CN113151479B - Kit for detecting lung adenocarcinoma cell cycle progression pathway related gene mutation - Google Patents
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Abstract
The invention relates to the technical field of molecular diagnosis, in particular to a composition for detecting lung adenocarcinoma cell cycle progression pathway related gene (CDKN2A, CCND1, CCNE1, CDK4 and RB1) mutation and application thereof, wherein the composition consists of reagents for detecting related gene expression quantity. The invention screens genes related to a cell cycle progression pathway by RNA sequencing, LASSO and binary Logistic regression, constructs Score, obtains a corresponding cut-off value of the Score in lung adenocarcinoma by ROC analysis, and can be used for predicting the mutation of the genes related to the cell cycle progression pathway of the lung adenocarcinoma. The specific gene mutation of the lung adenocarcinoma is predicted by utilizing the expression quantity of the genome composition, and the prediction method has the advantages of high accuracy and good specificity and has good application prospect according to the verification.
Description
Technical Field
The invention relates to the technical field of molecular diagnosis, in particular to a kit for detecting lung adenocarcinoma cell cycle progression pathway related gene mutation and application thereof.
Background
Lung cancer is the most common malignant tumor, and the mortality rate of lung cancer is high in all tumor leaders worldwide. Lung adenocarcinoma is the most common subtype of lung cancer, accounting for over 40% of lung cancer cases. The emergence of various targeted drugs aiming at lung adenocarcinoma obviously improves the prognosis of lung adenocarcinoma patients, so that the treatment of lung adenocarcinoma has entered the molecular era. How to accurately evaluate the molecular characteristics of the lung adenocarcinoma is of great significance to the accurate treatment of patients and the reduction of social burden.
The maintenance of persistent proliferation signals is one of tumor markers, and has important significance for promoting tumor growth and dissemination. In lung adenocarcinoma, CDKN2A, CCND1, CCNE1, CDK4 and RB1 genes have higher mutation rates and are highly correlated with cell cycle progression. The mutation of the gene can enable the tumor cells to get rid of the regulation and control of normal cell division signals and enter a continuous proliferation mode, thereby promoting the growth and the metastasis of tumors and influencing the prognosis of patients. Third-generation CDK (cyclin-dependent kinase) inhibitors have been approved by the fda (food and drug administration) in the united states for the treatment of specific types of breast cancer, which could benefit patients from them, showing great promise for cell cycle therapy in tumor therapy. At present, the detection of the lung adenocarcinoma cell cycle progression pathway related gene mutation is not a clinical routine project, the single detection is expensive, and a detection means with both accuracy and application value can be applied to the formulation of a personalized treatment scheme of a lung adenocarcinoma patient.
The kit for detecting the gene mutation related to the cell cycle progression pathway of the lung adenocarcinoma is not reported at present.
Disclosure of Invention
The invention aims to provide a composition for detecting genes related to cell cycle progression of lung adenocarcinoma and application thereof, aiming at the blank of the prior art.
In a first aspect, the invention provides an application of a detection reagent in preparing a kit for evaluating mutation of any one of lung adenocarcinoma cell cycle progression pathway-related genes CDKN2A, CCND1, CCNE1, CDK4 and RB1, wherein the detection reagent consists of reagents for detecting expression levels of the following genes: GFRA3, IGF2, CST2, PADI2, PLA2G12B, LRP4, DLX3, TRIM17, MYOZ1, FFAR2, CNIH2, ZNF713, GBP6, FGF9, CAMK2N2, EYA1, CRISP3, FBXO43, LIPK, PAGE5, TAF7L, SV2B, FER1L5, KIR2DL1, ASB11, C10orf82, ASTN1, NXPH1, GCG, PLA2G2F, MAGEB16, TMEM151B, CHRM2, CALML5, ZACN; the detection reagent is used as a kit for evaluating the only key component of the lung adenocarcinoma cell cycle progression pathway related gene mutation.
In certain embodiments, the kit further comprises instructions describing the following formula: score (-0.3591 × GFRA3) + (-0.2567 × IGF2) + (-0.4489 × CST2) + (0.3886 × PADI2) + (-0.6545 × PLA2G12B) + (0.5672 × LRP4) + (0.6257 × DLX3) + (-0.8741 × TRIM17) + (-0.7005 × MYOZ1) + (0.9237 × ar2) + (1.5149 × cnf 2) + (2.6282 × ZNF713) + (1.2654 × GBP6) + (-2.6386 × FGF9) + (-1.4028 × CAMK2N2) + (-1.3872 × EYA1) + (0.6539 × CRISP3) + (-1.8037 × FBXO43) + ((-43 × 363672) + ((-43 × 363672) + ((-43 × 363672 × 36363672) + (43 × 36363672 × 43) + (3636363672 × 36363672 × 3636363636363672) + ((-43 × 363672 × 36363636363636363672 × 3636363672 × 43) + (363672 × 363636363672 × 43 × 36363636363672) + (36363672 × 363636363672 × 36363636363672) + (363672 × 363636363672 × 36363672 × 363636363672) + (43 × 363672 × 3636363636363636363672) + (3636363672 × 3636363636363672 × 43 × 36363672 × 43) + (36363636363636363672 × 363672 × 43 × 3636363672 × 43 × 363672 × 43 × 36363636363672 × 43) + (43 × 36363636363636363672 × 43 × 3636363672 × 36363636363672 × 43 × 36363672 × 43 × 36363636363636363636369) + (363636363636363633) + (363636369).
In certain embodiments, the lung adenocarcinoma has a Score cutoff of 1.946.
In certain embodiments, the test sample is a fresh tissue tumor sample.
In certain embodiments, the assessment indicates that the sample has a greater likelihood of having a mutation in a gene associated with a cell cycle progression pathway when the test sample Score is ≧ 1.946; when the Score of the test sample is smaller than 1.946, the sample has less possibility of the mutation of the gene related to the cell cycle progression pathway.
In a second aspect, the invention provides a kit for evaluating mutation of any one of lung adenocarcinoma cell cycle progression pathway-related genes CDKN2A, CCND1, CCNE1, CDK4 and RB1, which comprises at least reagents for specifically detecting expression levels of the following genes: GFRA3, IGF2, CST2, PADI2, PLA2G12B, LRP4, DLX3, TRIM17, MYOZ1, FFAR2, CNIH2, ZNF713, GBP6, FGF9, CAMK2N2, EYA1, CRISP3, FBXO43, LIPK, PAGE5, TAF7L, SV2B, FER1L5, KIR2DL1, ASB11, C10orf82, ASTN1, NXPH1, GCG, PLA2G2F, MAGEB16, TMEM151B, CHRM2, CALML5, ZACN.
In a third aspect, the present invention provides a method for assessing mutations in any one of the lung adenocarcinoma cell cycle progression pathway-associated genes CDKN2A, CCND1, CCNE1, CDK4, RB1 for non-disease diagnostic and therapeutic purposes, comprising the steps of:
(1) detecting the following gene expression levels of the sample: GFRA3, IGF2, CST2, PADI2, PLA2G12B, LRP4, DLX3, TRIM17, MYOZ1, FFAR2, CNIH2, ZNF713, GBP6, FGF9, CAMK2N2, EYA1, CRISP3, FBXO43, LIPK, PAGE5, TAF7L, SV2B, FER1L5, KIR2DL1, ASB11, C10orf82, ASTN1, NXPH1, GCG, PLA2G2F, MAGEB16, TMEM151B, CHRM2, CALML5, ZACN;
(2) and (3) calculating: score (— xgra) + (× IGF) + (× CST) + (× PADI) + (× PLA2G 12) + (× LRP) + (× DLX) + (× TRIM) + (× MYOZ) + (× FFAR) + (× CNIH) + (× ZNF713) + (× GBP) + (× FGF) + (× CAMK 2N) + (× aya) + (× pasp) + (× FBXO) + (× LIPK) + (× PAGE) + (× TAF 7) + (sv2) + (× FER 1L) + (× kir2 DL) + (× ASB) + (× C10 orf) + (× ASTN) + (× zah) + (× GCG) + (× PLA2G 2) + (× MAGEB) + (× TMEM 151) + (× CHRM (-) -calm 0.1535) + (× clcn);
(3) and (3) judging: when the Score is more than or equal to 1.946, the sample has higher possibility of having the gene mutation related to the cell cycle progression pathway; when Score <1.946, it indicates that the sample has a low probability of having a mutation in a gene associated with a cell cycle progression pathway.
Compared with the prior art, the invention has the following technical effects:
1. the invention firstly provides that the genome composition can be used for evaluating whether the lung adenocarcinoma has the mutation of cell cycle progression pathway related genes (CDKN2A, CCND1, CCNE1, CDK4 and RB1), can effectively guide the individualized treatment of the lung adenocarcinoma patients, improves the clinical benefit, and avoids unnecessary medical resource waste.
2. The lung adenocarcinoma cell cycle progression pathway related gene mutation belongs to an unconventional detection project clinically at present, and the cost for detecting the gene mutation alone is high. The invention can judge whether the lung adenocarcinoma patients have the gene mutation or not through the clinically widely applied RNA sequencing technology, and has the advantages of economy, high accuracy, good sensitivity and good specificity.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.
FIG. 1 is a Receiver Operating Characteristic (ROC) curve of the prediction model
FIG. 2 shows 656 difference genes analyzed by TCGA database
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and embodiments.
While specific embodiments of the present invention or prior art will be described briefly in order to more clearly illustrate it, it should be apparent that the following description of the embodiments is illustrative of some embodiments of the present invention and that others can be devised by those skilled in the art without departing from the inventive concept.
Example 1 Scoring model construction of Gene mutation related to cell cycle progression pathway of Lung adenocarcinoma
1. Method of producing a composite material
We first obtained 510 lung adenocarcinoma sample data from the TCGA database, divided the samples into a mutation group (n-57) and a wild group (n-453) according to the presence or absence of cell cycle progression-related gene (CDKN2A, CCND1, CCNE1, CDK4, RB1) mutations, and further calculated and screened 656 differential genes. By performing LASSO regression analysis on expression quantity data [ log2(FPKM +1) form ] of the 656 difference genes of the 510 samples, we obtained genes significantly related to the mutation state of the genes related to the cell cycle progression pathway, and finally selected 35 genes as: GFRA3, IGF2, CST2, PADI2, PLA2G12B, LRP4, DLX3, TRIM17, MYOZ1, FFAR2, CNIH2, ZNF713, GBP6, FGF9, CAMK2N2, EYA1, CRISP3, FBXO43, LIPK, PAGE5, TAF7L, SV2B, FER1L5, KIR2DL1, ASB11, C10orf82, ASTN1, NXPH1, GCG, PLA2G2F, MAGEB16, TMEM151B, CHRM2, CALML5, ZACN.
Score model Score was constructed by binary Logistic regression as (— xgra) + (× IGF) + (× CST) + (× PADI) + (× PLA2G 12) + (× LRP) + (× DLX) + (× TRIM) + (× MYOZ) + (× FFAR) + (× CNIH) + (× znf713) + (× GBP) + (× FGF) + (× CAMK 2N) + (× aya) + (× CRISP) + (× FBXO) + (× LIPK) + (× PAGE) + (× TAF 7) + (× SV 2) + (× FGF) + (× CAMK2 DL) + (× ASB) + (× C10 orf) + (× ASTN) + (× NXPH) + (× GCG) + × PLA2G 2) + (× mage m 151) + (× CHRM m2 DL) + (× ASB) + (× SV2 1535 × CALML) + (× calm CALML).
Finally, we calculate the score of each sample based on the scoring model, and obtain the optimal threshold point, i.e., the cutoff value, of 1.946 through the ROC curve. The TCGA samples were divided into two groups based on cutoff values, above which the scores were assigned to the mutant groups and below which the scores were assigned to the wild groups.
The experimental results show that: the sensitivity of the prediction model was 0.877 and the specificity was 0.918.
Example 2 Effect verification
34 lung adenocarcinoma samples collected from thoracic surgery of Zhongshan Hospital affiliated at the university of Fudan were RNA sequenced and tested for mutations in cell cycle progression pathway-associated genes (CDKN2A, CCND1, CCNE1, CDK4, RB1), RNA expression data were put into the predictive model to obtain a Score for each sample, with scores above the cutoff value of 1.946 for the mutant group and below the cutoff value of 1.946 for the wild group.
The experimental results show that: the sensitivity of the prediction model was 0.833, and the specificity was 0.893.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (6)
1. The application of a detection reagent in preparing a kit for evaluating mutation of any one of genes related to cell cycle progression pathway of lung adenocarcinoma CDKN2A, CCND1, CCNE1, CDK4 and RB1 is characterized in that the detection reagent consists of reagents for detecting the expression levels of the following genes: GFRA3, IGF2, CST2, PADI2, PLA2G12B, LRP4, DLX3, TRIM17, MYOZ1, FFAR2, CNIH2, ZNF713, GBP6, FGF9, CAMK2N2, EYA1, CRISP3, FBXO43, LIPK, PAGE5, TAF7L, SV2B, FER1L5, KIR2DL1, ASB11, C10orf82, ASTN1, NXPH1, GCG, PLA2G2F, MAGEB16, TMEM151B, CHRM2, CALML5, ZACN; the detection reagent is used as a kit to evaluate the only key component of the gene mutation related to the cell cycle progression pathway of the lung adenocarcinoma.
2. The use of claim 1, further comprising instructions in the kit, the instructions describing the formula: score (= (-0.3591 × GFRA3) + (-0.2567 × IGF2) + (-0.4489 × CST2) + (0.3886 × PADI2) + (-0.6545 × PLA2G12B) + (0.5672 × LRP4) + (0.6257 × DLX3) + (-0.8741 × TRIM17) + (-0.7005 × MYOZ1) + (0.9237 × AR2) + (1.5149 × CNF 2) + (2.6282 × ZNF713) + (1.2654 × GBP6) + (-2.6386 × FGF9) + (-1.4028 × CAMK2N2) + (-1.3872 × EYA1) + (0.6539 × CRI SP3) + (-1.8037 × FBXFBX3672) + ((-43) + (-43 × 43 363672 × PAGE 43) + ((-43 × 363672) + ((-363672 × 43 × 363672 × 43) + (363672 × GCS 43) + ((-43 × 363636363672 × 43) + (363636363672 × 43 × 3636363636363672) + ((-43 × 3636363672 × PAGE 43) + (43 × 3636363672) + (36363672 × 363636363672) + (43 × SV).
3. The use according to claim 2, wherein the lung adenocarcinoma has a Score cut-off of 1.946.
4. The use of claim 2, wherein the assessment indicates that the sample has a greater probability of having a mutation in a gene associated with a cell cycle progression pathway when the Score of the test sample is greater than or equal to 1.946; when the Score of the test sample is smaller than 1.946, the sample has less possibility of the mutation of the gene related to the cell cycle progression pathway.
5. A kit for evaluating mutation of any one of genes related to lung adenocarcinoma cell cycle progression pathway CDKN2A, CCND1, CCNE1, CDK4 and RB1, which is characterized by at least comprising reagents for specifically detecting the expression levels of the following genes: GFRA3, IGF2, CST2, PADI2, PLA2G12B, LRP4, DLX3, TRIM17, MYOZ1, FFAR2, CNIH2, ZNF713, GBP6, FGF9, CAMK2N2, EYA1, CRISP3, FBXO43, LIPK, PAGE5, TAF7L, SV2B, FER1L5, KIR2DL1, ASB11, C10orf82, ASTN1, NXPH1, GCG, PLA2G2F, MAGEB16, TMEM151B, CHRM2, CALML5, ZACN.
6. A method of assessing mutations in any one of the lung adenocarcinoma cell cycle progression pathway-associated genes CDKN2A, CCND1, CCNE1, CDK4, RB1 for non-disease diagnostic and therapeutic purposes comprising the steps of:
(1) detecting the following gene expression levels in the sample: GFRA3, IGF2, CST2, PADI2, PLA2G12B, LRP4, DLX3, TRIM17, MYOZ1, FFAR2, CNIH2, ZNF713, GBP6, FGF9, CAMK2N2, EYA1, CRISP3, FBXO43, LIPK, PAGE5, TAF7L, SV2B, FER1L5, KIR2DL1, ASB11, C10orf82, ASTN1, NXPH1, GCG, PLA2G2F, MAGEB16, TMEM151B, CHRM2, CALML5, ZACN;
(2) and (3) calculating: score (= (-0.3591 × GFRA3) + (-0.2567 × IGF2) + (-0.4489 × CST2) + (0.3886 × PADI2) + (-0.6545 × PLA2G12B) + (0.5672 × LRP4) + (0.6257 × DLX3) + (-0.8741 × TRIM17) + (-0.7005 × MYOZ1) + (0.9237 × AR2) + (1.5149 × CNF 2) + (2.6282 × ZNF713) + (1.2654 × GBP6) + (-2.6386 × FGF9) + (-1.4028 × CAMK2N2) + (-1.3872 × EYA1) + (0.6539 × CRI SP3) + (-1.8037 × FBXFBX3672) + (-43) + (-43 × 43 363672) + (-363672 × PAGE 43) + ((-43 × 363672) + (-363672 × 43 × 36363672) + (43 × 363672 × 36363672) + (-3636363672 × 36363672) + (43 × GCX 3636363672) + (36363636363672 × 363672) + (43 × VESX 363672) + (43 × VESF 36363672) + (363672) + (3636363672) + (363672 × SV × 363672) + (3636363672) + (36363672 × 3636363672) + (43 × 363636363636363672) + (43) + (36363672 × VESK 43) + (43 × 43) + (36363672 × 43 × 363636363672) + (43 × 369) + (363672 × 369) + (36363672 × 43) + (43 × 369) + (43 × VESF);
(3) and (3) judging: when the Score is more than or equal to 1.946, the sample has higher possibility of having the gene mutation related to the cell cycle progression pathway; when Score <1.946, it indicates that the sample has a low probability of having a mutation in a gene associated with a cell cycle progression pathway.
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