CN108977533B - 一种用于预测慢性乙肝炎症损伤的miRNA组合物 - Google Patents
一种用于预测慢性乙肝炎症损伤的miRNA组合物 Download PDFInfo
- Publication number
- CN108977533B CN108977533B CN201811052248.7A CN201811052248A CN108977533B CN 108977533 B CN108977533 B CN 108977533B CN 201811052248 A CN201811052248 A CN 201811052248A CN 108977533 B CN108977533 B CN 108977533B
- Authority
- CN
- China
- Prior art keywords
- mir
- mirna
- hsa
- chronic hepatitis
- injury
- 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.)
- Expired - Fee Related
Links
- 208000000419 Chronic Hepatitis B Diseases 0.000 title claims abstract description 27
- 208000002672 hepatitis B Diseases 0.000 title claims abstract description 27
- 230000006378 damage Effects 0.000 title claims abstract description 21
- 208000027418 Wounds and injury Diseases 0.000 title claims abstract description 18
- 208000014674 injury Diseases 0.000 title claims abstract description 18
- 108091070501 miRNA Proteins 0.000 title abstract description 40
- 239000000203 mixture Substances 0.000 title abstract description 29
- 108091044802 Homo sapiens miR-1285-1 stem-loop Proteins 0.000 claims abstract description 13
- 108091069034 Homo sapiens miR-193a stem-loop Proteins 0.000 claims abstract description 13
- 108091070372 Homo sapiens miR-26a-1 stem-loop Proteins 0.000 claims abstract description 13
- 108091065428 Homo sapiens miR-26a-2 stem-loop Proteins 0.000 claims abstract description 13
- 238000013399 early diagnosis Methods 0.000 claims abstract description 4
- 239000002679 microRNA Substances 0.000 abstract description 34
- 239000000090 biomarker Substances 0.000 abstract description 8
- 210000004185 liver Anatomy 0.000 abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 6
- 201000010099 disease Diseases 0.000 abstract description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract 1
- 238000011529 RT qPCR Methods 0.000 abstract 1
- 238000001228 spectrum Methods 0.000 abstract 1
- 230000002757 inflammatory effect Effects 0.000 description 14
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 13
- 230000017074 necrotic cell death Effects 0.000 description 12
- 238000012163 sequencing technique Methods 0.000 description 12
- 238000012216 screening Methods 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 7
- 238000012549 training Methods 0.000 description 7
- 108091032955 Bacterial small RNA Proteins 0.000 description 6
- 101710142246 External core antigen Proteins 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 208000019423 liver disease Diseases 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 206010019799 Hepatitis viral Diseases 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 238000002123 RNA extraction Methods 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- 230000000840 anti-viral effect Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 238000007477 logistic regression Methods 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 201000001862 viral hepatitis Diseases 0.000 description 3
- 208000007848 Alcoholism Diseases 0.000 description 2
- 108091067995 Homo sapiens miR-192 stem-loop Proteins 0.000 description 2
- 206010067125 Liver injury Diseases 0.000 description 2
- 108091030146 MiRBase Proteins 0.000 description 2
- 239000013614 RNA sample Substances 0.000 description 2
- 102000039471 Small Nuclear RNA Human genes 0.000 description 2
- 108020004688 Small Nuclear RNA Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 231100000234 hepatic damage Toxicity 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 230000008818 liver damage Effects 0.000 description 2
- 210000005228 liver tissue Anatomy 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000010223 real-time analysis Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 206010019728 Hepatitis alcoholic Diseases 0.000 description 1
- 108091069016 Homo sapiens miR-122 stem-loop Proteins 0.000 description 1
- 108091032636 Homo sapiens miR-433 stem-loop Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108091007780 MiR-122 Proteins 0.000 description 1
- 108091028695 MiR-224 Proteins 0.000 description 1
- 108091028684 Mir-145 Proteins 0.000 description 1
- 208000003445 Mouth Neoplasms Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 208000002353 alcoholic hepatitis Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229940124405 anti-hepatitis b virus drug Drugs 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000012350 deep sequencing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003677 hemocyte Anatomy 0.000 description 1
- 229940000351 hemocyte Drugs 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000028974 hepatocyte apoptotic process Effects 0.000 description 1
- 230000002443 hepatoprotective effect Effects 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 238000002558 medical inspection Methods 0.000 description 1
- 108091051828 miR-122 stem-loop Proteins 0.000 description 1
- 108091025686 miR-199a stem-loop Proteins 0.000 description 1
- -1 miR-22l/222 Proteins 0.000 description 1
- 108091061970 miR-26a stem-loop Proteins 0.000 description 1
- 108091029119 miR-34a stem-loop Proteins 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000011022 operating instruction Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Communicable Diseases (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及一种用于预测慢性乙肝炎症损伤的miRNA组合物,所述组合物包括hsa‑miR‑1285‑5p、hsa‑miR‑193a‑5p和hsa miR‑26a‑5p,核苷酸序列分别见SEQ ID NO.1‑3。本发明解决了现有技术中采用肝组织学检查来评估慢性乙肝炎症损伤带来有创性和局限性的问题,通过qRT‑PCR对上述miRNA组合物进行检测,可以实现对慢性乙肝炎症损伤的早期诊断,miRNA作为生物学标志物是一种非侵入性取样,可以在无创条件下通过单独的miRNA或miRNA组合物的表达谱来辅助诊断被试者是否有慢性乙肝炎症损伤,提高了病情判断的准确率。
Description
技术领域
本发明涉及一种用于预测慢性乙肝炎症损伤的miRNA组合物,属于医学检验技术领域。
背景技术
微小RNA(microRNA)是一类内源性非编码单链小分子RNA,长度为18~24个核苷酸。近年对微小RNA生物功能的研究越来越多,发现其在机体生理及病理方面起重要作用,生理情况下参与细胞的增殖、分化过程,若表达及功能失调则可能导致包括白血病在内的肿瘤的发生,以及病毒、细菌感染等多种病理现象。
微小RNA普遍存在于人类的体液中,性质稳定,可以定量检测,且存在显著的疾病特异性。近年的研究表明,血液、唾液、尿液、乳汁及脑脊液等体液miRNA检测可应用于妊娠等生理状态的判断以及口腔癌、膀胱癌及阿尔海默茨病等疾病的诊断和预后判断。体液特异性miRNA检测的临床意义及应用前景已引起高度关注,miRNA作为一类非编码调节性的小分子RNA有可能取代传统的特异蛋白为代表的生物标志物。体液miRNA含量丰富且性质稳定,具备成为优异生物标志物的潜质,并且拥有以蛋白质为代表的传统生物标志物不具备的特性,无需抗体制备且易于精确定量,可能克服抗原抗体类生物标志物的制备瓶颈。
肝脏是人体内重要的器官之一,肝脏疾病尤其是病毒性肝炎、肝细胞癌、酒精性肝炎等病情重,治疗棘手,严重困扰着人类的健康,研究肝脏疾病的发病机制对其治疗至关重要。不断有研究发现,微小RNA与多种肝脏疾病相关,肝脏中的微小RNA表达异常或缺失可导致肝细胞凋亡、再生,甚至感染等,微小RNA参与众多肝脏疾病的病理发病过程。在众多肝脏疾病中,肝细胞癌和微小RNA的相互关系研究最多。近年研究发现,微小RNA失调和癌症发生发展密切相关,微小RNA参与癌症的病理发病过程。肝细胞癌的发生同样受微小RNA的调控,已发现在肝细胞癌中表达上调的微小RNA有miR-2l、miR-34a、miR-22l/222和miR-224等,而表达下调的有miR-122、miR-145和miR-199a等。
ALT是肝功能损害最敏感的检测指标,也是各种指南关于慢性乙肝抗病毒治疗和预后判断的重要指标,但很多ALT正常的慢性HBV感染者肝组织学均提示有严重的炎症反应损伤,一部分患者甚至出现肝硬化表现。因此2010年我国慢性乙肝防治指南明确建议对>40岁或有HCC家族史的男性,即使ALT正常建议肝组织学检查,以明确肝组织的损伤,以免耽误抗病毒治疗。肝组织学检查虽然是确切了解肝组织损伤的金标准,但其有创性和局限性,不能被大多患者接受。因此,寻找到新的诊断方法和更敏感的生物学标志是慢性乙肝迫切要解决的问题。
发明内容
本发明的目的是解决上述不足,提供一种用于预测慢性乙肝的miRNA组合物。
技术方案
一种用于预测慢性乙肝炎症损伤的miRNA组合物,所述组合物包括hsa-miR-1285-5p、hsa-miR-193a-5p和hsa miR-26a-5p。
进一步,所述hsa-miR-1285-5p的核苷酸序列如SEQ ID NO.1所示。
SEQ ID NO.1:GAUCUCACUUUGUUGCCCAGG。
进一步,所述hsa-miR-193a-5p的核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.2:UGGGUCUUUGCGGGCGAGAUGA。
进一步,所述hsa miR-26a-5p的核苷酸序列如SEQ ID NO.3所示。
SEQ ID NO.3:UUCAAGUAAUCCAGGAUAGGCU。
用于检测上述miRNA组合物的引物组,包括针对hsa-miR-1285-5p的特异性引物,针对hsa-miR-193a-5p的特异性引物以及针对hsa miR-26a-5p的特异性引物;所述针对hsa-miR-1285-5p的特异性引物包括SEQ ID NO.4所示的正向引物和SEQ ID NO.5所示的反向引物,所述针对hsa-miR-193a-5p的特异性引物包括SEQ ID NO.6所示的正向引物和SEQID NO.7所示的反向引物,所述针对hsa miR-26a-5p的特异性引物包括SEQ ID NO.8所示的正向引物和SEQ ID NO.9所示的反向引物。
上述用于检测miRNA组合物的引物组在预测或诊断慢性乙肝炎症损伤方面的应用。采用该引物组,通过qRT-PCR对上述miRNA组合物进行检测,可以实现对慢性乙肝炎症损伤的早期诊断,为慢性乙肝炎症损伤的早期发现和诊断提供参考。
一种慢性乙肝炎症损伤的诊断试剂盒,包括上述用于检测miRNA组合物的引物组。
进一步,所述miRNA组合物的筛选方法包括如下步骤:
(1)样品采集:采集患者新鲜血液,2h内分离,收集血清,并将血清转移至一次性使用无RNA酶的无菌微量离心管中,-80℃保存,备用;
(2)总RNA提取:采用LCS TRK1001试剂盒(LC Sciences)操作说明书进行;
(3)文库构建:使用Illumina Truseq Small RNA Preparation kit试剂盒参照试剂盒说明书Illumina’s TruSeq Small RNA Sample Preparation Guide构建小RNA文库,总RNA链接5'接头和3'接头后经RT-PCR扩增形成小分子RNA的cDNA文库,经过6%TBE变性胶电泳分离,将长度范围在147bp的小分子RNA切胶回收;
(4)第二代测序:cDNA经纯化后在Illumina’s Cluster Station上生成DNA簇后上机(Illumina GAIIx)进行测序,通过Illumina’s Sequencing Control Studio softwareversion 2.8(SCS v2.8)软件实时分析测序图片并使用Illumina's Real-Time Analysisversion 1.8.70(RTAv1.8.70)提取base-calling,提取的原始序列利用ACGT101-miR v4.2(LC Sciences)软件分析,生成RawData数据库,同时去除由于样品制备、测序化学与处理以及测序仪器的光学数码处理而产生的非纯序列,剩下的序列(长度在15和32bases)按照families进行分组,生成mappableReads。Mappable序列与最新版本的miRbase数据库以及测序物种基因组进行序列比对,鉴定该物种已知的miRNA;同时发现新的5p或者3p miRNA序列,鉴定在其它近源物种中已有报道,在该物种中崭新的miRNA序列。其中Mappable序列能与Rfam(ie rRNA,tRNA,snRNA,snoRNA and others),Repbase以及mRNA序列比对上的均被去除。除此之外,为了保证筛选到高质量的基因结果,把reads数小于10的基因剔除掉。最后分析实验组和对照组之间差异表达的miRNAs,最终获得hsa-miR-1285-5p、hsa-miR-193a-5p和hsamiR-26a-5p。
本发明的有益效果是:
本发明提供了一种用于预测慢性乙肝炎症损伤的miRNA组合物,通过qRT-PCR对该miRNA组合物进行定量检测,实现对慢性乙肝肝纤维化的早期诊断和预测,miRNA作为生物学标志物是一种非侵入性取样,可以在无创条件下通过单独的miRNA或miRNA组合物的表达谱来辅助诊断被试者是否有慢性乙肝炎症损伤,提高了病情判断的准确率。
附图说明
图1为本发明的miRNA组合物在验证集炎症坏死组的ROC曲线。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1慢性乙肝炎症损伤相关miRNA的筛选
采用四步筛选法:
(1)发现阶段,我们采用新一代深度测序技术(IlluminaHiseq 2000),对发现集混合样本进行检测,发现表达差异的miRNA,轻、中度以上炎症坏死分别选择6例;
(2)筛选阶段,使用qRT-PCR的方法,选定内参miRNA,建立均一的少量队列筛选集,2-△△Ct测定循环数(Ct),淘汰miRNACt value<35和detection rate>75%;p<0.05的miRNA,轻、中度以上炎症坏死分别选择20例;
(3)训练阶段,训练筛选阶段选出的目的miRNA,使用qRT-PCR对大样本训练集(轻、中度以上炎症坏死分别选择136例,201例)检验目的miRNA,并使用LogiticP建立miRNA模型。
(4)验证阶段,利用训练集建立的miRNA模型,对独立建立的队列验证集(轻、中度以上炎症坏死分别选择75例,125例)进行验证,评价其诊断价值。
具体如下:
病例选择:病例来自于镇江市第三人民医院2008年8月至2013年12月所有的慢性乙肝感染者进行肝穿刺病理检查的患者。病例入选标准:(1)HBsAg阳性至少6月;(2)HBVDNA>1000拷贝/毫升。排除标准如下:(1)合并其他病毒性肝炎和病毒性疾病,如HAV,HCV,HEV,HIV;(2)药物性肝损以及有规律嗜酒史;(3)标本不符合要求;(4)有使用抗乙肝病毒药物史或者肝穿刺前已开始抗病毒治疗;(5)肝穿刺前12月内有保肝药使用。共入选1210例进行过肝穿刺慢性乙肝病例,排除621例(合并其他病毒性肝炎,n=83;药物性肝损或者有规律嗜酒史,n=169;样本不符合要求,n=35;有使用抗病毒药物史,n=156;随访期间有保肝药使用,n=178)。共589例入选病例,PNALT共129例,其中HBeAg阳性48例,HBeAg阴性81例;PIALT(ALT1-2ULN,n=186),其中HBeAg阳性87例,HBeAg阴性99例;PIALT(ALT≥2ULN,n=274),其中HBeAg阳性86例,HBeAg阴性,168例。
1.样品采集
患者在首次门诊或住院时采集新鲜血液5ml于医用血清管中(无肝素抗凝),上下轻轻颠倒混匀,立即将全血置于4℃冰盒中保存,并在2h内4000g,离心10min。将上层血清转移至1.5ml离心管(RNase free)中,13000g,离心2min。最后将上清液转移至2ml旋盖尖底离心管中(RNase free),每个管子吸入250ul血清进行分装,弃去血细胞沉淀。置于-80℃长期保存。
2.总RNA提取及质检
血清RNA的抽提采用LCS TRK1001试剂盒(LC Sciences)操作说明书进行。随机抽取两个在血清中稳定表达的miRNA作为标准检测总RNA提取质量,分别为hsa-miR-16、hsa-miR-192。然后各取2μl,以上述引物对应的逆转录引物分别逆转录(反应体系为10μl);以1μl/孔的cDNA为模板,进行realtimePCR,反应体系为20μl,复孔为三个,同时做引物NTC(模板以水代替)。然后检测hsa-miR-16和hsa-miR-192PCR扩展曲线、溶解曲线以及CT值。
3.文库构建
使用Illumina Truseq Small RNA Preparation kit试剂盒参照试剂盒说明书Illumina’s TruSeq Small RNA Sample Preparation Guide构建小RNA文库。总RNA链接5’接头和3’接头后经RT-PCR扩增形成小分子RNA的cDNA文库,经过6%TBE变性胶电泳分离,将长度范围在147bp的小分子RNA切胶回收。
4.第二代测序
cDNA经纯化后在Illumina’s Cluster Station上生成DNA簇后上机(IlluminaGAIIx)进行测序。通过Illumina’s Sequencing Control Studio software version 2.8(SCS v2.8)软件实时分析测序图片并使用Illumina's Real-Time Analysis version1.8.70(RTAv1.8.70)提取base-calling。提取的原始序列利用ACGT101-miR v4.2(LCSciences)软件分析,生成RawData数据库,同时去除由于样品制备、测序化学与处理以及测序仪器的光学数码处理而产生的非纯序列。剩下的序列(长度在15和32bases)按照families进行分组,生成mappableReads。Mappable序列与最新版本的miRbase数据库以及测序物种基因组进行序列比对,鉴定该物种已知的miRNA;同时发现新的5p或者3p miRNA序列,鉴定在其它近源物种中已有报道,在该物种中崭新的miRNA序列。其中Mappable序列能与Rfam(ierRNA,tRNA,snRNA,snoRNA and others),Repbase以及mRNA序列比对上的均被去除。除此之外,为了保证筛选到高质量的基因结果,我们把reads数小于10的基因剔除掉。最后分析实验组和对照组之间差异表达的miRNAs。
通过上述测序分析,轻度炎症坏死和中度以上炎症坏死组经过初级分析后得到8,580,434和7,485,507条原始序列,经过去除冗余后剩余659,447和441,182条可比对序列。对三数据归一化后比较,在炎症坏死不同损伤,血清miRNA在MPCHB和SPCHB两者的表达差异。P<0.05表示有统计学差异。共发现143条有差异显著性的miRNAs,其中表达上调的有105条,表达下调的有38条。符合表达差异倍数2倍以上,P<0.05的14个miRNA为上调基因,3个下调miRNA表达基因,见表1:
表1慢性乙肝炎症损伤轻中度分组的miRNA的差异表达
5、表达差异的miRNA进一步筛选:对上述表达差异的17个差异miRNA进一步筛选,筛选条件为:Ct值〈35,检测率〉75%,共筛选出5个候选miRNA,分别为:hsa-miR-1285-5p,hsa-miR-122-5p,hsa-miR-193a-5p,hsa-miR-433-3p,和hsa miR-26a-5p。见表2:
表2慢性乙肝轻中度炎症损伤在训练集miRNA的差异表达
6、训练集RT-PCR进一步筛选目的miRNA
对上阶段筛选出的5个候选miRNA使用新的训练集进行qRT-PCR检验,发现炎症坏死组比较有3个差异表达miRNA,分别为:hsa-miR-1285-5p,hsa-miR-193a-5p,和hsamiR-26a-5p。Logistic回归分析建立联合预测因子,经过逐步Logistic回归建立LogitP。炎症坏死组,有3个miRNA进入方程,Logit P1=-4.0262-0.28851*miR_1285-0.50803*miR_193+0.85701*miR_26a,见表3:
表3训练集慢性乙肝轻中度炎症损伤的Logistic回归分析
验证集验证miRNA组合:
本发明的miRNA组合物在验证集炎症坏死组的ROC曲线见图1,可以看出,ACU为0.887(95%CI=0.775–0.878,灵敏度=70.8%,特异度=83.4%),说明miRNA组合物可以作为预测慢性乙肝炎症损伤的生物标志物。
序列表
<110> 镇江市第三人民医院
<120> 一种用于预测慢性乙肝炎症损伤的miRNA组合物
<140> 2018110522487
<141> 2018-09-10
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> miRNA组合物(hsa-miR-1285-5p)
<400> 1
gaucucacuu uguugcccag g 21
<210> 2
<211> 22
<212> RNA
<213> miRNA组合物(hsa-miR-193a-5p)
<400> 2
ugggucuuug cgggcgagau ga 22
<210> 3
<211> 22
<212> RNA
<213> miRNA组合物(hsa miR-26a-5p)
<400> 3
uucaaguaau ccaggauagg cu 22
<210> 4
<211> 21
<212> DNA
<213> miRNA组合物(hsa-miR-1285-5p)
<400> 4
tcgctggatc tcactttgtt g 21
<210> 5
<211> 20
<212> DNA
<213> miRNA组合物(hsa-miR-1285-5p)
<400> 5
cagtgcaggg tccgaggtat 20
<210> 6
<211> 22
<212> DNA
<213> miRNA组合物(hsa-miR-193a-5p)
<400> 6
ggtacccggg gttttgaggg cg 22
<210> 7
<211> 20
<212> DNA
<213> miRNA组合物(hsa-miR-193a-5p)
<400> 7
cagtgcaggg tccgaggtat 20
<210> 8
<211> 22
<212> DNA
<213> miRNA组合物(hsa miR-26a-5p)
<400> 8
gccgccttca agtaatccag ga 22
<210> 9
<211> 20
<212> DNA
<213> miRNA组合物(hsa miR-26a-5p)
<400> 9
cagtgcaggg tccgaggtat 20
Claims (1)
1.一种用于早期诊断慢性乙肝炎症损伤的试剂盒,其特征在于,包括针对hsa-miR-1285-5p的特异性引物,针对hsa-miR-193a-5p的特异性引物,针对hsa miR-26a-5p的特异性引物;所述针对hsa-miR-1285-5p的特异性引物包括SEQ ID NO.4所示的正向引物和SEQID NO.5所示的反向引物,所述针对hsa-miR-193a-5p的特异性引物包括SEQ ID NO.6所示的正向引物和SEQ ID NO.7所示的反向引物,所述针对hsa miR-26a-5p的特异性引物包括SEQ ID NO.8所示的正向引物和SEQ ID NO.9所示的反向引物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811052248.7A CN108977533B (zh) | 2018-09-10 | 2018-09-10 | 一种用于预测慢性乙肝炎症损伤的miRNA组合物 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811052248.7A CN108977533B (zh) | 2018-09-10 | 2018-09-10 | 一种用于预测慢性乙肝炎症损伤的miRNA组合物 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108977533A CN108977533A (zh) | 2018-12-11 |
CN108977533B true CN108977533B (zh) | 2021-09-17 |
Family
ID=64546037
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201811052248.7A Expired - Fee Related CN108977533B (zh) | 2018-09-10 | 2018-09-10 | 一种用于预测慢性乙肝炎症损伤的miRNA组合物 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108977533B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110057954B (zh) * | 2019-04-30 | 2021-02-23 | 中国医学科学院病原生物学研究所 | 血浆代谢标志物在诊断或监测hbv的应用 |
CN114525331B (zh) * | 2021-11-23 | 2024-04-26 | 中山大学 | 一种快速识别重症肺炎的检测产品 |
CN115029347B (zh) * | 2022-05-11 | 2024-02-20 | 珠海中科先进技术研究院有限公司 | 识别和调控肝肾细胞纤维化的分子监测序列、重组质粒、抑制病毒 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102023218A (zh) * | 2009-09-16 | 2011-04-20 | 复旦大学 | 一种乙肝病毒感染者血清标志物及其用途 |
CN104293914A (zh) * | 2014-09-05 | 2015-01-21 | 镇江市第三人民医院 | 用于检测原发性肝细胞癌的血清miRNA标志物组合及应用 |
CN104903468A (zh) * | 2012-11-16 | 2015-09-09 | 西门子公司 | 用于帕金森氏病的新诊断MiRNA标志物 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014164253A1 (en) * | 2013-03-09 | 2014-10-09 | Moderna Therapeutics, Inc. | Heterologous untranslated regions for mrna |
-
2018
- 2018-09-10 CN CN201811052248.7A patent/CN108977533B/zh not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102023218A (zh) * | 2009-09-16 | 2011-04-20 | 复旦大学 | 一种乙肝病毒感染者血清标志物及其用途 |
CN104903468A (zh) * | 2012-11-16 | 2015-09-09 | 西门子公司 | 用于帕金森氏病的新诊断MiRNA标志物 |
CN104293914A (zh) * | 2014-09-05 | 2015-01-21 | 镇江市第三人民医院 | 用于检测原发性肝细胞癌的血清miRNA标志物组合及应用 |
Non-Patent Citations (3)
Title |
---|
"A Serum MicroRNA Panel as Potential Biomarkers for Hepatocellular Carcinoma Related with Hepatitis B Virus";Youwen Tan等;《PLOS ONE》;20140919;第9卷(第9期);e107986 * |
"Differential Expression Profiles of MicroRNAs between de novo and Complete Response Severe Aplastic Anemia";SHAO Ying-Qi等;《中国实验血液学杂志》;20180220;第26卷(第1期);第213-218页 * |
"MicroRNA-1285-5p influences the proliferation and metastasis of non-small-cell lung carcinoma cells via downregulating CDH1 and Smad4";Shixia Zhou等;《Tumor Biology》;20170630;第39卷(第6期);第1-10页 * |
Also Published As
Publication number | Publication date |
---|---|
CN108977533A (zh) | 2018-12-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108977533B (zh) | 一种用于预测慢性乙肝炎症损伤的miRNA组合物 | |
JP2022533033A (ja) | 胃がんを診断するためのmiRNAマーカーの組み合わせ物、およびキット | |
CN107034301A (zh) | 一种检测肺结节为良性或恶性的试剂盒及其应用 | |
CN104293914A (zh) | 用于检测原发性肝细胞癌的血清miRNA标志物组合及应用 | |
CN111826466B (zh) | 乙型肝炎感染患者或携带者外泌体miRNA分子标志物组合及筛查试剂盒 | |
CN108660215B (zh) | 检测circMAN1A2和circRNF13试剂的应用及试剂盒 | |
CN107022605A (zh) | 一种活动性肺结核的生物标志物 | |
CN108796074B (zh) | 检测环状RNA circRNF13的试剂在制备肿瘤辅助诊断制剂上的应用及试剂盒 | |
CN109022569B (zh) | 一种用于预测慢性乙肝肝纤维化的miRNA组合物 | |
CN108220416B (zh) | 一种用于检测阴虚上火体质血清特异性miRNA的试剂盒及其应用 | |
CN108148908B (zh) | 动脉粥样硬化性肾动脉狭窄诊断分子标记物的应用 | |
CN105154533B (zh) | 诊断早期肝癌的miRNA组合及其试剂盒 | |
CN105671179B (zh) | 血清microRNA在肝癌诊断中的应用及诊断试剂盒 | |
CN110511995B (zh) | 一组结核病标志物及其应用 | |
Li et al. | Characterization of MicroRNA cargo of extracellular vesicles isolated from the plasma of Schistosoma japonicum-infected mice | |
CN116769892A (zh) | circRNA生物标志物在抑郁症诊断中的应用 | |
CN113234817B (zh) | 利用CpG位点甲基化水平检测早期肝癌的标志物 | |
CN102021169A (zh) | 一种血清/血浆miRNA组合物及其应用 | |
CN115261454A (zh) | 一种新的let-7d-5p和miR-140-5p的生物标志物面板诊断方法 | |
KR102229647B1 (ko) | 신장이식 환자의 급성거부반응 진단용 miRNA 바이오 마커 및 이의 용도 | |
CN108424960B (zh) | 一种LncRNA作为深静脉血栓形成诊断标志物的应用 | |
CN110699450A (zh) | miRNA生物标志物在肝脏疾病诊断和预后判断中的应用 | |
CN113801936B (zh) | 用于肺癌诊断的试剂盒、装置及方法 | |
CN113755598B (zh) | miRNA联合标志物在制备诊断或检测HBV+且LC-原发性HCC的试剂盒中的应用 | |
CN117248029A (zh) | 一种基于外泌体miRNA的肝癌诊断标志物及其应用 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210917 |