Combination of 3'-end-2'-O-methylated miRNA markers related to gastric cancer diagnosis and its application

Abstract

The present invention discloses a 3'-terminal-2'-O-methylation modified miRNA marker combination related to gastric cancer diagnosis and its application. The 3'-terminal-2'-O-methylation modified miRNA combination at least includes: miR-451, miR-1 and let-7f. The combination of the markers of the present invention for clinical detection of gastric cancer has the characteristics of being economical, rapid, sensitive and specific. It can be used for a wide range of health screening and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and relates to a 3' end-2'-O methylation modified miRNA marker combination related to gastric cancer diagnosis and an application thereof. Background Art

[0002] Gastric cancer (GC) is a malignant tumor originating from the gastric mucosal epithelium. It is recognized as the fifth most common cancer and the third leading cause of cancer death in the world. Most cases of gastric cancer occur in developing countries, with the highest incidence in Southeast Asia, among which China and Japan have the highest incidence of gastric cancer. Gastric cancer has an insidious onset, and most patients have already metastasized to distant sites when they develop symptoms, which seriously affects the prognosis of gastric cancer patients. The current screening methods for gastric cancer patients, such as gastroscopy or traditional plasma tumor marker tests, are painful for patients and lack strong diagnostic sensitivity and specificity. Therefore, it is particularly important to discover molecular markers for early diagnosis of tumors.

[0003] miRNA (microRNA) is a type of single-stranded RNA molecule with non-coding properties, with a length of about 18-22nt. It can stably exist in almost all body fluid environments. It has been found that miRNA plays an important role in almost every process of individual growth and development. In gastric cancer, miRNA has also been found to play an important role in tumor cell proliferation, metastasis, and drug resistance. Circulating tumor miRNA secreted by tumor cells into the plasma has also received increasing attention as a non-invasive diagnostic marker. However, due to the high false positive rate, obvious individual differences, and the lack of appropriate internal reference correction in miRNA detection, its clinical application has been slow.

[0004] Recent studies have shown that there are many modifications such as methylation and acetylation in miRNA, which affect the generation, stability and binding of miRNA to target genes. 3'-2'-O methylation is a methylation modification method that occurs at the 3' end of RNA. As early as 2005, researchers discovered that 2'-O methylation modification of the 3' end occurs during the maturation of plant miRNA, which can make plant miRNA more resistant to degradation by RNase. Recent studies have shown that a variety of small RNA molecules in insects and mammals also have methylation modification at the 3' end. Methylation modification of RNA molecules can protect them from attacks by a variety of enzymes such as exonucleases, ligases, terminal transferases and polymerases in cells, thus allowing them to exist stably in the body environment for a long time. However, since the current methylation detection methods mostly require mass spectrometry and sequencing methods, the operation is cumbersome and the cost is high, which does not meet the needs of clinical screening, thus limiting the development of methylation detection in clinical applications. Summary of the invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide a 3'-end-2'-O methylated modified miRNA marker or a combination thereof that is relevant to the diagnosis of gastric cancer.

[0006] Another object of the present invention is to provide a 3' end-2'-O methylation modified miRNA marker or a combination thereof related to the diagnosis of gastric cancer.

[0007] Another object of the present invention is to provide a gastric cancer auxiliary diagnosis kit.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] The 3' end-2'-O methylation modified miRNA marker associated with gastric cancer diagnosis is selected from any one or more of the following miRNAs: miR-451, miR-1, let-7f, miR-92a, miR-10a, miR-21.

[0010] Table 1. Differential miRNAs screened by the present invention.

[0011]

[0012] A combination of 3' end-2'-O methylation modified miRNA markers associated with gastric cancer diagnosis, including miR-451, miR-1 and let-7f.

[0013] The 3' end-2'-O methylation modified miRNA marker combination related to gastric cancer diagnosis of the present invention preferably also includes any one or more of miR-92a, miR-10a, and miR-21.

[0014] The 3' end-2'-O methylation modified miRNA marker combination associated with gastric cancer diagnosis preferably consists of miR-451, miR-1, let-7f and miR-92a.

[0015] The 3' end-2'-O methylation modified miRNA marker combination associated with gastric cancer diagnosis preferably consists of miR-451, miR-1, let-7f and miR-10a.

[0016] The 3' end-2'-O methylation modified miRNA marker combination associated with gastric cancer diagnosis preferably consists of miR-451, miR-1, let-7f and miR-21.

[0017] The 3' end-2'-O methylation modified miRNA marker combination associated with gastric cancer diagnosis preferably consists of miR-451, miR-1, let-7f, miR-92a and miR-21.

[0018] The 3' end-2'-O methylation modified miRNA marker combination associated with gastric cancer diagnosis preferably consists of miR-451, miR-1, let-7f, miR-92a and miR-10a.

[0019] The 3' end-2'-O methylation modified miRNA marker combination associated with gastric cancer diagnosis preferably consists of miR-451, miR-1, let-7f, miR-10a and miR-21.

[0020] The 3' end-2'-O methylation modified miRNA marker combination associated with gastric cancer diagnosis preferably consists of miR-451, miR-1, let-7f, miR-92a, miR-10a and miR-21.

[0021] The 3' end-2'-O methylated modified miRNA marker or a combination thereof of the present invention is used as a diagnostic target in the preparation of a gastric cancer auxiliary diagnosis kit.

[0022] Use of a reagent for detecting the 3' end-2'-O methylated modified miRNA marker or a combination thereof in the preparation of a gastric cancer auxiliary diagnosis kit.

[0023] A plasma kit for auxiliary diagnosis of gastric cancer, comprising a reagent for detecting the 3' end-2'-O methylated modified miRNA marker or its combination by the stem-loop method and a reagent for detecting the 3' end-2'-O methylated modified miRNA marker or its combination by the tailing method.

[0024] The reagent for detecting the 3' end-2'-O methylated modified miRNA marker or its combination by the stem-loop method comprises a TaqMan probe for detecting the miRNA marker or its combination; the reagent for detecting the 3' end-2'-O methylated modified miRNA marker or its combination by the tailing method comprises a primer for detecting the miRNA marker or its combination by the fluorescent quantitative PCR method.

[0025] The kit also contains enzymes and reagents commonly used in PCR reactions, such as reverse transcriptase, buffer, dNTPs, MgCl 2 and Taq enzyme, etc.; it also includes standards and / or control samples of the corresponding miRNA.

[0026] Beneficial effects:

[0027] The present invention provides a group of miRNAs combinations with significantly increased 2'-O methylation at the 3' end of the plasma of gastric cancer patients compared with normal people. The auxiliary diagnosis of gastric cancer using the combination as a target has the characteristics of high sensitivity, strong specificity and no need for internal reference correction, and can achieve non-invasive diagnosis of gastric cancer in the general population.

[0028] The gastric cancer auxiliary diagnosis kit developed based on the combination of the present invention has the characteristics of being economical, rapid, sensitive and specific for clinical detection of gastric cancer, can be used for a wide range of health screening, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The distribution of the detection results of 2'-O methylation at the 3' end of 6 miRNAs in the plasma of gastric cancer patients compared with the plasma of normal subjects.

[0030] Figure 2 Diagnostic ROC curves for different combinations of screened miRNAs.

[0031] The panel 1 Represents the combined detection of the above six indicators;

[0032] The panel 2 It represents the combined detection of miR-451, miR-1 and let-7f.

[0033] The panel 3 represents the combined detection of miR-451, miR-1, let-7f and miR-10a.

[0034] The panel 4 represents the combined detection of miR-451, miR-1, let-7f, and miR-21;

[0035] The panel 5 represents the combined detection of miR-451, miR-1, let-7f, miR-21, and miR-92a;

[0036] The panel 6 represents the combined detection of miR-451, miR-1, let-7f, miR-92a, and miR-10a;

[0037] The panel 7 Represents the combined detection of miR-451, miR-1, let-7f, miR-10a, and miR-21. DETAILED DESCRIPTION

[0038] The technical solutions and technical effects of the present invention are described below in conjunction with specific embodiments and drawings.

[0039] The present invention determines the difference in 2'-O methylation ratio of the 3' end of plasma miRNA between gastric cancer patients and normal subjects as a diagnostic marker for gastric cancer patients, aiming to design a simpler and faster method for screening gastric cancer patients.

[0040] Example 1 Screening of differentially 2'-O-methylated miRNAs at the 3' end of specific plasma samples from gastric cancer patients

[0041] (1) The subjects were gastric cancer patients and healthy controls. Plasma from gastric cancer patients and healthy controls was collected from Nanjing Eastern Theater Command General Hospital. The blood collection was approved by the Ethics Committee of the Eastern Theater Command General Hospital after the consent of the patients and healthy volunteers. The samples consisted of 60 primary gastric cancer patients and 60 normal healthy volunteers. All gastric cancer patients were diagnosed by biopsy and had not undergone surgery, chemotherapy, radiotherapy or other tumor-related treatments before blood collection. The age of the samples ranged from 41 to 69 years old, and there was no statistical difference in the age composition between the two groups (P = 0.83). The experiment of the present invention consisted of a test set (n = 12) and a validation set (n = 48). The test set was used to verify the sequencing screening results and preliminarily evaluate the diagnostic efficacy of the stem-loop tailing method for verifying the 2'-O methylation of the 3' end of gastric cancer plasma miRNA. The validation set was used to further expand the sample verification of the diagnostic accuracy of the 2'-O methylation detection of the 3' end of miRNA.

[0042] (2) In the morning, draw 2 mL of peripheral blood from lung cancer patients and healthy volunteers on an empty stomach, place in anticoagulant tubes and place in a 4°C refrigerator to separate plasma within 2 hours. Plasma separation is 3000 g, 10 min. Carefully aspirate the upper liquid phase and store in a -80°C refrigerator for later use.

[0043] (3) Total RNA was extracted using the Trizol method, and both groups of RNA were divided into two equal parts according to the previously established method, and one of the parts was oxidized with sodium periodate.

[0044] (4) Sequencing the oxidized miRNA. The final sequencing results are sorted according to the change factor F = (TO / T) / (NO / N) between the two groups after oxidation, and the miRNA with the most significant difference in 2'-O methylation level at the 3' end of the miRNA between gastric cancer patients and normal people is obtained.

[0045] Table 2. Preliminary screening of 3' end 2'-O methylated differential miRNAs by miRNA sequencing

[0046]

[0047] The data are the number of reads, T (Tumor) is the plasma group of gastric cancer patients, TO is the plasma group of gastric cancer patients oxidized by sodium periodate, N (Normal) is the plasma group of healthy controls, and NO is the plasma oxidation group of healthy controls.

[0048] Example 2

[0049] Verification of sequencing results using the kit of the present invention

[0050] The principle of determining the 2'-O methylation degree at the 3' end of miRNA by the stem-loop tailing method: The methylation modification of the 3' end of miRNA can affect the tailing efficiency of the tailing method in the kit due to the presence of steric hindrance, thereby reducing the measured miRNA content and increasing the Ct value. However, the 2'-O methylation at the 3' end of miRNA does not affect the miRNA content determined by the stem-loop method, and the Ct value remains unchanged. Therefore, two methods are used to detect the content of miRNA in plasma, and the Ct values ​​measured by the two methods are subtracted. The Ct difference can reflect the difference in the degree of methylation in plasma miRNA, and explore its use in screening and diagnosis of gastric cancer. The specific experimental methods are:

[0051] (1) Stem-loop method to screen the relative expression of miRNA

[0052] The steps of the stem-loop method for determining miRNA content are as follows:

[0053]

[0054] The reaction procedure was: 16°C, 30 min→42°C, 30 min→85°C, 30 min→storage at 4°C.

[0055]

[0056] After mixing all reaction components, set up 3 replica wells and perform the following procedure on the fluorescence quantitative PCR instrument:

[0057] 95℃,10min→(95℃,15s→60℃,1min)*40cycles.

[0058] The final result is the Ct value calculated by the instrument software.

[0059] (2) Tailing method to determine the relative expression of screened miRNAs

[0060] The kits used were: miScript SYBR Green PCR Kit (Qiagen), miScript II RT Kit (Qiagen). The experimental steps were:

[0061]

[0062] The reaction procedure was: 37°C, 1h→85°C, 5min→storage at 4°C.

[0063]

[0064] After mixing all reaction components, set up 3 replica wells and perform the following procedure on the fluorescence quantitative PCR instrument:

[0065] 95℃,10min→(95℃,15s→60℃,1min)*40cycles.

[0066] (5) Data statistical analysis: The results of the same sample measured by stem-loop method and tailing method qPCR were Ct 1 and Ct 2 , with ΔCt=Ct 2 -Ct 1 The difference in content was measured by the two methods and represented the plasma miRNA 3' end-2'-O methylation level.

[0067] Experimental Results

[0068] (1) We first verified the 10 miRNAs with a change greater than 20 in the sequencing results according to the change factor P = (TO / T) / (NO / N) in 12 samples, and finally screened out 6 miRNAs with significant differences in 3' end-2'-O methylation after excluding the miRNAs with Ct values ​​greater than 35 and no statistical significance between the two groups. Then we expanded the sample to detect the methylation levels of the 6 different miRNAs between 48 gastric cancer patients and healthy controls, and the results are as follows:

[0069] Table 3: Data of 6 screened miRNA stem-loop tailing methods:

[0070]

[0071] Example 3

[0072] ROC curve analysis

[0073] In order to clarify the diagnostic efficiency of the methylation detection of the 6 miRNAs we screened in gastric cancer patients, we performed ROC curve analysis on the experimental results. The results are shown in Table 3. The areas under the ROC curve of the 6 miRNAs using the stem-loop tailing method to detect 3'-end-2'-O methylation to distinguish gastric cancer patients from the normal control group are: miR-451, 0.743; miR-1, 0.722; let-7f, 0.840; miR-92a, 0.679; miR-10a, 0.685; miR-21, 0.691. The area under the curve of the combined detection of the 6 miRNAs can reach 0.947, indicating that the combined detection of the 3'-end-2'-O methylation level of the 6 miRNAs by the stem-loop tailing method can significantly screen gastric cancer patients.

[0074] Table 3: ROC curve analysis of 6 miRNAs.

[0075]

[0076]

[0077] *The panel 1 Represents the combined detection of the above six indicators;

[0078] The panel 2 It represents the combined detection of miR-451, miR-1 and let-7f.

[0079] The panel 3 represents the combined detection of miR-451, miR-1, let-7f and miR-10a.

[0080] The panel 4 represents the combined detection of miR-451, miR-1, let-7f, and miR-21;

[0081] The panel 5 represents the combined detection of miR-451, miR-1, let-7f, miR-21, and miR-92a;

[0082] The panel 6 represents the combined detection of miR-451, miR-1, let-7f, miR-92a, and miR-10a;

[0083] The panel 7 Represents the combined detection of miR-451, miR-1, let-7f, miR-10a, and miR-21.

[0084] The above description is only the preferred embodiment of the present invention, which is only illustrative and not restrictive to the present invention. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes, modifications, and even minor changes to the present invention, but they will all fall within the scope of protection of the present invention. Sequence Listing <110> Nanjing University <120> Combination of 3'-end-2'-O-methylated miRNA markers related to gastric cancer diagnosis and its application <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> RNA <213> Artificial Sequence <400> 1 aaaccguuac cauuacugag uu 22 <210> 2 <211> twenty two <212> RNA <213> Artificial Sequence <400> 2 uggaauguaa agaaguaugu au 22 <210> 3 <211> twenty two <212> RNA <213> Artificial Sequence <400> 3 ugagguagua gauuguauag uu 22 <210> 4 <211> twenty two <212> RNA <213> Artificial Sequence <400> 4 uauugcacuu gucccggccu gu 22 <210> 5 <211> twenty three <212> RNA <213> Artificial Sequence <400> 5 uacccuguag auccgaauuu gug 23 <210> 6 <211> twenty two <212> RNA <213> Artificial Sequence <400> 6 uagcuuauca gacugauguu ga 22

Claims

1. A combination of 3'-end-2'-O-methylated miRNA markers associated with gastric cancer diagnosis, Features The combination consists of miR-451, miR-1, let-7f, miR-92a, miR-10a and miR-21.

2. Use of a reagent for detecting the 3' end-2'-O methylated modified miRNA marker combination associated with gastric cancer diagnosis as claimed in claim 1 in the preparation of a gastric cancer auxiliary diagnosis kit.

3. A kit for auxiliary diagnosis of gastric cancer, Features The kit comprises a reagent for detecting the miRNA marker combination according to claim 1 by a stem-loop method and a reagent for detecting the miRNA marker combination according to claim 1 by a tailing method.

4. The kit according to claim 3, Features The reagent for detecting the miRNA marker combination by the stem-loop method includes a TaqMan probe for detecting the miRNA marker combination; the reagent for detecting the miRNA marker combination by the tailing method includes a primer for detecting the miRNA marker combination by the fluorescent quantitative PCR method.

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