A miRNA kit for detecting mutations of atm / atr genes of laryngeal squamous cell carcinoma and application thereof

Abstract

The present application relates to a kind of kit for detecting laryngeal squamous cell carcinoma radiotherapy sensitivity related gene ATM and ATR gene mutation and its application, the kit is composed of the reagent for detecting the expression amount of related gene.By using miRNA sequencing, binary classification Logistic regression and ROC curve, the gene related to ATM, ATR mutation is analyzed, and score Score is constructed, which can be used for the prediction of ATM and ATR gene mutation in laryngeal squamous cell carcinoma.The present application predicts specific gene mutation related to laryngeal squamous cell carcinoma radiotherapy sensitivity by using the expression amount of miRNA composition, and it is found that the detection method has the advantages of simple operation, high accuracy and good specificity, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics technology, and more specifically, to a miRNA kit for detecting ATM / ATR gene mutations in laryngeal squamous cell carcinoma and its application. Background Technology

[0002] Laryngeal cancer is a common tumor of the head and neck, and laryngeal squamous cell carcinoma (LSCC) is its most common pathological type. The onset age of laryngeal cancer is concentrated between 50 and 70 years old. Etiological studies have confirmed a clear correlation between smoking and the occurrence of laryngeal cancer; therefore, laryngeal cancer is more common in men, with a male-to-female ratio of 4:1. The laryngeal structure is mostly composed of pseudostratified ciliated columnar epithelium, therefore 90% of laryngeal cancer pathological types are squamous cell carcinoma. Radiotherapy is one of the main treatment methods for laryngeal squamous cell carcinoma, and it has a very good therapeutic effect on most patients. However, in some patients with laryngeal cancer, the effect of radiotherapy is poor. If the radiosensitivity of the patient's tumor can be determined as early as possible, it is possible to accurately determine whether the patient is suitable for radiotherapy, thereby guiding clinical practice, greatly improving patient prognosis, and reducing unnecessary expenses and waste of medical resources.

[0003] ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3 related) occupy a crucial position at the top of DNA damage repair, which is a key factor influencing the killing effect of radiation on tumor cells. ATM and ATR possess phosphorylated kinase activity; upon radiation damage, they sense DNA strand breaks, leading to increased expression and rapid activation. They act on corresponding sites of downstream target proteins, resulting in cell cycle arrest and DNA repair. When ATM or ATR is mutated, cells cannot repair radiation-damaged DNA in a timely manner, thus exhibiting higher radiosensitivity. Detecting ATM and ATR-related mutations is of great significance in determining a patient's suitability for radiotherapy.

[0004] Our previous invention patent CN113755596A, published on December 7, 2021, disclosed a kit for detecting mutations in the ATM and ATR genes, which are related to radiosensitivity in laryngeal squamous cell carcinoma, and its application. The kit consists of reagents that detect the expression levels of VSTM2L, TSPAN8, RDH10, PRODH, PITX2, ODC1, MASP1, KLHDC8A, GPRC5D, CTAG2, CCL26, and ABCA13. This invention predicts specific gene mutations related to radiosensitivity in laryngeal squamous cell carcinoma by utilizing the expression levels of these gene combinations. Verification has shown that this detection method is simple to operate, highly accurate, and has good specificity, demonstrating promising application prospects. Currently, the detection of ATM and ATR gene mutations in laryngeal squamous cell carcinoma is not a routine clinical procedure, and individual testing is expensive. Therefore, developing new detection kits has greater research value.

[0005] Currently, there are no reports on the miRNA kit for detecting ATM / ATR gene mutations in laryngeal squamous cell carcinoma, as described in this invention, or its application. Summary of the Invention

[0006] The first objective of this invention is to address the gaps in the prior art by providing a method for detecting ATM / ATR gene mutations in laryngeal squamous cell carcinoma and its application.

[0007] In a first aspect, the present invention provides the application of a reagent for detecting miRNA expression levels in the preparation of a kit for detecting ATM or ATR gene mutations in laryngeal squamous cell carcinoma. The detection reagent is composed of reagents for detecting the expression levels of the following miRNAs: miR-26a-5p, miR-100-5p, miR-210-3p, miR-200b-3p, miR-149-5p, miR-584-5p, miR-20a-3p, miR-24-2-5p, let-7g-3p, and miR-744-3p.

[0008] As a preferred example, the reagent for measuring miRNA expression is a key component of the kit used to detect mutations in the radiosensitivity-related genes ATM and ATR in laryngeal squamous cell carcinoma.

[0009] More preferably, the kit also includes an instruction manual containing the following formula: Score = 10.423*miR-26a-5p - 4.073*miR-100-5p + 1.975*miR-210-3p + 3.517*miR-200b-3p + 3.61*miR-149-5p - 1.858*miR-584-5p - 2.056*miR-20a-3p - 4.72*miR-24-2-5p - 4.5*let-7g-3p - 2.26*miR-744-3p – 88.172, where the miRNA expression level is expressed in log2(TPM+1).

[0010] More preferably, the above-mentioned test sample is a fresh laryngeal squamous cell carcinoma tumor tissue sample.

[0011] More preferably, when the test sample score is ≤0, the sample does not have an ATM or ATR gene mutation; when the test sample score is >0, the sample has an ATM or ATR gene mutation.

[0012] Secondly, the present invention provides a kit for detecting the presence of ATM or ATR gene mutations in laryngeal squamous cell carcinoma, the kit containing reagents for detecting the expression levels of miR-26a-5p, miR-100-5p, miR-210-3p, miR-200b-3p, miR-149-5p, miR-584-5p, miR-20a-3p, miR-24-2-5p, let-7g-3p, and miR-744-3p.

[0013] Thirdly, the present invention provides a kit for predicting the radiosensitivity of laryngeal squamous cell carcinoma, the kit containing reagents for detecting the expression levels of miR-26a-5p, miR-100-5p, miR-210-3p, miR-200b-3p, miR-149-5p, miR-584-5p, miR-20a-3p, miR-24-2-5p, let-7g-3p, and miR-744-3p.

[0014] The advantages of this invention are:

[0015] 1. This invention is the first to propose that this miRNA composition can be used to detect mutations in the radiosensitivity-related genes ATM / ATR in the tumor tissue of patients with laryngeal squamous cell carcinoma. This can effectively guide individualized treatment for patients with laryngeal squamous cell carcinoma, improve clinical benefits, and avoid unnecessary waste of medical resources.

[0016] 2. ATM / ATR gene mutations are currently not routinely tested in clinical practice, and testing for these gene mutations alone is expensive. This invention can determine whether patients with laryngeal squamous cell carcinoma have the above-mentioned gene mutations using miRNA sequencing technology, which is widely used in clinical practice. It has the advantages of being economical, accurate, sensitive, and specific. Attached Figure Description

[0017] Appendix Figure 1 This is the receiver operating characteristic (ROC) curve of the prediction model of this invention.

[0018] Appendix Figure 2 These are 14 differentially expressed miRNAs obtained from the TCGA database analysis of this invention. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0020] Example 1: Construction of a scoring model for ATM / ATR mutations in laryngeal squamous cell carcinoma

[0021] 1. Method

[0022] We first obtained data from 98 laryngeal squamous cell carcinoma samples with both mutation and miRNA expression data from the TCGA database. Based on the presence or absence of ATM or ATR gene mutations, the samples were divided into a mutant group (n=10) and a wild-type group (n=88). Then, using the limma package in R, we calculated and screened 14 differentially expressed miRNAs (see [link to R documentation]). Figure 2 By performing logistic regression on the expression levels of the above 14 differentially expressed miRNAs [in log2(TPM+1) form] from 98 samples, we obtained miRNAs significantly associated with ATM / ATR gene mutations. Finally, we selected 10 miRNAs as follows: miR-26a-5p, miR-100-5p, miR-210-3p, miR-200b-3p, miR-149-5p, miR-584-5p, miR-20a-3p, miR-24-2-5p, let-7g-3p, and miR-744-3p (Table 1).

[0023] Table 1. Logistic Regression Results

[0024]

[0025] A scoring model was constructed using binary logistic regression: 10.423*miR-26a-5p-4.073*miR-100-5p+1.975*miR-210-3p+3.517*miR-200b-3p+3.61*miR-149-5p-

[0026] 1.858*miR-584-5p-2.056*miR-20a-3p-4.72*miR-24-2-5p-4.5*let-7g-3p-2.26*miR-744-3p–91.384. The miRNA expression level in the above formula is in log2(TPM+1).

[0027] We calculated the score for each sample based on this scoring model and obtained the optimal threshold point, which was -2.672, using the ROC curve. Integrating this value into the scoring model, we obtained the final scoring model: Score = 10.423*miR-26a-5p-4.073*miR-100-5p+1.975*miR-210-3p+3.517*miR-200b-3p+3.61*miR-149-5p-1.858*miR-584-5p-2.056*miR-20a-3p-4.72*miR-24-2-5p-4.5*let-7g-3p-2.26*miR-744-3p-88.172.

[0028] Based on the scoring model, TCGA samples were divided into two groups: those with a score less than or equal to 0 were classified as wild-type samples, and those with a score greater than 0 were classified as mutant samples.

[0029] 2. Results

[0030] The sensitivity and specificity results of the prediction model are shown in Table 2. The sensitivity is 10 / 10 = 100%, and the specificity is 76 / 88 = 86.3%.

[0031] Table 2. Sensitivity and specificity of this method in predicting laryngeal squamous cell carcinoma in the TCGA database.

[0032]

[0033] Example 2 Effect Verification

[0034] 1. Method

[0035] We performed miRNA sequencing on 50 samples of laryngeal squamous cell carcinoma collected from the Eye, Ear, Nose and Throat Hospital affiliated with Fudan University and detected ATM / ATR gene mutations.

[0036] ① Use the miRcute miRNA Isolation Kit to extract miRNA from tumor tissue samples;

[0037] ② miRNA library construction: Completed using the TruSeq miRNA Sample Prep Kitv2;

[0038] ③ Cluster generation: Completed using the TruSeq SR Cluster Kitv3-cBot–HS kit;

[0039] ④ Illumina Hiseq2000 sequencing: The Hiseq2000 was used to convert the raw sequencing data into Fastq format;

[0040] ⑤ Data Analysis: After removing adapter sequences from the original Fastq file data, the quality and length of the sequencing fragments were examined, and reliable sequencing fragments were screened. The sequencing results were compared and filtered with the miRbase database to identify known human miRNA data. The expression levels of the identified miRNAs were statistically analyzed. The miRNA expression level was calculated using TPM (transcripts per million), and the TPM formula was = (number of reads aligned to each miRNA) / (total number of aligned reads in the sample) × 106, which was then converted to Log2(TPM+1).

[0041] ⑥ Substitute the miRNA expression data into the prediction model to obtain the Score for each sample. When the Score of the detected sample is ≤0, it is considered that the sample does not have ATM or ATR gene mutations; when the Score of the detected sample is >0, it means that the sample has ATM or ATR gene mutations.

[0042] 2 Results

[0043] The experimental results are shown in Table 2. The sensitivity of the prediction model is 8 / 9 = 88.9%, and the specificity is 37 / 41 = 90.2%.

[0044] Table 2. Sensitivity and specificity of this method in predicting laryngeal squamous cell carcinoma in this department.

[0045]

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting miRNA expression levels in the preparation of a kit for detecting ATM or ATR gene mutations in laryngeal squamous cell carcinoma, characterized in that, The miRNAs include: miR-26a-5p, miR-100-5p, miR-210-3p, miR-200b-3p, miR-149-5p, miR-584-5p, miR-20a-3p, miR-24-2-5p, let-7g-3p, and miR-744-3p.

2. The application according to claim 1, characterized in that, The reagent used to detect miRNA expression levels is a key component of the kit for detecting mutations in ATM and ATR genes, which are related to radiosensitivity in laryngeal squamous cell carcinoma.

3. The application according to claim 1, characterized in that, The kit also includes an instruction manual, which states the following formula: Score = 10.423 * miR-26a-5p - 4.073 * miR-100-5p + 1.975 * miR-210-3p + 3.517 * miR-200b-3p + 3.61 * miR-149-5p - 1.858 * miR-584-5p - 2.056 * miR-20a-3p - 4.72 * miR-24-2-5p - 4.5 * let-7g-3p - 2.26 * miR-744-3p – 88.172, where the miRNA expression level is in log2(TPM+1).

4. The application according to claim 3, characterized in that, The kit is used to test samples, and the test samples are fresh laryngeal squamous cell carcinoma tumor tissue samples.

5. The application according to claim 4, characterized in that, When the score is ≤ 0, the sample does not have an ATM or ATR gene mutation; when the score is > 0, the sample has an ATM or ATR gene mutation.

6. A kit for detecting the presence of ATM or ATR gene mutations in laryngeal squamous cell carcinoma, characterized in that, The kit contains reagents for detecting the expression levels of miR-26a-5p, miR-100-5p, miR-210-3p, miR-200b-3p, miR-149-5p, miR-584-5p, miR-20a-3p, miR-24-2-5p, let-7g-3p, and miR-744-3p.

7. A kit for predicting radiosensitivity of laryngeal squamous cell carcinoma, characterized in that, The kit contains reagents for detecting the expression levels of miR-26a-5p, miR-100-5p, miR-210-3p, miR-200b-3p, miR-149-5p, miR-584-5p, miR-20a-3p, miR-24-2-5p, let-7g-3p, and miR-744-3p.

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