CN109652549B - Application of circular RNA as diagnosis biomarker and treatment target of gastric cancer and colorectal cancer - Google Patents
Application of circular RNA as diagnosis biomarker and treatment target of gastric cancer and colorectal cancer Download PDFInfo
- Publication number
- CN109652549B CN109652549B CN201910052745.5A CN201910052745A CN109652549B CN 109652549 B CN109652549 B CN 109652549B CN 201910052745 A CN201910052745 A CN 201910052745A CN 109652549 B CN109652549 B CN 109652549B
- Authority
- CN
- China
- Prior art keywords
- circ
- lrp11
- colorectal cancer
- gastric
- cancer
- 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
- 208000005718 Stomach Neoplasms Diseases 0.000 title claims abstract description 46
- 208000001333 Colorectal Neoplasms Diseases 0.000 title claims abstract description 45
- 206010017758 gastric cancer Diseases 0.000 title claims abstract description 43
- 201000011549 stomach cancer Diseases 0.000 title claims abstract description 43
- 206010009944 Colon cancer Diseases 0.000 title claims abstract description 42
- 108091028075 Circular RNA Proteins 0.000 title claims abstract description 10
- 238000003745 diagnosis Methods 0.000 title claims abstract description 10
- 239000000090 biomarker Substances 0.000 title abstract description 9
- 101000984624 Homo sapiens Low-density lipoprotein receptor-related protein 11 Proteins 0.000 claims abstract description 5
- 102100027119 Low-density lipoprotein receptor-related protein 11 Human genes 0.000 claims abstract description 5
- 230000002441 reversible effect Effects 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 108700024394 Exon Proteins 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims 1
- 210000003917 human chromosome Anatomy 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 238000007363 ring formation reaction Methods 0.000 claims 1
- 230000002496 gastric effect Effects 0.000 abstract description 17
- 206010028980 Neoplasm Diseases 0.000 abstract description 13
- 230000014509 gene expression Effects 0.000 abstract description 13
- 108020004459 Small interfering RNA Proteins 0.000 abstract description 10
- 239000004055 small Interfering RNA Substances 0.000 abstract description 9
- 238000011529 RT qPCR Methods 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 5
- 230000001225 therapeutic effect Effects 0.000 abstract description 4
- 238000012404 In vitro experiment Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract 1
- 230000002452 interceptive effect Effects 0.000 abstract 1
- 231100000590 oncogenic Toxicity 0.000 abstract 1
- 230000002246 oncogenic effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 14
- 210000001519 tissue Anatomy 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 108010087230 Sincalide Proteins 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004784 molecular pathogenesis Effects 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- 101100128886 Homo sapiens LRP11 gene Proteins 0.000 description 2
- 101150097651 LRP11 gene Proteins 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 230000033607 mismatch repair Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 description 1
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 102000038594 Cdh1/Fizzy-related Human genes 0.000 description 1
- 108091007854 Cdh1/Fizzy-related Proteins 0.000 description 1
- 208000037051 Chromosomal Instability Diseases 0.000 description 1
- 108091029523 CpG island Proteins 0.000 description 1
- 230000030933 DNA methylation on cytosine Effects 0.000 description 1
- 102100034157 DNA mismatch repair protein Msh2 Human genes 0.000 description 1
- 102100021147 DNA mismatch repair protein Msh6 Human genes 0.000 description 1
- 208000002699 Digestive System Neoplasms Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 101710113436 GTPase KRas Proteins 0.000 description 1
- 206010019375 Helicobacter infections Diseases 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101001134036 Homo sapiens DNA mismatch repair protein Msh2 Proteins 0.000 description 1
- 101000968658 Homo sapiens DNA mismatch repair protein Msh6 Proteins 0.000 description 1
- 101000848199 Homo sapiens Protocadherin Fat 4 Proteins 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 208000032818 Microsatellite Instability Diseases 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 108700019961 Neoplasm Genes Proteins 0.000 description 1
- 102000048850 Neoplasm Genes Human genes 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 1
- 108050003267 Prostaglandin G/H synthase 2 Proteins 0.000 description 1
- 102100034547 Protocadherin Fat 4 Human genes 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 1
- 108700020471 RNA-Binding Proteins Proteins 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000001793 Wilcoxon signed-rank test Methods 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 201000002758 colorectal adenoma Diseases 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000001819 effect on gene Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 201000003782 large intestine adenoma Diseases 0.000 description 1
- 201000011061 large intestine cancer Diseases 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 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 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 230000006508 oncogene activation Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000007427 paired t-test Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000037425 regulation of transcription Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 238000010200 validation analysis Methods 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
- C12Q1/68—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
- C12Q2600/158—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
- 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)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Veterinary Medicine (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Hospice & Palliative Care (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Oncology (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (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)
Abstract
The invention discloses application of a circular RNA LRP11(circ-LRP11) as a new potential diagnosis biomarker and therapeutic target of gastric cancer and colorectal cancer. circ-LRP11(hsa _ circ _0007771| chr 6: 150147399-. The invention prepares the qRT-PCR primer of circ-LRP11 and small interfering RNA (siRNA) interfering with circ-LRP11 in vitro. As compared to normal controls, circ-LRP11 expression was significantly upregulated in both tumor and plasma samples from patients with gastric and colorectal cancer. In vitro experiments showed that circ-LRP11 plays a oncogenic role in gastric and colorectal cancer cells. The research result of the invention shows that circ-LRP11 is a new potential biomarker and therapeutic target for diagnosing gastric cancer and colorectal cancer.
Description
Technical Field
The invention belongs to the technical field of medicine, and relates to application of a circular RNA LRP11 as a diagnosis biomarker and a treatment target of gastric cancer and colorectal cancer.
Background
Gastrointestinal tumors are the most common and predominant tumor types, including gastric, colon, and rectal cancers, among others. According to the latest global cancer statistical report of 2018, the stomach cancer mortality rate is third worldwide and the incidence rate is fifth worldwide in all malignant tumors; colorectal cancer mortality is second worldwide and morbidity is third worldwide. Research shows that the occurrence of gastric cancer is related to regional environment, diet and life factors, helicobacter pylori infection, precancerous lesion, genetic factors and genetic factors. The occurrence of large intestine cancer is related to high fat low cellulose diet, chronic inflammation of large intestine, large intestine adenoma, genetic factors and other factors (such as environmental factors, smoking, etc.). Specifically to molecular pathogenesis, the molecular pathogenesis of gastric cancer includes gene mutation (P53, AR1D1A, FAT4, CDH1), abnormal signal pathways related to tumorigenesis (Wnt, RTK, PI3K signal pathways), chromosome instability (somatic copy number change, chromosome translocation), epigenetics (mismatch repair gene CpG island cytosine methylation, histone modification), microsatellite instability; molecular pathogenesis of colorectal cancer includes oncogene activation (K-ras, c-myc, EGFR), oncogene inactivation (APC, DCC, P53), mismatch repair gene mutation (hMLH1, hMSH2, hMSH6, hPMS2), and gene overexpression (COX-2, CD44v), among others. However, only a small fraction of the above molecular targets have been applied in clinical practice for gastric and colorectal cancer. Therefore, there is a need to explore other potential pathogenesis leading to the development of gastric and colorectal cancer.
Circular RNA (circular RNA/circular RNA) is a large class of noncoding RNA that covalently bind to form a circular structure. Circular RNA was first discovered in 1976 in plant viruses and was originally thought to be a class of mis-spliced and unexplored RNA. Subsequent research finds that the circRNA has an important regulation effect on gene expression in organisms, and the circRNA has a structural characteristic of a closed ring structure without a 5 'cap end and a 3' tail end. This loop is formed by the reverse splicing of the upstream 3 'splice acceptor to the downstream 5' splice donor. This covalently bound circular closed structure results in a circRNA that is not affected by exonuclease and is more stable than linear RNA. In recent years, research shows that the circRNA has the following basic functions: adsorbing microRNA as competitive endogenous RNA (cepRNA); regulation of transcription and alternative splicing; acting on RNA binding proteins; translated into protein. Based on the above functions of circRNA, related studies have demonstrated that circRNA is closely associated with human diseases, including tumors. Therefore, the deep research on the action mechanism of the circRNA in gastrointestinal tract tumors has potential great significance for the diagnosis and treatment of the gastrointestinal tract tumors.
Disclosure of Invention
The invention provides application of circRNA LRP11(circ-LRP11) in diagnosis and treatment of gastric cancer and colorectal cancer. The invention discloses a circ-LRP11 as a new potential diagnosis biomarker and therapeutic target point of gastric cancer and colorectal cancer: circ-LRP11 was significantly up-regulated in tumor and plasma samples from patients with gastric and colorectal cancer; a Receiver Operating Characteristic (ROC) curve shows that circ-LRP11 has good potential for diagnosing gastric cancer and colorectal cancer; circ-LRP11 promotes the proliferation of gastric and colorectal cancer cells.
The first objective of the invention is to provide a potential diagnosis biomarker and a treatment target of gastric cancer and colorectal cancer, which is circ-LRP11(hsa _ circ _0007771| chr 6: 150147399-. circ-LRP11 was formed by reverse splicing of exons 2-6 of the LRP11 gene, with the circularized sequence of 735 bases.
It is a second object of the present invention to provide a primer pair specifically recognizing circ-LRP11, comprising an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown as SEQ ID No. 2; the nucleotide sequence of the downstream primer is shown as SEQ ID No. 3.
The third purpose of the invention is to disclose the expression of circ-LRP11 in tumor tissues of patients with gastric cancer and colorectal cancer.
A fourth object of the present invention is to disclose the expression of circ-LRP11 in plasma of patients with gastric and colorectal cancer, and the corresponding ROC curve for evaluating the diagnostic ability of circ-LRP 11.
A fifth object of the invention is to disclose the function of circ-LRP11 in gastric and colorectal cancer cells.
The invention has the following beneficial effects: 1) the circ-LRP11 is found to be used as a biomarker for diagnosing gastric cancer and colorectal cancer and a drug treatment target for the first time; 2) circ-LRP11 was significantly upregulated in both tumor and plasma samples from both gastric and colorectal cancer patients compared to normal controls; 3) the ROC curve shows that circ-LRP11 has good ability to diagnose gastric cancer and colorectal cancer. 4) The result of the invention shows that the interference with circ-LRP11 can inhibit the proliferation of gastric cancer and colorectal cancer cells, and shows that circ-LRP11 plays a role of promoting cancer genes in the occurrence and development of gastric cancer and colorectal cancer, thereby providing a new target for clinical treatment of gastric cancer and colorectal cancer.
Drawings
FIG. 1 is a schematic diagram of the biological synthesis and structure of circ-LRP 11.
FIG. 2 is a graph showing the results of examination of tissue expression levels of gastric cancer patients using the circ-LRP11 primer. Represents p value < 0.01.
FIG. 3A is a graph showing the results of plasma expression measurements of gastric cancer patients and healthy examiners using the circ-LRP11 primer; b is the ROC curve for evaluating the potential of circ-LRP11 in diagnosing gastric cancer. Represents p value < 0.0001.
FIG. 4 is a graph showing the results of examination of the tissue expression level of a patient with colorectal cancer using the circ-LRP11 primer. Denotes p value < 0.0001.
FIG. 5A is a graph showing the results of plasma expression levels of colorectal cancer patients and healthy subjects using the circ-LRP11 primer; b is the ROC plot for the evaluation of the colorectal cancer diagnosis potential of circ-LRP 11. Represents a p value < 0.001.
FIG. 6 is a qRT-PCR validation graph of the transfection knockdown efficiency of circ-LRP11 small interfering RNA (siRNA) in both HGC-27 and SW480 cell lines. si-NC stands for negative control group; si-circLRP11# 1, si-circLRP11# 2 represent groups of siRNAs that were transfected with two reverse splice sites targeting circ-LRP11, respectively; represents a p value < 0.01, represents a p value < 0.001, and represents a p value < 0.0001.
FIG. 7 is a graph showing the results of changes in cell proliferation of gastric and colorectal cancers after knockdown of circ-LRP 11. Represents a p value < 0.05, represents a p value < 0.01, represents a p value < 0.001, represents a p value < 0.0001.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The embodiment is as follows:
1. experimental materials and methods:
clinical samples: tumor tissues and paracancerous normal mucosal tissues of 30 patients with gastric and 60 colorectal cancers were collected during 2016-. The tissue specimen is quickly frozen by liquid nitrogen and then is transferred to a refrigerator at the temperature of minus 80 ℃. Preoperative blood was collected from 30 gastric cancer and 60 colorectal cancer patients. Blood from 45 healthy examinees age-and-gender-matched was collected as a control. Plasma was obtained by centrifugation and stored in a freezer at-80 ℃.
Cell lines and cell culture: human gastric cancer cell line HGC-27 and colorectal cancer cell line SW480, purchased from American Type Culture Collection (ATCC). Cell culture in DMEM (Invitrogen, Carlsbad, Calif., USA) medium containing 10% fetal bovine serum (Gibco, NY, USA), 100U/ml penicillin and 100. mu.g/ml streptomyces were addedPlain (Gibco, NY, USA). At 37 deg.C, contains 5% CO 2 Culturing the cells in the environment of (a).
RNA extraction and real-time fluorescent quantitative PCR (qRT-PCR): extracting total RNA from cells and tissues by Trizol (Invitrogen, Carlsbad, Calif., USA); using TRlzol TM LS (Invitrogen, Carlsbad, Calif., USA) extracts total RNA from plasma. The reverse transcription kit adopts PrimeScript TM RT reagent Kit (TaKaRa, Dalian, China); the fluorescent quantitative PCR kit adopts TB Green TM Premix Ex Taq TM II (TaKaRa, Dalian, China): PCR reaction was performed using ABI 7500 real-time fluorescent quantitative PCR instrument (Applied Biosystems, Foster City, Calif., USA); adopting 18S rRNA as an internal reference; by means of 2 -ΔΔCt Calculating the relative expression quantity of RNA; the primers were synthesized by Biotechnology engineering (Shanghai) GmbH; the primer sequences are shown in Table 1.
TABLE 1 primer sequences for qRT-PCR
Transfection: specific small interfering RNA (siRNA) targeting the reverse splice site of circ-LRP11 and a negative control (si-NC) were synthesized from the Gima gene (Shanghai); 50nM siRNA was transfected into gastric and colorectal cancer cells using Lipofectamine 3000(Invitrogen, Carlsbad, Calif., USA); the siRNA sequences are shown in Table 2.
TABLE 2 siRNA sequences
CCK-8 experiment: cell proliferation experiments were performed using CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan). About 1000 cells were seeded in 96-well plates. At 0, 24, 48, 72, 96 hours, 10. mu.l CCK-8 reagent was added to the 96-well plate. After two hours of incubation, 450nm Optical Density (OD) values were read using a multifunctional microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Each group was tested for 5 replicates.
Statistical analysis: the results were statistically analyzed using SPSS 23.0 software. Plots were made using GraphPad Prism 7.0 software. Statistical analysis was performed using paired t-test or Wilcoxon signed rank test as appropriate. Data are presented as mean ± standard deviation of at least three independent experiments, all P values are bilateral and P < 0.05 is considered statistically significant.
2. The experimental results are as follows:
as shown in FIG. 1, circ-LRP11 was formed by reverse splicing of exons 2-6 of the LRP11 gene.
As shown by the qRT-PCR results in FIG. 2, circ-LRP11 expression was significantly upregulated in gastric cancer tumor tissue compared to normal tissue. Represents p value < 0.01.
As shown by the qRT-PCR results in FIG. 3A, circ-LRP11 was significantly up-regulated in the plasma of gastric cancer patients compared to healthy examiners. The area under the ROC curve (AUC) in FIG. 3B was 0.739, indicating that circ-LRP11 has good potential for diagnosing gastric cancer patients. Represents p value < 0.0001.
As shown in figure 4 by the qRT-PCR results, circ-LRP11 expression was significantly upregulated in tumor tissue in colorectal cancer patients compared to normal tissue. Denotes p value < 0.0001.
As shown by the qRT-PCR results in FIG. 5A, circ-LRP11 was significantly up-regulated in the plasma of colorectal cancer patients compared to healthy examiners. The AUC in FIG. 5B was 0.830, indicating that circ-LRP11 has good potential for diagnosing colorectal cancer patients. Represents a p value < 0.001.
As shown in FIG. 6, after transfection of si-circLRP11# 1 and si-circLRP11# 2 in HGC-27 gastric cancer and SW480 colorectal carcinoma cell line, the expression level of circ-LRP11 was significantly reduced, while the corresponding expression level of LRP11 mRNA was not significantly changed. Represents a p value < 0.01, represents a p value < 0.001, and represents a p value < 0.0001.
As shown in FIG. 7, after transfection of si-circLRP11# 1, the proliferation capacity of gastric and colorectal cancer cells was significantly decreased. Represents a p value < 0.05, represents a p value < 0.01, represents a p value < 0.001, represents a p value < 0.0001.
The above results indicate that circ-LRP11 is up-regulated in tumor tissues and plasma of patients with gastric and colorectal cancer; the ROC curve shows that circ-LRP11 has good ability to diagnose gastric cancer and colorectal cancer. In vitro experiments show that circ-LRP11 promotes the proliferation of gastric and colorectal cancer cells, indicating that circ-LRP11 plays a role of a oncogene in gastric and colorectal cancers; the circ-LRP11 is expected to become a new biomarker and therapeutic target for diagnosing gastric cancer and colorectal cancer.
The above-described embodiments are merely exemplary and are merely preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and those skilled in the art can make various modifications and variations without departing from the technical principle of the present invention, and these modifications and variations should be construed as the scope of the present invention.
Claims (4)
1. The application of circular RNA circ-LRP11 in preparing a gastric cancer and colorectal cancer diagnosis product is characterized in that: the circBase ID of the circular RNA circ-LRP11 is hsa _ circ _0007771, is derived from human chromosome 6, is generated by reverse splicing and cyclization of exons 2 to 6 of an LRP11 host gene, the length of a cyclized mature sequence of the cyclized mature sequence is 735bp, and the nucleotide sequence is shown as SEQ ID No. 1.
2. Use according to claim 1, characterized in that: the product is selected from a formulation, a chip or a kit.
3. Use according to claim 1, characterized in that: the product comprises a primer pair specifically recognizing circ-LRP 11.
4. Use according to claim 3, characterized in that: the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 2; the nucleotide sequence of the downstream primer is shown as SEQ ID No. 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910052745.5A CN109652549B (en) | 2019-01-21 | 2019-01-21 | Application of circular RNA as diagnosis biomarker and treatment target of gastric cancer and colorectal cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910052745.5A CN109652549B (en) | 2019-01-21 | 2019-01-21 | Application of circular RNA as diagnosis biomarker and treatment target of gastric cancer and colorectal cancer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109652549A CN109652549A (en) | 2019-04-19 |
| CN109652549B true CN109652549B (en) | 2022-08-02 |
Family
ID=66119012
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910052745.5A Active CN109652549B (en) | 2019-01-21 | 2019-01-21 | Application of circular RNA as diagnosis biomarker and treatment target of gastric cancer and colorectal cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109652549B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107447033A (en) * | 2017-09-15 | 2017-12-08 | 东南大学 | A kind of diagnosis of colorectal carcinoma biomarker and its application |
| CN109055563A (en) * | 2018-08-27 | 2018-12-21 | 新乡医学院 | The related cyclic annular rna gene of colorectal cancer, colorectal cancer molecular marker and its application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120252028A1 (en) * | 2009-08-14 | 2012-10-04 | Michael Shtulman | Target genes for cancer therapy |
-
2019
- 2019-01-21 CN CN201910052745.5A patent/CN109652549B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107447033A (en) * | 2017-09-15 | 2017-12-08 | 东南大学 | A kind of diagnosis of colorectal carcinoma biomarker and its application |
| CN109055563A (en) * | 2018-08-27 | 2018-12-21 | 新乡医学院 | The related cyclic annular rna gene of colorectal cancer, colorectal cancer molecular marker and its application |
Non-Patent Citations (2)
| Title |
|---|
| Circular RNA hsa_circ_0000745 may serve as a diagnostic marker for gastric cancer;Mei Huang等;《World Journal of Gastroenterology》;20170914(第34期);147-155 * |
| 环状RNA与肿瘤关系研究进展;左等;《中华实用诊断与治疗杂志》;20181210(第12期);94-97 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109652549A (en) | 2019-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | CSTF2-induced shortening of the RAC1 3′ UTR promotes the pathogenesis of urothelial carcinoma of the bladder | |
| CN109609643B (en) | Application of circular RNA as diagnosis biomarker and treatment target of gastric cancer and colorectal cancer | |
| CN109576373A (en) | Application of the circ-VAPA as gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target | |
| CN109652548B (en) | Application of circ-CCNB1 as diagnosis biomarker and treatment target of gastric cancer and colorectal cancer | |
| CN107760784B (en) | Application of circular RNA circ-FOXP1 | |
| CN112280857B (en) | Biomarker for hepatocellular carcinoma diagnosis and application thereof | |
| CN106701900A (en) | Long-chain noncoding RNA HERC2P3 gene and application thereof in gastric cancer | |
| Zhang et al. | MALAT1 inhibits the Wnt/β-catenin signaling pathway in colon cancer cells and affects cell proliferation and apoptosis | |
| Yu et al. | A novel LncRNA, AC091729. 7 promotes sinonasal squamous cell carcinomas proliferation and invasion through binding SRSF2 | |
| Chen et al. | Fra-2/AP-1 regulates melanoma cell metastasis by downregulating Fam212b | |
| Ren et al. | MiR-135b-5p affected malignant behaviors of ovarian cancer cells by targeting KDM5B. | |
| Zhao et al. | TRIM36 suppresses cell growth and promotes apoptosis in human esophageal squamous cell carcinoma cells by inhibiting Wnt/β-catenin signaling pathway | |
| CN114107492B (en) | Molecular marker for tumor molecular typing and therapeutic drug evaluation, and detection primer and kit thereof | |
| CN113718035A (en) | Application of circular RNA hsa _ circ _0003552 and kit for detecting circular RNA hsa _ circ _0003552 | |
| CN109652549B (en) | Application of circular RNA as diagnosis biomarker and treatment target of gastric cancer and colorectal cancer | |
| CN109652550B (en) | Application of circ-HUWE1 as diagnosis biomarker and treatment target of gastric cancer and colorectal cancer | |
| Yang et al. | LncRNA MORT negatively regulates FGF1 to suppress malignant progression of breast cancer. | |
| CN114934048B (en) | circMTMR14 for tumor treatment target and diagnosis biomarker, kit and application | |
| He et al. | PPM1D accelerates proliferation and metastasis of osteosarcoma by activating PKP2 | |
| CN109486817A (en) | A kind of application of the long-chain non-coding RNA and combinations thereof in diagnoses and treatment cholangiocarcinoma | |
| CN105457041B (en) | Application of miR-26a in non-small cell lung cancer | |
| CN111088357B (en) | Tumor marker for ESCC and application thereof | |
| CN110628791B (en) | Application of tRNA (transfer RNA) modified enzyme gene in non-small cell lung cancer | |
| CN109593855A (en) | Application of the circ-NOTCH3 as gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target | |
| CN113122625B (en) | Application of SMCO2 gene as marker in diagnosis and treatment of endometrial cancer |
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 |