的情况下独立打分。
根据染色评分结果对数据进行统计分析。结果显示，Kin17在肺正常组织及良性病变组织中的表达较低，而在肺癌标本中表达明显异常升高（P＜0.01）；肿瘤细胞增殖越旺盛的组织部位，Kin17的表达越强（图1）。用Westernblot方法检测Kin17在肺癌患者新鲜组织与A549细胞中的表达，发现Kin17在肺癌标本的癌巢组织与A549细胞系中的表达明显高于癌旁正常组织（图2）。
因此，采用免疫组化、免疫荧光和实时定量PCR等方法对患者的肺病变组织中的Kin17蛋白或基因表达进行检测，有助于鉴别诊断肺良性病变与肺癌病情诊断。
Kin17蛋白的制备
纯化Kin17蛋白是制备Kin17抗体及相关的诊断试剂的重要材料。为了研发基于Kin17表达的肺癌诊断试剂，采用如下方法制备Kin17蛋白。
1.先用引物P1（5'-CGGGATCCATGGGGAAGTCGGATTTTCT-3'，SEQIDNO：3）和P2（5'–CGTAAGCTTTCAGGCAAGTTTAGAAATGTCTTC-3'，SEQIDNO：4）从细胞中PCR扩增KIN17基因开放阅读框全长，经用内切酶BamHI和Hind分别进行双酶切反应后，插入到pET32a-c(+)载体中，测序分析（图3）证明成功构建pET32a-KIN17重组质粒；
2.将重组质粒转化入大肠杆菌表达菌BL21中，再用IPTG诱导表达携带His标签的Kin17蛋白，经SDS－PAGE电泳分离及考马斯亮蓝染色后，发现目的蛋白的表达较好（图4）；
3.接着利用Ni-NTAHis-Bind柱进行亲和层析来纯化Kin17蛋白。
纯化终产物经SDS-PAGE电泳分析显示，一个和His标签Kin17蛋白大小一致的蛋白条带纯度很高，达到95%以上（如图5左）；该主要条带经Westernblot方法鉴定为Kin17目的蛋白（图5右）。
Kin17基因或蛋白应用于肺癌患者的预后评估的方案：
收集肺癌患者及肿瘤组织的病理参数的数据信息，统计分析病理组织中Kin17表达与这些参数的相关性。结果显示，患者肺癌标本中Kin17表达水平高低（评分＞100或者≤100）与肿瘤大小（P＝0.037，T-test）、肿瘤分化程度（P＝0.028，χ2检验）、Ki-67表达水平（P＝0.024，χ2检验）及对化疗药物敏感性（P＝0.031，χ2检验）密切相关。通过患者的随访调查发现，经治疗后死亡的肺癌患者的组织中，Kin17的表达比没有死亡的肺癌患者要高（P＝0.045，χ2检验）。
因此，对Kin17基因或蛋白的检测有助于评估与预测肺癌患者的疾病进展状况、抗癌治疗效果及预后情况。
Kin17基因或蛋白应用于肺癌的临床治疗的方案：
按照以下步骤进行RNA干扰试验：肺癌A549细胞经血清饥饿2h后，用脂质体Lipofectamine2000转染Kin17特异性的siRNA（siRNA_Kin17）与相对应的对照siRNA（siRNA_NC）到细胞中；48h后，收集两组细胞进行Westernblot分析及细胞生长曲线比较，发现转染siRNA_Kin17的细胞中Kin17表达被明显下调，而且其生长速度明显减慢（P＜0.05，图6）。因此，沉默肺癌细胞中Kin17表达，抑制能够肺癌细胞的增殖活性。
转染siRNA_Kin17与siRNA_NC到A549细胞48h后，在两组细胞中各加入肺癌常用化疗药卡铂，然后比较两组细胞的存活率变化；结果显示，转染siRNA_Kin17的A549细胞存活率下降得比对照组要更快（图7）。因此，沉默肺癌细胞中与DNA复制、修复相关的Kin17表达，能增强其对化疗药卡铂的敏感性。
因此Kin17可以作为肺癌治疗的靶点，采用特异性的siRNA、shRNA、Kin17特异性的抗体或者小分子药物，沉默或者降低肿瘤组织中的Kin17的表达，可用于肺癌的治疗。
<110>涛,曾
<120>Kin17基因或蛋白在制备肺癌诊断及治疗药剂中的应用
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<150>CN201310164095.6
<151>2013-05-07
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taaagtgttttatctatgacaatttagaatcatgtaattggaggaaatccttcatacact1560
ctatattttcagtagtagagtagtaaagttgaactctaagtgtgactaaattttaaatgt1620
caaactataaatatagaagcagctttattgtggtaataattcatataccatacagttcac1680
tcatttaaaatatacatacaattcagtggtttttagtatttttgcagaattgtgcagcca1740
tcaccactgtctgattttaaaacactttcataccccaaaatgaaaccctttaatagtcaa1800
tcgctatttttcccctctgccctagccctagacagccactaatgtactttctgtctctat1860
ggatttgccctttctggacatttcttataaatggaatcatataata1906
<210>2
<211>393
<212>PRT
<213>human
<400>2
MetGlyLysSerAspPheLeuThrProLysAlaIleAlaAsnArgIle
151015
LysSerLysGlyLeuGlnLysLeuArgTrpTyrCysGlnMetCysGln
202530
LysGlnCysArgAspGluAsnGlyPheLysCysHisCysMetSerGlu
354045
SerHisGlnArgGlnLeuLeuLeuAlaSerGluAsnProGlnGlnPhe
505560
MetAspTyrPheSerGluGluPheArgAsnAspPheLeuGluLeuLeu
65707580
ArgArgArgPheGlyThrLysArgValHisAsnAsnIleValTyrAsn
859095
GluTyrIleSerHisArgGluHisIleHisMetAsnAlaThrGlnTrp
100105110
GluThrLeuThrAspPheThrLysTrpLeuGlyArgGluGlyLeuCys
115120125
LysValAspGluThrProLysGlyTrpTyrIleGlnTyrIleAspArg
130135140
AspProGluThrIleArgArgGlnLeuGluLeuGluLysLysLysLys
145150155160
GlnAspLeuAspAspGluGluLysThrAlaLysPheIleGluGluGln
165170175
ValArgArgGlyLeuGluGlyLysGluGlnGluValProThrPheThr
180185190
GluLeuSerArgGluAsnAspGluGluLysValThrPheAsnLeuSer
195200205
LysGlyAlaCysSerSerSerGlyAlaThrSerSerLysSerSerThr
210215220
LeuGlyProSerAlaLeuLysThrIleGlySerSerAlaSerValLys
225230235240
ArgLysGluSerSerGlnSerSerThrGlnSerLysGluLysLysLys
245250255
LysLysSerAlaLeuAspGluIleMetGluIleGluGluGluLysLys
260265270
ArgThrAlaArgThrAspTyrTrpLeuGlnProGluIleIleValLys
275280285
IleIleThrLysLysLeuGlyGluLysTyrHisLysLysLysAlaIle
290295300
ValLysGluValIleAspLysTyrThrAlaValValLysMetIleAsp
305310315320
SerGlyAspLysLeuLysLeuAspGlnThrHisLeuGluThrValIle
325330335
ProAlaProGlyLysArgIleLeuValLeuAsnGlyGlyTyrArgGly
340345350
AsnGluGlyThrLeuGluSerIleAsnGluLysThrPheSerAlaThr
355360365
IleValIleGluThrGlyProLeuLysGlyArgArgValGluGlyIle
370375380
GlnTyrGluAspIleSerLysLeuAla
385390
<210>3
<211>28
<212>DNA
<213>人工引物
<400>3
cgggatccatggggaagtcggattttct28
<210>4
<211>33
<212>DNA
<213>人工引物
<400>4
cgtaagctttcaggcaagtttagaaatgtcttc33

Claims (2)

1.用于检测Kin17基因表达水平的试剂在制备肺癌诊断或预后评估试剂中的应用。
2.检测Kin17蛋白表达水平的试剂在制备肺癌诊断或预后评估试剂中的应用。
CN201410188632.5A 2013-05-07 2014-05-06 Kin17基因或蛋白在制备肺癌诊断及治疗药剂中的应用 Active CN104004831B (zh)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
D. S. F. Biard et al.."Ectopic Expression of MmKin17 Protein Inhibits Cell Proliferation of Human Tumor-Derived Cells".《Experimental Cell Research》.1999,(第250期), *
曾涛等."KIN17在28例肺癌标本中的表达分析".《检验医学与临床》.2013,第10卷(第12期), *

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