[go: up one dir, main page]

CN107058556A - A method for assessing the cancer-promoting risk of endosulfan - Google Patents

A method for assessing the cancer-promoting risk of endosulfan Download PDF

Info

Publication number
CN107058556A
CN107058556A CN201710322541.XA CN201710322541A CN107058556A CN 107058556 A CN107058556 A CN 107058556A CN 201710322541 A CN201710322541 A CN 201710322541A CN 107058556 A CN107058556 A CN 107058556A
Authority
CN
China
Prior art keywords
endosulfan
prl
prostate cancer
cancer
risk
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710322541.XA
Other languages
Chinese (zh)
Inventor
徐丹
郭瑜冰
林丽梅
孙野青
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dalian Maritime University
Original Assignee
Dalian Maritime University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dalian Maritime University filed Critical Dalian Maritime University
Priority to CN201710322541.XA priority Critical patent/CN107058556A/en
Publication of CN107058556A publication Critical patent/CN107058556A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57434Specifically defined cancers of prostate
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/142Toxicological screening, e.g. expression profiles which identify toxicity
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/34Genitourinary disorders
    • G01N2800/342Prostate diseases, e.g. BPH, prostatitis

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention provides a method for assessing the risk of endosulfan promoted cancer, the method comprising the step of detecting the expression of a biomarker, which is PRL-3, in a biological sample. The method is simple and convenient, has high correlation, and can quickly evaluate the cancer promotion risk of the endosulfan. According to the invention, PRL-3 is screened out through bioinformatics analysis and is a key gene related to endosulfan and prostate cancer, and cytology proves that endosulfan can improve migration and migration capacity of prostate cancer cells by promoting PRL-3 expression, thus suggesting that endosulfan has risk of promoting prostate cancer development. The invention provides important reference value for searching key genes of human diseases caused by environmental pollutants and provides important clues for the future targeted treatment of the human diseases caused by the environmental pollutants.

Description

一种用于评估硫丹促癌风险的方法A method for assessing the cancer-promoting risk of endosulfan

技术领域technical field

本发明属于生物技术领域,涉及一种用于环境污染物促癌风险的方法,尤其是涉及一种评估硫丹暴露引起前列腺癌的方法。The invention belongs to the field of biotechnology, and relates to a method for promoting cancer risk by environmental pollutants, in particular to a method for evaluating prostate cancer caused by endosulfan exposure.

背景技术Background technique

随着科学技术的进步、工业化水平的提高、各类化学制剂正不断地进入人们的生活,其中持久性有机环境污染物(POPs)对环境的危害和人体健康的影响已经不容忽视,包括二嗯英(PCDD/Fs)、多环芳烃(PAHs)、多氯联苯(PCBs)、有机氯农药硫丹等。With the advancement of science and technology, the improvement of the level of industrialization, various chemical agents are constantly entering people's lives, among which the environmental hazards of persistent organic pollutants (POPs) and the impact on human health cannot be ignored, including two British (PCDD/Fs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorine pesticide endosulfan, etc.

硫丹是一种有机氯农药,作为杀虫剂广泛用于农业生产中,具有高生物毒性、潜在的致癌性和环境雌激素效应等特点,它对环境的污染和对生物的毒性作用近几年来引起了广泛的关注。作为一种持久性有机污染物,目前关于硫丹与人类疾病的关系研究仅限于流行病学调查,缺少实验室的数据和生物信息学的分析,硫丹与癌症的关系如何、涉及的关键因子及分子机制至今尚未完全阐明。Endosulfan is an organochlorine pesticide, which is widely used in agricultural production as an insecticide. It has the characteristics of high biological toxicity, potential carcinogenicity and environmental estrogen effect. has attracted widespread attention in recent years. As a persistent organic pollutant, the current research on the relationship between endosulfan and human diseases is limited to epidemiological investigations, lacking laboratory data and bioinformatics analysis, how is the relationship between endosulfan and cancer, and the key factors involved And the molecular mechanism has not yet been fully elucidated.

环境污染物暴露与人类多种疾病尤其是癌症的发生发展密切相关,为了研究环境污染物的毒性特点与基因改变的关系,揭示毒性作用的分子机制,基因芯片技术以其良好的检测基础和分析标准已经应用于环境污染物的疾病机制分析研究。通过对基因表达谱数据进行分析,能够揭示不同组织或不同时间基因表达差异的情况,有利于疾病的分子机制分析、诊断、预后和风险评估。Exposure to environmental pollutants is closely related to the occurrence and development of various human diseases, especially cancer. In order to study the relationship between the toxicity characteristics of environmental pollutants and gene changes, and reveal the molecular mechanism of toxicity, gene chip technology has a good detection basis and analysis Standards have been applied to the analysis of disease mechanisms of environmental pollutants. Analysis of gene expression profile data can reveal differences in gene expression in different tissues or at different times, which is beneficial to molecular mechanism analysis, diagnosis, prognosis and risk assessment of diseases.

基因芯片技术在环境毒理学领域被用来分析污染物对某些组织或器官引起损伤的分子机制、预测和评估人类疾病的健康风险,但如何筛选致病关键基因,确定该基因表达改变与疾病的关系尚无有效可靠的方法。Gene chip technology is used in the field of environmental toxicology to analyze the molecular mechanism of damage caused by pollutants to certain tissues or organs, and to predict and evaluate the health risks of human diseases. There is no effective and reliable method for the relationship.

NextBio和Metacore软件是可以实现人类疾病预测和基因分析的两个软件,目前已得到广泛的应用,两种数据库都能够挖掘基因表达谱中的关键信息,然而二者却有各自的优缺点。NextBio数据库中的研究多为临床医学上的研究,其结果与临床上的状况较为相近,但是受研究数目的限制,使得所分析出结果的准确性受到影响。Metacore数据库具有较多的功能分析,结果可靠性且准确度高,擅长分析转录因子调控网,但是只能实现人、大鼠、小鼠这三个物种的基因芯片分析。NextBio and Metacore software are two softwares that can realize human disease prediction and gene analysis, and have been widely used at present. Both databases can mine key information in gene expression profiles, but both have their own advantages and disadvantages. Most of the studies in the NextBio database are clinical studies, and the results are relatively similar to the clinical situation. However, due to the limitation of the number of studies, the accuracy of the analyzed results is affected. The Metacore database has more functional analysis, the results are reliable and accurate, and it is good at analyzing the transcription factor regulatory network, but it can only realize gene chip analysis of three species: human, rat, and mouse.

发明内容Contents of the invention

鉴于上述作为持久性有机污染物硫丹的生物毒性检测的现有技术的不足,本发明提供一种用于评估环境污染物硫丹促癌风险的方法,尤其是提供检测硫丹促癌风险的生物标志物。本发明发现PRL-3能够参与硫丹对前列腺癌细胞促细胞游走和侵润的作用,是硫丹暴露与前列腺癌最相关的关键基因。In view of the deficiencies in the prior art for the detection of the biological toxicity of endosulfan as a persistent organic pollutant, the present invention provides a method for assessing the cancer-promoting risk of endosulfan, an environmental pollutant, especially a method for detecting the cancer-promoting risk of endosulfan. Biomarkers. The present invention finds that PRL-3 can participate in the effect of endosulfan on promoting cell migration and invasion of prostate cancer cells, and is the key gene most related to endosulfan exposure and prostate cancer.

本发明首先探讨硫丹暴露对血管内皮细胞基因表达谱的影响,利用硫丹暴露所引起的差异表达基因进行生物信息学的分析,即利用Metacore软件分析转录因子调控网,利用Nextbio数据库比较已知疾病的基因表达谱,获得表达变化趋势一致的基因。在对前列腺癌差异表达基因的比较分析中发现有68个基因呈共同上调或下调的变化,PRL-3表达升高最明显,并受到三种转录因子CREB1、p53、ESR1的调控。经文献调研发现其参与多种癌的转移和进展,进一步细胞学实验也证实PRL-3能够参与硫丹对前列腺癌细胞促细胞游走和侵润的作用。本研究筛选出了硫丹促进前列腺癌的关键因子PRL-3,为揭示硫丹与前列腺癌的关系提供了重要的实验依据。The present invention first explores the influence of endosulfan exposure on the gene expression profile of vascular endothelial cells, and uses the differentially expressed genes caused by endosulfan exposure to perform bioinformatics analysis, that is, uses Metacore software to analyze the transcription factor regulatory network, and uses the Nextbio database to compare known Gene expression profiles of diseases, to obtain genes with consistent expression changes. In the comparative analysis of differentially expressed genes in prostate cancer, 68 genes were found to be up-regulated or down-regulated, and the expression of PRL-3 was the most obvious, and it was regulated by three transcription factors CREB1, p53, and ESR1. Through literature research, it is found that it is involved in the metastasis and progression of various cancers, and further cytological experiments have also confirmed that PRL-3 can participate in the role of endosulfan in promoting cell migration and invasion of prostate cancer cells. This study screened out the key factor PRL-3 that endosulfan promotes prostate cancer, which provides an important experimental basis for revealing the relationship between endosulfan and prostate cancer.

本发明的技术方案如下:Technical scheme of the present invention is as follows:

一种用于评估硫丹促癌风险的方法,包括检测生物样品中生物标志物表达的步骤,所述生物标志物为PRL-3。A method for evaluating the cancer-promoting risk of endosulfan, comprising the step of detecting the expression of a biomarker in a biological sample, and the biomarker is PRL-3.

进一步地,在上述技术方案中,所述生物标志物为PRL-3蛋白或PRL mRNA中的一种或两种。Further, in the above technical solution, the biomarker is one or both of PRL-3 protein or PRL mRNA.

进一步地,在上述技术方案中,所述检测基于PRL-3的过表达。优先地,所述PRL-3的过表达为PRL-3蛋白的过表达、PRL mRNA的过表达或PRL-3蛋白和PRL mRNA的过表达。Further, in the above technical solution, the detection is based on the overexpression of PRL-3. Preferably, the overexpression of PRL-3 is overexpression of PRL-3 protein, overexpression of PRL mRNA or overexpression of PRL-3 protein and PRL mRNA.

进一步地,在上述技术方案中,所述生物样品为培养的细胞、细胞裂解产物,组织、组织裂解产物、全血、血浆、血清。血管内皮细胞具有多种生理功能,可产生和分泌多种生物活性物质,参与机体的正常调节,在维持血管稳态方面起重要的作用,与癌症的发生发展密切相关,血管内皮细胞可以作为本发明优选的生物样品。Further, in the above technical solution, the biological sample is cultured cells, cell lysates, tissues, tissue lysates, whole blood, plasma, serum. Vascular endothelial cells have a variety of physiological functions, can produce and secrete a variety of biologically active substances, participate in the normal regulation of the body, play an important role in maintaining vascular homeostasis, and are closely related to the occurrence and development of cancer. Invention of preferred biological samples.

本发明还提供所述的生物标志物PRL-3在评估硫丹促癌风险中的应用。The present invention also provides the application of the biomarker PRL-3 in evaluating the cancer-promoting risk of endosulfan.

本发明的有益效果:Beneficial effects of the present invention:

本发明提供了一种用于评估环境污染物硫丹促癌风险的方法,提供检测硫丹促癌风险的生物标志物PRL-3。本发明的方法简便,相关性较高,可以快速评估硫丹的促癌风险。The invention provides a method for evaluating the cancer-promoting risk of environmental pollutant endosulfan, and provides a biomarker PRL-3 for detecting the cancer-promoting risk of endosulfan. The method of the invention is simple and has high correlation, and can quickly evaluate the cancer-promoting risk of endosulfan.

本研究利用基因表达谱分析了硫丹与疾病的关系,利用生物信息学方法筛选出了硫丹促进前列腺癌的关键因子,从基因层面探讨了硫丹的促癌作用与PRL-3表达升高变化有关。本发明首次提出硫丹暴露与前列腺癌密切相关,且首次提出硫丹暴露与前列腺癌最相关的关键基因为PRL-3。本发明为寻找环境污染物引起人类疾病的关键基因提供重要的参考价值,并为将来针对环境污染物所引起的人类疾病的靶向治疗提供了重要的线索。In this study, the relationship between endosulfan and diseases was analyzed by gene expression profiling, and the key factors of endosulfan promoting prostate cancer were screened out by bioinformatics methods. change related. The present invention proposes for the first time that endosulfan exposure is closely related to prostate cancer, and for the first time it is proposed that the key gene most related to endosulfan exposure and prostate cancer is PRL-3. The invention provides important reference value for searching key genes of human diseases caused by environmental pollutants, and provides important clues for targeted treatment of human diseases caused by environmental pollutants in the future.

附图说明Description of drawings

图1为利用Metacore软件预测分析与硫丹暴露相关的人类疾病。Figure 1 is the use of Metacore software to predict and analyze human diseases related to endosulfan exposure.

图2为利用Nextbio软件预测分析与硫丹暴露相关的癌症。Figure 2 is the predictive analysis of cancers related to endosulfan exposure using Nextbio software.

图3为一项关于前列腺癌的基因表达谱的研究。Figure 3 is a study on the gene expression profile of prostate cancer.

图4为硫丹暴露与前列腺癌差异表达基因变化的比较。Figure 4 is a comparison between endosulfan exposure and differentially expressed gene changes in prostate cancer.

图5为硫丹引起PC3细胞PRL-3表达水平升高(P*<0.05vs D)。Figure 5 shows that endosulfan increases the expression level of PRL-3 in PC3 cells (P*<0.05vs D).

图6为PRL-3抑制剂干扰硫丹暴露对PC3细胞迁移能力的影响(P**<0.01)。Figure 6 shows the effect of PRL-3 inhibitors interfering with endosulfan exposure on the migration ability of PC3 cells (P**<0.01).

图7为PRL-3抑制剂干扰硫丹暴露对PC3细胞侵润能力的影响(P**<0.01)。Figure 7 shows the effect of PRL-3 inhibitors interfering with endosulfan exposure on the invasion ability of PC3 cells (P**<0.01).

具体实施方式detailed description

以下的实施例便于更好的理解本发明,但并不限定本发明。另外,下述实施例中,如无特殊说明,所使用的实验方法均为常规方法,所用材料、试剂等均可从生物或化学试剂公司购买。The following examples facilitate a better understanding of the present invention, but do not limit the present invention. In addition, in the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

实施例1Example 1

(1)硫丹引起血管内皮细胞差异表达基因变化:(1) Endosulfan causes changes in differentially expressed genes in vascular endothelial cells:

对血管内皮细胞(HUVEC-C)进行硫丹暴露处理,实验分为两组:DMSO对照组(二甲基亚砜,D),硫丹实验组(endosulfan,ES)。对照组DMSO 0.1%浓度,硫丹暴露浓度为20μM,暴露时间为48小时,收集细胞提取总RNA进行基因芯片[Agilent Human Gene ExpressionProfiling,上海康成生物有限公司,Whole Human Genome Oligo Microarray(4x44K,Agilent Technologies)]分析,通过基因表达谱分析发现硫丹暴露时相对于对照组有457个基因表达下调而319个基因表达上调。Endosulfan exposure treatment was performed on vascular endothelial cells (HUVEC-C), and the experiment was divided into two groups: DMSO control group (dimethyl sulfoxide, D) and endosulfan experimental group (endosulfan, ES). The concentration of DMSO in the control group was 0.1%, the exposure concentration of endosulfan was 20 μM, and the exposure time was 48 hours. The cells were collected to extract total RNA for gene chip [Agilent Human Gene Expression Profiling, Shanghai Kangcheng Biological Co., Ltd., Whole Human Genome Oligo Microarray (4x44K, Agilent Technologies )] analysis, through gene expression profiling analysis, it was found that compared with the control group, 457 genes were down-regulated and 319 genes were up-regulated when exposed to endosulfan.

(2)硫丹暴露引起人类疾病的预测分析:(2) Predictive analysis of human diseases caused by endosulfan exposure:

对于上述(1)中筛选出的下调基因和上调基因,利用Metacore软件预测与硫丹相关的前十位人类疾病,其相关性按-log10(P-Value)的数值排列,数值越大则代表与该疾病相关性越高。结果如图1,图1表明,与硫丹最相关的疾病为男性生殖系统疾病,前列腺癌、前列腺疾病等。For the down-regulated genes and up-regulated genes screened in (1) above, use Metacore software to predict the top ten human diseases related to endosulfan, and their correlations are arranged according to the value of -log 10 (P-Value). The higher the correlation with the disease. The results are shown in Figure 1. Figure 1 shows that the diseases most related to endosulfan are male reproductive system diseases, prostate cancer, and prostate diseases.

对于上述(1)中筛选出的下调基因和上调基因,通过NextBio软件预测与硫丹相关的人类癌症,按照评分的高低排序,评分的分数表示疾病与硫丹暴露的相关性程度。结果如图2,图2表明,与硫丹相关的前十位癌症,包括胃癌、肝癌、脑癌、肠癌、前列腺癌等。For the down-regulated genes and up-regulated genes screened in (1) above, the human cancers related to endosulfan are predicted by NextBio software, and they are sorted according to the high and low scores, and the scores of the scores indicate the degree of correlation between the disease and endosulfan exposure. The results are shown in Figure 2. Figure 2 shows that the top ten cancers related to endosulfan include gastric cancer, liver cancer, brain cancer, intestinal cancer, and prostate cancer.

通过Metacore和Nextbio软件分析结果,确定硫丹暴露与前列腺癌密切相关。The results were analyzed by Metacore and Nextbio software, and it was determined that endosulfan exposure was closely related to prostate cancer.

实施例2Example 2

(1)人类前列腺癌的基因表达谱分析:(1) Gene expression profiling analysis of human prostate cancer:

为了研究硫丹与前列腺癌之间的相关性,在NextBio数据库按照研究物种为人、数据类型为基因芯片的数据、共同差异基因相关性P值小于0.05的标准筛选出一项关于前列腺癌基因表达谱的临床研究数据(来自Stanford Microarray Database,GSE3933;URLhttp://genome-www5.stanford.edu/stanford university,文献截图如图3)。该数据由斯坦福大学数据库提供,是包含了62个前列腺癌组织、41个正常的前列腺组织和9个淋巴结转移癌的基因表达谱,整个研究中探测了约26,000个基因,其中前列腺癌组织与正常前列腺组织相比有7767个基因发生差异性表达变化。In order to study the correlation between endosulfan and prostate cancer, a study on the gene expression profile of prostate cancer was screened out in the NextBio database according to the research species of human, the data type of gene chip data, and the common differential gene correlation P value less than 0.05. The clinical research data (from Stanford Microarray Database, GSE3933; URL http://genome-www5.stanford.edu/stanford university, the document screenshot is shown in Figure 3). The data is provided by the Stanford University database, which contains the gene expression profiles of 62 prostate cancer tissues, 41 normal prostate tissues and 9 lymph node metastatic cancers. About 26,000 genes were detected in the whole study, and the prostate cancer tissues were compared with the normal ones. Compared with prostate tissue, there were 7767 genes with differential expression changes.

(2)硫丹暴露引起的差异表达基因与前列腺癌差异表达基因变化的比较分析:(2) Comparative analysis of differentially expressed genes caused by endosulfan exposure and differentially expressed genes in prostate cancer:

利用NextBio软件将实施例1得到的硫丹暴露的基因表达谱(Bs1)与前列腺癌的基因表达谱(Bs2)进行比较分析,结果如图4,发现共同差异表达的基因有142个,具有相同变化趋势的基因有68个,其中18个基因共同表达上调,50个基因共同表达下调。其中PTP4A3基因变化倍数最大,PTP4A3编码的蛋白称为PRL-3。The gene expression profile (Bs1) exposed to endosulfan obtained in Example 1 was compared with the gene expression profile (Bs2) of prostate cancer using NextBio software. As shown in Figure 4, it was found that there were 142 genes with the same differential expression. There were 68 genes with changing trends, among which 18 genes were co-expressed up-regulated and 50 genes were co-expressed down-regulated. Among them, PTP4A3 gene had the largest fold change, and the protein encoded by PTP4A3 was called PRL-3.

(3)硫丹暴露的转录因子调控网分析:(3) Analysis of the regulatory network of transcription factors exposed to endosulfan:

将步骤(2)中筛选得到的68个具有相同变化趋势的共同差异基因上传至Metacore数据库,进行转录因子调控网分析。共得到三十个相关的转录因子调控网络,其中前五名最相关的转录因子,其顺序按照P-Value的大小排列,如表1所示,硫丹暴露与前列腺癌相关的转录因子分别为CREB1、p53、SP1、c-Myc、ESR1。通过比较五个转录因子调控网络,发现PTP4A3在三个转录因子调控网络中,受到CREB1,p53,ESR1的直接调控。The 68 common differential genes with the same variation trend screened in step (2) were uploaded to the Metacore database for transcription factor regulatory network analysis. A total of 30 relevant transcription factor regulatory networks were obtained, among which the top five most relevant transcription factors were arranged according to the size of P-Value, as shown in Table 1, the transcription factors related to endosulfan exposure and prostate cancer were CREB1, p53, SP1, c-Myc, ESR1. By comparing the five transcription factor regulatory networks, it was found that PTP4A3 was directly regulated by CREB1, p53, and ESR1 in the three transcription factor regulatory networks.

表1.硫丹暴露与前列腺癌最相关的五个转录因子Table 1. Five transcription factors most associated with endosulfan exposure and prostate cancer

实施例3Example 3

硫丹暴露引起PRL-3表达水平的变化:Endosulfan Exposure Causes Changes in PRL-3 Expression Levels:

对前列腺癌PC3细胞进行硫丹暴露处理,实验分为两组:DMSO对照组(二甲基亚砜,D),硫丹实验组(endosulfan,ES)。对照组DMSO 0.1%浓度,硫丹暴露浓度为20μM,暴露时间为48小时,收集DMSO组和硫丹实验组的细胞,用Trizol法提取总RNA,测定RNA浓度并进行RNA质检。采用SYBR green(Applied Biosystems,ABI)方法进行qRT-PCR反应,设计引物(PRL-3和GAPDH,见表2),以GAPDH为内参,用2-ΔΔCt方法将mRNA表达差异通过处理样本相对于未处理样本的倍数来表示。结果如图5所示,硫丹暴露(20μM)可以引起PRL-3mRNA表达升高,具有统计学意义。Prostate cancer PC3 cells were exposed to endosulfan, and the experiment was divided into two groups: DMSO control group (dimethyl sulfoxide, D) and endosulfan experimental group (endosulfan, ES). The concentration of DMSO in the control group was 0.1%, the exposure concentration of endosulfan was 20 μM, and the exposure time was 48 hours. The cells in the DMSO group and the endosulfan experimental group were collected, and the total RNA was extracted by Trizol method, and the RNA concentration was determined and RNA quality inspection was carried out. The SYBR green (Applied Biosystems, ABI) method was used for qRT-PCR reaction, primers (PRL-3 and GAPDH, see Table 2) were designed, GAPDH was used as an internal reference, and the difference in mRNA expression was measured by the 2- ΔΔCt method by comparing the treated samples with the untreated samples. Multiples of processed samples are represented. The results are shown in Figure 5, endosulfan exposure (20 μM) can cause an increase in the expression of PRL-3 mRNA, which is statistically significant.

表2.引物序列及相关信息Table 2. Primer sequences and related information

实施例4Example 4

PRL-3干扰硫丹暴露引起PC3细胞游走和侵润能力的变化:PRL-3 interferes with changes in the migration and invasion abilities of PC3 cells induced by endosulfan exposure:

对前列腺癌PC3细胞进行硫丹暴露处理,实验分为四组:正常对照组(C),DMSO对照组(D),硫丹实验组(20μM ES),硫丹加PRL-3抑制剂组(20μM ES+PRL-3 inhibitor),PRL-3inhibitor浓度为10μM,暴露时间为48小时,测定各组细胞游走和侵润能力的变化。Prostate cancer PC3 cells were exposed to endosulfan, and the experiment was divided into four groups: normal control group (C), DMSO control group (D), endosulfan experimental group (20μM ES), endosulfan plus PRL-3 inhibitor group ( 20 μM ES+PRL-3 inhibitor), the concentration of PRL-3 inhibitor was 10 μM, the exposure time was 48 hours, and the changes of cell migration and invasion ability in each group were measured.

为了测定细胞游走迁移能力,用无血清的培养液制备100μL PC3细胞悬液放入上层小室中,将500μL完全性培养基(含10%胎牛血清)放在下层小室里,在37℃、5%CO2孵箱中孵育24h,PBS清洗,4%PFA固定后用棉签轻擦掉微孔膜上表面的细胞,染色干燥,取下微孔膜置于载玻片上进行镜下观察,以有游走能力迁移进入下层的细胞占总细胞数的百分比表示(图6)。细胞侵润能力测定提前将无血清培养基配置的Matrigel(BD Biosciences)加入上层小室中,覆盖小室微孔膜,其他与迁移实验相同,培养48h后观察结果,细胞侵润能力以有侵润能力进入下层的细胞占总细胞数的百分比表示(图7)。In order to measure the ability of cell migration and migration, 100 μL of PC3 cell suspension was prepared with serum-free culture medium and placed in the upper chamber, and 500 μL of complete medium (containing 10% fetal bovine serum) was placed in the lower chamber, at 37 ° C, Incubate in a 5% CO2 incubator for 24 hours, wash with PBS, fix with 4% PFA, wipe off the cells on the upper surface of the microporous membrane with a cotton swab, stain and dry, remove the microporous membrane and place it on a glass slide for observation under a microscope. Cells with migratory ability to migrate into the lower layer are expressed as a percentage of the total number of cells ( FIG. 6 ). Determination of cell invasion ability Matrigel (BD Biosciences) prepared in serum-free medium was added to the upper chamber in advance to cover the microporous membrane of the chamber. Others were the same as the migration experiment, and the results were observed after 48 hours of culture. The cell invasion ability was infiltrating ability Cells entering the lower layer were expressed as a percentage of the total number of cells (Figure 7).

结果显示,硫丹暴露能够促进PC3细胞游走和侵润能力,而PRL-3抑制剂处理后抑制了硫丹的促PC3游走和侵润作用,提示PRL-3作为关键因子参与了硫丹的促癌作用。The results showed that endosulfan exposure could promote PC3 cell migration and invasion, while PRL-3 inhibitor treatment inhibited endosulfan's ability to promote PC3 migration and invasion, suggesting that PRL-3 was involved in endosulfan as a key factor. cancer-promoting effect.

综上,本发明通过生物信息学分析筛选出PRL-3是硫丹与前列腺癌相关的关键基因,在细胞学方面证实硫丹可以通过促进PRL-3表达,提高前列腺癌细胞的迁移游走能力,提示硫丹具有促进前列腺癌发展的风险。这些分析和实验结果为确定PRL-3是硫丹促癌作用的关键因子提供了重要的参考依据。In summary, the present invention screens out PRL-3 as the key gene related to endosulfan and prostate cancer through bioinformatics analysis, and it is confirmed in cytology that endosulfan can improve the migration and migration ability of prostate cancer cells by promoting the expression of PRL-3 , suggesting that endosulfan has the risk of promoting the development of prostate cancer. These analysis and experimental results provide an important reference for determining that PRL-3 is the key factor for the carcinogenic effect of endosulfan.

Claims (6)

1. a kind of method for being used to assess 5a,6,9,9a-hexahydro-6,9-methano-2,4 rush cancer risk, including in detection biological sample the step of biomarker expression, Characterized in that, the biomarker is PRL-3.
2. according to the method described in claim 1, it is characterised in that the biomarker is PRL-3 albumen or PRL mRNA One or both of.
3. according to the method described in claim 1, it is characterised in that the overexpression of the detection based on PRL-3.
4. method according to claim 3, it is characterised in that the overexpression of the PRL-3 is PRL-3 albumen or PRL One or both of mRNA overexpression.
5. according to the method described in claim 1, it is characterised in that the biological sample cracks production for cell, the cell of culture Thing, tissue, Tissue Lysis product, whole blood, blood plasma, serum.
6. biomarker PRL-3 as claimed in claim 1 is assessing the application during 5a,6,9,9a-hexahydro-6,9-methano-2,4 promotees cancer risk.
CN201710322541.XA 2017-05-09 2017-05-09 A method for assessing the cancer-promoting risk of endosulfan Pending CN107058556A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710322541.XA CN107058556A (en) 2017-05-09 2017-05-09 A method for assessing the cancer-promoting risk of endosulfan

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710322541.XA CN107058556A (en) 2017-05-09 2017-05-09 A method for assessing the cancer-promoting risk of endosulfan

Publications (1)

Publication Number Publication Date
CN107058556A true CN107058556A (en) 2017-08-18

Family

ID=59597539

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710322541.XA Pending CN107058556A (en) 2017-05-09 2017-05-09 A method for assessing the cancer-promoting risk of endosulfan

Country Status (1)

Country Link
CN (1) CN107058556A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040010045A1 (en) * 2001-09-07 2004-01-15 Taolin Yi Therapeutic compositions comprised of pentamidine and methods of using same to treat cancer
WO2016024918A1 (en) * 2014-08-13 2016-02-18 Agency For Science, Technology And Research Diagnosis of cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040010045A1 (en) * 2001-09-07 2004-01-15 Taolin Yi Therapeutic compositions comprised of pentamidine and methods of using same to treat cancer
WO2016024918A1 (en) * 2014-08-13 2016-02-18 Agency For Science, Technology And Research Diagnosis of cancer

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ELISABETH FEIK等: ""Integrative Analysis of Prostate Cancer Aggressiveness"", 《THE PROSTATE》 *
ESTEN N. VANDSEMB等: ""Phosphatase of regenerating liver 3 (PRL-3) is overexpressed in human prostate cancer tissue and promotes growth and migration"", 《J TRANSL MED》 *
PIERRE R. BAND等: ""Prostate Cancer Risk and Exposure to Pesticides in British Columbia Farmers"", 《THE PROSTATE》 *
林丽梅: ""硫丹暴露HUVEC-C细胞的基因表达谱和疾病预测分析"", 《中国学位论文全文数据库》 *

Similar Documents

Publication Publication Date Title
Andersen et al. Integrative metabolic and transcriptomic profiling of prostate cancer tissue containing reactive stroma
Krauskopf et al. The human circulating miRNome reflects multiple organ disease risks in association with short-term exposure to traffic-related air pollution
Nakagawa et al. Molecular markers of tubulointerstitial fibrosis and tubular cell damage in patients with chronic kidney disease
Lauwen et al. Omics biomarkers in ophthalmology
Rotem et al. ALS along the axons–expression of coding and noncoding RNA differs in axons of ALS models
US11142800B2 (en) Biomarkers of cancer
Journe et al. TYRP1 mRNA expression in melanoma metastases correlates with clinical outcome
Jia et al. Sets of serum exosomal microRNAs as candidate diagnostic biomarkers for Kawasaki disease
KR20130129347A (en) Lung cancer biomarkers and uses thereof
Huang et al. Increased STAT1 signaling in endocrine-resistant breast cancer
Ning et al. miRNAs deregulation in serum of mice is associated with lung cancer related pathway deregulation induced by PM2. 5
Gao et al. Specific long non-coding RNAs response to occupational PAHs exposure in coke oven workers
EP2867375A1 (en) Use of markers in the diagnosis and treatment of prostate cancer
Zhan et al. A three‐gene signature from protein–protein interaction network of LOXL 2‐and actin‐related proteins for esophageal squamous cell carcinoma prognosis
Cheng et al. Single-cell analysis reveals urothelial cell heterogeneity and regenerative cues following cyclophosphamide-induced bladder injury
Mee et al. Maintaining breast cancer specimen integrity and individual or simultaneous extraction of quality DNA, RNA, and proteins from Allprotect-stabilized and nonstabilized tissue samples
Boujedidi et al. Housekeeping gene variability in the liver of alcoholic patients
JP2010115131A (en) Evaluation method and method for screening skin ageing improving agent and skin aging preventive
Abdalla et al. Promising urinary protein biomarkers for the early detection of hepatocellular carcinoma among high-risk hepatitis C virus Egyptian patients
Domenyuk et al. Poly-ligand profiling differentiates trastuzumab-treated breast cancer patients according to their outcomes
Wang et al. Identification of proteins implicated in the development of pancreatic cancer-associated diabetes mellitus by iTRAQ-based quantitative proteomics
Chang et al. Link between plasminogen activator inhibitor-1 and cardiovascular risk in chronic hepatitis C after viral clearance
KR102087373B1 (en) Protein biomarker panel for detecting non-small cell lung cancer and method for diagnosing a non-small cell lung cancer by the use thereof
KR20250005003A (en) Biomarker for diagnosing atopic dermatitis and use thereof
Li et al. A panel of three serum microRNA can be used as potential diagnostic biomarkers for nasopharyngeal carcinoma

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20170818