CN113755599B - Grb7基因在结直肠癌的诊断、预后检测评价和药物制备中的应用 - Google Patents
Grb7基因在结直肠癌的诊断、预后检测评价和药物制备中的应用 Download PDFInfo
- Publication number
- CN113755599B CN113755599B CN202111199124.3A CN202111199124A CN113755599B CN 113755599 B CN113755599 B CN 113755599B CN 202111199124 A CN202111199124 A CN 202111199124A CN 113755599 B CN113755599 B CN 113755599B
- Authority
- CN
- China
- Prior art keywords
- colorectal cancer
- grb7
- treatment
- gene
- prognosis
- 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.)
- Active
Links
- 101150052409 GRB7 gene Proteins 0.000 title claims abstract description 67
- 206010009944 Colon cancer Diseases 0.000 title claims abstract description 60
- 208000001333 Colorectal Neoplasms Diseases 0.000 title claims abstract description 58
- 239000003814 drug Substances 0.000 title claims abstract description 20
- 238000004393 prognosis Methods 0.000 title abstract description 14
- 238000003745 diagnosis Methods 0.000 title abstract description 11
- 238000001514 detection method Methods 0.000 title abstract description 10
- 238000011156 evaluation Methods 0.000 title abstract description 6
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 238000011282 treatment Methods 0.000 claims abstract description 36
- 230000006907 apoptotic process Effects 0.000 claims description 13
- 230000035755 proliferation Effects 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 239000002829 mitogen activated protein kinase inhibitor Substances 0.000 claims description 5
- 229940122498 Gene expression inhibitor Drugs 0.000 claims description 4
- 229940124647 MEK inhibitor Drugs 0.000 claims description 3
- 239000002246 antineoplastic agent Substances 0.000 claims description 3
- 230000001737 promoting effect Effects 0.000 claims description 3
- LIRYPHYGHXZJBZ-UHFFFAOYSA-N trametinib Chemical group CC(=O)NC1=CC=CC(N2C(N(C3CC3)C(=O)C3=C(NC=4C(=CC(I)=CC=4)F)N(C)C(=O)C(C)=C32)=O)=C1 LIRYPHYGHXZJBZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 229960004066 trametinib Drugs 0.000 claims description 2
- 229940079593 drug Drugs 0.000 abstract description 12
- 239000003550 marker Substances 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 230000006872 improvement Effects 0.000 abstract description 3
- 239000002773 nucleotide Substances 0.000 abstract description 2
- 125000003729 nucleotide group Chemical group 0.000 abstract description 2
- 108700031843 GRB7 Adaptor Proteins 0.000 description 43
- 102000053331 GRB7 Adaptor Human genes 0.000 description 40
- 210000004027 cell Anatomy 0.000 description 27
- 230000005723 MEK inhibition Effects 0.000 description 25
- 229940125374 mitogen-activated extracellular signal-regulated kinase inhibitor Drugs 0.000 description 25
- 230000014509 gene expression Effects 0.000 description 22
- 206010028980 Neoplasm Diseases 0.000 description 15
- 108091027967 Small hairpin RNA Proteins 0.000 description 11
- 239000004055 small Interfering RNA Substances 0.000 description 11
- 201000011510 cancer Diseases 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 230000002018 overexpression Effects 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 206010059866 Drug resistance Diseases 0.000 description 3
- 102000043136 MAP kinase family Human genes 0.000 description 3
- 108091054455 MAP kinase family Proteins 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- CYOHGALHFOKKQC-UHFFFAOYSA-N selumetinib Chemical compound OCCONC(=O)C=1C=C2N(C)C=NC2=C(F)C=1NC1=CC=C(Br)C=C1Cl CYOHGALHFOKKQC-UHFFFAOYSA-N 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 241000700605 Viruses Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000003021 clonogenic effect Effects 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000002626 targeted therapy Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 102000000412 Annexin Human genes 0.000 description 1
- 108050008874 Annexin Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 206010052358 Colorectal cancer metastatic Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 208000007433 Lymphatic Metastasis Diseases 0.000 description 1
- 229940126560 MAPK inhibitor Drugs 0.000 description 1
- 102000004232 Mitogen-Activated Protein Kinase Kinases Human genes 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 229960005395 cetuximab Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 238000011281 clinical therapy Methods 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 230000001447 compensatory effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 235000006694 eating habits Nutrition 0.000 description 1
- 230000000547 effect on apoptosis Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000011242 molecular targeted therapy Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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
- C12Q1/68—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
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- C12Q2600/118—Prognosis of disease development
-
- 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
- C12Q2600/136—Screening for pharmacological compounds
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Pathology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Hospice & Palliative Care (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种GRB7基因在结直肠癌的诊断、预后检测评价和药物制备中的应用。该GRB7基因的核苷酸序列如SEQ ID NO.1所示。本发明提供一种新的结直肠癌诊断标志物GRB7基因，并能够特异性指示结直肠癌患者的靶向治疗敏感性及预后判断，有助于临床治疗方案更精准的个体化选择及改善预后；还可作为结直肠癌治疗靶点、用于制备结直肠癌治疗药物。
Description
技术领域
本发明属于生物医药技术领域，具体涉及GRB7基因在结直肠癌的诊断、治疗、预后检测评价和药物制备中的应用。
背景技术
结直肠癌(Colorectal Cancer，CRC)是全球范围内常见的恶性肿瘤。2018 年全球癌症统计报告结果表明，在常见肿瘤中，结直肠癌的发生率和死亡率均位居前三位。结直肠癌的发生除了遗传因素外，还很大程度上与人们的饮食习惯和生活方式有关。
早期结直肠癌患者的治疗方式通常采用外科切除手术，再加上一些辅助药物治疗就能有较好的治疗效果；疾病晚期或者被诊断为不可切除的患者通常采用化疗、分子靶向治疗等方式，但在治疗过程中，患者经常出现耐药问题，所以结直肠癌的临床预后仍然不尽人意，尤其是当CRC患者出现淋巴结转移时，预后更差。
在结直肠癌靶向治疗中，EGFR单抗药物Cetuximab最早被批准用于转移性结直肠癌的治疗。它可作为KRAS野生型结直肠癌患者的临床一线治疗药物。但是在结直肠癌患者中，KRAS突变的患者约占50％，临床研究表明携带这种突变的患者对EGFR相关抑制剂的治疗响应率极低。结直肠癌靶向治疗耐药导致的低反应率严重制约了结直肠癌靶向治疗的临床效果，成为临床治疗急需解决的问题。由于基因突变引起的各种诱导细胞增殖和存活的代偿传导途径的激活，以及信号传导网络的复杂，导致针对结直肠癌患者的靶向MAPK疗法临床获益率不高。结直肠癌的耐药问题亟需解决。
发明内容
针对现有技术中的上述不足，本发明提供一种GRB7基因在结直肠癌的诊断、治疗、预后检测评价和药物制备方面的应用，提供一种新的结直肠癌诊断标志物GRB7基因，并能够特异性指示结直肠癌患者的靶向治疗敏感性及预后判断，有助于临床治疗方案更精准的个体化选择及改善预后；还可作为结直肠癌治疗靶点、用于制备结直肠癌治疗药物。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
GRB7作为标志物、诊断靶点在制备治疗结直肠癌药物中的应用，该GRB7 基因的核苷酸序列如SEQ ID NO.1所示。
一种检测GRB7基因的试剂在结直肠癌诊断或预后中的应用。
进一步地，该试剂可特异性识别GRB7基因，并能通过测序技术、核酸杂交技术、核酸扩增技术或免疫测定等方法检测GRB7基因的表达量。
进一步地，该试剂可用于制备检测GRB7基因表达量的试剂盒。
一种GRB7基因表达抑制剂或敲除试剂在制备治疗结直肠癌药物中的应用，该药物能够抑制结直肠癌细胞的增殖、促进细胞凋亡。
一种GRB7基因表达抑制剂或敲除试剂在制备提高结直肠癌治疗效果的辅助药物中的应用，所述辅助药物能够抑制结直肠癌细胞的增殖、促进细胞凋亡。
进一步地，结直肠癌为KRAS突变的结直肠癌。
一种用于结直肠癌诊断或预后检测的试剂盒，试剂盒包括检测GRB7基因表达量的试剂。
进一步地，试剂盒包括特异性识别GRB7基因的试剂。
进一步地，特异性识别GRB7基因的试剂选自：特异性扩增GRB7基因的引物；或特异性识别GRB7基因的探针。
一种治疗结直肠癌的联合药物，包括GRB7基因表达抑制剂或敲除试剂，以及结直肠癌抑制剂。
进一步地，结直肠癌抑制剂为MEK抑制剂或MAPK抑制剂。
进一步地，抑制GRB7基因表达能够降低细胞对MEK抑制剂或MAPK抑制剂的耐受性；过表达GRB7基因则会提升细胞对MEK抑制剂或MAPK抑制剂的耐受性。
进一步地，联合药物是能够抑制结直肠癌细胞的增殖、促进细胞凋亡的药物。
本发明的有益效果：
本发明提供一种新的结直肠癌诊断标志物GRB7基因，并能够特异性指示结直肠癌患者的靶向治疗敏感性及预后判断，有助于临床治疗方案更精准的个体化选择及改善预后；还可作为结直肠癌治疗靶点、用于制备结直肠癌治疗药物。
附图说明
图1为结直肠癌样本中基因GRB7的mRNA表达量；
图2为结直肠癌样本中基因GRB7的蛋白表达量；
图3为GRB7的DFS生存曲线；
图4为qPCR检测GRB7的shRNA敲除效率；
图5为敲减GRB7后对细胞HCT116增殖的影响；
图6为敲减GRB7后对细胞HCT116凋亡的影响；
图7为GRB7在MEKi处理之后的表达量差异；
图8为MEKi处理改变了GRB7在细胞内的分布情况；
图9为敲减GRB7后协同MEKi促进细胞增殖；
图10为敲减GRB7后协同MEKi促进细胞凋亡；
图11为过表达GRB7促进细胞对MEKi的耐受。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
结直肠癌细胞系HCT116和HEK293T购买于美国典型培养物保藏中心 (ATCC)。
实施例1证明GRB7的含量在结直肠癌患者中含量显著升高
1、用TCGA(The Cancer Genome Atlas癌症基因图谱)数据库中结直肠癌病人的正常组织(normal)和肿瘤组织(cancer)的基因表达量进行了统计分析 (图1，****p<0.0001)，结果表明不仅在KRAS突变的结直肠癌中，甚至在整个数据库的结直肠癌样本中，GRB7在肿瘤中的表达量高于正常组织。
2、在结直肠癌患者的正常和肿瘤组织中，用免疫组织化学的方式检测GRB7 的表达量(图2)，再对染色的强度进行量化。结果也证明GRB7在肿瘤中的表达量显著高于正常组织
3、用TCGA(The Cancer Genome Atlas癌症基因图谱)数据库中结直肠癌病人的正常组织(normal)和肿瘤组织(cancer)基因表达量和临床信息进行DFS (无疾病生存期)分析，结果表明GRB7表达高的患者预后较差(图3，p＝0.0062)，提示GRB7作为结直肠癌预后标志物的作用。
实施例2证明GRB7能调节肿瘤细胞的增殖和凋亡
1、构建靶向GRB7的shRNA
从选择了两条靶向GRB7的shRNA序列，通过分子克隆的方式将其克隆在载体PLKO上，并通过慢病毒系统包装表达shRNA，对GRB7进行靶向敲减。利用荧光定量PCR(q-PCR)检测GRB7的敲减效率。如图4所示，两株敲减后的细胞GRB7相对表达量均低于30％，表明shRNA靶向性较好，敲减效率符合要求。
2、用克隆形成方法检测敲减GRB7对细胞增殖的影响
将HCT116在常规下培养过夜，然后加入表达shRNA的病毒颗粒进行感染。感染24h之后加入嘌呤霉素筛选稳定株。将筛选得到的稳定株进行克隆形成实验，大约2周之后，进行结晶紫染色，从而得到不同条件下细胞的增殖情况。如图5所示，敲减GRB7之后显著降低了细胞的增殖。
3、检测敲减GRB7对细胞凋亡的影响
将HCT116感染表达shRNA的病毒颗粒24小时之后，更换新鲜培养基继续培养48小时，用标记了FITC的Annexin V和PI对凋亡细胞进行染色，采用流式细胞仪检测细胞凋亡。图6结果显示敲减GRB7之后显著促进了细胞的凋亡。
实施例3GRB7参与了MEKi的耐药过程
1、MEKi处理影响了GRB7的表达量以及胞内分布情况
利用GEO数据库信息，我们用生物信息学的方法分析了MEKi处理前后 GRB7的mRNA表达量，结果如图7显示，MEKi处理之后，显著增加了GRB7 的表达量。此外，我们检测了MEKi处理是否对GRB7的分布产生影响。将 HCT116铺到细胞培养皿中，分别用1μM AZD6244处理0，24，48，96小时。处理之后，利用蛋白核质分离试剂盒分别提取细胞核和细胞浆蛋白。之后用western blot的方法检测GRB7的表达量。同时利用细胞核和细胞浆蛋白内参指示核质分离效果。结果如图8所示，MEKi处理之后，显著增加了细胞浆中的蛋白表达量，可能预示着MEKi处理促进了GRB7在细胞浆的作用。
2、敲减GRB7协同MEKi抑制细胞增殖
用长期克隆形成实验检测敲减GRB7是否能协同MEKi发挥作用。将 HCT116过夜培养，用表达GRB7的shRNA或者载体病毒颗粒24小时之后，加入药物1μM AZD6244处理两周左右，知道对照组长满。用0.05％的结晶紫染液对细胞进行染色，由此检测细胞的增殖情况。结果如图9所示，与对照组相比，敲减GRB7显著增加了HCT116对MEKi的敏感性。
3、敲减GRB7协同MEKi促进细胞凋亡
GSK1120212(曲美替尼)是MEK的另外一种小分子抑制剂，目前已被FDA 批准用于多种肿瘤的败选哪个治疗。靶向GRB7的shRNA协同MEKi促进了细胞的凋亡。感染shRNA和对照的HC116细胞，分别用不同浓度(0，30nM，60nM) 的GSK1120212处理24小时后，利用Annexin V-PI的凋亡检测试剂盒检测细胞的凋亡水平，结果如图10所示。结果表明，shRNA处理之后，显著增加了MEKi 对肿瘤细胞的杀伤作用。
4、过表达GRB7显著增加了细胞对MEKi的耐受
我们在体外构建GRB7过表达的慢病毒载体，通过慢病毒感染将外源GRB7 引入HCT116细胞。首先通过检测细胞中GRB7蛋白表达情况来鉴定过表达效率。如图11A所示，与对照(CPPT)细胞相比，过表达后的细胞GRB7蛋白水平显著升高，说明GRB7的过表达效率很高。
接下来我们用不同浓度(0、0.01、0.1、1、10μM)的MEKi分别处理CPPT 和过表达GRB7的细胞，药物处理72小时后，用MTS法检测药物对细胞增殖的影响。如图11B所示，在实验组(过表达GRB7)细胞中，MEKi对细胞的半数抑制浓度相比于对照组升高，表明过表达GRB7的细胞对MEKi的耐受性有所增加。在长期克隆形成实验中，我们用不同浓度(0、0.125、0.25、0.5、1、5μM) 的MEKi隔天处理细胞，图11C的结果显示，实验组相较于对照CPPT组在AZD6244 1μM、5μM时对药物的耐受性更强。以上实验结果表明，过表达GRB7 有助于增加结直肠癌细胞对MEKi的耐受性。
综合上述失去功能和获得功能实验结果，表明GRB7的表达变化显著影响 HCT116细胞对MEKi的耐受性。
序列表
<110> 四川大学
<120> GRB7基因在结直肠癌的诊断、预后检测评价和药物制备中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1599
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggagctgg atctgtctcc acctcatctt agcagctctc cggaagacct ttgcccagcc 60
cctgggaccc ctcctgggac tccccggccc cctgataccc ctctgcctga ggaggtaaag 120
aggtcccagc ctctcctcat cccaaccacc ggcaggaaac ttcgagagga ggagaggcgt 180
gccacctccc tcccctctat ccccaacccc ttccctgagc tctgcagtcc tccctcacag 240
agcccaattc tcgggggccc ctccagtgca agggggctgc tcccccgcga tgccagccgc 300
ccccatgtag taaaggtgta cagtgaggat ggggcctgca ggtctgtgga ggtggcagca 360
ggtgccacag ctcgccacgt gtgtgaaatg ctggtgcagc gagctcacgc cttgagcgac 420
gagacctggg ggctggtgga gtgccacccc cacctagcac tggagcgggg tttggaggac 480
cacgagtccg tggtggaagt gcaggctgcc tggcccgtgg gcggagatag ccgcttcgtc 540
ttccggaaaa acttcgccaa gtacgaactg ttcaagagct ccccacactc cctgttccca 600
gaaaaaatgg tctccagctg tctcgatgca cacactggta tatcccatga agacctcatc 660
cagaacttcc tgaatgctgg cagctttcct gagatccagg gctttctgca gctgcggggt 720
tcaggacgga agctttggaa acgctttttc tgcttcttgc gccgatctgg cctctattac 780
tccaccaagg gcacctctaa ggatccgagg cacctgcagt acgtggcaga tgtgaacgag 840
tccaacgtgt acgtggtgac gcagggccgc aagctctacg ggatgcccac tgacttcggt 900
ttctgtgtca agcccaacaa gcttcgaaat ggccacaagg ggcttcggat cttctgcagt 960
gaagatgagc agagccgcac ctgctggctg gctgccttcc gcctcttcaa gtacggggtg 1020
cagctgtaca agaattacca gcaggcacag tctcgccatc tgcatccatc ttgtttgggc 1080
tccccaccct tgagaagtgc ctcagataat accctggtgg ccatggactt ctctggccat 1140
gctgggcgtg tcattgagaa cccccgggag gctctgagtg tggccctgga ggaggcccag 1200
gcctggagga agaagacaaa ccaccgcctc agcctgccca tgccagcctc cggcacgagc 1260
ctcagtgcag ccatccaccg cacccaactc tggttccacg ggcgcatttc ccgtgaggag 1320
agccagcggc ttattggaca gcagggcttg gtagacggcc tgttcctggt ccgggagagt 1380
cagcggaacc cccagggctt tgtcctctct ttgtgccacc tgcagaaagt gaagcattat 1440
ctcatcctgc cgagcgagga ggagggccgc ctgtacttca gcatggatga tggccagacc 1500
cgcttcactg acctgctgca gctcgtggag ttccaccagc tgaaccgcgg catcctgccg 1560
tgcttgctgc gccattgctg cacgcgggtg gccctctga 1599
Claims (2)
1.一种GRB7基因表达抑制剂或敲除试剂在制备治疗结直肠癌的联合药物中的用途，其特征在于，包括GRB7基因表达抑制剂或敲除试剂，以及结直肠癌抑制剂，所述结直肠癌抑制剂为MEK抑制剂，所述MEK抑制剂为曲美替尼。
2.根据权利要求1所述的用途，其特征在于，所述联合药物是能够抑制结直肠癌细胞的增殖、促进凋亡的药物。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111199124.3A CN113755599B (zh) | 2021-10-14 | 2021-10-14 | Grb7基因在结直肠癌的诊断、预后检测评价和药物制备中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111199124.3A CN113755599B (zh) | 2021-10-14 | 2021-10-14 | Grb7基因在结直肠癌的诊断、预后检测评价和药物制备中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113755599A CN113755599A (zh) | 2021-12-07 |
| CN113755599B true CN113755599B (zh) | 2023-12-26 |
Family
ID=78799514
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111199124.3A Active CN113755599B (zh) | 2021-10-14 | 2021-10-14 | Grb7基因在结直肠癌的诊断、预后检测评价和药物制备中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113755599B (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500687A (zh) * | 2020-05-12 | 2020-08-07 | 深圳市草履虫生物科技有限公司 | 一种乳腺癌21基因检测试剂盒 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8609410B2 (en) * | 2005-09-08 | 2013-12-17 | Medinet Co., Ltd. | Method for activation treatment of antigen-presenting cell |
| US7939255B2 (en) * | 2006-07-03 | 2011-05-10 | Catholic University Industry Academy Cooperation Foundation | Diagnostic methods for colorectal cancer |
| WO2018161081A1 (en) * | 2017-03-03 | 2018-09-07 | Board Of Regents, The University Of Texas System | Gene signatures to predict drug response in cancer |
-
2021
- 2021-10-14 CN CN202111199124.3A patent/CN113755599B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500687A (zh) * | 2020-05-12 | 2020-08-07 | 深圳市草履虫生物科技有限公司 | 一种乳腺癌21基因检测试剂盒 |
Non-Patent Citations (5)
| Title |
|---|
| Hechtman JF等. Identification of Targetable Kinase Alterations in Patients with Colorectal Carcinoma That are Preferentially Associated with Wild-Type RAS/RAF.Mol Cancer Res.2015,第14卷(第3期),296-301. * |
| Nadler Y等.Growth factor receptor-bound protein-7 (Grb7) as a prognostic marker and therapeutic target in breast cancer.Ann Oncol.2010,第21卷(第3期),466-473. * |
| Pei-Yu Chu等.EGF-Activated Grb7 Confers to STAT3-Mediated EPHA4 Gene Expression in Regulating Lung Cancer Progression.biorxiv.2020,第1-22页. * |
| Yu C等.Genome-wide CRISPR-cas9 knockout screening identifies GRB7 as a driver for MEK inhibitor resistance in KRAS mutant colon cancer.Oncogene.2021,第41卷(第2期),191-203. * |
| 李毅 ; 孙涛 ; .胃肠黏膜G蛋白偶联受体5阳性细胞研究进展.生物技术通讯.2013,(第02期),全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113755599A (zh) | 2021-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Circular RNA CircNOLC1, upregulated by NF-KappaB, promotes the progression of prostate cancer via miR-647/PAQR4 axis | |
| EP1888787B1 (en) | Small interference rna for multiple myeloma | |
| KR20100015529A (ko) | 화학요법 약물-내성 상피성 종양을 치료하기 위한 ｓｉＲＮＡ-매개 유전자 침묵화 | |
| Schütz et al. | Lung adenocarcinomas and lung cancer cell lines show association of MMP-1 expression with STAT3 activation | |
| Li et al. | MiR-410 affects the proliferation and apoptosis of lung cancer A549 cells through regulation of SOCS3/JAK-STAT signaling pathway. | |
| US20220112498A1 (en) | Methods for diagnosing and treating metastatic cancer | |
| Liu et al. | LncRNA MNX1-AS1 sustains inactivation of Hippo pathway through a positive feedback loop with USP16/IGF2BP3 axis in gallbladder cancer | |
| KR20190087106A (ko) | 위암의 항암제 치료 반응성 예측용 바이오마커 및 이의 용도 | |
| Hou et al. | LncRNA EPIC1 promotes tumor angiogenesis via activating the Ang2/Tie2 axis in non-small cell lung cancer | |
| US20200326343A1 (en) | Gene and its expression product promoting the occurrence and development of cancer and application | |
| Hirao et al. | Transcription factor homeobox D9 is involved in the malignant phenotype of cervical cancer through direct binding to the human papillomavirus oncogene promoter | |
| CN110358834B (zh) | 一种lncRNA的应用以及试剂盒和药物 | |
| US20120225076A1 (en) | Fra-1 target genes as drug targets for treating cancer | |
| CN113755599B (zh) | Grb7基因在结直肠癌的诊断、预后检测评价和药物制备中的应用 | |
| CN110938698A (zh) | Sox9在预测三阴性乳腺癌对CDK7抑制剂THZ1敏感性中的应用 | |
| Zhang et al. | Deregulated lncRNA expression profile in the mouse lung adenocarcinomas with KRAS‐G12D mutation and P53 knockout | |
| WO2016040167A1 (en) | Compositions and methods for detecting and treating small cell lung cancer | |
| WO2019031637A1 (ko) | p53-비돌연변이 암에 대한 암 마커 유전자 및 치료제 스크리닝 방법 | |
| CN112375810A (zh) | GnT-II基因下调表达作为肝癌预后标志物中的应用 | |
| US10865415B2 (en) | Prevention, diagnosis and treatment of cancer overexpressing GPR160 | |
| CN114231625B (zh) | Qser1基因的用途及其相关药物 | |
| WO2023082242A1 (zh) | Ctd-2256p15.2及其编码微肽作为靶点在开发肿瘤治疗药物中的应用 | |
| CN116676385B (zh) | Linc02096在作为预测食管癌免疫治疗效果、预后的标志物及治疗靶点中的应用 | |
| CN111647658B (zh) | Linc00963相关致癌轴在前列腺癌远处转移的早期诊断和治疗中的应用 | |
| CN113789381B (zh) | 人CLDN10-AS1 lncRNA在评估肺腺癌患者预后中的应用及检测试剂盒 |
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 |