A miRNA marker for nasopharyngeal carcinoma diagnosis and its application

Abstract

The present invention provides a mi RNA marker for nasopharyngeal carcinoma diagnosis and its application. The expression level of miR‑376a‑5p in the blood of nasopharyngeal carcinoma patients is significantly higher than that in healthy people, and the area under the AUC curve is 0.850 after calculation and analysis. The diagnostic sensitivity of miR‑376a‑5p can reach 89.6%, and the specificity is as high as 90.1%. It was found that knocking down miR‑376a‑5p can significantly inhibit the expression of miR‑376a‑5p in nasopharyngeal carcinoma cells and can effectively inhibit the proliferation, metastasis and invasion of nasopharyngeal carcinoma cells. It suggests the feasibility of using siRNA to target and knock down miR‑376a‑5p for the treatment of nasopharyngeal carcinoma.

Description

Technical Field

[0001] The present invention relates to the technical field of auxiliary diagnosis of nasopharyngeal carcinoma, and in particular to a miRNA marker for nasopharyngeal carcinoma diagnosis and application thereof. Background Art

[0002] Nasopharyngeal carcinoma (NPC) refers to a malignant tumor that occurs on the top and side walls of the nasopharynx. It is one of the most common malignant tumors in my country, and its incidence rate ranks first among malignant tumors of the ear, nose and throat. Common clinical symptoms include nasal congestion, blood in mucus, stuffy ears, hearing loss, diplopia and headache.

[0003] At present, radiotherapy is the treatment of choice for nasopharyngeal carcinoma. It has a very high cure rate for early patients and significantly improves the overall survival rate. However, many patients have no symptoms in the early stages of the disease and are already in the middle and late stages when they seek medical treatment. The efficacy of radiotherapy alone is poor, which is not conducive to disease control. At present, research on the molecular and clinical characteristics of nasopharyngeal carcinoma continues to deepen, and nasopharyngeal endoscopy, pathological examination, and imaging examination have become important means of clinical screening. However, these methods are either too dependent on the experience of the tester, or are invasive and unsuitable for routine screening, or are too expensive to promote, or the detection sensitivity and specificity need to be strengthened.

[0004] Therefore, there is an urgent need to develop new, reliable, non-invasive early diagnostic markers to promote early intervention and treatment of NPC and prolong the survival of NPC patients. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides an application of circular RNA in the preparation of an auxiliary diagnostic reagent for nasopharyngeal carcinoma. The present invention uses miR-376a-5p as an auxiliary diagnostic marker for nasopharyngeal carcinoma, and can quickly achieve auxiliary detection of nasopharyngeal carcinoma by analyzing its expression level. In addition, miR-376a-5p can be used as a therapeutic target, and targeted inhibition of its expression can achieve the treatment of nasopharyngeal carcinoma patients, which has a broad application prospect.

[0006] One of the purposes of the present invention is to provide the use of miR-376a-5p as a diagnostic marker in the preparation of reagents for auxiliary diagnosis and / or evaluation of nasopharyngeal carcinoma, wherein the nucleotide sequence of miR-376a-5p is shown in SEQ ID No.1.

[0007] Preferably, the expression level of miR-376a-5p in nasopharyngeal carcinoma patients is detected to assist in the diagnosis and / or assessment of nasopharyngeal carcinoma.

[0008] One of the purposes of the present invention is to provide a kit for assisting in the diagnosis and / or evaluation of nasopharyngeal carcinoma, characterized in that it contains primers for specifically amplifying miR-376a-5p.

[0009] Preferably, the primers for specifically amplifying miR-376a-5p are: the reverse transcription primer sequence is SEQ ID No.2, the upstream primer sequence is SEQ ID No.3, and the downstream primer sequence is SEQ ID No.4.

[0010] Further preferably, the test sample in the kit is blood.

[0011] One of the purposes of the present invention is to use miR-376a-5p in preparing a kit for auxiliary diagnosis and / or evaluation of nasopharyngeal carcinoma.

[0012] One of the purposes of the present invention is to use miR-376a-5p as a therapeutic target in the preparation of a drug for treating nasopharyngeal carcinoma.

[0013] Preferably, the use of miR-376a-5p siRNA in the preparation of a drug for treating nasopharyngeal carcinoma, wherein the drug uses miR-376a-5p as a therapeutic target, and the miR-376a-5p siRNA comprises a nucleotide sequence as shown in SEQ ID No.6.

[0014] One of the purposes of the present invention is a drug for treating nasopharyngeal carcinoma, characterized in that the drug contains a nucleic acid, a biologically active functional fragment or a variant having a sequence as shown in SEQ ID No.5.

[0015] Preferably, the drug comprises a pharmaceutically acceptable carrier or excipient.

[0016] The advantages of the present invention are as follows: The present invention found that the expression level of miR-376a-5p in the blood of nasopharyngeal carcinoma patients was significantly higher than that of healthy people, and the area under the AUC curve was calculated to be 0.850, and the diagnostic sensitivity of miR-376a-5p could reach 89.6%, and the specificity was as high as 90.1%. It is confirmed that the miR-376a-5p gene has good application prospects in the diagnosis of nasopharyngeal carcinoma patients. In addition, it was found that knocking down miR-376a-5p would significantly inhibit the expression of miR-376a-5p in nasopharyngeal carcinoma patients and effectively restore the hormone levels of patients. The above findings show the importance of miR-376a-5p to nasopharyngeal carcinoma, and suggest the feasibility of using siRNA to target and knock down miR-376a-5p for the treatment of nasopharyngeal carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 .The expression of miR-376a-5p gene in patients with nasopharyngeal carcinoma;

[0018] Figure 2. Validation analysis of miR-376a-5p gene in a large number of nasopharyngeal carcinoma patients;

[0019] Figure 3 . Interference with the expression of miR-376a-5p gene before and after siRNA treatment;

[0020] Figure 4 .Analysis of nasopharyngeal carcinoma cell proliferation ability before and after interference siRNA treatment;

[0021] Figure 5 .Analysis of nasopharyngeal carcinoma cell migration ability before and after siRNA interference treatment;

[0022] Figure 6 .Analysis of nasopharyngeal carcinoma cell invasion ability before and after interference siRNA treatment. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0024] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0025] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0026] Example 1 The expression of miR-376a-5p in nasopharyngeal carcinoma patients was analyzed.

[0027] 1. Preparation of experimental materials:

[0028] All test subjects were patients diagnosed with nasopharyngeal carcinoma by clinicians from May to December 2022. Blood samples from 35 patients were collected for backup; blood samples from 35 healthy people were collected as controls during the same period.

[0029] 2. Test methods

[0030] 1) Blood total RNA extraction: Use blood total RNA extraction kit ( Blood RNA kit was used to extract total RNA from blood samples of patients and healthy subjects and stored at -20℃ for future use;

[0031] 2) Reverse transcription cDNA synthesis: The total RNA in step 1) was reverse transcribed using a reverse transcription kit PrimeScript RT reagent Kit to obtain cDNA, which was stored at -20°C for future use; wherein the reverse transcription primer sequence is shown in SEQ ID NO.2.

[0032] 3) Real-time quantitative PCR: Specific primers were designed according to the nucleic acid sequence of miR-376a-5p (SEQ ID NO.1), wherein the upstream primer and downstream primer of miR-376a-5p were SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0033] The 25 μL PCR reaction system included: 10 μL 10× PCR Buffer, 2 μL SYBR Green I fluorescent dye, 4 μL dNTP, 1 μL each of the upstream and downstream primers of miR-376a-5p, 2 μL Taq DNA polymerase and 5 μL DNA; the PCR reaction program was 94°C pre-denaturation for 2 min; 94°C for 45 s, 60°C for 30 s, 72°C extension for 45 s, for a total of 40 cycles; 72°C extension for 5 min;

[0034] The specificity of the reaction was determined based on the melting curve, and the relative expression of miR-376a-5p was calculated using the formula 2-ΔΔCt. Figure 1 .Depend on Figure 1 It can be seen that compared with healthy people, the expression level of miR-376a-5p detected in 35 pairs of nasopharyngeal carcinoma patient blood samples was significantly upregulated.

[0035] Example 2 Analysis of the diagnostic value of miR-376a-5p gene in a large number of nasopharyngeal carcinoma patients

[0036] 1. Preparation of experimental materials:

[0037] All test subjects were patients diagnosed with nasopharyngeal carcinoma by clinicians from January to October 2023. Blood samples from 100 patients were collected for backup; blood samples from 100 healthy people were collected as controls during the same period.

[0038] 2. Test methods

[0039] The method steps were the same as in Example 1, and the sensitivity and specificity were calculated by AUC curve area. Figure 2As shown, similar to the results of Example 1, the expression of miR-376a-5p gene in nasopharyngeal carcinoma patients is generally high, and the area under the AUC curve is 0.850, and its diagnostic sensitivity can reach 89.6%, and the specificity is as high as 90.1%. The experimental data analysis further confirms that the miR-376a-5p gene has a good application prospect in the diagnosis of nasopharyngeal carcinoma patients.

[0040] Example 3 Analysis of the effect of inhibiting the expression of miR-376a-5p on the improvement of nasopharyngeal carcinoma

[0041] 1. Preparation of experimental materials:

[0042] Human nasopharyngeal carcinoma cell CNE-1 was activated in RMPI-1640 culture medium containing 10% FBS, subcultured at 37°C and 5% CO2 for later use.

[0043] 2. Test methods

[0044] 1) Design of miR-376a-5p interference fragment: The siRNA sequence was synthesized in Shanghai Jima Biotechnology, wherein the siRNA sequence is shown in SEQ ID NO.5.

[0045] 2) Interference efficiency verification: Experimental group: CNE-2 cells were transfected with siRNA according to the lipofectamine RNAimax operating instructions; the control group was not transfected during the same period; one week after the transfection, the relative expression level of miR-376a-5p was quantitatively analyzed by the method described in Example 1.

[0046] 3) Cell proliferation ability analysis: MTT cell proliferation and cytotoxicity detection kit was used to analyze the proliferation ability of CNE-2 before and after siRNA transfection. The specific analysis method can be found in the kit manual.

[0047] 4) Cell invasion and migration assay: 24 h after siRNA transfection, the experimental and control groups were collected, resuspended in complete culture medium, and the cells were counted. 100 μL of cell suspension (RMPI-1640 culture medium containing 10% FBS, about 4×10 cells per well) was added to the Transwell chamber without or with pre-coated matrix gel. 4cells), put the chamber into a 24-well cell culture plate, add 600 μL of RMPI-1640 culture medium containing 20% ​​FBS to the lower layer, and culture the cells for 24 hours at 37°C, 5% CO2 concentration, and saturated humidity. Take out the chamber, discard the culture medium in the 24-well plate, add 600 μL of 90% ethanol to fix for 10 minutes, gently wipe the residual ethanol and cells inside the chamber with a sterile cotton swab, add 600 μL of 0.1% crystal violet staining solution after air drying, and stain for 10 minutes. Using an inverted microscope, under low power, randomly select 5 to 8 fields of view in each chamber for observation, photography, and counting.

[0048] result Figure 3 It can be seen that the siRNA fragments interfering with the expression of miR-376a-5p can significantly reduce the expression of miR-376a-5p in human nasopharyngeal carcinoma cell CNE-1, and the proliferation ability of MTT cells is also significantly reduced ( Figure 4 ), in addition, the inventors unexpectedly found that after interfering with miR-376a-5p expression, the number of CNE-1 cells undergoing migration and invasion was significantly reduced ( Figure 5 and Figure 6 ), suggesting that siRNA interfering with miR-376a-5p expression can significantly inhibit the migration and invasion ability of NPC cells.

[0049] The above results show that the siRNA targeting miR-376a-5p provided by the present invention has a good interference effect, can significantly inhibit the proliferation, invasion and migration ability of nasopharyngeal carcinoma cells after transfection, and has a broad application prospect.

[0050] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Use of a reagent for detecting the expression level of miR-376a-5p in the preparation of a reagent for auxiliary diagnosis and / or evaluation of nasopharyngeal carcinoma, characterized in that: The nucleotide sequence of miR-376a-5p is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The reagent is used to assist in the diagnosis and / or evaluation of nasopharyngeal carcinoma by detecting the expression level of miR-376a-5p in nasopharyngeal carcinoma patients.

3. Use of a kit for assisting diagnosis and / or evaluating nasopharyngeal carcinoma in preparing a reagent for assisting diagnosis and / or evaluating nasopharyngeal carcinoma, characterized in that: The kit contains primers for specifically amplifying miR-376a-5p, and the primers detect the expression level of miR-376a-5p. The nucleotide sequence of miR-376a-5p is shown in SEQ ID No.

1.

4. The use according to claim 3, characterized in that The primers for specifically amplifying miR-376a-5p are: the upstream primer sequence is SEQ ID No.3, and the downstream primer sequence is SEQ ID No.

4.

5. The use according to claim 3, characterized in that The test sample in the kit is blood.

6. Use of a reagent for detecting the expression level of miR-376a-5p in the preparation of a kit for auxiliary diagnosis and / or evaluation of nasopharyngeal carcinoma, wherein the nucleotide sequence of miR-376a-5p is shown in SEQ ID No.

1.

7. Use of a reagent for targeted knockdown of miR-376a-5p in the preparation of a drug for treating nasopharyngeal carcinoma, characterized in that: The reagent is siRNA targeting miR-376a-5p, the nucleotide sequence of the siRNA is the nucleotide sequence shown in SEQ ID No.5, and the nucleotide sequence of the miR-376a-5p is shown in SEQ ID No.

1.

8. The use according to claim 7, characterized in that The medicine includes a pharmaceutically acceptable carrier or excipient.

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