WO2021043340A2 - 一种肿瘤标志物aquaporin 2蛋白及其应用 - Google Patents

一种肿瘤标志物aquaporin 2蛋白及其应用 Download PDF

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WO2021043340A2
WO2021043340A2 PCT/CN2020/126029 CN2020126029W WO2021043340A2 WO 2021043340 A2 WO2021043340 A2 WO 2021043340A2 CN 2020126029 W CN2020126029 W CN 2020126029W WO 2021043340 A2 WO2021043340 A2 WO 2021043340A2
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aqp2
cells
tumor
protein
aquaporin
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WO2021043340A3 (zh
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徐寒梅
李梦玮
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南京安吉生物科技有限公司
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Priority to US17/640,380 priority patent/US20220299516A1/en
Priority to EP20859995.1A priority patent/EP4006166A4/en
Priority to AU2020342299A priority patent/AU2020342299B2/en
Priority to JP2022514787A priority patent/JP7323965B2/ja
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Definitions

  • the invention belongs to the field of tumor detection and molecular targeted therapy, and more specifically, relates to a transmembrane protein AQUAPORIN 2 (abbreviated as AQP2 in the text) and applications thereof.
  • AQP2 transmembrane protein AQUAPORIN 2
  • Tumors are currently the most serious type of disease that endangers human health. Studies have found that the occurrence of tumors is a complex process that gradually accumulates by gene mutations. The development of modern medical technology and molecular biology has enabled tumor treatment to enter the era of individualization, which greatly increases the remission rate of tumor treatment. Therefore, finding specific targets for the early diagnosis, treatment and prognosis of tumors is currently the key bottleneck restricting the clinical efficacy of tumors.
  • Head and neck cancers include neck tumors (thyroid tumors, etc.), otolaryngology tumors (laryngeal cancer, nasopharyngeal cancer, paranasal sinus cancer, etc.), and oral and maxillofacial tumors (tongue cancer, gum cancer, cheek cancer, etc.), of which more than 90 % Of head and neck tumors are squamous cell carcinoma.
  • Squamous cell carcinoma of the head and neck is the sixth most common cancer in the world. There are more than 500,000 new cases worldwide each year, and the 5-year survival rate does not exceed 40%.
  • the current treatment methods are still focused on radiotherapy, chemotherapy, and surgery, and the clinical prognosis is not good. Therefore, in-depth study of the pathogenesis of head and neck squamous cell carcinoma and the discovery of new biomarkers are of great significance for the targeted therapy of head and neck squamous cell carcinoma and the prognosis of patients.
  • Renal cell carcinoma also known as renal cell carcinoma, originates from renal tubular epithelial cells and is the most common renal parenchymal malignant tumor. There are about 208,500 new cases worldwide each year, and the incidence in my country is about 4.5/100,000. At present, the etiology of kidney cancer is not clear, and clinical treatment has found that most kidney cancer patients are not sensitive to radiotherapy and chemotherapy, and mostly rely on surgery. resection. Therefore, improving the accuracy of early diagnosis is helpful for timely treatment of kidney cancer patients.
  • Prostate cancer refers to epithelial malignant tumors that occur in the prostate, which mainly include adenocarcinoma (acinar adenocarcinoma), ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma, and adenosquamous carcinoma.
  • adenocarcinoma acinar adenocarcinoma
  • ductal adenocarcinoma ductal adenocarcinoma
  • urothelial carcinoma squamous cell carcinoma
  • squamous cell carcinoma adenosquamous carcinoma
  • Tumor metastasis and invasion is an important feature of malignant tumors, and it is also the culprit that causes most tumors to recur.
  • Studies have found that tumor metastasis and invasion is a continuous dynamic process involving multiple genes, in which proto-oncogenes and tumor suppressor genes play an equally important role.
  • the role of a large number of proto-oncogenes such as PTEN, MYC, RAS, PIK3CA, and AKT1 in malignant tumors including head and neck squamous cell carcinoma has been deeply revealed, while studies on tumor suppressor genes other than TP53 are rarely reported.
  • bioinformatics methods such as high-throughput screening and big data analysis, the discovery of tumor suppressor genes with important functions is very important for revealing the pathogenesis of tumors and proposing more comprehensive diagnosis and treatment plans.
  • Aquaporin-2 (AQP2), as a member of the aquaporin family, is mainly distributed in the luminal membrane of the main cell of the collecting duct and intracellular vesicles. It is an vasopressin-sensitive aquaporin.
  • Current studies have found that AQP2 is mainly expressed in kidney tissues and is involved in the pathological process of diseases including neurological diabetes insipidus and polycystic kidney disease.
  • the expression and function of AQP2 in tumors have not yet been reported in the literature. This study found for the first time the expression level and potential biological functions of AQP2 in different types of tumors, which is important for the development of the application value of AQP2 in tumor detection and treatment. significance.
  • the present invention provides a tumor marker AQP2 protein, and successfully applied it in tumor detection and treatment, by combining bioinformatics methods, Clinical tumor samples and biological function experiments have found new biomarkers closely related to the occurrence, development, and metastasis of head and neck squamous cell carcinoma, kidney cancer and prostate cancer.
  • a transmembrane protein is used in the preparation of tumor therapeutic drugs or as a tumor marker.
  • the marker is the transmembrane protein AQP2, and its amino acid sequence is shown in SEQ ID NO.2.
  • the tumors to be detected by the tumor marker include head and neck squamous cell carcinoma, kidney cancer and prostate cancer.
  • a kit for detecting the expression of the above-mentioned markers comprising a specific primer pair designed for the nucleotide sequence encoding AQP2 (shown in SEQ ID NO. 1).
  • the reagent for detecting the expression of the biomarker can be applied to a tool for prognosticating tumor subjects.
  • the prognostic method described herein includes: obtaining a test sample from a tumor; determining the expression level of a biomarker in the test sample; and analyzing the expression level to generate a risk score, wherein the risk score can be used to provide a subject’s Prognosis.
  • the test samples used in the prognosis are fresh, frozen or paraffin-embedded tissues.
  • the aforementioned detection reagent is a reagent containing an anti-AQP2 protein antibody, or a composition detection reagent containing an anti-AQP2 protein antibody.
  • the above-mentioned biomarker detection method is characterized in that: specific primers are designed to detect the expression of the transmembrane protein AQP2 in tissue cells by PCR, and the primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4 .
  • a recombinant vector for realizing the overexpression of the transmembrane protein AQP2, and the recombinant vector can be used in the preparation of drugs for treating tumors.
  • the recombinant vector is an overexpression plasmid, lentivirus or cell strain containing the nucleotide sequence shown in SEQ ID NO. 1, which has the following functions (al)-(a3):
  • transmembrane protein AQP2 plays an important role in tumor diagnosis, prognosis and treatment, and can be used as a tumor marker including head and neck squamous cell carcinoma, kidney cancer and prostate cancer.
  • the present invention found that the expression level of the transmembrane protein AQP2 in head and neck squamous cell, renal cell and prostate cancer cells was significantly lower than that of normal epithelial cells. Overexpression of AQP2 can significantly inhibit head and neck squamous cell, renal cell and prostate cancer cells.
  • AQP2 has the potential to be used as a target for drug design, for example, anti-tumor substances targeting AQP2 (containing its coding nuclear
  • anti-tumor substances targeting AQP2 containing its coding nuclear
  • the overexpression plasmid vector, lentivirus or transgenic cell line etc. can be used to prepare drugs against head and neck squamous cell carcinoma, kidney cancer and prostate cancer.
  • the present invention uses GAPDH as an internal reference gene to detect the expression level of AQP2. It is found that the expression level of AQP2 protein in head and neck squamous cell carcinoma cells SCC4, kidney cancer cell 786-O, and prostate cancer cell DU145 is significantly reduced, which proves that AQP2 can As a new biomarker to diagnose malignant tumors including head and neck squamous cell carcinoma, kidney cancer and prostate cancer.
  • Figure 1 is a comparison of the expression level of AQP2 gene in human head and neck squamous cell carcinoma tissues and adjacent tissues. The data comes from the TCGA database;
  • Figure 2 is a comparison of the expression level of AQP2 gene in human renal papillary cell carcinoma tissues and paracancerous tissues, the data comes from the TCGA database;
  • Figure 3 is a comparison of the expression level of AQP2 gene in human renal clear cell carcinoma tissue and adjacent tissues, the data comes from the TCGA database;
  • Figure 4 is a comparison of the expression levels of AQP2 gene in human renal chromophobe cell carcinoma tissues and adjacent tissues, the data comes from the TCGA database;
  • Figure 5 is a comparison of the expression level of AQP2 gene in human prostate cancer tissues and adjacent tissues, the data comes from the TCGA database;
  • Figure 6 is a comparison of AQP2 gene expression in three types of tumor cells and normal cells
  • Figure 7 is a comparison of AQP2 protein expression of AQP2 gene in three kinds of tumor cells and normal cells;
  • Figure 8 is a map of the lentiviral overexpression vector of AQP2
  • Figure 9 shows the effect of overexpression of AQP2 on AQP2 gene and protein expression in head and neck squamous cell carcinoma cells
  • Figure 10 shows the effect of overexpression of AQP2 on AQP2 gene and protein expression in renal cancer cells
  • Figure 11 shows the effect of overexpression of AQP2 on the expression of AQP2 gene and protein in prostate cancer cells
  • Figure 12 is a graph showing the effect of overexpression of AQP2 on the proliferation of head and neck squamous cell carcinoma cells SCC4;
  • Figure 13 is a graph showing the effect of overexpression of AQP2 on the proliferation ability of renal cancer cell 786-O;
  • Figure 14 is a graph showing the effect of overexpression of AQP2 on the proliferation of prostate cancer cells DU145.
  • Figure 15 is a graph showing the effect of overexpression of AQP2 on tumor growth in head and neck squamous cell carcinoma cells SCC4;
  • Figure 16 is a graph showing the effect of overexpression of AQP2 on the tumor growth of renal cancer cell 786-O in vivo;
  • Figure 17 is a graph showing the effect of overexpression of AQP2 on the tumor growth of prostate cancer cell DU145 in vivo.
  • the Tumor Genome Atlas (TCGA) project was jointly launched in 2006 by the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI) in the United States. It uses large-scale sequencing-based genome analysis technology to conduct large-scale analysis of 36 cancers.
  • NCI National Cancer Institute
  • NHGRI National Human Genome Research Institute
  • GCC Genome Analysis Center
  • the fluorescent quantitative PCR method was used to detect the expression of AQP2 in tumor cells and normal epithelial cells.
  • the above six types of cells were cultured in an incubator at 37°C and 5% CO 2 respectively. When the density reached 90%, the cells were digested and collected with trypsin. The cells were resuspended in culture medium and counted under a microscope, and the cell concentration was adjusted to 5 ⁇ 10 5 cells/mL, then inoculate the adjusted cell suspension into a 6-well plate with 2 mL per well, and continue to incubate in a 37°C, 5% CO 2 incubator for 24 hours.
  • RNA of head and neck squamous cell carcinoma cell SCC4 and human normal oral epithelial cell HIOEC, kidney cancer cell 786-O and human renal epithelial cell HEK293T, prostate cancer cell DU145 and human normal prostate epithelial cell RWPE-1 were extracted according to the Trizol instructions of Life Company. , And then use NanoDrop ND-1000 nucleic acid quantifier to quantify the purity and concentration of the extracted RNA, and agarose quality inspection to ensure the integrity of the extracted RNA.
  • the kit contains gDNAEraser DNase, which can effectively remove confounding genomic DNA.
  • the upstream primer and downstream primer of AQP2 are SEQ ID NO. 3 and SEQ ID NO. 4, respectively, and the upstream primer and downstream primer of GAPDH are SEQ ID NO. 5 and SEQ, respectively. ID NO.6.
  • the reaction system is as follows:
  • the immunoblotting method was used to detect the expression of AQP2 protein in tumor cells and normal epithelial cells.
  • Example 2 The six cells in Example 2 were collected by trypsin digestion when the growth density reached 90%, and the cells were resuspended in culture medium for expansion and culture, and then the cells were collected at 80% confluence, centrifuged and discarded the supernatant, rinsed with PBS Twice, discard the supernatant. Add RIPA lysis buffer and lyse on ice for 20 min. The supernatant was collected by centrifugation at 12000g for 10 min. Add 1XSDS loading buffer, mix by pipetting and boil for 5min to denature. The total protein was separated on 10% SDS-PAGE gel, and then transferred to PVDF membrane.
  • a full-length cDNA targeting AQP2 (see SEQ ID NO. 1 for the specific sequence) was synthesized and introduced into the plvx-CMV-ZsGreen1 plasmid (see Figure 8 for the map).
  • the above plasmids, the packaging plasmid psPAX2 and the envelope plasmid pMD2.G were co-transformed into 293T cells to produce the virus. After 48 hours of transfection, the viral supernatant of the cells was collected and infected with SCC4 head and neck squamous cell carcinoma cells and 786-O renal carcinoma cells respectively. , DU145 prostate cancer cells.
  • This example shows the effect of overexpression of AQP2 on the proliferation ability of human tumor cells.
  • the head and neck squamous cell carcinoma cell SCC4, renal carcinoma cell 786-O, prostate cancer cell DU145 were stably transfected with empty vector and AQP2 overexpressing cells were cultured in an incubator at 37°C and 5% CO 2 to a density of 90% with trypsin Digest and collect, resuspend the cells in culture medium and count under a microscope, adjust the cell concentration to 3.0 ⁇ 10 4 cells/mL, inoculate the cell suspension into a 96-well plate, 100 ⁇ L per well, and place it at 37°C, 5% Culture in a CO 2 incubator for 24h, 48h, 72h.
  • This example shows the effect of overexpression of AQP2 on the migration ability of human tumor cells.
  • the complete medium stimulated cell migration, cultured in 5% CO 2 at 37°C for 24h. Discard the culture medium in the well, fix it with 90% alcohol at room temperature for 30 minutes, stain with 0.1% crystal violet at room temperature for 10 minutes, rinse with water, gently wipe off the upper layer of non-migrated cells with a cotton swab, observe under a microscope and select four fields of view to take pictures and count.
  • MIR migration inhibition rate
  • N test is the number of cell migration in the test group (plvx-AQP2)
  • N control is the number of cell migration in the blank control group (plvx-ctrl).
  • the experiment was repeated 3 times independently, the results obtained from the experiment were calculated as mean ⁇ SD, and statistical t-test was performed.
  • the data of the two or more groups were compared by one-way analysis of variance (One-way ANOVA), and the P value was used to indicate statistical significance, and P ⁇ 0.05 was Significant difference, P ⁇ 0.01 is a very significant difference.
  • This example shows the effect of overexpression of AQP2 on the growth of human tumor cells in vivo.
  • Each nude mouse (order 4-6 weeks old females weighing 14-16g, and adaptively reared in an SPF animal rearing room for 1 week) inoculate 100 ⁇ l of the cell suspension of the corresponding group in the left armpit, and inject The amount of cells is 5 ⁇ 10 6 ;
  • tumor volume (Tumor volume, TV) calculation formula is as follows:
  • Tumor volume 0.5 ⁇ a ⁇ b ⁇ 2
  • a is the length of the transplanted tumor (mm)
  • b is the width of the transplanted tumor (mm).

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Abstract

本发明公开了一种肿瘤标志物AQUAPORIN 2蛋白及其应用，属于肿瘤检测和分子靶向治疗领域。本发明包括AQUAPORIN 2蛋白在如下功能中的应用：(1)作为新的肿瘤标志物用于包括头颈鳞癌、肾癌和前列腺癌在内的恶性肿瘤的早期诊断及预后效果的判定；(2)抑制肿瘤细胞增殖；(3)抑制肿瘤细胞迁移；(4)抑制动物移植瘤模型的生长；本发明以AQUAPORIN 2蛋白作为生物标志物的靶向疗法为头颈鳞癌、肾癌和前列腺癌等恶性肿瘤的治疗提供新的思路。

Description

一种肿瘤标志物AQUAPORIN 2蛋白及其应用 技术领域
本发明属于肿瘤检测和分子靶向治疗领域，更具体地说，涉及一种跨膜蛋白AQUAPORIN 2(文中简写为AQP2)及其应用。
背景技术
肿瘤是目前危害人类健康最严重的一类疾病。研究发现肿瘤的产生是一个由基因突变逐渐积累的复杂过程，现代医学技术和分子生物学的发展，使得肿瘤治疗进入个体化时代，大大增加肿瘤治疗的缓解率。因此找到特异性的靶点对于肿瘤早期诊断、治疗及预后是目前制约肿瘤临床疗效的关键瓶颈。
头颈癌包括颈部肿瘤(甲状腺肿瘤等)、耳鼻喉科肿瘤(喉癌、鼻咽癌、副鼻窦癌等)以及口腔颌面部肿瘤(舌癌、牙龈癌、颊癌等)，其中超过90％的头颈部肿瘤为鳞状细胞癌。头颈鳞癌为全球第六大常见癌症，全球每年新发病例超过50万例，且5年生存率不超过40％。目前治疗手段仍集中在放疗、化疗、手术三种，临床预后不佳。因此深入研究头颈鳞癌的发病机制、发现新型的生物标志物对于头颈鳞癌的靶向治疗和患者预后具有重要意义。
肾癌又称肾细胞癌，起源于肾小管上皮细胞，是最常见的肾脏实质恶性肿瘤。全球每年约有208,500的新增病例，在我国发病率约4.5/10万，目前对肾癌的病因了解并不清楚，且临床治疗发现肾癌患者多数对放化疗均不敏感，多依赖于手术切除。因此提高早期诊断的准确性，有助于肾癌患者进行及时治疗。
前列腺癌是指发生在前列腺的上皮性恶性肿瘤，主要包括腺癌(腺泡腺癌)、导管腺癌、尿路上皮癌、鳞状细胞癌、腺鳞癌等。发病率随着年龄的增长而增长，高峰年龄是70～80岁。前列腺癌发病有明显的地区和种族差异，据统计中国人最低，欧洲人最高。近年来随着生活条件的改善，人群寿命的延长，我国前列腺癌的发病率也逐年增加。
肿瘤转移侵袭是恶性肿瘤的重要特征，也是引起大多数肿瘤复发的元凶。研究发现肿瘤转移侵袭是一个连续的、由多基因参与的动态过程，其中原癌基因与抑癌基因发挥同等重要的作用。大量原癌基因如PTEN、MYC、RAS、PIK3CA、AKT1在包括头颈鳞癌在内的恶性肿瘤中的作用被深入揭示，而抑癌基因除TP53以外的研究鲜有报道。借助高通量筛选及大数据分析等生物信息学手段，发现具有重要功能的抑癌基因对揭示肿瘤的发病机制、提出更全面的诊疗方案十分重要。
水通道蛋白-2(AQP2)作为水通道蛋白家族成员之一，主要分布在集合管主细胞管腔膜及细胞内囊泡中，是一种抗利尿激素敏感性水通道蛋白。目前研究发现AQP2主要表达在肾脏组织，参与包括神经性尿崩症、多囊肾等疾病的病理过程。但AQP2在肿瘤中的表达和功能还未有文献资料报道，本研究首次发现AQP2在不同种类的肿瘤中表达水平和潜在的生物学功能，对AQP2在肿瘤检测和治疗中应用价值的开发具有重要意义。
发明内容
针对现有的头颈鳞癌等恶性肿瘤缺乏关系密切的生物标志物的问题，本发明提供一种肿瘤标志物AQP2蛋白，并成功将其应用在肿瘤检测和治疗中，通过结合生物信息学手段、临床肿瘤样本以及生物功能实验，发现与头颈鳞癌、肾癌和前列腺癌的发生发展、转移有密切关系的新型生物标志物。
本发明所采用的技术方案如下：
一种跨膜蛋白在制备肿瘤治疗药物或作为肿瘤标志物中的应用，所述的标志物为跨膜蛋白AQP2，其氨基酸序列如SEQ ID NO.2所示。
优选地，该肿瘤标志物用于检测的肿瘤包括头颈鳞癌、肾癌和前列腺癌。
一种检测上述标志物表达的试剂盒，所述的检测试剂盒包括针对编码AQP2的核苷酸序列(为SEQ ID NO.1所示)设计的特异性引物对。
优选地，检测生物标志物表达的试剂可以应用于对肿瘤对象进行预后的工具中。本文中所述的预后的方法包括：从肿瘤中获得测试样品；确定所述测试样品中生物标志物的表达水平；和分析所述表达水平以产生风险评分，其中该风险评分可用于提供对象的预后。需要说明的是，预后中用到的测试样品是新鲜的、冷冻的或石蜡固定包埋的组织。
优选地，上述的检测试剂，为含有抗AQP2蛋白抗体的试剂，也可以是含有抗AQP2蛋白抗体的组合物检测试剂。
上述生物标志物的检测方法，其特征在于：设计特异性引物利用PCR方法检测组织细胞中跨膜蛋白AQP2的表达量，所述的引物序列为SEQ ID NO.3和SEQ ID NO.4所示。
一种实现跨膜蛋白AQP2过表达的重组载体，所述的重组载体可以在制备用于治疗肿瘤的药物中应用。
优选地，所述的重组载体为含有SEQID NO.1所示核苷酸序列的过表达质粒、慢病毒或细胞株，其具有如以下(al)-(a3)功能：
(a1)为抑制肿瘤生长；
(a2)为抑制肿瘤细胞增殖；
(a3)为抑制肿瘤细胞迁移。
相比于现有技术，本发明的有益效果为：
(1)首次发现跨膜蛋白AQP2在肿瘤诊断、预后和治疗方面具有重要作用，可以作为包括头颈鳞癌、肾癌和前列腺癌的肿瘤标志物。
(2)本发明发现跨膜蛋白AQP2在头颈鳞癌细胞、肾癌细胞和前列腺癌细胞中的表达水平显著低于正常上皮细胞，AQP2过表达能显著抑制头颈鳞癌细胞、肾癌细胞和前列腺癌细胞的增殖、迁移和体内肿瘤生长，这证明AQP2对肿瘤生长和转移的重要性，说明AQP2具有作为靶标进行药物设计的潜能，例如，以AQP2为靶点的抗肿瘤物质(含有其编码核苷酸的过表达质粒载体、慢病毒或转基因细胞系等)可用于制备抗头颈鳞癌、肾癌和前列腺癌的药物。
(3)本发明以GAPDH为内参基因，对AQP2表达水平进行了检测，发现AQP2蛋白在头颈鳞癌细胞SCC4、肾癌细胞786-O、前列腺癌细胞DU145中的表达量显著降低，证明AQP2可作为一个新的生物标志物来诊断包括头颈鳞癌、肾癌和前列腺癌在内的恶性肿瘤。
附图说明
附图1为AQP2基因在人头颈鳞癌组织和癌旁组织中的表达量比较，数据来自TCGA数据库；
附图2为AQP2基因在人肾乳头状细胞癌组织和癌旁组织中的表达量比较，数据来自TCGA数据库；
附图3为AQP2基因在人肾透明细胞癌组织和癌旁组织中的表达量比较，数据来自TCGA数据库；
附图4为AQP2基因在人肾嫌色细胞癌组织和癌旁组织中的表达量比较，数据来自TCGA数据库；
附图5为AQP2基因在人前列腺癌组织和癌旁组织中的表达量比较，数据来自TCGA数据库；
附图6为AQP2基因在三种肿瘤细胞和正常细胞中的AQP2基因表达量比较；
附图7为AQP2基因在三种肿瘤细胞和正常细胞中的AQP2蛋白表达量比较；
附图8为AQP2的慢病毒过表达载体图谱；
附图9为过表达AQP2对头颈鳞癌细胞中AQP2基因和蛋白表达量的影响；
附图10为过表达AQP2对肾癌细胞中AQP2基因和蛋白表达量的影响；
附图11为过表达AQP2对前列腺癌细胞中AQP2基因和蛋白表达量的影响；
附图12为过表达AQP2对头颈鳞癌细胞SCC4增殖能力的影响结果图；
附图13为过表达AQP2对肾癌细胞786-O增殖能力的影响结果图；
附图14为过表达AQP2对前列腺癌细胞DU145增殖能力的影响结果图。
附图15为过表达AQP2对头颈鳞癌细胞SCC4体内肿瘤生长的影响结果图；
附图16为过表达AQP2对肾癌细胞786-O体内肿瘤生长的影响结果图；
附图17为过表达AQP2对前列腺癌细胞DU145体内肿瘤生长的影响结果图。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1
AQP2在人不同肿瘤组织与癌旁组织的表达谱芯片分析
肿瘤基因组图谱(TCGA)计划由美国National Cancer Institute(NCI)和National Human Genome Research Institute(NHGRI)于2006年联合启动的项目，利用大规模测序为主的基因组分析技术，针对36种癌症进行大规模实验，TCGA基因组分析中心(GCC)比对肿瘤和正常组织，寻找与各癌症或者亚型相关的基因突变、扩增或者缺失。为理解癌症的分子机制，提高人们对癌症发病分子基础的科学认识提供帮助。
利用TCGA标准方法下载36种肿瘤和其癌旁组织的全基因表达谱数据及临床信息，采用R语言(3.1.1版本)软件过滤掉不含AQP2表达信息的肿瘤类型，发现共在20种肿瘤中检测到AQP2表达。
表1.TCGA数据库中AQP2在不同肿瘤中的表达水平分析
Figure PCTCN2020126029-appb-000001
Figure PCTCN2020126029-appb-000002
利用graphpad Prism 7对20种肿瘤组织和对应癌旁组织中AQP2的表达水平进行统计学分析，所有数据进行统计t检验，*P＜0.05为显著性差异，**P＜0.01为极显著性差异。具体分析结果见表1，与对应的癌旁组织相比，AQP2在头颈鳞癌(图1)、三种肾癌亚型(肾乳头状细胞癌、肾透明细胞癌、肾嫌色细胞癌，图2-4)和前列腺癌(图5)中显著低表达。
实施例2
本实施例中利用荧光定量PCR方法检测肿瘤细胞和正常上皮细胞中AQP2的表达量。
1.材料
头颈鳞癌细胞SCC4和人正常口腔上皮细胞HIOEC，肾癌细胞786-O和人肾上皮细胞HEK293T，前列腺癌细胞DU145和人正常前列腺上皮细胞RWPE-1；上述细胞均购自美国ATCC细胞库。
2.方法
2.1肿瘤细胞和正常上皮细胞中总RNA的提取
上述六种细胞分别在37℃、5％CO 2的培养箱中培养，培养至密度为90％时用胰蛋白酶消化收集，用培养液重悬细胞并在显微镜下计数，将细胞浓度调整为5×10 5个/mL，然后将浓度调整好的细胞悬液接种到6孔板中，每孔2mL，并继续于37℃，5％CO 2培养箱中培养24h。
按life公司的Trizol说明书分别提取头颈鳞癌细胞SCC4和人正常口腔上皮细胞HIOEC，肾癌细胞786-O和人肾上皮细胞HEK293T，前列腺癌细胞DU145和人正常前列腺上皮细胞RWPE-1的总RNA，再用NanoDrop ND-1000核酸定量仪定量所提取的RNA的纯度和浓度，琼脂糖质检确保提取RNA的完整性。
2.2 RNA反转录合成第一链cDNA
采用TaKaRa试剂盒PrimeScriptTM RT reagent Kit with gDNA Eraser(Perfect Real Time)对提取的总RNA反转录合成cDNA。该试剂盒含gDNAEraser DNase，可有效去除混杂的基因组DNA。
2.3实时定量PCR
根据AQP2和GAPDH的核酸序列设计特异性引物，采用TaKaRa试剂盒
Figure PCTCN2020126029-appb-000003
Premix Ex Taq TM II(TliRNaseH Plus)进行PCR反应，AQP2的上游引物和下游引物分别为SEQ ID NO.3和SEQ ID NO.4，GAPDH的上游引物和下游引物分别为SEQ ID NO.5和SEQ ID NO.6。反应体系如下表：
表2.PCR反应体系
Figure PCTCN2020126029-appb-000004
Figure PCTCN2020126029-appb-000005
将上述组份混合均匀后按以下程序进行实时定量PCR：95℃30s预变性，40个循环；95℃5s，60℃30s。
根据熔解曲线判断反应的特异性，由公式2 -ΔΔCt计算AQP2的mRNA表达量。结果见图6，与人正常上皮细胞相比，AQP2在头颈鳞癌细胞SCC4、肾癌细胞786-O、前列腺癌细胞DU145中的表达量显著降低，与临床样本的分析结果一致。
实施例3
本实施例中，采用免疫印迹方法检测肿瘤细胞和正常上皮细胞中AQP2蛋白的表达量。
将实例2中的六种细胞在生长密度达到90％时用胰蛋白酶消化收集，用培养液重悬细胞进行扩增培养，然后收集80％汇合度时细胞，离心后弃上清，PBS润洗两次，弃上清。加入RIPA裂解液，冰上裂解20min。12000g离心10min收集上清。加入1XSDS上样缓冲液，吹打混匀后煮沸变性5min。10％SDS-PAGE凝胶分离总蛋白，然后转移到PVDF膜。5％BSA室温封闭2h，与AQP2抗体(abcam)4℃孵育过夜，TBST洗涤3次。二抗室温孵育1h，TBST洗涤3次。ECL超敏化学发光液显影，经Tannon成像系统成像，以GAPDH作为内参比较不同细胞中AQP2蛋白的表达水平。
结果见图7，与AQP2mRNA表达差异一致，AQP2蛋白在头颈鳞癌细胞SCC4、肾癌细胞786-O、前列腺癌细胞DU145中的表达量显著降低。
实施例4
本实施例中进行过表达AQP2载体的制备和病毒转染效率检测。
同时合成针对AQP2的全长cDNA(具体序列见SEQ ID NO.1)，导入plvx-CMV-ZsGreen1质粒(图谱见图8)中。将上述质粒同包装质粒psPAX2和包膜质粒pMD2.G共转入293T细胞中以产生病毒，转染48h后，收集细胞的病毒上清，分别感染SCC4头颈鳞癌细胞、786-O肾癌细胞、DU145前列腺癌细胞。感染48h后，加入嘌呤霉素筛选两周后，获得稳定促进AQP2基因表达的细胞株。收集三种细胞空白载体和过表达载体的总RNA和总蛋白，通过qPCR(具体方法同实施例2)和免疫印迹方法(具体方法同实施例3)，比较AQP2基因和蛋白的表达量变化。
结果见图9-11，过表达AQP2使头颈鳞癌细胞SCC4(图9)、肾癌细胞786-O(图10)、前列腺癌细胞DU145(图11)中的AQP2基因和蛋白的表达量显著升高。
实施例5
本实施例为过表达AQP2对人肿瘤细胞增殖能力的影响。
将头颈鳞癌细胞SCC4、肾癌细胞786-O、前列腺癌细胞DU145稳定转染空载体和AQP2过表达的细胞在37℃、5％CO 2的培养箱中培养至密度90％时用胰蛋白酶消化收集，用培养液重悬细胞并在显微镜下计数，将细胞浓度调整为3.0×10 4个/mL，将细胞悬液接种到96孔板中，每孔100μL，并于37℃，5％CO 2培养箱中分别培养24h，48h，72h。向96孔板中每孔加入20μL 5mg/mL的MTT，继续培养4h。吸去培养基，每孔加入100μL DMSO溶解。用酶标仪在检测波长为570nm，参比波长为630nm处测定吸光值，并计算生长抑制率(proliferation inhibition，PI)，公式为：
PI(％)＝1-给药组/阴性组
试验独立重复3次，试验得到的结果以mean±SD表示，并进行统计t检验，两组以上数据比较采用单因素方差分析(One-way ANOVA)，利用P值表示统计学意义，其中P＜0.05为显著性差异，P＜0.01为极显著性差异。
结果见图12-14，与空载体细胞(plvx-crtl)相比，具有AQP2过表达量的细胞(plvx-AQP2)头颈鳞癌细胞SCC4(图12)、肾癌细胞786-O(图14)、前列腺癌细胞DU145(图15)的增殖速度明显减弱。表明AQP2过表达能显著抑制头颈鳞癌细胞SCC4、肾癌细胞786-O、前列腺癌细胞DU145细胞的增殖，进一步证实AQP2作为抑癌基因的重要性。
实施例6
本实施例为过表达AQP2对人肿瘤细胞迁移能力的影响。
将头颈鳞癌细胞SCC4、肾癌细胞786-O、前列腺癌细胞DU145稳定转染空载体和AQP2过表达的细胞接种到transwell小室中，每孔100μL，在transwell下室加入0.6mL含10％FBS的完全培养基刺激细胞迁移，于5％CO 2，37℃培养24h。弃去孔中培液，用90％酒精常温固定30min，0.1％结晶紫常温染色10min，清水漂净，用棉签轻轻擦掉上层未迁移细胞，显微镜下观察并选择四个视野拍照计数。按照公式计算迁移抑制率(migration inhibition rate，MIR)：
Figure PCTCN2020126029-appb-000006
其中N test为测试组(plvx-AQP2)的细胞迁移数，N control为空白对照组(plvx-ctrl)的细胞迁移数。试验独立重复3次，试验得到的结果计算mean±SD，并进行统计t检验，两组以上数据比较采用单因素方差分析(One-way ANOVA)，利用P值表示统计学意义，P＜0.05为显著性差异，P＜0.01为极显著性差异。
表3过表达AQP2对人肿瘤细胞迁移能力的抑制作用
Figure PCTCN2020126029-appb-000007
表中：*表示P值＜0.05，**表示P值＜0.01。
结果如表3所示，发现上调AQP2表达后，头颈鳞癌细胞SCC4、肾癌细胞786-O、前列腺癌细胞DU145细胞的迁移能力明显减弱。
实施例7
本实施例为过表达AQP2对人肿瘤细胞体内生长的影响。
(1)大量培养头颈鳞癌细胞SCC4、肾癌细胞786-O、前列腺癌细胞DU145稳定转染空载体和AQP2过表达的细胞，采用0.25％胰酶溶液消化，终止消化后细胞悬液1000rpm离心5min，用无血清的DMEM培养基重悬细胞后计数，调整细胞浓度至5×10 7个/ml；
(2)每只裸鼠(订购4-6周龄、体重在14-16g的雌性，在SPF级动物饲养室适应性饲养1周)左侧腋下接种100μl对应组别的细胞悬液，注射细胞量为5×10 6个；
(3)接种后密切观察裸鼠接种部位的肿瘤生长情况，每隔两日测量并记录移植瘤体积，肿瘤体积(Tumor volume，TV)计算公式如下：
肿瘤体积＝0.5×a×b^2
其中，a为移植瘤长度(mm)，b为移植瘤宽度(mm)。
与空载体对照组(plvx-ctrl)相比，AQP2过表达量的细胞(plvx-AQP2)(头颈鳞癌细胞SCC4(图15)、肾癌细胞786-O(图16)、前列腺癌细胞DU145(图17)的裸鼠内肿瘤生长速度更慢，体内成瘤能力明显降低，说明过表达AQP2可以抑制恶性肿瘤细胞的体内生长能力。
Figure PCTCN2020126029-appb-000008
Figure PCTCN2020126029-appb-000009
Figure PCTCN2020126029-appb-000010
Figure PCTCN2020126029-appb-000011

Claims (7)

  1. 一种跨膜蛋白在制备肿瘤治疗药物或作为肿瘤标志物中的应用，其特征在于：所述的跨膜蛋白为AQUAPORIN 2，其氨基酸序列如SEQ ID NO.2所示。
  2. 根据权利要求1所述的应用，其特征在于：所述的肿瘤治疗是通过在肿瘤中过表达SEQ ID NO.1所示的核苷酸序列而实现。
  3. 一种肿瘤标志物的检测方法，其特征在于：所述的检测方法为a或b中的任一种：
    (a)利用PCR方法检测组织或细胞中跨膜蛋白AQUAPORIN 2的基因表达量；
    (b)利用免疫印迹方法检测组织或细胞中跨膜蛋白AQUAPORIN 2的表达量。
  4. 根据权利要求3所述的检测方法，其特征在于：所述的PCR方法包括针对SEQ ID NO.1序列设计的特异性引物对。
  5. 根据权利要求4所述的检测方法，其特征在于：所述特异性引物对序列为SEQ ID NO.3和SEQ ID NO.4。
  6. 根据权利要求1或2所述的应用及权利要求3-5所述的检测方法，其特征在于：所述的肿瘤包括头颈鳞癌、肾癌和前列腺癌。
  7. 一种重组载体，其特征在于：所述的重组载体含有所述SEQ ID NO.1核苷酸序列。
PCT/CN2020/126029 2019-09-05 2020-11-03 一种肿瘤标志物aquaporin 2蛋白及其应用 WO2021043340A2 (zh)

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