Method for rapidly assisting in identifying MPLC cells and IPM cells and application of method

Abstract

The invention discloses a method for rapidly assisting in identifying MPLC cells and IPM cells and application of the method, and belongs to the technical field of tumor treatment. The method comprises the following steps: S1, acquiring lung cancer tissue cells; s2, detecting the mRNA expression level of the CXCL1 gene and / or the CXCL8 gene in the lung cancer tissue cells to obtain mRNA expression level data of the CXCL1 gene and / or the CXCL8 gene; s3, judging the type of the lung cancer tissue cell according to mRNA expression level data of the CXCL1 gene and / or CXCL8 gene of the lung cancer tissue cell; according to the method, different expression levels of the CXCL1 gene and the CXCL8 gene in lung cancer MPLC cells and IPM cells are utilized, and the expression levels of the CXCL1 gene and the CXCL8 gene in the lung cancer cells are specifically detected, so that a more accurate and rapid auxiliary means can be provided for identifying the types of the lung cancer cells according to the obtained expression level data of the CXCL1 gene and the CXCL8 gene; and accurate formulation of a lung cancer patient treatment scheme is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and in particular to a method for rapidly assisting in identifying MPLC cells and IPM cells and an application thereof. Background Art

[0002] Lung cancer is one of the leading causes of cancer-related deaths worldwide. The emergence of early lung cancer screening methods, especially low-dose computed tomography (LDCT), has facilitated the early detection of multiple lung lesions associated with lung cancer. These lung lesions are mainly divided into two categories according to their origin: multiple primary lung cancer (MPLC) and intrapulmonary metastatic lung cancer (IPM). MPLC refers to two or more primary lung cancers occurring in different lobes or segments of one or both lungs of the same patient; while IPM refers to malignant tumors originating in other parts of the body metastasize to the lungs through the blood or lymphatic system to form new tumor foci. According to the time of occurrence, MPLC can be further divided into synchronous MPLC (sMPLC) or metachronous MPLC (mMPLC). Accurately distinguishing whether lung cancer cells belong to MPLC or IPM is not only of great guiding significance for tumor staging, treatment strategies and prognosis, but also crucial for doctors to tailor precise and personalized treatment plans for lung cancer patients.

[0003] The Martini-Melamed (MM) criteria were proposed in 1975 and are the earliest classification guidelines for distinguishing MPLC from IPM. The criteria use parameters such as tumor location, pathological morphology, and the presence of distant metastases to distinguish MPLC from IPM. Although this criterion remains the most widely used diagnostic framework in clinical practice, it has also been found in clinical practice that patients diagnosed with primary lung cancer (stage I and II) according to the MM criteria may actually be misdiagnosed as lung metastases (T4 or M1a). Subsequently, several new diagnostic criteria for MPLC have been proposed and revised several times in clinical practice. For example, the 2013 American College of Chest Physicians (ACCP) guidelines are one of these efforts. However, it is worth noting that the ACCP guidelines point out that many diagnostic criteria for MPLC can only be used as a reference, and the comprehensive analysis of clinical manifestations and imaging features is the main basis for judgment. This also shows that there is currently a lack of a universally accepted gold standard for distinguishing MPLC from IPM, and the accuracy of judging MPLC still relies on the input of experts from multiple fields, especially the opinions of radiologists and pathologists.

[0004] With the advancement of science and technology, genetic testing technology has been widely used in the treatment and diagnosis of diseases. At present, the application of genetic testing technology in disease classification is mainly focused on the specificity of analyzing cell DNA. In the case of lung cancer, a variety of molecular technologies in genetic research methods have been used to analyze the genetic characteristics of MPLC, such as array comparative genomic hybridization (aCGH), microsatellite analysis (MSA), next-generation sequencing (NGS) and whole exome sequencing (WES). However, due to the differences in the number of genes included in different tests, as well as the significant differences in sensitivity and specificity, the existing genetic testing technology for disease classification still has diagnostic bias. Therefore, the invention of a set of effective and low-cost auxiliary means to distinguish IPM and MPLC is of great significance to improve the speed of diagnosis and improve the efficiency of diagnosis. Summary of the invention

[0005] The purpose of the present invention is to address the shortcomings of the existing methods for distinguishing MPLC cells from IPM cells, such as poor accuracy and long time, and to propose a method for rapid auxiliary identification of MPLC cells from IPM cells and its application.

[0006] In order to achieve the above object, the present invention provides a method for rapidly assisting in distinguishing MPLC cells from IPM cells, comprising the following steps:

[0007] S1: Obtain lung cancer tissue cells;

[0008] S2: detecting the mRNA expression level of CXCL1 gene and / or CXCL8 gene in lung cancer tissue cells to obtain mRNA expression level data of CXCL1 gene and / or CXCL8 gene;

[0009] S3: Determine the type of lung cancer tissue cells based on the mRNA expression level data of CXCL1 gene and / or CXCL8 gene in lung cancer tissue cells.

[0010] The present invention discloses a method for rapidly assisting in distinguishing MPLC cells from IPM cells. The method utilizes the difference in expression levels of CXCL1 and CXCL8 genes between lung cancer MPLC cells and IPM cells, and specifically detects the expression levels of CXCL1 and CXCL8 genes in lung cancer cells. Thus, according to the obtained CXCL1 and CXCL8 gene expression level data, a more accurate and rapid auxiliary means is provided for distinguishing lung cancer cell types, which is beneficial to the accurate formulation of treatment plans for lung cancer patients. Compared with large-scale NGS sequencing, the identification method has the advantages of simple operation and low detection cost, and is suitable for large-scale promotion and application.

[0011] Preferably, in step S2, the mRNA expression level detection technology is one of RNA next-generation sequencing (NGS) technology and real-time fluorescence quantitative chain amplification reaction (qRTPCR) technology.

[0012] Preferably, when the mRNA expression level data is obtained by RNA second-generation sequencing (NGS) technology, the mRNA expression level data is TPM data (Transcripts Per Kilobase of exon model per Million mapped reads); the TPM data is the transcripts per kilobase of transcription per million mapped reads, which is one of the quantitative indicators of mRNA expression in second-generation sequencing.

[0013] Among them, preferably, the specific method of using RNA second-generation sequencing (NGS) technology to detect the mRNA expression level of CXCL1 gene and / or CXCL8 gene in lung cancer tissue cells includes the following steps: (1) RNA extraction; (2) RNA quantitative detection; (3) transcription and synthesis of cDNA; (4) cDNA fragmentation; (5) ligation of sequencing adapters; (6) library amplification; (7) library sequencing to obtain the TPM value.

[0014] Preferably, when the mRNA expression level data is obtained by real-time fluorescence quantitative chain amplification reaction (qRTPCR) technology, the mRNA expression level data is 2 -△△CT Data; where CT is the cycle threshold of lung cancer cells; 2 -△△CT Indicates the relative expression change of the target gene.

[0015] Preferably, in step S3, the judgment standard of the lung cancer tissue cell type is: when the log(TPM+1) of CXCL1 in the lung cancer tissue cell is less than 7, the lung cancer cell is an MPLC cell, and when the log(TPM+1) of CXCL1 is ≥7, the lung cancer cell is an IPM cell; or, when the log(TPM+1) of CXCL8 in the lung cancer tissue cell is less than 2.68, the lung cancer cell is an MPLC cell, and when the log(TPM+1) of CXCL8 is ≥2.68, the lung cancer cell is an IPM cell;

[0016] Or, when CXCL12 in lung cancer tissue cells -△△CT When CXCL1 is 2.305, lung cancer cells are MPLC cells. -△△CT When ≥2.305, lung cancer cells are IPM cells; or, when CXCL8 2 -△△CT When CXCL8 is 2 -△△CT When ≥0.235, lung cancer cells are IPM cells.

[0017] Among them, preferably, the method of detecting the mRNA expression level of CXCL1 gene and / or CXCL8 gene in lung cancer tissue cells using real-time fluorescence quantitative chain amplification reaction (qRTPCR) technology includes the following steps: A. RNA extraction; B. RNA quantitative detection; C. transcription and synthesis of cDNA; D. amplification using primers; E. preparation of amplification curve; F. determination of Ct value based on the amplification curve.

[0018] Among them, preferably, the forward primer of CXCL1 is: 5′-AAGTCATAGCCACACTCAAG-3′, and the reverse primer is: 5′-TCAGTTGGATTTGTCACTGT-3′; the forward primer of CXCL8 is: 5′-GAGAGTGATTGAGAGTGGAC-3′, and the reverse primer is: 5′-GAATTCTCAGCCCTCTTCAA-3′; the forward primer of β-actin (internal reference gene, used to assist in the quantification of CXCL1 and CXCL8) is: 5′-GACATTAAGGAGAAGCTGTG-3′, and the reverse primer is: 5′-CATGATGGAGTTGAA GGTAG-3′.

[0019] Furthermore, the present invention also provides a method for quickly assisting in the identification of MPLC cells and IPM cells and its application in assisting in the formulation of a lung cancer treatment plan; the lung cancer treatment plan includes at least one of a medication plan, a surgical plan and / or a health care plan.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The method of the present invention for rapid auxiliary identification of MPLC cells and IPM cells utilizes the difference in expression levels of CXCL1 gene and CXCL8 gene in lung cancer MPLC cells and IPM cells, and specifically detects the expression levels of CXCL1 gene and CXCL8 gene in lung cancer cells. Thus, based on the obtained CXCL1 gene and CXCL8 gene expression level data, a more accurate and rapid auxiliary means can be provided for the identification of lung cancer cell types, which is beneficial to the accurate formulation of treatment plans for lung cancer patients.

[0022] (2) The method for rapid auxiliary identification of MPLC cells and IPM cells of the present invention has the advantages of simple operation and low detection cost, and is suitable for large-scale promotion and application. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with specific embodiments. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits used in the experiments are all commercially available unless otherwise specified.

[0025] Test subjects: Tissue samples of at least 2 lesions were collected from 34 lung cancer patients who met the MM criteria, for a total of 100 samples; at the same time, a comprehensive diagnostic method (combining histopathological evaluation, multi-gene sequencing (NGS) technology and whole-genome sequencing technology) was used (each sample took 3 days and the test cost was about 2,000) to identify the 100 samples separately and accurately determine the lung cancer cell types of the 100 samples, including 48 MPLC lung cancer cell samples and 52 IPM lung cancer cell samples.

[0026] Embodiment 1:

[0027] RNA next-generation sequencing (NGS) technology was used to randomly detect the CXCL1 gene expression level in 100 samples. The specific steps included:

[0028] (1) RNA extraction: RNA was extracted using the AmoyDx Tissue DNA / RNA Kit (AmoyDx, Xiamen, China);

[0029] (2) RNA quantitative detection: The concentration of the extracted RNA was quantitatively detected using a Quantus fluorometer and RNA HS kit (Cat. No. E3310, Promega);

[0030] (3) cDNA synthesis by transcription: cRNA is obtained by transcription from a strand-specific DNA library;

[0031] (4) cDNA fragmentation: cRNA samples were fragmented at 95°C. The fragmentation time was determined by the DV200 value estimated by the Agilent 2100 bioanalyzer system. The fragment length was evaluated by the Agilent 2100 bioanalyzer.

[0032] (5) Library preparation: Use Ultra TM IIDirectional RNA Library Prep Kit forI (Cat. No. E7760L, NEB) reverse transcription, complementary RNA synthesis and strand-specific library preparation were performed on the fragmented RNA;

[0033] (6) Library sequencing: sequencing was performed on an Illumina NovaSeq 6000 instrument to obtain the TPM value;

[0034] (7) Calculate the log(TPM+1) value of the CXCL1 gene in the sample, and judge the type of lung cancer tissue cells based on the log(TPM+1) value. The judgment standard is: when the log(TPM+1) of CXCL1 in the lung cancer tissue cells is less than 7, the lung cancer cells are MPLC cells, and when the log(TPM+1) of CXCL1 is ≥7, the lung cancer cells are IPM cells. The judgment results are: 45 MPLC lung cancer cell samples were correctly judged, 3 were wrongly judged, 29 IPM lung cancer cell samples were correctly judged, 23 were wrongly judged, and the total judgment was wrong. 26 samples were wrongly judged, and the judgment accuracy was 74%. Among them, each sample test took 1 day (can be carried out simultaneously), and the cost was RMB 500.

[0035] Example 2

[0036] RNA next-generation sequencing (NGS) technology was used to randomly detect the CXCL8 gene expression level in 100 samples. The specific steps included:

[0037] (1) RNA extraction: RNA was extracted using the AmoyDx Tissue DNA / RNA Kit (AmoyDx, Xiamen, China);

[0038] (2) RNA quantitative detection: The concentration of the extracted RNA was quantitatively detected using a Quantus fluorometer and RNA HS kit (Cat. No. E3310, Promega);

[0039] (3) cDNA synthesis by transcription: cRNA is obtained by transcription from a strand-specific DNA library;

[0040] (4) cDNA fragmentation: cRNA samples were fragmented at 95°C. The fragmentation time was determined by the DV200 value estimated by the Agilent 2100 bioanalyzer system. The fragment length was evaluated by the Agilent 2100 bioanalyzer.

[0041] (5) Library preparation: Use Ultra TM II Directional RNA Library Prep Kit for I (Cat. No. E7760L, NEB) reverse transcription, complementary RNA synthesis and strand-specific library preparation were performed on the fragmented RNA;

[0042] (6) Library sequencing: sequencing was performed on an Illumina NovaSeq 6000 instrument to obtain the TPM value;

[0043] (7) Calculate the log(TPM+1) value of the CXCL8 gene in the sample, and determine the type of lung cancer tissue cells based on the log(TPM+1) value. The judgment standard is: when the log(TPM+1) of CXCL8 in the lung cancer tissue cells is less than 2.68, the lung cancer cells are MPLC cells; when the log(TPM+1) of CXCL8 is greater than or equal to 2.68, the lung cancer cells are IPM cells. The judgment results are: 36 MPLC lung cancer cell samples were correctly judged, 12 were wrongly judged, and 40 IPM lung cancer cell samples were correctly judged (12 were wrongly judged, 24 were wrongly judged in total, and the judgment accuracy was 76%). Among them, each sample detection took 1 day (can be performed simultaneously), and the cost was RMB 500.

[0044] Example 3

[0045] The CXCL1 gene expression levels in 100 samples were randomly detected using real-time fluorescence quantitative chain amplification reaction (qRTPCR) technology. The specific steps include:

[0046] A. RNA extraction: RNA was extracted from samples using TRIzol reagent (Thermo Fisher Scientific Inc.);

[0047] B. RNA quantitative detection: The concentration of the extracted RNA was quantitatively detected using a Quantus fluorometer and an RNA HS kit (Cat. No. E3310, Promega);

[0048] C, transcribe to synthesize cDNA; RNA through IIQ Select RT SuperMix for qPCR (R232-01; Vazyme) was used to transcribe cDNA; the transcription conditions were 55°C for 15 min and 85°C for 15 min.

[0049] D. Amplification using primers; SYBR Green qPCR Master Mix (Q712-02; Vazyme) was used for amplification. The forward primer for CXCL1 was 5′-AAGTCATAGCCACACTCAAG-3′, and the reverse primer was 5′-TCAGTTGGATTTGTCACTGT-3′; the forward primer for CXCL8 was 5′-GAGAGTGATTGAGAGTGGAC-3′, and the reverse primer was 5′-GAATTCTCAGCCCTCTTCAA-3′; the forward primer for β-actin was 5′-GACATTAAGGAGAAGCTGTG-3′, and the reverse primer was 5′-CATGATGGAGTTGAAGGTAG-3′; the initial condition was 95°C for 3 minutes, followed by 42 cycles of 95°C for 10 seconds, 60°C for 3 minutes, and 72°C for 1 minute, and the final extension step was 72°C for 10 minutes;

[0050] E. Prepare an amplification curve; plot the fluorescence intensity against the cycle number, and use the qPCR instrument to generate an amplification curve;

[0051] F. Determine the Ct value based on the amplification curve;

[0052] G. Calculate the 2 of CXCL1 gene in the sample -△△CT value, and according to 2 -△△CT The value of CXCL1 in lung cancer tissue cells is used to determine the type of lung cancer tissue cells. The judgment standard is: when the 2 -△△CT When CXCL1 is 2.305, lung cancer cells are MPLC cells. -△△CT When ≥2.305, lung cancer cells are IPM cells; the judgment results are: 35 MPLC lung cancer cell samples were correctly judged, 13 were wrongly judged, 43 IPM lung cancer cell samples were correctly judged, 9 were wrongly judged, and the total wrong judgment was 22, with a judgment accuracy of 78%. Among them, each sample test took 1 day (can be performed simultaneously) and cost 200 yuan.

[0053] Example 4

[0054] The CXCL8 gene expression levels in 100 samples were randomly detected using real-time fluorescence quantitative chain amplification reaction (qRTPCR) technology. The specific steps include:

[0055] A. RNA extraction: RNA was extracted from samples using TRIzol reagent (Thermo Fisher Scientific Inc.);

[0056] B. RNA quantitative detection: The concentration of the extracted RNA was quantitatively detected using a Quantus fluorometer and an RNA HS kit (Cat. No. E3310, Promega);

[0057] C, transcribe to synthesize cDNA; RNA through IIQ Select RT SuperMix for qPCR (R232-01; Vazyme) was transcribed into cDNA; the transcription conditions were 55°C for 15 min and 85°C for 15 min;

[0058] D. Amplification using primers; SYBR Green qPCR Master Mix (Q712-02; Vazyme) was used for amplification. The forward primer for CXCL1 was 5′-AAGTCATAGCCACACTCAAG-3′, and the reverse primer was 5′-TCAGTTGGATTTGTCACTGT-3′; the forward primer for CXCL8 was 5′-GAGAGTGATTGAGAGTGGAC-3′, and the reverse primer was 5′-GAATTCTCAGCCCTCTTCAA-3′; the forward primer for β-actin was 5′-GACATTAAGGAGAAGCTGTG-3′, and the reverse primer was 5′-CATGATGGAGTTGAAGGTAG-3′; the initial condition was 95°C for 3 minutes, followed by 42 cycles of 95°C for 10 seconds, 60°C for 3 minutes, and 72°C for 1 minute, and the final extension step was 72°C for 10 minutes;

[0059] E. Prepare an amplification curve; plot the fluorescence intensity against the cycle number, and use the qPCR instrument to generate an amplification curve;

[0060] F. Determine the Ct value based on the amplification curve.

[0061] G. Calculate the 2 of CXCL8 gene in the sample -△△CT value, and according to 2 -△△CT The value of CXCL8 in lung cancer tissue cells is used to determine the type of lung cancer tissue cells. The judgment standard is: when the 2 -△△CT When CXCL8 is 2 -△△CT When ≥0.235, lung cancer cells are IPM cells; the judgment results are: 35 MPLC lung cancer cell samples were correctly judged, 13 were wrongly judged, 35 IPM lung cancer cell samples were correctly judged, 17 were wrongly judged, and the total wrong judgment was 30, with a judgment accuracy of 70%. Among them, each sample test took 1 day (can be performed simultaneously) and cost 200 yuan.

[0062] It can be seen from the judgment results of Examples 1-4 that the method of the present invention for rapid identification of MPLC cells and IPM cells has the advantages of high accuracy, short time and low cost, and can be used on a large scale for auxiliary identification of MPLC cells and IPM cells.

Claims

1. A method for rapid auxiliary identification of MPLC cells and IPM cells, characterized in that: The following steps are involved: S1: Obtain lung cancer tissue cells; S2: detecting the mRNA expression level of CXCL1 gene and / or CXCL8 gene in lung cancer tissue cells to obtain the mRNA expression level data of CXCL1 gene and / or CXCL8 gene; S3: Determine the type of lung cancer tissue cells based on the mRNA expression level data of CXCL1 gene and / or CXCL8 gene in lung cancer tissue cells.

2. The method for rapid auxiliary identification of MPLC cells and IPM cells according to claim 1, characterized in that: In step S2, the mRNA expression level detection technology is one of RNA second-generation sequencing technology and real-time fluorescence quantitative chain amplification reaction technology.

3. The method for rapid auxiliary identification of MPLC cells and IPM cells according to claim 2, characterized in that: When the mRNA expression level data is obtained by RNA second-generation sequencing technology, the mRNA expression level data is TPM data.

4. The method for rapid auxiliary identification of MPLC cells and IPM cells according to claim 3, characterized in that: In step S3, the judgment standard of the lung cancer tissue cell type is: when the log(TPM+1) of CXCL1 in the lung cancer tissue cell is less than 7, the lung cancer cell is an MPLC cell, and when the log(TPM+1) of CXCL1 is greater than or equal to 7, the lung cancer cell is an IPM cell; or, when the log(TPM+1) of CXCL8 in the lung cancer tissue cell is less than 2.68, the lung cancer cell is an MPLC cell, and when the log(TPM+1) of CXCL8 is greater than or equal to 2.68, the lung cancer cell is an IPM cell.

5. The method for rapid auxiliary identification of MPLC cells and IPM cells according to claim 3, characterized in that: The specific method for detecting the mRNA expression level of CXCL1 gene and / or CXCL8 gene in lung cancer tissue cells using RNA second-generation sequencing technology comprises the following steps: (1) RNA extraction; (2) RNA quantitative detection; (3) transcription and synthesis of cDNA; (4) cDNA fragmentation; (5) ligation of sequencing adapters; (6) library amplification; and (7) library sequencing to obtain a TPM value.

6. The method for rapid auxiliary identification of MPLC cells and IPM cells according to claim 2, characterized in that: When the mRNA expression level data is obtained by real-time fluorescence quantitative chain amplification reaction technology, the mRNA expression level data is 2 -△△CT data.

7. The method for rapid auxiliary identification of MPLC cells and IPM cells according to claim 6, characterized in that: In step S3, the criterion for judging the type of lung cancer tissue cells is: when the 2- △△CT When CXCL1 is 2.305, lung cancer cells are MPLC cells. -△△CT When ≥2.305, lung cancer cells are IPM cells; or, when CXCL8 2 -△△CT When CXCL8 is 2 -△△CT When ≥0.235, lung cancer cells are IPM cells.

8. The method for rapid auxiliary identification of MPLC cells and IPM cells according to claim 6, characterized in that: The method for detecting the mRNA expression level of CXCL1 gene and / or CXCL8 gene in lung cancer tissue cells by using real-time fluorescence quantitative chain amplification reaction technology comprises the following steps: A. RNA extraction; B. RNA quantitative detection; C. transcription synthesis of cDNA; D. amplification using primers; E. preparation of amplification curve; F. determination of Ct value based on the amplification curve.

9. The method for rapid auxiliary identification of MPLC cells and IPM cells according to claim 8, characterized in that: The forward primer of CXCL1 is: 5′-AAGTCATAGCCACACTCAAG-3′, and the reverse primer is: 5′-TCAGTTGGATTTGTCACTGT-3′; the forward primer of CXCL8 is: 5′-GAGAGTGATTGAGAGTGGAC-3′, and the reverse primer is: 5′-GAATTCTCAGCCCTCTTCAA-3′; the forward primer of β-actin is: 5′-GACATTAAGGAGAAGCTGTG-3′, and the reverse primer is: 5′-CATGATGGAGTTGAAGGTAG-3′.

10. Use of the method for rapid auxiliary identification of MPLC cells and IPM cells according to any one of claims 1 to 9 in assisting in formulating a lung cancer treatment plan.