Marker, detection kit and medicine for detecting gastric cancer

Abstract

The invention discloses a marker for detecting gastric cancer. The marker comprises miRNA-204-5p (micro Ribonucleic Acid). According to the present invention, qPCR is adopted to verify that the diagnosis efficiency is 96.78%, the sensitivity is 95.79%, the specificity is 91.67% and the detection limit is 101 copy / mL in the serum of 100 gastric cancer patients and healthy people, such that the non-invasive and rapid detection method is provided for the gastric cancer patients, and the important significance is provided for the clinical gastric cancer diagnosis.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to a marker, a detection kit and a medicine for detecting gastric cancer. Background Art

[0002] Gastric cancer (GC) is one of the common malignant tumors of the digestive system. According to the data released by the National Cancer Center in 2023, the incidence and mortality of GC in my country ranked second and third among tumors respectively, and the number of cases accounted for 40% of the global GC cases, among which the incidence rate was higher in plateau areas, which seriously affected the health of the people. Although the diagnosis and treatment methods of GC have been constantly innovating in recent years, the incidence and mortality of GC in my country are still high. The main reason is that the pathogenesis of GC is complex and is a multi-step and multi-factorial pathological process. In addition to the influence of geographical environment and diet, it also involves changes in its own coding genes and non-coding genes. Therefore, it is particularly important to further explore and improve the molecular mechanism of GC and provide more valuable target molecules for the early diagnosis and treatment of GC. Summary of the invention

[0003] To solve the above problems, the present invention aims to provide a marker, a detection kit and a drug for detecting gastric cancer. The marker miRNA-204-5p alone shows a significantly high diagnostic performance in the plasma of patients with early gastric cancer. A detection kit and a drug are also provided.

[0004] The present invention is achieved through the following technical solutions:

[0005] A marker for detecting gastric cancer includes miRNA-204-5p, and its miRNA sequence is shown as SEQ ID No.1.

[0006] The above-mentioned markers also include any one or any combination of miRNA-3622a-5p, miRNA-503-5p, miRNA-671-5p, miRNA-3065-3p, miRNA-502-3p, miRNA-7-1-3P, miRNA-548bc, miRNA-200a-5p, and miNRA-378d, and the above-mentioned miRNA sequences are shown in SEQ ID No. 2-10 respectively.

[0007] The miRNA included in the above-mentioned markers is miRNA in plasma.

[0008] Use of the markers as described above in drugs or kits for early gastric cancer diagnosis, efficacy or prognosis evaluation.

[0009] A detection kit for early gastric cancer screening or diagnosis, comprising the markers as described above.

[0010] A drug for early gastric cancer screening, diagnosis or prognosis evaluation, comprising the aforementioned marker. The present invention found that the expression level of miRNA-204-5p in gastric cancer plasma was significantly downregulated by 2.10 times, so the use of miRNA-204-5p alone can effectively screen or diagnose early gastric cancer.

[0011] The detection kit of the present invention uses RNA-seq technology to screen the miRNA marker miRNA-204-5p for detecting gastric cancer. Compared with the normal population, the expression of miRNA-204-5p is significantly downregulated in early gastric cancer and advanced gastric cancer. The specific detection method is: collecting plasma, extracting, reversing, designing specific primers for miRNA-204-5p for amplification, and detecting its expression level in miRNA, which can realize rapid screening of early gastric cancer and make up for the shortcomings of poor specificity and low sensitivity of existing early gastric cancer diagnostic biomarkers.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0013] The present invention uses qPCR to verify the diagnostic efficacy of 96.78%, the sensitivity of 95.79%, the specificity of 91.67% and the detection limit of 10^1copy / mL in the sera of 100 gastric cancer patients and healthy people, providing a non-invasive and rapid detection method for gastric cancer patients, which is of great significance for the clinical diagnosis of gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0015] Figure 1 Differential expression analysis of whole transcriptome sequencing between early gastric cancer plasma and normal plasma (n=3).

[0016] Figure 2 Expression levels of miRNA-204-5p in normal plasma samples and plasma of gastric cancer patients.

[0017] Figure 3 Minimum detection limit detection amplification curve.

[0018] Figure 4 Dissolution curve with minimum detection limit.

[0019] Figure 5 The detection method has the lowest detection limit.

[0020] Figure 6The ROC curve of the detection method was drawn. DETAILED DESCRIPTION

[0021] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0022] Example 1

[0023] In the practice of the present invention, kits and drugs can be prepared, such as a detection kit for early gastric cancer screening or diagnosis, including the markers described above; a drug for early gastric cancer screening, diagnosis or prognosis evaluation, including the markers described above.

[0024] The screening process of plasma miRNAs biomarkers for early gastric cancer is as follows:

[0025] (1) 5 mL of peripheral blood was collected from 3 healthy subjects and 3 patients with early gastric cancer. The blood was centrifuged at 4°C and 3000 rpm for 5 min to separate the plasma and extract the total RNA. The high-throughput sequencing was performed using the Illumina platform and combined with bioinformatics analysis. A total of 1725 miRNAs were detected by sequencing, and the significant difference threshold was set at log2Fold. Change>2, P<0.05 screening differentially expressed miRNAs, found a total of 124 differentially expressed miRNAs in the plasma of early gastric cancer and normal people, further expanded the screening conditions to P<0.001, 11 miRNAs were screened, of which miRNA-204-5p, miRNA-3622a-5p, miRNA-503-5p, miRNA-671-5p, miRNA-3065-3p, miRNA-502-3p, miRNA-7-1-3P, miRNA-548bc, miRNA-200a-5p, miNRA-378d were the 10 molecules with the largest change multiples, and P<0.001. Among them, miRNA-204-5p was significantly downregulated in gastric cancer plasma, and the gastric cancer group was 2.10 times lower than the normal group.

[0026] Figure 1 A: A volcano plot of differential expression of known miRNAs in early gastric cancer plasma and normal plasma. Black represents miRNAs that are highly expressed in early gastric cancer, and gray represents miRNAs that are downregulated in early gastric cancer. B: Heat map of differential expression of miRNAs in early gastric cancer plasma and normal plasma. T represents early gastric cancer plasma, and N represents normal plasma.

[0027] (2) 30 healthy subjects and 30 patients with early gastric cancer were selected for qPCR validation. Peripheral blood was collected according to the method described in step (1) to extract total RNA. Then, a reverse transcription kit (purchased from Novozymes Biotech Co., Ltd.) was used to synthesize cDNA by tailing method and qPCR amplification was performed. Then, based on the Ct values ​​of the internal reference genes U6 and mi-204-5p, the relative expression level of miRNA was calculated according to the 2-ΔΔCt formula. The results showed that compared with the plasma of the normal population, the expression level of miRNA-204-5p in the plasma of patients with early gastric cancer was significantly downregulated.

[0028] The primer sequences are as follows:

[0029] Table 1 U6 and miRNA-204-5p gene primer sequences

[0030]

[0031] The amplification system is as follows:

[0032] Table 2 Real-time fluorescence quantitative PCR reaction system

[0033]

[0034] The amplification procedure is as follows:

[0035] Table 3 Real-time fluorescence quantitative PCR reaction program

[0036]

[0037] Figure 2 The expression levels of miRNA-204-5p in normal plasma samples and plasma of gastric cancer patients are shown. A: expression levels of miRNA-204-5p in early gastric cancer plasma and normal plasma (n=30), T represents early gastric cancer plasma, and N represents normal plasma.

[0038] (4) Minimum detection limit

[0039] Construct the recombinant miRNA-204-5p plasmid and dilute it to 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 copy / mL, amplification detection was performed using the method in step 2, and the results were as follows Figure 3-5 As shown, the minimum detection limit of the detection method of the present invention can reach 10 1 copy / mL.

[0040] (5) SPSS software was used for statistical analysis and ROC curve was drawn. Figure 6 As shown, the diagnostic efficacy of candidate mi-RNA-204-5p in early gastric cancer was evaluated, and the difference was statistically significant with bilateral P<0.05. By drawing the ROC curve, the AUC of miR-204-5p was 0.9678, and the sensitivity was 95.79% when the specificity was 91.67%, indicating that the biomarker described in the present invention showed significantly high diagnostic performance when used alone in the plasma of patients with early gastric cancer.

[0041] The sequence numbers of the miRNA markers of the present invention are shown in Table 1 below:

[0042] Table 1 Sequence Listing

[0043] sequence SEQ ID No.1 5'-uucccuuugucauccuaugccu-3' SEQ ID No.2 5'-caggcacgggagcucaggugag-3' SEQ ID No.3 5'-uagcagcgggaacaguucugcag-3' SEQ ID No.4 5'-aggaagcccuggaggggcuggag-3' SEQ ID No.5 5'-uaaggcaugaucugagucuau-3' SEQ ID No.6 5'-aaugcaccugggcaaggauuca-3' SEQ ID No.7 5'-uggaagacuagugauuuuguu-3' SEQ ID No.8 5'-aaaaacugugauuacuuuugc-3' SEQ ID No.9 5'-caucuuaccggacagugcugga-3' SEQ ID No.10 5'-acuggacuuggagucagaaa-3'

[0044] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A marker for detecting gastric cancer, characterized in that: Including miRNA-204-5p, its miRNA sequence is shown as SEQ ID No.

1.

2. The marker according to claim 1, characterized in that It also includes any one or any combination of miRNA-3622a-5p, miRNA-503-5p, miRNA-671-5p, miRNA-3065-3p, miRNA-502-3p, miRNA-7-1-3P, miRNA-548bc, miRNA-200a-5p, and miNRA-378d, and the above miRNA sequences are shown in SEQ ID No.2-10 respectively.

3. The marker according to claim 2, characterized in that For miRNA in plasma.

4. Use of the marker according to any one of claims 1 to 3 in a drug or kit for diagnosis, efficacy or prognosis evaluation of early gastric cancer.

5. A detection kit for early gastric cancer screening or diagnosis, characterized in that: The invention comprises the marker according to any one of claims 1 to 3.

6. A drug for early gastric cancer screening, diagnosis or prognosis assessment, characterized in that: The invention comprises the marker according to any one of claims 1 to 3.

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