CN114657250A

CN114657250A - Application of miRNA in preparation of tool for diagnosing non-small cell lung cancer

Info

Publication number: CN114657250A
Application number: CN202210343420.4A
Authority: CN
Inventors: 张强; 王新立
Original assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Current assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-06-24

Abstract

The invention discloses an application of miRNA in preparing a tool for diagnosing non-small cell lung cancer, wherein the miRNA comprises hsa-miR-26b and/or hsa-miR-378. When two miRNAs of hsa-miR-26b and hsa-miR-378 are jointly applied, the AUC is closer to 1 than that of a single index. By detecting the miRNA, the non-small cell lung cancer can be diagnosed accurately, with high specificity and high sensitivity.

Description

Application of miRNA in preparation of tool for diagnosing non-small cell lung cancer

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of miRNA in diagnosing non-small cell lung cancer.

Background

The Lung Cancer (Lung Cancer) is the most common respiratory malignant tumor and the most common primary malignant tumor of the Lung all over the world, has the first morbidity and mortality all over the world, is the 'gold medal killer' of malignant tumor patients, and seriously threatens the life health of human beings. It is reported that the death of lung cancer accounts for 29% of the death of global malignant tumors, and the death of lung cancer is on a gradual rise, and new cases of lung cancer account for 12.4% of global malignant tumors every year. Currently, lung cancer is mainly classified into Non-Small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC), wherein about 85% of lung cancer is NSCLC.

Compared with small cell carcinoma, NSCLC has slower growth and division of cancer cells, and thus has relatively later time for diffusion and metastasis. At present, early clinical symptoms of the non-small cell lung cancer mainly comprise fever, chest pain, short breath, cough, blood sputum and the like, but the symptoms are difficult to be understood and regarded by people, and the disease condition of at least a plurality of people is not accurately diagnosed. In clinical diagnosis, the main effective basis is the comprehensive analysis of various imaging results, but the final diagnosis of the patients needs to rely on pathological histology or cytology diagnosis to confirm that the patients are NSCLC. In recent years, researches show that the prognosis of the middle and late non-small cell lung cancer is poor, and the non-small cell lung cancer has the characteristics of easy relapse and micrometastasis, so that the death rate is high. Therefore, in order to prolong the life cycle of a lung cancer patient and improve the life quality of the patient, the medical community needs to continuously explore the relevant mechanisms of lung cancer and search for markers for early diagnosis of lung cancer.

Disclosure of Invention

The first purpose of the invention is to provide a marker for the early diagnosis of non-small cell lung cancer;

it is a second object of the present invention to provide a system for diagnosing non-small cell lung cancer.

In order to realize the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a marker for diagnosing non-small cell lung cancer, and the marker comprises hsa-miR-26b and/or hsa-miR-378.

The term "and/or" as used herein in phrases such as "a and/or B" is intended to include both a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

In a preferred embodiment, the markers include hsa-miR-26b and hsa-miR-378.

In another aspect, the present invention provides the use of an agent for detecting the expression level of the aforementioned marker in a sample for the preparation of a tool for diagnosing non-small cell lung cancer.

The term "level of expression" or "expression level" generally refers to the amount of a marker in a biological sample. "expression" generally refers to the process by which information (e.g., gene coding and/or epigenetic information) is converted into structures present and operating in a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Transcribed polynucleotides, translated polypeptides, or fragments of a polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered expressed, whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of a polypeptide (e.g., by proteolysis). "expressed genes" include genes that are transcribed into a polynucleotide (e.g., mRNA) and then translated into a polypeptide, as well as genes that are transcribed into RNA but not translated into a polypeptide (e.g., transport and ribosomal RNA, miRNA, IncRNA). As a preferred embodiment, "expressed gene" refers to a gene that is transcribed into RNA but not translated into a polypeptide.

"expression up-regulated" refers to increased expression or increased level of a marker in an individual relative to a control, such as an individual who does not have a disease or disorder (e.g., cancer), an internal control (e.g., a housekeeping marker), or the median expression level of the marker in a sample from one patient group/population.

"downregulating expression" refers to reduced expression or reduced level of a marker in an individual relative to a control, such as an individual without a disease or disorder (e.g., cancer) or an internal control (e.g., a housekeeping marker), or the median expression level of the marker in a sample from one patient group/population. In some embodiments, the reduced expression is little or no expression.

As a preferred embodiment, the reagent comprises:

a probe that specifically recognizes the marker; or

Primers for specific amplification of the marker.

In the present invention, the term "probe" refers to a nucleic acid fragment corresponding to several bases to several hundreds of bases capable of specifically binding to mRNA, for example, RNA or DNA, etc. Because of the labeling, the presence or absence of a specific mRNA can be confirmed. The probe can be produced in the form of an oligonucleotide (oligonucleotide) probe, a single-stranded dna (single stranded dna) probe, a double-stranded dna (double stranded dna) probe, an RNA probe, or the like. In the present invention, hybridization is performed using a probe complementary to the marker gene, and the expression level of the gene can be diagnosed by whether hybridization is performed or not. The selection of an appropriate probe and hybridization conditions may be changed based on techniques known in the art, and there is no particular limitation in the present invention.

The term "primer" refers to a synthetic oligonucleotide that, upon forming a duplex with a polynucleotide template, is capable of acting as a point of initiation of nucleic acid synthesis and extends from its 3' end along the template to form an extended duplex. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Typically, the primer is extended by a DNA polymerase. The length of the primer is typically compatible with its use in the synthesis of primer extension products, and is typically between 8 and 100 nucleotides in length, e.g., 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, etc. Typical primers may be between 10-50 nucleotides in length, e.g., 15-45, 18-40, 20-30, 21-25, etc., and any length between the stated ranges. In some embodiments, the primer is generally no more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.

The primer or probe of the present invention can be chemically synthesized by using a phosphoramidite solid phase support method or other known methods. Such nucleic acid sequences may be modified by a variety of means well known in the art. Non-limiting examples of such variations include methylation, encapsulation, substitution of more than one homolog of the natural nucleotide, and variations between nucleotides, for example, variations to uncharged linkers (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkers (e.g., phosphorothioates, phosphorodithioates, etc.).

As a preferred embodiment, the sample comprises blood, plasma, serum, saliva, urine, tears, cerebrospinal fluid or organ tissue.

In a more preferred embodiment, the sample is blood.

In another aspect, the present invention provides a system for diagnosing non-small cell lung cancer, including the following units:

1) a detection unit: comprises a marker detection module;

2) an analysis unit: the expression level of the marker detected by the detection unit is used as an input variable and is input into a diagnosis model of the non-small cell lung cancer for analysis;

3) an evaluation unit: outputting a risk value of the individual corresponding to the sample for the non-small cell lung cancer;

the marker comprises hsa-miR-26b and/or hsa-miR-378.

A "model" is any mathematical equation, algorithm, analytical or programmed process or statistical technique that takes one or more continuous or categorical inputs and calculates an output value, sometimes referred to as an "index," index value, "" predictor, "" predicted value, "" probability, "or" probability score. Non-limiting examples of "formulas" include sums, ratios, and regression operators, such as coefficients or indices, marker value conversions and normalizations, rules and guidelines, statistical classification models, and neural networks trained on historical populations. Of particular interest in group (panel) and combinatorial constructs are structural and syntactic statistical classification algorithms, as well as risk index construction methods that utilize pattern recognition features, including established techniques such as cross-correlation, Principal Component Analysis (PCA), factor rotation, log regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), Support Vector Machines (Support Vector Machines, SVMs), Random Forest (Random Forest, RF), recursive partition Trees (RPART), xgboost (xgb), and other related Decision tree classification techniques, shrunken centroids (sc), stepic, Nearest-Neighbor, Boosting, Decision Trees (Decision Trees), neural networks, bayesian networks, Support Vector Machines, and Hidden markov models (Hidden markov, etc. Many such algorithmic techniques are further implemented to perform feature (locus) selection and regularization (regularization) regularization, for example in ridge regression, lasso and elastic net, among others. Other techniques may be used in time to event hazard analysis (time to event hazard analysis), including Cox, Weibull, Kaplan-Meier, and Greenwood models, which are well known to those skilled in the art. Many of these techniques can be used in conjunction with marker selection techniques, such as forward selection, backward selection or step-wise selection, complete enumeration of all potential marker sets or groups of a given size, genetic algorithms or themselves can include marker selection methods. These can be used in conjunction with Information criteria, such as Akaike's Information Criterion (AIC) or Bayesian Information Criterion (BIC), to quantify tradeoffs between other markers and model improvements and to help minimize overfitting. The generated diagnostic models can be validated in other studies, or cross-validated in studies in which they were originally trained, using techniques such as Bootstrap, Leave-One-out (LOO) and 10-Fold cross-validation (10-Fold cross-validation) (10-Fold CV). At various steps, the false discovery rate may be estimated by value permutation according to techniques known in the art.

In another aspect, the invention provides the use of a marker comprising hsa-miR-26b and/or hsa-miR-378 in the construction of a system as described above.

In another aspect, the present invention provides a kit for diagnosing non-small cell lung cancer, the kit comprising a reagent for detecting the expression level of the aforementioned marker in a sample.

As a preferred embodiment, the means comprises a chip or kit.

In the present invention, "chip", also referred to as "array", refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.

A "microarray" is an ordered array of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The matrix may be a solid matrix, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid matrix, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any permutation thereof.

The present invention provides a kit for diagnosing non-small cell lung cancer in a subject, the kit being for determining the expression level of the aforementioned marker. The kit may comprise materials and reagents suitable for selectively detecting the presence of a marker or a set of markers for diagnosing non-small cell lung cancer in a sample derived from a subject. For example, in one embodiment, the kit can include reagents that specifically hybridize to the marker. Such reagents may be suitable for measuring the mRNA expression level of the marker gene at the mRNA level, for example, probes or primers. The kit may include reagents for performing an assay to detect one or more markers.

In further embodiments, the kit may contain instructions for appropriate operating parameters in the form of labels or product inserts. For example, the instructions may include information or guidance on how to collect the sample, how to determine the level of one or more markers in the sample, or how to correlate the level of one or more markers in the sample with the subject's development of non-small cell lung cancer.

In another embodiment, the kit may contain one or more containers with a sample of the marker to be used as a reference standard, a suitable control, or for calibration of the assay to detect the marker in the test sample.

The invention has the advantages and beneficial effects that:

the invention discloses that the expression levels of hsa-miR-26b and hsa-miR-378 are related to non-small cell lung cancer for the first time, whether a subject suffers from non-small cell lung cancer and risks of suffering from the non-small cell lung cancer can be judged by detecting the expression levels of hsa-miR-26b and hsa-miR-378 in a sample of the subject, and the application prospect is good.

Drawings

FIG. 1 is a boxplot of differential expression of hsa-miR-26b in non-small cell lung cancer;

FIG. 2 is a boxplot of differential expression of hsa-miR-378 in non-small cell lung cancer;

FIG. 3 is a boxplot of differential expression of hsa-let-7b in non-small cell lung cancer;

FIG. 4 is a ROC plot of hsa-miR-26b for diagnosing non-small cell lung cancer;

FIG. 5 is a ROC plot of hsa-miR-378 for diagnosing non-small cell lung cancer;

FIG. 6 is a ROC plot of hsa-let-7b for diagnosing non-small cell lung cancer;

FIG. 7 is a ROC plot of hsa-miR-26b and hsa-miR-378 in combination for diagnosing non-small cell lung cancer;

FIG. 8 is a ROC plot of hsa-let-7b and hsa-miR-26b in combination for diagnosing non-small cell lung cancer;

FIG. 9 is a graph of the results of qPCR validation of differential expression of hsa-miR-26 b;

FIG. 10 is a graph of the results of qPCR validation of differential expression of hsa-miR-378;

FIG. 11 is a ROC plot of the diagnostic performance of qPCR validation of hsa-miR-26 b;

FIG. 12 is a ROC plot of the diagnostic efficacy of qpCR validation of hsa-miR-378.

Detailed Description

The following detailed description of embodiments of the present application will be made with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present application, are given by way of illustration and explanation only, and are not intended to limit the present application.

Example 1 non-Small cell Lung cancer marker screening

The GSE17681 data was downloaded from the GEO database, the data set comprising blood samples of 17 non-small cell lung cancer (NSCLC) patients and 19 non-cancer controls, and after log-taking processing the data set was differentially analyzed using the R language limma package to obtain 260 differentially expressed genes, with the screening criteria: value < 0.05. The expression of hsa-miR-26b, hsa-miR-378 and hsa-let-7b is shown in table 1 and figures 1-3.

TABLE 1 data on differential expression of genes to which the invention relates

Gene	AveExpr	t	P.Value
				hsa-miR-26b	6.937	-2.632	0.012
hsa-miR-378	3.933	3.547	0.001
				hsa-let-7b	8.958	-2.306	0.027

Example 2 diagnostic Performance analysis

Receiver Operating Curves (ROCs) were plotted using the R package "pROC" (version 1.15.0), AUC values, sensitivity and specificity were analyzed, and the diagnostic efficacy of the markers alone or in combination was judged. When the diagnosis efficiency of the index combination is judged, logistic regression is carried out on the expression level of each gene, the probability of whether each individual suffers from cancer is calculated through a fitted regression curve, different probability division threshold values are determined, and the sensitivity, specificity, accuracy and the like of the combined detection scheme are calculated according to the determined probability division threshold values.

As a result:

the diagnosis efficiencies of hsa-miR-26b, hsa-miR-378 and hsa-let-7b are shown in the table 2 and the figures 4-6, and the results show that hsa-miR-26b, hsa-miR-378 and hsa-let-7b have good diagnosis effects on non-small cell lung cancer.

TABLE 2 diagnostic potency of individual genes

Gene	AUC	Sensitivity of the composition	Specificity of
				hsa-miR-26b	0.817	1.000	0.632
hsa-miR-378	0.741	1.000	0.526
				hsa-let-7b	0.721	0.412	0.947

The data of the gene combined diagnosis are shown in a table 3, a figure 7 and a figure 8, and the results show that the combined diagnosis effect of hsa-miR-26b + hsa-miR-378 is better than that of a single marker.

TABLE 3 diagnostic potency of Gene combinations

Genome	AUC
		hsa-miR-26b+hsa-miR-378	0.950
hsa-let-7b+hsa-miR-26b	0.751

Example 3 qPCR validation

The first experiment method comprises the following steps:

1. sample collection

Blood samples of 30 healthy persons and blood samples of 30 patients with non-small cell lung cancer were collected, and detailed information of the samples was written, and all the samples were obtained with the approval of the ethical committee.

2. RNA extraction

Total RNA was extracted using a blood RNA extraction kit from Promega corporation.

3. Reverse transcription:

1) reverse transcription reaction

Preparing 25 mu l of reaction system, taking 1 mu g of total RNA of each sample as template RNA, and respectively adding the following components into a PCR tube: DEPC water, 5 Xreverse transcription buffer, 10mM dNTP, 0.1mM DTT, 30. mu.M Oligo dT, 200U/. mu. l M-MLV, template RNA.

Reaction conditions are as follows: incubate at 42 ℃ for 1h, 72 ℃ for 10min, and centrifuge briefly.

2) Primer design

QPCR amplification primers were designed based on the coding sequences of hsa-miR-26b, hsa-miR-378 and U6 genes.

3) QPCR amplification assay:

prepare 25. mu.l reaction system: SYBR Green polymerase chain reaction system 12.5. mu.l, forward and reverse primers (5. mu.M) 1. mu.l each, template cDNA 2.0. mu.l, 8.5. mu.l enzyme-free water. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the results. All operations were performed on ice.

SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent real-time quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, and relative quantification is carried out through a delta CT method.

4. Statistical method

The experiment adopts 3 times of repeated experiments, the result data are all expressed in a mode of mean value plus or minus standard deviation, statistical analysis is carried out by using SPSS13.0 statistical software, the difference analysis between the two is carried out by adopting a t test, and the difference is considered to have statistical significance when P is less than 0.05.

Second, experimental results

As shown in FIGS. 9 and 10, compared with healthy people, hsa-miR-26b is significantly downregulated in non-small cell lung cancer patients, and hsa-miR-378 is significantly upregulated in non-small cell lung cancer patients, and the difference has statistical significance (P < 0.05). As shown in Table 4, Table 5, FIG. 11 and FIG. 12, hsa-miR-26b has high diagnostic efficacy (AUC of 0.800) and hsa-miR-378 has high diagnostic efficacy (AUC of 0.831), which indicates that hsa-miR-26b and hsa-miR-378 can be used for diagnosing non-small cell lung cancer.

TABLE 4 hsa-miR-26b region under ROC Curve for diagnosing non-small cell lung cancer

a. By nonparametric assumptions

b. The original assumption is that: true area of 0.5

TABLE 5 region under ROC Curve for hsa-miR-378 diagnosing non-Small cell Lung cancer

a. By nonparametric assumptions

b. The original assumption is that: true area of 0.5

The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications may be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications all belong to the protection scope of the present application.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in the present application.

In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.

Claims

1. A marker for diagnosing non-small cell lung cancer, wherein the marker comprises hsa-miR-26b and/or hsa-miR-378.

2. The marker of claim 1, wherein the marker comprises hsa-miR-26b and hsa-miR-378.

3. Use of an agent that detects the expression level of the marker of claim 1 or 2 in a sample for the preparation of a tool for diagnosing non-small cell lung cancer.

4. The use of claim 3, wherein said agent comprises:

a probe that specifically recognizes the marker; or

Primers for specific amplification of the marker.

5. The use of claim 3, wherein the sample comprises blood, plasma, serum, saliva, urine, tears, cerebrospinal fluid, or organ tissue.

6. The use of claim 5, wherein the sample is blood.

7. A system for diagnosing non-small cell lung cancer, comprising the following units:

1) a detection unit: comprises a marker detection module;

3) an evaluation unit: outputting the risk value of the individual corresponding to the sample to suffer from the non-small cell lung cancer;

the marker comprises hsa-miR-26b and/or hsa-miR-378.

8. Use of a marker in the construction of a system according to claim 7, wherein the marker comprises hsa-miR-26b and/or hsa-miR-378.

9. A kit for diagnosing non-small cell lung cancer, comprising a reagent for detecting the level of expression of the marker of claim 1 or 2 in a sample.

10. The kit of claim 9, wherein the kit comprises a chip or kit.