WO2020220994A1 - 一种检测胃癌的miRNA标志物组合及试剂盒 - Google Patents

一种检测胃癌的miRNA标志物组合及试剂盒 Download PDF

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WO2020220994A1
WO2020220994A1 PCT/CN2020/084687 CN2020084687W WO2020220994A1 WO 2020220994 A1 WO2020220994 A1 WO 2020220994A1 CN 2020084687 W CN2020084687 W CN 2020084687W WO 2020220994 A1 WO2020220994 A1 WO 2020220994A1
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mirna
gastric cancer
marker combination
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邹瑞阳
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Mirxes Lab Pte Ltd
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Mirxes Lab Pte Ltd
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Priority to US17/594,805 priority patent/US20220186320A1/en
Priority to JP2021564879A priority patent/JP7676321B2/ja
Priority to AU2020265027A priority patent/AU2020265027A1/en
Priority to KR1020217039294A priority patent/KR20220009970A/ko
Priority to MX2021013424A priority patent/MX2021013424A/es
Priority to SG11202112095VA priority patent/SG11202112095VA/en
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    • 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
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    • 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

Definitions

  • the invention belongs to the field of molecular biology, and specifically relates to a miRNA marker combination and a kit for detecting gastric cancer.
  • Gastric cancer is one of the most common malignant tumors in the world and one of the malignant tumors with the highest mortality rate. Most patients with gastric cancer have missed the best timing of diagnosis and treatment when they are diagnosed, leading to disease progression, tumor metastasis, and even end-stage. From the perspective of TMN staging of gastric cancer, the 5-year survival rate of gastric cancer is 97.6% in the first stage, 94.9% in the first stage, 70.49% in the second stage, 56.7% in the third stage, and the third stage in the later stage. It was 31.9%, and the fourth period was 6.5%. It can be seen that early diagnosis of gastric cancer is necessary.
  • Endoscope is currently the most advantageous tool for diagnosing gastric cancer.
  • the endoscopic manifestations of early gastric cancer include abnormal mucosal color, disappearance of blood vessels on the mucosal surface, depression or thickening of the mucosal layer, irregular nodules, and abnormal mucosal folds around the ulcer. If necessary, some tissues can be cut for biopsy.
  • Detection of protein markers in the blood can be used as a reference for the diagnosis of gastric cancer.
  • Commonly used protein tumor markers for gastric cancer include: such as carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 72-4 (CA72-4) and carbohydrate antigen 50 (CA50) and stomach Protease etc.
  • Biopsy after endoscopy is the gold standard for gastric cancer detection.
  • this method is invasive and can cause discomfort and fear in patients.
  • asymptomatic patients usually do not undergo endoscopy.
  • Conventional protein tumor markers are often used for gastrointestinal cancer detection, but due to lack of sensitivity and/or specificity, they are not recommended for gastric cancer diagnosis.
  • miRNA is a class of non-coding single-stranded small RNA molecules with a length of about 19-24 nt.
  • the vast majority of miRNAs can complement the 3'UTR region of the target gene to inhibit the translation of the target gene into protein, and then at the cell, tissue or individual level Affect the growth and development of organisms and participate in a variety of disease processes.
  • the expression profile of miRNA has obvious tissue specificity and a specific expression pattern in different tumors. These characteristics make it possible for miRNA to become a new biological marker and therapeutic target for tumor diagnosis.
  • qPCR is the most commonly used method to detect the expression of known miRNAs.
  • the purpose of the present invention is to provide a combination of markers and kits for detecting early gastric cancer to distinguish between the serum of patients with gastric cancer and the serum of healthy people, and to distinguish the serum of patients with gastric cancer from the serum of patients with gastritis, which can be simple, effective and effective.
  • Non-invasive detection of gastric cancer is to provide a combination of markers and kits for detecting early gastric cancer to distinguish between the serum of patients with gastric cancer and the serum of healthy people, and to distinguish the serum of patients with gastric cancer from the serum of patients with gastritis.
  • the present invention adopts the following technical solutions:
  • the applicant used RT-qPCR technology to screen and obtain 12 miRNAs as biomarkers and combinations for gastric cancer detection, specifically hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa- miR-93-5p, hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-142-5p, hsa-miR- 126-3p, hsa-miR-183-5p, hsa-miR-340-5p.
  • the miRNA marker combination of the present invention was used to test 4566 subjects, compared with the Helicobacter pylori test and the pepsinogen test, and the results showed good agreement with the clinical gold standard of endoscopy (AUC of 0.84) It is significantly better than the two existing biomarkers of pepsinogen 1/2 ratio and Helicobacter pylori test respectively (AUC is 0.62 and 0.64).
  • a combination of miRNA markers for detecting gastric cancer selected from hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa-miR-181a- 5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-142-5p, hsa-miR-126-3p, hsa-miR-183-5p or At least 4 miRNA markers in hsa-miR-340-5p.
  • the miRNA marker combination is selected from hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa- miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, has-miR-142-5p, hsa-miR-126-3p, hsa-miR- At least 5 miRNA markers in 183-5p or hsa-miR-340-5p.
  • the miRNA marker combination is selected from hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa- miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, has-miR-142-5p, hsa-miR-126-3p, hsa-miR- At least 6 miRNA markers in 183-5p or hsa-miR-340-5p.
  • the miRNA marker combination is selected from hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa- miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, has-miR-142-5p, hsa-miR-126-3p, hsa-miR- At least 7 miRNA markers in 183-5p or hsa-miR-340-5p.
  • the miRNA marker combination is selected from hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa- miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, has-miR-142-5p, hsa-miR-126-3p, hsa-miR- At least 8 miRNA markers in 183-5p or hsa-miR-340-5p.
  • the miRNA marker combination is selected from hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa- miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, has-miR-142-5p, hsa-miR-126-3p, hsa-miR- At least 9 miRNA markers in 183-5p or hsa-miR-340-5p.
  • the miRNA marker combination is selected from hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa- miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, has-miR-142-5p, hsa-miR-126-3p, hsa-miR- At least 10 miRNA markers in 183-5p or hsa-miR-340-5p.
  • the miRNA marker combination is selected from hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa- miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, has-miR-142-5p, hsa-miR-126-3p, hsa-miR- At least 11 miRNA markers in 183-5p or hsa-miR-340-5p.
  • the miRNA marker combination for detecting gastric cancer in a peripheral blood sample comprises hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93 -5p, hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-142-5p, hsa-miR-126-3p , Hsa-miR-183-5p and hsa-miR-340-5p.
  • the miRNA marker combination consists of hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa-miR- 181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-142-5p, hsa-miR-126-3p, hsa-miR-183- 5p and hsa-miR-340-5p consist of 12 miRNA markers.
  • the miRNA marker combination is composed of hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa-miR -181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-126-3p, hsa-miR-183-5p and hsa-miR-340 -5p consists of 11 miRNA markers.
  • the miRNA marker combination is composed of hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa-miR-181a-5p, hsa-miR- 21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-126-3p, hsa-miR-183-5p and hsa-miR-340-5p consist of 10 miRNA markers .
  • the miRNA marker combination is composed of hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR- 140-5p, hsa-miR-30e-5p, hsa-miR-126-3p, hsa-miR-183-5p and hsa-miR-340-5p consist of 9 miRNA markers.
  • the miRNA marker combination is composed of hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR- 140-5p, hsa-miR-30e-5p, hsa-miR-126-3p and hsa-miR-340-5p consist of 8 miRNA markers.
  • the miRNA marker combination is composed of hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR- 140-5p, hsa-miR-30e-5p and hsa-miR-340-5p consist of 7 miRNA markers.
  • the miRNA marker combination is composed of hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR- 140-5p and hsa-miR-340-5p consist of 6 miRNA markers.
  • the miRNA marker combination is composed of hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p and hsa-miR- 340-5p consists of 5 miRNA markers.
  • the miRNA marker combination consists of four miRNA markers: hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21-5p and hsa-miR-340-5p ⁇ Composition.
  • the present invention also provides a method for identifying subjects who are at risk of suffering from gastric cancer, including:
  • methods for determining miRNA expression levels include the use of quantitative RT-PCR (for example, using SYBR-Green or Tagman-based chemical methods), chip or sequencing.
  • the biomarker can be detected by Northern blot, droplet digital PCR, mass spectrometry, electrochemiluminescence, or other methods known in the art.
  • a linear regression model is used to calculate the risk score.
  • the linear regression model used is logistic regression.
  • the linear regression model is as follows:
  • miRNA1, miRNA2, miRNA3... are selected from hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa-miR-181a-5p , Hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-142-5p, hsa-miR-126-3p, hsa-miR-183-5p or hsa -miR-340-5p;
  • CT is the relative expression level of each miRNA detected by qPCR
  • K is the coefficient of each miRNA marker.
  • the invention also provides a kit for detecting gastric cancer, which includes a reagent for specifically detecting the miRNA marker combination.
  • the reagent in the kit of the present invention is a qPCR method detection reagent, a chip method detection reagent or a sequencing method detection reagent.
  • the reagent for specifically detecting the miRNA marker combination in the kit includes at least one oligonucleotide, wherein at least a part of the oligonucleotide is related to the peripheral gastric cancer detection system described above.
  • the miRNA markers in the blood miRNA marker combination specifically bind.
  • the reagent for specifically detecting the miRNA marker combination in the kit of the present invention is a qPCR detection reagent.
  • the qPCR detection reagent in the kit of the present invention includes the reverse transcription primer and/or qPCR amplification primer of the miRNA marker combination.
  • the kit includes stem-loop reverse transcription primers and/or semi-nested qPCR primers for amplifying the miRNA markers in the peripheral blood miRNA marker combination for detecting gastric cancer.
  • the qPCR detection reagents in the kits of the present invention include hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93 -5p, hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-142-5p, hsa-miR-126-3p , Hsa-miR-183-5p and hsa-miR-340-5p, the reverse transcription primers and/or qPCR amplification primers of each miRNA of the miRNA marker combination consisting of 12 miRNA markers.
  • the qPCR detection reagents in the kits of the present invention include hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93 -5p, hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-126-3p, hsa-miR-183-5p Reverse transcription primers and/or qPCR amplification primers of each miRNA combined with a miRNA marker consisting of 11 miRNA markers in total with hsa-miR-340-5p.
  • the qPCR detection reagents in the kit of the present invention include hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa-miR-181a -5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-126-3p, hsa-miR-183-5p and hsa-miR-340-5p Reverse transcription primers and/or qPCR amplification primers for each miRNA of a miRNA marker combination consisting of a total of 10 miRNA markers.
  • the qPCR detection reagents in the kits of the present invention include hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21 -5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-126-3p, hsa-miR-183-5p and hsa-miR-340-5p consisting of 9 miRNA markers
  • the reverse transcription primers and/or qPCR amplification primers of each miRNA of the miRNA marker combination consisting of 9 miRNA markers.
  • the qPCR detection reagents in the kits of the present invention include hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21 -5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-126-3p and hsa-miR-340-5p, a total of 8 miRNA markers composed of miRNA marker combinations of each miRNA Reverse transcription primers and/or qPCR amplification primers.
  • the qPCR detection reagents in the kits of the present invention include hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21 -5p, hsa-miR-140-5p, hsa-miR-30e-5p, and hsa-miR-340-5p.
  • the reverse transcription primer and/or qPCR amplification of each miRNA of the miRNA marker combination consisting of 7 miRNA markers Increase primer.
  • the qPCR detection reagents in the kits of the present invention include hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21 -5p, hsa-miR-140-5p, and hsa-miR-340-5p, which consist of 6 miRNA markers including reverse transcription primers and/or qPCR amplification primers for each miRNA.
  • the qPCR detection reagent in the kit of the present invention includes sa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR-140 -5p and hsa-miR-340-5p, the reverse transcription primers and/or qPCR amplification primers of each miRNA in the miRNA marker combination consisting of 5 miRNA markers.
  • the qPCR detection reagent in the kit of the present invention includes hsa-miR-103a-3p, hsa-miR-181a-5p, hsa-miR-21-5p, and hsa-miR-340 -5p
  • the reverse transcription primers and/or qPCR amplification primers of each miRNA of the miRNA marker combination consisting of a total of 4 miRNA markers.
  • the qPCR detection reagents in the kit of the present invention further include positive quality control products, negative quality control products, reverse transcriptase, dNTPs, reverse transcription buffer, and nuclease-free water , QPCR buffer, magnesium chloride, DNA polymerase, at least one of SYBR Green fluorescent dye.
  • the present invention also provides a use of the miRNA marker combination in preparing a gastric cancer diagnostic reagent that predicts the development of the subject or the possibility of gastric cancer by the following method, the method comprising:
  • the score based on the previously measured expression level of miRNA is used to predict the likelihood that the subject will develop or have gastric cancer.
  • the peripheral blood is serum or plasma.
  • the expression level of the miRNA is scored by using a linear regression algorithm to construct a simple linear regression model.
  • a linear regression model was used to calculate the risk score.
  • Logistic regression is an example of a linear regression method that can be used for this purpose. However, any relevant person skilled in the art will understand that other forms of linear regression calculation can be used to derive the score. Determine the critical value of the risk score based on clinical needs, and use two critical values to define the population as a high-risk, low-risk and uncertain population.
  • the subjects include, but are not limited to, Chinese, Malays, and Indians.
  • the miRNA marker combination for detecting gastric cancer of the present invention is selected from hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p, hsa-miR- 181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-142-5p, hsa-miR-126-3p, hsa-miR-183- At least 4 miRNA markers in 5p or hsa-miR-340-5p.
  • the combination of markers for detecting gastric cancer to detect the serum of a subject can distinguish the serum of a patient with gastric cancer from that of a healthy person, distinguish the serum of a patient with gastric cancer from that of a gastritis patient, and use the miRNA marker combination of the present invention to perform
  • the test is compared with the Helicobacter pylori test and the pepsinogen test.
  • the results show good agreement with the clinical gold standard of endoscopy, and are significantly better than the pepsinogen 1/2 ratio and the Helicobacter pylori test.
  • the kit for detecting gastric cancer of the present invention has a simple composition and can detect gastric cancer simply, effectively and non-invasively.
  • Figure 1 shows the roadmap for clinical verification of gastric cancer serum miRNA
  • Figure 2 shows the ROC characteristics of different blood markers
  • Figure 3 shows the ROC characteristics of men and women
  • Figure 4 shows the ROC characteristics of subjects of different races in this kit
  • Figure 5 shows the ROC characteristics of the kit for subjects with early gastric cancer and late gastric cancer
  • Figure 6 shows the ROC feature map under different miRNA numbers combined linear regression algorithm.
  • the invention discloses a miRNA marker combination and kit for detecting gastric cancer. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method and product of the present invention have been described in the preferred embodiments. It is obvious that relevant personnel can make changes or appropriate changes and combinations to the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invent technology.
  • the reagents involved in the embodiments of the present invention are all commercially available products, which can be purchased through commercial channels.
  • the design method of the reverse transcription primers and RT-qPCR primers of miRNA in the present invention is in accordance with the U.S. Patent Publication No. US9850527B2 and Wan G, Lim Q', Too H P. High-performance quantification of mature microRNAs by real-time RT -PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers[J].Rna-a Publication of the Rna Society,2010,16(7):1436-45. All miRNA sequences disclosed in the present invention have been stored in the miRBase database (http://www.mirbase.org/).
  • RT-qPCR technology was used to detect sera of 236 gastric cancer patients and 236 non-cancer control subjects for miRNA.
  • 191 miRNAs expression level ⁇ 500 copies/ml
  • 75 of the 191 miRNAs are differentially expressed between the control group and cancer patients (FDR P value ⁇ 0.01).
  • 51 are up-regulated in gastric cancer patients and 24 are down-regulated.
  • RT-qPCR technology was used to detect the serum miRNA of 94 gastric cancer patients and 116 non-cancer control subjects. There is a good correlation between the change of miRNA expression fold between the development stage and the verification stage.
  • 12 miRNAs were further selected as biomarkers and combinations for gastric cancer detection, specifically hsa-miR-29c-3p, hsa-miR-424-5p, hsa-miR-103a-3p, hsa-miR-93-5p , Hsa-miR-181a-5p, hsa-miR-21-5p, hsa-miR-140-5p, hsa-miR-30e-5p, hsa-miR-142-5p, hsa-miR-126-3p, hsa -miR-183-5p, hsa-miR-340-5p.
  • the miRNA sequences and miRBase database registration numbers are shown in Table
  • AUC is used as an optimization index to construct a simple linear regression model.
  • a linear regression model was used to calculate the risk score.
  • a logistic regression model is used. Determine the critical value of the risk score based on clinical needs, and use two critical values to define the population as a high-risk, low-risk and uncertain population. The Logistic score of gastric cancer population was significantly higher than that of healthy population.
  • the linear regression model calculation formula is as follows:
  • K is the coefficient of each miRNA marker, and the coefficient of each marker is different for different combinations of markers.
  • Table 2 is a possible embodiment.
  • CT is the relative expression of each miRNA marker. That is, the Ct value obtained by qPCR.
  • test samples collecting blood and separating serum
  • the reverse transcription product (cDNA) is amplified by qPCR using qPCR buffer, DNA polymerase and a qPCR plate containing miRNA sequence-specific primers under specific reaction conditions and temperatures.
  • the Ct value of each miRNA is obtained by setting a threshold.
  • RT-qPCR Before RT-qPCR, a set of artificially synthesized miRNA (3) controls was added to each sample to monitor and standardize the technical differences in this process.
  • each miRNA was added with 6 logarithmically diluted synthetic templates, negative controls and mixed human serum RNA reference materials, and each isolated serum RNA sample was reverse-transcribed and quantified by qPCR. These quality control measures help to monitor and standardize technical differences in pipetting and measurement efficiency during RT, cDNA amplification and qPCR.
  • the serum sample is lysed by phenol/guanidine, and the total RNA of the serum sample is separated by silica purification column.
  • the corresponding miRNA reverse transcription stem-loop primers were used to reverse transcribe 12 miRNAs in each sample into cDNA. Then use sequence-specific forward PCR primers and semi-nested sequence-specific reverse PCR primers for qPCR, and use SYBR Green I dye for detection.
  • Example 2 5282 subjects from the National University Hospital of Singapore and Tan Tock Seng Hospital of Singapore were selected for clinical testing. The specific verification route is shown in Figure 1. A total of 5282 subjects entered the clinical verification.
  • the 12 miRNA biomarkers described in Example 1 were selected for miRNA test, Helicobacter pylori test, pepsinogen test, and gastroscopy for all subjects.
  • the 12-miR qPCR assay is developed and manufactured according to the ISO13485 medical equipment quality management system. In the absence of endoscopic and histopathological test results, miRNA testing is performed in a CAP/ISO certified laboratory. Western blot assay was used to determine the Helicobacter pylori antibodies in serum samples.
  • the levels of pepsinogen I and II were determined with a latex agglutination turbidimetric immunoassay kit. Both determinations are performed when the clinical results are unknown. After screening, 4566 subjects were finally used for data analysis, 125 subjects were diagnosed with gastric cancer, and 4441 subjects were confirmed as healthy people without cancer. Table 3 shows the analysis results of clinical characteristics data of 4566 subjects.
  • the miRNA marker combination test method, Helicobacter pylori test and pepsinogen test of the present invention are compared, and the results are shown in Figure 2 and Table 4.
  • the miRNA marker combination test of the present invention showed consistent AUC in male and female subjects ( Figure 3 and Table 5).
  • the clinical validation data is further analyzed to determine whether the miRNA marker combination test of the present invention performs differently on different ethnic groups.
  • Singapore In line with Singapore’s demographic data, the majority of subjects participating in this clinical trial are Chinese (76.43%), with Malays, Indians and other ethnic groups each accounting for about 8% (Table 3). The results showed that the AUC of Chinese subjects was higher than that of other races ( Figure 4, Table 6).
  • the performance of the miRNA marker combination test of the present invention is further evaluated by cancer staging.
  • the miRNA marker combination test of the present invention showed that the AUC of early (stage 0, 1 and 2) and advanced (stage 3 and 4) cancers were 0.83 and 0.85, respectively (Figure 5, Table 7).
  • Example 2 in accordance with the important order of miRNA, using the cancer and control subject samples described in Example 3, sequentially select 12, 11, 10, 9, 8, 7, 6, and 12 described in Example 1. 5. The combination of 4 miRNAs was tested, and the ROC feature map was obtained ( Figure 6, Table 9).
  • the Logistic algorithm uses different scores as the dividing line between cancer and non-cancer, it shows different diagnostic performance and is suitable for different diagnostic approaches (Table 8).
  • Table 8 For example, when a dividing line of 40 points is used, that is, a patient with a score of 40 or more is defined as a cancer patient, the sensitivity will be significantly higher than that of 50 points, and the specificity and accuracy will be significantly lower than that of 50 points. Therefore, the lower Logistic boundary is suitable for gastric cancer screening in the population, and the higher Logistic boundary is suitable for auxiliary diagnosis.
  • miRNA can be used as a marker for the diagnosis of cancer and other diseases, there is no consensus on specific miRNA expression profiles for specific diseases.
  • the use of miRNAs as diagnostic or prognostic markers may face certain challenges (Tiberio et al, 2015). Even for the same disease, the miRNA markers screened by different studies may be very different (Leidner et al, 2013). Due to the selection of research objects in the existing literature, the lack of consistency in sample collection and processing steps, and detection methods may result in the suboptimal choice of miRNA biomarkers for diagnosis or prognosis. And most of the research results in these documents have not been verified in larger research cohorts.
  • the subject's health, treatment, environmental and genetic factors may all have an impact on the miRNA expression profile.
  • miRNA analysis is based only on a single study cohort or samples from the same location, it may cause bias in the results. Therefore, it is necessary to sample a large enough population and design a well-designed and controllable data analysis workflow to screen miRNA markers that can be applied to large-scale populations.
  • the present invention fully validates the currently selected 12 miRNA biomarkers, and these validations are performed on different patient cohorts from different research locations. From the initial development stage, the screening and verification of miRNA biomarkers in different cohorts. The 12 selected miRNAs were subsequently further validated in a larger prospective clinical study involving 5282 subjects. The 12-clock miRNA screened and verified in a large number of subjects from multiple independent research cohorts ensures its robustness as a biomarker and can be applied to a wide range of populations to identify subjects at risk of gastric cancer .
  • the miRNA profile may also be affected by technical aspects, such as sample collection, processing steps, and methods for detecting miRNA expression in samples.
  • the sensitivity and specificity of detecting changes in miRNA expression may vary significantly due to different detection methods. In most published studies, sample collection and processing methods can be very different. In addition, there are differences in the reagents and methods used in the research. In this paper, a semi-nested primer was designed based on the principle of Wan et al, 2010, using a highly sensitive and specific RT-qPCR method and an optimized miRNA expression profile and data analysis workflow, using internal controls to monitor and control miRNA extraction, Differences in the efficiency of steps such as reverse transcription and qPCR.
  • the clinical validation study of 5282 subjects adopted methods that require more clinical validation of medical diagnostic tests (Wilson et al, 2008; Mattocks et al, 2010).
  • the test kit is produced and executed under strict quality control requirements, which is not common in clinical research.
  • the technical method of the present invention further ensures the effectiveness and robustness of 12 miRNA markers for identifying the risk of gastric cancer.
  • the miRNAs of 5 or more miRNA combinations have a better AUC for the diagnosis of gastric cancer (AUC of 5-miRNA group is 0.8029, AUC of 12-miRNA group is 0.8423).
  • AUC of miRNA detection composed of 5 or more miRNAs is very close to that of gastroscopy (AUC is 0.84), which is currently the gold standard for gastric cancer diagnosis.
  • Subjects who are determined to be at risk of gastric cancer may need further tests, such as gastroscopy, biopsy, or diagnostic imaging tests, such as magnetic resonance imaging (MRI) or computed tomography (CT). Patients diagnosed with gastric cancer will receive appropriate treatment.
  • the treatment of gastric cancer generally includes one or more of the following interventions: surgery, radiotherapy, chemotherapy or immunotherapy, or the use of targeted therapies such as trastuzumab and lamotizumab.
  • Potential drugs for gastric cancer treatment include small molecules, antibodies, vaccines or peptides.
  • Chemotherapy drugs used for the treatment of gastric cancer include 5-fluorouracil, capecitabine, carboplatin, cisplatin, docetaxel, epirubicin, irinotecan, oxaliplatin, paclitaxel, trifluridine, and tipir Lacy.
  • Immunotherapy drugs used to treat gastric cancer include immune checkpoint inhibitors, such as pembrolizumab.
  • the treatment option for early gastric cancer is usually surgery. For cancers that are detected early, endoscopic resection is also possible. It is generally believed that the treatment effect of patients with early gastric cancer is significantly better than that of patients with advanced gastric cancer (Lello et al, 2007). Therefore, a reliable and convenient detection method is of great significance for the early diagnosis of gastric cancer patients.
  • the present invention provides detailed verification data of a large-scale clinical study of 12 miRNA biomarker combinations.
  • the miRNA marker combination can provide an AUC equivalent to that of gastroscopy in the diagnosis of gastric cancer, but without the invasiveness of gastroscopy and the risk of related complications.
  • a key principle of population cancer screening is to insist on regular screening to facilitate early detection of cancer. In this regard, compared with more invasive tests, blood-based tests that only require a single blood draw and provide reliable conclusions will be a more promising development direction.

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