Application of miR-21-based 3'terminal 2 '-O-methylation as lung cancer diagnosis marker and application of miR-21-based 3' terminal 2 '-O-methylation

Abstract

The invention belongs to the technical field of biological monitoring, and particularly relates to application of 3'terminal 2 '-O-methylation based on serum miR-21 as a detection marker for non-small cell lung cancer, especially KRAS G12C, and application of the detection marker. According to the application of the 3'terminal 2 '-O-methylation of the miR-21 in the serum as the non-small cell lung cancer, especially KRAS G12C detection marker, KRAS G12C mutation can be accurately detected by detecting the 3' terminal 2 '-O-methylation level of the miR-21 in the serum, so that the non-small cell lung cancer is diagnosed and screened based on a liquid biopsy technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection and diagnosis, and particularly relates to the use and application of the 3'-terminal 2'-O-methylation of serum miR-21 as a biomarker for the detection of non-small cell lung cancer, especially KRAS G12C. Background Art

[0002] Non-small cell lung cancer is a highly malignant tumor, and its incidence and mortality rates are both high globally. Although significant progress has been made in the treatment of non-small cell lung cancer in recent years, given the importance of early diagnosis, precise treatment, and prognosis assessment in improving the survival rate and quality of life of patients, the early diagnosis of non-small cell lung cancer still has crucial significance. Therefore, it is very necessary to establish a rapid and reliable method for the early diagnosis of non-small cell lung cancer.

[0003] Recent studies have shown that 3'-terminal methylation exists in a variety of small RNAs in mammals, such as some carcinogenic microRNAs. The 3'-terminal 2'-O-methylation modification of microRNA can not only enhance the stability of miRNA but also increase its binding affinity with AGO2. The 3'-terminal methylation of these small RNAs can protect them from the attack of various exonucleases, ligases, terminal transferases, polymerases, etc. in cells that can act on the 3'-terminal hydroxyl group of nucleic acids, thus protecting the stability of small RNAs.

[0004] In fact, the role of miRNA methylation abnormalities in cancer is multi-faceted. On the one hand, miRNA methylation abnormalities may lead to the down-regulation of the expression of certain tumor-suppressive miRNAs, thereby losing their negative regulatory effects on cancer-related genes and further promoting the development of tumors. On the other hand, miRNA methylation abnormalities may also lead to the up-regulation of the expression of some oncogenic miRNAs, thereby enhancing their positive regulatory effects on cancer-related genes, such as the KRAS G12C mutation, and further promoting the development of tumors.

[0005] It is reported that KRAS is one of the common oncogenes in humans, which can participate in key processes such as cell proliferation, differentiation, and survival. The gene mutation of KRAS is one of the most common genetic alterations in non-small cell lung cancer, and G12C is the most common mutation type. This mutation will increase the GTPase activity of the KRAS protein, making it in an active state, and then continuously activating downstream signaling pathways (such as the MAPK and PI3K pathways), promoting cell proliferation and survival. Therefore, the KRAS G12C mutation is closely related to the malignancy, prognosis, and drug treatment sensitivity of tumors.

[0006] Clinically, KRAS mutations have become important molecular markers for guiding tumor treatment strategies. Currently, the commonly used methods for detecting KRAS G12C mutations in clinical practice usually require tissue biopsies to obtain tissue samples, often through procedures such as surgery or puncture to obtain tumor tissue, which is an invasive process for patients and may bring complications such as pain, infection, and bleeding. In addition, due to the possible heterogeneity between different regions of the tumor, the information obtained from a single local tissue sample may not comprehensively reflect the characteristics of the entire tumor. Moreover, tissue biopsies usually take a certain amount of time to complete, including surgical waiting time, laboratory processing, and pathological analysis, etc., which may lead to delays in diagnosis and treatment decisions.

[0007] Liquid biopsy is a non-invasive method for tumor diagnosis and monitoring. By analyzing tumor-related markers in body fluids (such as serum), such as exosomes and circulating tumor RNA (ctRNA), etc., it can obtain tumor information. Compared with traditional tissue biopsies, liquid biopsy has many advantages:

[0008] Non-invasive: Liquid biopsy can collect routine body fluid samples such as blood, urine, saliva, etc., without the need for invasive surgery or tissue biopsies, reducing patient discomfort and risks and providing a more convenient way of sample collection;

[0009] Systemic: Liquid biopsy can provide systemic tumor information, not limited to a specific tumor site. It can detect circulating tumor markers throughout the body, reflecting the systemic characteristics and changes of the tumor;

[0010] Dynamic monitoring: Since liquid biopsy can repeatedly collect and analyze samples, it can achieve dynamic monitoring of tumors. It can be used to detect residual lesions after treatment, monitor tumor recurrence and metastasis, and evaluate treatment effects and drug resistance, etc.;

[0011] Consideration of spatial heterogeneity: Liquid biopsy can overcome the limitations of local heterogeneity in tissue biopsies because circulating tumor markers can reflect the genetic and expression characteristics of tumors throughout the body;

[0012] Provide individualized treatment strategies: Liquid biopsy can help select the best treatment plan and formulate individualized treatment strategies based on the molecular characteristics and variability of tumors.

[0013] In summary, liquid biopsy, as a non-invasive method for tumor diagnosis and monitoring, has advantages such as non-invasiveness, systemic nature, dynamic monitoring, and individualized treatment, providing new opportunities and options for the diagnosis and treatment of tumor patients. Therefore, there is an expectation in this field to develop a simple and non-invasive method for detecting KRAS G12C mutations in non-small cell lung cancer based on liquid biopsy technology, which is of great significance for the formulation of clinical diagnosis and treatment strategies. Summary of the Invention

[0014] To this end, the technical problem to be solved by the present invention is to provide the use of 3'-terminal 2'-O-methylation of serum miR-21 as a detection biomarker for non-small cell lung cancer, especially KRAS G12C, which can accurately detect KRAS G12C mutations by detecting the 3'-terminal 2'-O-methylation level of miR-21 in serum, thus solving the defects of traditional tissue biopsy methods such as invasiveness, local heterogeneity, and time lag;

[0015] The second technical problem to be solved by the present invention is to provide the application of the above-mentioned 3'-terminal 2'-O-methylation of serum miR-21 as a detection biomarker for non-small cell lung cancer, especially KRAS G12C, and further provide a rapid, non-invasive, and systemic method for diagnosing and detecting non-small cell lung cancer based on liquid biopsy technology.

[0016] To solve the above technical problems, the present invention relates to the use of 3'-terminal 2'-O-methylation of miR-21 for preparing a lung cancer diagnostic biomarker.

[0017] Specifically, the lung cancer includes non-small cell lung cancer.

[0018] Specifically, the non-small cell lung cancer includes KRAS G12C mutations.

[0019] Specifically, the 3'-terminal 2'-O-methylation of miR-21 is derived from serum.

[0020] The present invention also discloses the use of a substance and / or reagent for detecting the 3'-terminal 2'-O-methylation level of miR-21 for preparing a product for diagnosing or assisting in diagnosing, screening or assisting in screening non-small cell lung cancer, especially non-small cell lung cancer.

[0021] The present invention also discloses the use of a substance and / or reagent for detecting the expression level of 3'-terminal 2'-O-methylation of miR-21 for preparing a product for detecting the correlation and / or mutation expression level of KRAS G12C mutations.

[0022] The present invention also discloses a product for diagnosing or assisting in diagnosing, screening or assisting in screening non-small cell lung cancer, and the product includes a reagent for detecting the correlation and / or mutation level of KRAS G12C mutations based on RT-qPCR method.

[0023] Specifically, the product for diagnosing or assisting in diagnosing, screening or assisting in screening non-small cell lung cancer includes a plasma kit.

[0024] Specifically, the product for the diagnosis or auxiliary diagnosis, screening or auxiliary screening of non-small cell lung cancer, the kit includes a stem-loop fluorescence quantitative PCR kit or a tailing fluorescence quantitative PCR kit.

[0025] Specifically, the optional stem-loop fluorescence quantitative PCR kit of the present invention can use the stem-loop primers of Thermo Fisher Scientific Company for reverse transcription. Its detection system is as shown in Table 1 below, and the PCR reaction program is as shown in Table 2 below.

[0026] Table 1 Stem-loop fluorescence quantitative PCR system

[0027] Reagent Volume (μL) AMV 0.5 5×AMV Buffer 2 dNTP Mix (2.5 mM) 4 DEPC 1.5 Stem-loop RT Primer 1 RNA 1 Total 10

[0028] Table 2 Reaction program

[0029]

[0030]

[0031] After the above reverse transcription is completed, quantitative detection is carried out using the corresponding qPCR probe. Specifically, the q-PCR system is as shown in Table 3 below, and the qPCR reaction program is as shown in Table 4 below.

[0032] Table 3 q-PCR system

[0033] Reagent Volume (μL) <![CDATA[ddH2O]]> 14.77 10×buffer 2 <![CDATA[MgCl2]]> 1.2 dNTP (10 mM) 0.4 rTaq 0.3 TM Probe 0.33 cDNA 1 Total 20

[0034] Table 4 The qPCR reaction program is as follows:

[0035]

[0036] Specifically, the optional tailing fluorescence quantitative PCR kit of the present invention can use the miRCURY LNART Kit reverse transcription kit of QIAGEN Company for reverse transcription. Its PCR system is as shown in Table 5 below, and the reaction program is as shown in Table 6 below.

[0037] Table 5 Reaction system

[0038] Reagent Volume (μL) 10x miRCURY RT Enzyme Mix 0.5 5x miRCURY Reaction Buffer 1 Nuclease free water 0.5 RNA 3 Total 5

[0039] Table 6 Reaction program

[0040] Temperature (℃) Time (min) 42 60 95 5

[0041] After the above reaction, the cDNA obtained by reverse transcription is added with 15 μl of DEPC water, diluted and mixed evenly, and then the subsequent q-PCR reaction is carried out. The specific reaction system is as shown in Table 7 below, and the reaction program is as shown in Table 8 below.

[0042] Table 7 q-PCR reaction system

[0043] Reagent Volume (μL) SYBR Green Master Mix 2x concentrate2 10 PCR Primer 6Mix 2 RNase-free water 6 cDNA 2 Total 20

[0044] Table 8 q-PCR reaction procedure

[0045]

[0046] In the present invention, the method for calculating the standard curve of methylated and unmethylated standards is as follows:

[0047] (1) Take 1 nmol of miR-21 standard, add 100 μL of DEPC water, prepare 10 μM Standard RNA storage solution, and store in aliquots;

[0048] (2) Take 5 nmol of 2'-O-methylated miR-21 standard, add 500 μL of DEPC water, prepare 10 μM Standard RNA storage solution, and store in aliquots;

[0049] (3) Gradient dilution: Take 5 μL of Standard RNA storage solution, add 45 μL of RNase free water, and prepare a gradient 1 sample with a concentration of 1 μM; take 5 μL of gradient 1 sample, add 45 μL of RNase free water, and prepare a gradient 2 sample with a concentration of 100 nM; take 5 μL of gradient (N) sample, add 45 μL of RNase free water, and prepare a gradient N+1 sample with a concentration one order of magnitude lower; the specific gradient dilution concentrations are shown in Table 9;

[0050] (4) Gradients 4-10 were selected for stem-loop and tailing qRT-PCR experiments, respectively, with concentration as the horizontal axis and Ct value as the vertical axis to draw the standard curve and calculate the difference.

[0051] Table 9 Sample concentration dilution gradient

[0052] 4 5 6 7 8 9 10 1 nM 100 pM 10 pM 1 pM 100 fM 10 fM 1 fM

[0053] The present invention also discloses a system for diagnosing or assisting in diagnosis, screening or assisting in screening of non-small cell lung cancer, comprising:

[0054] A detection device for determining KRAS G12C mutation relevance and / or mutation level in a biological sample;

[0055] A judgment device, which performs diagnosis or auxiliary diagnosis, screening or auxiliary screening of whether the person to be tested has non-small cell lung cancer based on whether KRAS G12C in the biological sample is mutated or the expression amount of the mutation.

[0056] The present invention provides the use of 2'-O-methylation at the 3'-end of miR-21 in serum as a biomarker for detecting non-small cell lung cancer, especially KRAS G12C. It can accurately detect KRAS G12C mutations by detecting the 2'-O-methylation level at the 3'-end of miR-21 in serum, effectively solving the problem of difficult early diagnosis and treatment selection for highly lethal non-small cell lung cancer, thereby realizing the diagnosis and screening of non-small cell lung cancer based on liquid biopsy technology and solving the defects of traditional tissue biopsy methods such as invasiveness, local heterogeneity, and time lag.

[0057] The present invention further provides the application of 2'-O-methylation at the 3'-end of miR-21 in serum as a biomarker for detecting non-small cell lung cancer, especially KRAS G12C, and further provides a rapid, non-invasive, and systemic method for diagnosing and detecting non-small cell lung cancer based on liquid biopsy technology, which is of great significance for the diagnosis and treatment of non-small cell lung cancer.

[0058] By detecting the 2'-O-methylation level at the 3'-end of miR-21 in the serum of patients, a liquid biopsy method for detecting KRAS G12C mutations in non-small cell lung cancer is established. This method has the following advantages:

[0059] Non-invasive: The detection of the 2'-O-methylation level at the 3'-end of miR-21 can be carried out by collecting blood samples from patients without invasive surgery or tissue biopsy. This non-invasive detection method is more convenient and acceptable for patients.

[0060] KRAS mutation specificity: The upregulation of the 2'-O-methylation level at the 3'-end of miR-21 has a high correlation with KRAS mutations in non-small cell lung cancer. Therefore, by detecting the expression level of the 2'-O-methylation level at the 3'-end of miR-21, the presence or absence of KRAS mutations can be indirectly inferred.

[0061] High sensitivity: The upregulation of the 2'-O-methylation level at the 3'-end of miR-21 is common in non-small cell lung cancer and is related to KRAS mutations. Therefore, the detection of the 2'-O-methylation level at the 3'-end of miR-21 can provide high-sensitivity screening and diagnosis of KRAS mutations in non-small cell lung cancer.

[0062] Dynamic monitoring: Due to the characteristics of liquid biopsy, the detection of the 2'-O-methylation level at the 3'-end of miR-21 can achieve dynamic monitoring of KRAS mutations in non-small cell lung cancer. By repeatedly collecting samples, the changes in KRAS mutations, including residual lesions, recurrence, and metastasis after treatment, can be monitored in a timely manner.

[0063] Potential prognostic value: Some studies have shown that the upregulation of the 2'-O-methylation level at the 3' end of miR-21 is associated with poor prognosis in patients with non-small cell lung cancer; therefore, the detection of the 2'-O-methylation level at the 3' end of miR-21 may help evaluate the prognostic risk of patients and provide a reference for the formulation of individualized treatment plans.

[0064] The present application further provides a method for detecting KRAS G12C mutations in patients with non-small cell lung cancer by using the expression level of the 2'-O-methylation level at the 3' end of serum miR-21, and detecting the 2'-O-methylation at the 3' end of circulating miR-21 in the serum of patients with non-small cell lung cancer to reflect the KRAS G12C mutations in patients with non-small cell lung cancer and predict the prognosis and survival of patients. This method has the advantages of non-invasiveness, high sensitivity and specificity, convenience and rapidity, and has the potential for popularization and application in clinical practice. Brief Description of the Drawings

[0065] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to specific embodiments of the present invention in conjunction with the drawings, wherein,

[0066] Figure 1 To compare the results of the 2'-O-methylation level of miR-21 in the sera of healthy volunteers, sera of other mutant non-small cell lung cancer patients, and sera of non-small cell lung cancer patients with KRAS G12C mutations in Example 1;

[0067] Figure 2 To show the survival analysis results (survival curves) of non-small cell lung cancer patients with KRAS G12C mutations with high expression of the 2'-O-methylation level of miR-21 at the 3' end in serum and non-small cell lung cancer patients with other mutant types with low expression of the 2'-O-methylation level of miR-21 at the 3' end in serum in Example 2;

[0068] Figure 3 To show the results based on miRNA target prediction software in Example 3. Detailed Description of the Invention

[0069] Example 1

[0070] In this example, serum samples of patients with non-small cell lung cancer were collected, and miRNAs in the serum were extracted by appropriate technical means. The specific method is as follows.

[0071] Separation of plasma: Collect anticoagulated blood samples (EDTA anticoagulant) from normal individuals and colorectal cancer patients. Centrifuge the blood samples at 3000 rpm for 10 minutes. Carefully aspirate the supernatant plasma (avoiding aspiration of blood cells) into a 1.5 mL EP tube. Store the plasma in an ultra-low temperature freezer at -80 °C.

[0072] Extraction of plasma small RNAs (TIANGEN miRcute Serum / Plasma miRNA Extraction and Isolation Kit):

[0073] (1) Aspirate 200 μL of plasma into a new 1.5 mL EP tube. Add 900 μL of lysis buffer MZA. Vortex for 30 s until completely homogenized, and invert to mix. After complete homogenization and before inverting to mix, add exogenous miR-2911 (at a concentration of 1 μM, add 1 μL).

[0074] (2) Incubate at room temperature for 5 min to completely separate the nucleic acid-protein complexes. Add 200 μL of chloroform. Vortex vigorously for 15 s to thoroughly mix the solution, and then incubate at room temperature for 5 min. Centrifuge at 12000 rpm at 4 °C for 15 min. After centrifugation, the sample will be divided into three layers: a yellow organic phase, a white intermediate layer, and a colorless aqueous phase. RNA is mainly in the aqueous phase. Transfer the top aqueous phase to a new 1.5 mL EP tube.

[0075] (3) Measure the volume when transferring the aqueous phase, and then slowly add 2 volumes of absolute ethanol (e.g., add 1 mL of absolute ethanol to 500 μL of the transfer solution). Slowly invert to mix.

[0076] (4) Transfer the obtained solution and precipitate into the adsorption column miRelute. Incubate at room temperature for 2 min, and then centrifuge at 12000 rpm at room temperature for 30 s. Discard the filtrate.

[0077] (5) Add 700 μL of protein removal solution MRD (previously added with ethanol) to the adsorption column miRelute. Let it stand at room temperature for 2 min. Centrifuge at 12000 rpm at room temperature for 30 s. Discard the waste liquid.

[0078] (6) Add 500 μL of washing solution RW (previously added with ethanol) to the adsorption column miRelute. Let it stand at room temperature for 2 min. Centrifuge at 12000 rpm at room temperature for 30 s. Discard the waste liquid, and repeat the washing once.

[0079] Centrifuge for 2 min at room temperature and 12,000 rpm, and discard the collection tube. Transfer the adsorption column miRelute into a new RNase-Free 1.5 mL centrifuge tube, add 20 μL of DEPC water to the center of the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge at 12,000 rpm (about 13,400 g) at room temperature for 2 min. Store the RNA in an ultra-low temperature freezer at -80 °C.

[0080] In this example, the RT-qPCR method was used to measure the 2'-O-methylation level at the 3' end of miR-21 in serum. Specifically, the Ct values of miR-21 were measured by RT-qPCR using the aforementioned stem-loop method and tailing method. The Ct difference between the stem-loop method and the tailing method was substituted into the fitted standard curve, and then the difference was calculated to obtain the expression level of the 2'-O-methylation level at the 3' end of miR-21. The 2'-O-methylation levels at the 3' end of miR-21 in the sera of healthy volunteers, other mutant non-small cell lung cancer patients, and non-small cell lung cancer patients with KRAS G12C mutation were compared. The results are shown in the appendix Figure 1 .

[0081] The results showed that the 2'-O-methylation level at the 3' end of miR-21 in the sera of non-small cell lung cancer patients with KRAS G12C mutation was significantly upregulated compared with that of healthy individuals and other mutant non-small cell lung cancer patients. It is indicated that the detection of the 2'-O-methylation level at the 3' end of serum miR-21 can better reflect the mutation status of KRAS G12C in non-small cell lung cancer patients.

[0082] Example 2

[0083] In this example, the correlation between the expression level of the 2'-O-methylation level at the 3' end of miR-21 and the KRAS G12C mutation was analyzed, and the presence or absence of the KRAS G12C mutation in non-small cell lung cancer patients was determined according to the expression level of the 2'-O-methylation level at the 3' end of miR-21.

[0084] In this example, non-small cell lung cancer patients with KRAS G12C mutation with high expression of the 2'-O-methylation level at the 3' end of miR-21 in serum and non-small cell lung cancer patients with other mutant types with low expression of the 2'-O-methylation level at the 3' end of miR-21 in serum were selected for survival analysis.

[0085] In this example, KRAS G12C mutation was diagnosed by histopathological examination, and the patient's survival time was defined as the period from the date of cancer diagnosis to the last follow-up or death. Whether and when the patient died was determined based on hospitalization and outpatient records, the patient's family, or follow-up calls. The data were analyzed using GraphPad Prism software. An independent sample t-test was used for inter-group comparisons, and repeated measures analysis of variance was performed for comparisons of each time point within the group; Log-Rank test was used for survival analysis, and P<0.05 was considered statistically significant. The results are attached. Figure 2 .

[0086] The results showed that the 3'-end 2'-O-methylation level of highly expressed miR-21 in serum was significantly correlated with the poor prognosis of patients with non-small cell lung cancer.

[0087] Example 3

[0088] In this example, the miRNA target prediction software miRanda v3.3a was used to verify the relationship between miR-21 and KRAS transcript NM_001369786.1. In the software settings, the Score Threshold was set to 120, the Energy Threshold was set to -1.000000 kcal / mol, and the lowest Energy Threshold, that is, the lowest binding free energy, was selected. The specific operations and results are shown in the attached Figure 3 shown.

[0089] The results showed that there was a potential binding site between miR-21 and KRAS, and the binding free energy was -11.740000 kCal / Mol, which was relatively stable, suggesting the regulatory correlation between miR-145 and KRAS.

[0090] In summary, the present invention verifies the use of 2'-O-methylation of the 3' end of miR-21 in serum as a detection marker for non-small cell lung cancer, especially KRAS G12C. KRAS G12C mutations can be accurately detected by detecting the 3' end 2'-O-methylation level of miR-21 in serum, which effectively solves the problem of difficulty in early diagnosis and treatment selection of highly lethal non-small cell lung cancer, thereby realizing the diagnosis and screening of non-small cell lung cancer based on liquid biopsy technology.

[0091] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of 2'-O-methylation at the 3'-end of miR-21 for preparing a biomarker for lung cancer diagnosis.

2. The use according to claim 1, characterized in that, The lung cancer includes non-small cell lung cancer.

3. The use according to claim 2, characterized in that, The non-small cell lung cancer includes KRAS G12C mutation.

4. The use according to any one of claims 1 to 3, characterized in that, The 2'-O-methylation at the 3'-end of miR-21 is derived from serum.

5. Use of a substance and / or reagent for detecting the 2'-O-methylation level of the 3'-end of miR-21 for preparing a product for diagnosing or assisting in diagnosing, screening or assisting in screening non-small cell lung cancer, especially non-small cell lung cancer.

6. Use of a substance and / or reagent for detecting the expression level of 2'-O-methylation at the 3'-end of miR-21 for preparing a product for detecting the correlation and / or mutation expression level of KRAS G12C mutation.

7. A product for the diagnosis or auxiliary diagnosis, screening or auxiliary screening of non-small cell lung cancer, characterized in that, The product includes a reagent for detecting the correlation and / or mutation level of KRAS G12C mutation based on RT-qPCR method.

8. The product for diagnosing or assisting in the diagnosis, screening or assisting in the screening of non-small cell lung cancer according to claim 7, characterized in that, The product includes a plasma kit.

9. The product for the diagnosis or auxiliary diagnosis, screening or auxiliary screening of non-small cell lung cancer according to claim 8, characterized in that, The kit includes a stem-loop fluorescence quantitative PCR kit or a tailing fluorescence quantitative PCR kit.

10. A system for diagnosing or assisting in the diagnosis, screening or assisting in the screening of non-small cell lung cancer, characterized in that, Comprising: A detection device for determining the correlation and / or mutation level of KRAS G12C mutation in a biological sample; A judgment device for diagnosing or assisting in diagnosing, screening or assisting in screening whether a person to be tested has non-small cell lung cancer based on whether KRAS G12C in the biological sample is mutated or the mutation expression amount.

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