CN115896291A - Marker for diagnosing lung adenocarcinoma and application thereof - Google Patents

Abstract

The invention discloses a marker for diagnosing lung adenocarcinoma and application thereof, and relates to the technical field of biomedicine, wherein experimental analysis shows that platelet has-mi R-199b-3p has higher sensitivity, specificity and accuracy for diagnosing benign and malignant lung nodules.

Description

Marker for diagnosing lung adenocarcinoma and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a marker for diagnosing lung adenocarcinoma and application thereof.

Background

Lung adenocarcinoma is now the most common subset of lung cancers worldwide, accounting for approximately 40% of all lung cancer cases. With advances in diagnosis, surgery, radiation therapy, immunotherapy, and molecular therapy, the clinical outcome of patients with lung adenocarcinoma has improved dramatically. However, the 5-year survival rate of lung adenocarcinoma patients is still at a low level. It has been demonstrated that aggressive surgery can provide a good prognosis for patients with early stage lung adenocarcinoma. However, diagnosing lung adenocarcinoma at an early stage is a clinical challenge, since there are no specific clinical symptoms, whereas CT behaves similar to benign nodules. The M i RNA is a small non-coding RNA containing about 19-25 nucleotides, which has been recently shown to be useful as a disease biomarker. At present, platelet mi RNA has been demonstrated as a liquid biopsy and is a growing research hotspot due to its potential diagnostic value for early detection of tumors. Thus, the present study performed mi RNA-seq assays on isolated peripheral platelets and investigated the differences in platelet mi RNA expression and the relationship of platelet mi RNA expression to lung adenocarcinoma.

In summary, there is a need for a diagnostic marker for lung adenocarcinoma and applications thereof, which solve the problem that lung adenocarcinoma is difficult to diagnose by CT at present.

Disclosure of Invention

The invention aims to: provides a marker for diagnosing lung adenocarcinoma and application thereof, and solves the problem that the lung adenocarcinoma is difficult to diagnose through CT at present.

In order to achieve the above object, the present invention provides the following technical solutions:

a diagnostic marker for lung adenocarcinoma, which is has-mi R-199b-3p.

Application of a diagnostic marker for lung adenocarcinoma in preparation of a reagent or a kit for lung adenocarcinoma diagnosis.

Application of a diagnostic marker for lung adenocarcinoma in preparing a reagent or a kit for judging and prognosing lung adenocarcinoma.

Application of a diagnostic marker for lung adenocarcinoma in preparation of a medicament for treating lung adenocarcinoma is provided.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the platelet has-mi R-199b-3p has higher sensitivity, specificity and accuracy to diagnosis of benign and malignant lung nodules, and in the actual diagnosis process, the content of the has-mi R-199b-3p in the platelet is analyzed through corresponding detection equipment by collecting blood of a patient, so that whether the patient has lung adenocarcinoma is judged. Compared with the existing diagnosis by methods such as CT, the diagnosis result is more accurate by judging by detecting the content of the platelet has-mi R-199b-3p in the blood.

Description of the drawings:

FIG. 1 is a flowchart of an assay demonstrating that has-mi R-199b-3p can be used as a marker for lung adenocarcinoma in accordance with the present invention;

FIG. 2 is a graph of platelet-differential miRNAs generated by sequencing results and screening conditions in an assay of the present invention;

FIG. 3 is a graph showing the results of the expression level of has-mi R-199b-3p in the test of the present invention;

FIG. 4 is a graph showing the evaluation analysis of the diagnostic ability of has-mi R-199b-3p in the test of the present invention.

Detailed Description

The present invention will be described in further detail with reference to test examples and specific embodiments. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.

As shown in FIG. 1, the test validation analysis is performed on the marker that has-mi R-199b-3p can be used for detecting the lung adenocarcinoma in the embodiment, and the specific test validation process is as follows:

1. selection of participants in the test

All participants were from tumor hospitals in Sichuan province. Wherein the subject comprises three groups: healthy Donors (HD), benign lung nodules (BPN) and Lung Adenocarcinoma (LAC); specifically, subjects who included BNP and LAC were as follows: 1) All patients who found lung nodules in LDCT for the first time and received surgical treatment or histological examination in tumor hospital of Sichuan province were grouped; 2) LAC group: LAC patients confirmed histologic type and staging using the international cancer control consortium (ui CC) 8 th TNM lung cancer classification; BNP group: post-operative pathology was determined for non-lung cancer subjects; 3) Participants in the LAC and BPN groups who did not receive any treatment (including chemotherapy or radiotherapy). 4) The subjects had basic clinical characteristics.

Participants excluded 1) participants who received treatments affecting platelets (such as blood transfusions and aspirin); 2) A pregnant woman; 3) Those who are infected; follow-up by low-dose helical CT screening may exclude healthy subjects with LAC, without other critical illness. The study was approved by the Hospital medical ethics Committee for tumors in Sichuan province (SCCHEC-02-2020-043).

2. Platelet extraction and purity assessment

1.5 ml of EDTA anticoagulant was added to a 2ml EP tube. Platelet rich plasma was separated from nucleated blood cells by centrifugation at 120g for 20 minutes. To avoid contamination with white and red blood cells, platelets were further collected using only the upper 4/5 of the PRP. Platelets were separated from PRP by centrifugation at 360g for 20 minutes. Platelets are located at the bottom of the EP tube. Platelet purity by morphometric experiments and platelet count standards is less than 5 white blood cells per million platelets. In addition, upper-layer platelet poor plasma was transferred to a new EP tube for exosome isolation. Wherein, in order to minimize the degradation of platelet RNA due to time effects, the isolation should be completed within 2 hours after blood collection.

3. Platelet miRNA sequencing

Platelet total RNA (lung adenocarcinoma 3, healthy donor 3) was isolated using a total RNA rapid extraction kit for blood samples (spin column type) (biotake, china) according to the manufacturer's instructions. Concentration and quality assessment of total RNA was performed by the seemer hewler scientific NanoDrop 2000 spectrophotometer (american seemer hewler science). Sequencing libraries were generated from the QIAseq miRNA library kit (Qi gen, freder i ck, MD) according to the manufacturer's recommendations. Library quality was assessed by agilent bioanalyzer 2100 and qPCR. Clustering of index-coded samples was performed on an acBot cluster generation system using TruSeq PE C l super kit v3-cBot-HS (il l umina, san diego, ca, usa) according to the manufacturer's instructions. After cluster generation, library preparations were sequenced on the I l umi na Hi seq platform and paired-end reads generated.

4. Quantitative analysis of miRNA and differential miRNA screening

Clean data is obtained by removing low quality reads and reads containing adapters or p l oy-N from the raw data, and then sequence aligned with the si l va database, gtRNAdb database, rfam database and the Repbase database, respectively, using a soft bow tie tool. The ncRNA such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snorRNA) and the like, and the repetitive sequence were filtered. Finally, the remaining reads were used to detect known and new mirnas, compared to known mirnas of miRbase and human genome (GRCh 38), respectively. The per million Transcripts (TPM) were calculated by comparing the number of reads per miRNA. Platelet differential mi RNA analysis was performed using the DESeq 2R package. The screening standard is that the value of | og2FOLD | is more than or equal to 0.5, and the value of P is less than or equal to 0.05.

5. Platelet miRNA extraction, cDNA synthesis and qRT-PCR analysis

Blood samples (centrifugal column type) mi RNA rapid extraction kit is adopted to extract platelet mi RNA of 71 lung adenocarcinoma patients, 32 lung benign nodule patients and 31 healthy people. And reverse transcription was performed using the M ir-XTM mi RNA first strand synthesis kit (TaKaRa Bi o, dalian China). Platelet differential mi RNA expression levels were then determined by RT-qPCR.

6. Data analysis

Comparison of the differential expression levels of mi RNA in lung adenocarcinoma, benign tubercular lung patients, healthy human platelets using the t-test or non-parametric test, P values <0.05 were considered statistically significant. Diagnostic values were assessed by Receiver Operating Characteristics (ROC) analysis. And the diagnostic performance of platelet-differential mi RNA was assessed by ROC analysis compared to other biomarkers and clinical models.

7. Data analysis results

TABLE 1 specific clinical characteristics of the enrolled participants

1. Sequencing results showed that has-mi R-199b-3p and has-mi-142-5 p were significantly differentially expressed in lung adenocarcinoma.

According to the screening standard, the sequencing result finally screens out the first ten up-regulated miRNAs and the first 10 down-regulated miRNAs with expression quantity in the lung adenocarcinoma blood platelets, and total 20 differential miRNAs, (has-mi R-223-3p, has-mi R-142-5p, has-mi R-1-3p, has-mi R-101-3p, has-mi R-223-5p, has-mi R-335-5p, has-mi R-19a-3p, has-mi R-19b-3p, has-mi R-301a-3p, has-mi R-4433b-3p, has-mi R-199b-3p, has-mi R-148a-3p, has-mi R-125a-5p, has-mi R-143-3p, has-mi R-99b-5p, has-mi R-379-mi R-125a-5p, has-mi R-127 a-mi R-143-3p, has-mi R-99b-5p, has-mi R-379-mi-134 a-mi-3p, has-2-map, the 20 platelet-differential mi RNAs clearly distinguished lung adenocarcinoma patients from healthy individuals (fig. 2A) the candidate differential mi RNAs we selected were has-mi R-199b-3p and has-mi R-142-5p. A total of 30 subjects, including 15 lung adenocarcinomas and 15 healthy donors (table 1), were first validated for inclusion according to exclusion and inclusion criteria preliminary RT-qPCR results show, the relative expression of has-mi R-199b-3p was statistically different in healthy donors and lung adenocarcinoma platelets (p = 0.0034), see fig. 3A, while the relative expression of has-mi R-142-5p was not statistically different in healthy donors and lung adenocarcinoma platelets (p = 0.23). Therefore, we finally selected has-mi R-199b-3p for further large-scale validation.

Has-mi R-199b-3p can be used as a biomarker for diagnosing lung cancer and benign and malignant pulmonary nodules.

Further validation of platelet has-mi R-199b-3p on a large scale by exclusion and inclusion criteria 70 lung adenocarcinoma patients and 31 healthy subjects and 32 BPN patients were included (Table 1). The results showed that the expression level of platelet has-mi R-199B-3p in the healthy donor group was significantly higher than that in the lung adenocarcinoma group (p = 0.0004) (fig. 3B), consistent with the sequencing results. Surprisingly, the results showed that expression of has-miR-199b-3p was significantly higher in the lung benign nodule patient group than in the lung adenocarcinoma group (p = 0.0016) (fig. 3C). In the present clinic, it is difficult to distinguish stage I lung adenocarcinoma with only a single lesion of less than 3cm from benign lung nodules. We further evaluated the expression level of platelet has-miR-199b-3p between LAC stage I patients with only a single lesion less than 3cm and BPN patients. To our surprise, the results showed that the expression of has-mi R-199b-3p was significantly higher in the lung benign nodule patient group than in stage I (p = 0.0098), see fig. 3D.

3. The diagnostic value of the platelet has-miR-199b-3p.

We used the ROC curve to analyze the ability of platelets has-miR-199b-3p to distinguish between benign and malignant lung nodules. The results of the analysis showed that platelet has-miR-199b-3p has good diagnostic accuracy in distinguishing the lung adenocarcinoma group from the benign nodule group of lung group, and the area under the curve (AUC) of ROC was 0.73 (95% CI,0.5574 to 0.7850), as shown in FIG. 4A. The results showed that the AUC of ROC was 0.72 (95% CI, 0.6285-0.8255) in the diagnosis of the benign tubercle group and the stage I lung adenocarcinoma group (FIG. 4B). Based on the complete clinical and laboratory information of the participants. The performance of has-miR-199b-3p in diagnosing benign and malignant lung nodules was compared to other clinical models and CEA. The results showed that the AUC of platelet has-miR-199b-3p in LAC was 0.67 (95% CI,0.4889 to 0.8576), while the AUC of CEA in LAC was 0.55 (95% CI,0.3722 to 0.7352). The diagnostic performance of benign and malignant lung nodules of other clinical models (including VA model (AUC =0.58, 95% ci,0.4034 to 0.7523), BU model (AUC =0.55, 95% ci,0.3735 to 0.7361) and MC model (AUC =0.53, 95% ci,0.3499 to 0.7028) were also limited (fig. 4℃) our results indicate that platelet has-miR-199b-3p has higher sensitivity, specificity and accuracy for LAC diagnosis and benign and malignant lung nodule diagnosis, showing significant clinical value.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (4)

1. A diagnostic marker for lung adenocarcinoma, which is has-miR-199b-3p.

2. Use of a diagnostic marker for lung adenocarcinoma according to claim 1 for the preparation of a reagent or a kit for the diagnosis of lung adenocarcinoma.

3. Use of the diagnostic marker for lung adenocarcinoma according to claim 1 in the preparation of a reagent or a kit for prognosis of lung adenocarcinoma.

4. Use of a diagnostic marker for lung adenocarcinoma according to claim 1 for the preparation of a medicament for the treatment of lung adenocarcinoma.

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