CN114107492B

CN114107492B - Molecular marker for tumor molecular typing and therapeutic drug evaluation, and detection primer and kit thereof

Info

Publication number: CN114107492B
Application number: CN202110016858.7A
Authority: CN
Inventors: 林舒晔; 黄家强
Original assignee: Beijing Chest Hospital; Beijing Tuberculosis and Thoracic Tumor Research Institute
Current assignee: Beijing Chest Hospital; Beijing Tuberculosis and Thoracic Tumor Research Institute
Priority date: 2021-01-07
Filing date: 2021-01-07
Publication date: 2024-04-26
Anticipated expiration: 2041-01-07
Also published as: CN114107492A

Abstract

The invention discloses a molecular marker for tumor molecular typing and therapeutic drug evaluation, and a detection primer and a kit thereof. The invention provides application of MUC22 gene expression difference as a molecular marker in any one of the following: preparing a product for molecular typing of lung squamous carcinoma and lung adenocarcinoma in non-small cell lung cancer; preparing a product for diagnosing, targeted therapy monitoring and/or medication prognosis evaluation of non-small cell lung cancer; and preparing a product for tumor diagnosis and/or prognosis detection. The invention provides a molecular basis for effectively identifying the function of MUC22 in tumors, and is beneficial to the transformation application of MUC22 genes in the molecular diagnosis and treatment of tumors. The researches find that not only provides a new mechanism for the occurrence and development of tumors, but also provides a new clue for the clinical application of MUC22 in tumor personalized diagnosis and treatment, thus having important scientific value and clinical significance.

Description

Molecular marker for tumor molecular typing and therapeutic drug evaluation, and detection primer and kit thereof

Technical Field

The invention relates to the technical field of biology, in particular to a molecular marker for tumor molecular typing and therapeutic drug evaluation, and a detection primer and a kit thereof.

Background

Malignant tumor seriously threatens the health of people and affects the social economy [1]. Due to the lack of effective monitoring means, malignant tumors are relatively hidden and progress rapidly, so that disturbance and difference of gene expression are often caused, abnormal cancer suppressor gene inactivation and activation of the cancer genes are finally presented, cell proliferation is out of control, the organism is avoided from being eliminated, and the phenotype can be greatly different, namely, tumor heterogeneity (Tumor heterogeneity) [2]. Recent progress has shown that tumor heterogeneity is a significant cause of failure in treatment due to tumor detection and drug resistance [2]. Tumor prevention and control depends on a convenient, rapid, accurate and reliable detection means and a path [3].

Tumor markers are an important approach to tumor screening and monitoring [4]. So far, a plurality of tumor markers are applied to clinical detection, treatment and the like, but often have low sensitivity and specificity, cause misdiagnosis, and are difficult to be easily applied to the actual clinical needs of crowd screening, dynamic detection, accurate drug administration evaluation and the like. Therefore, the screening of the effective tumor biomarker is not only beneficial to improving the specificity and sensitivity of tumor detection, but also is of great importance to realizing personalized and accurate parting and staging, drug resistance and curative effect evaluation and the like of tumors, so that the method has important clinical application value and social benefit, and is also the main content of medical transformation research in the current accurate medical treatment.

Reference is made to:

[1]Bray Freddie,FerlayJacques,SoerjomataramIsabelle,Siegel Rebecca L,Torre Lindsey A,JemalAhmedin.Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.[J].CA:a cancer journal for clinicians,2018,68(6).

[2]amón y Cajal,S.,Sesé,M.,Capdevila,C.et al.Clinical implications of intratumor heterogeneity:challenges and opportunities.J Mol Med 98,161–177(2020).

[3]O'Dowd Emma L,Baldwin David R.Early diagnosis pivotal to survival in lung cancer.[J].The Practitioner,2014,258(1776).

[4]Henry,N.Lynn,Hayes,Daniel F.,(2012),Cancer biomarkers,Molecular Oncology,6,doi:10.1016/j.molonc.2012.01.010.

Disclosure of Invention

The invention aims to provide a molecular marker for tumor molecular typing and therapeutic drug evaluation, and a detection primer and a kit thereof.

In a first aspect, the invention claims the use of a differential MUC22 gene expression as a molecular marker in any of the following:

P1, preparing a product for molecular typing of lung squamous cell carcinoma and lung adenocarcinoma in non-small cell lung cancer or molecular typing of lung squamous cell carcinoma and lung adenocarcinoma in non-small cell lung cancer;

P2, preparing a product for diagnosing, monitoring targeted therapy and/or evaluating medication prognosis of the non-small cell lung cancer, or diagnosing, monitoring targeted therapy and/or evaluating medication prognosis of the non-small cell lung cancer;

p3, preparing a product for tumor diagnosis and/or prognosis detection, or tumor diagnosis and/or prognosis detection.

In a second aspect, the present invention claims the use of a substance for detecting the expression level of the MUC22 gene in any of the following:

Wherein, the substance for detecting the expression level of the MUC22 gene may be a primer for amplifying the MUC22 gene. In a specific embodiment of the present invention, the substance for detecting the expression level of the MUC22 gene is specifically a primer pair or a primer pair set described in the fourth aspect or a kit described in the fifth aspect.

In a third aspect, the invention claims the use of the MUC22 gene as a target for pharmaceutical action in the manufacture of a product for the treatment and/or prophylaxis of non-small cell lung cancer.

In a fourth aspect, the invention claims a primer pair or primer pair set having the function as shown in any one of P1 to P3:

p1, molecular typing is carried out on lung squamous carcinoma and lung adenocarcinoma in non-small cell lung cancer;

P2, diagnosing the non-small cell lung cancer, monitoring targeted therapy and/or evaluating medication prognosis;

Wherein the primer pair is a primer pair for amplifying MUC22 gene.

Further, the primer pair is a primer pair A, a primer pair B or a primer pair C; the primer pair group consists of the primer pair A, the primer pair B and the primer pair C.

The primer pair A is used for amplifying the 342-580 th bit of the cDNA sequence of the MUC22 gene; the primer pair B is used for amplifying the 217 th-286 th position of the cDNA sequence of the MUC22 gene; the primer pair C is used for amplifying 5178-5349 of the cDNA sequence of the MUC22 gene.

Further, the cDNA sequence of the MUC22 gene is shown as SEQ ID No. 1.

More specifically, the primer pair A consists of two single-stranded DNAs shown as SEQ ID No.2 and SEQ ID No. 3; the primer pair B consists of two single-stranded DNA shown in SEQ ID No.4 and SEQ ID No. 5; the primer pair C consists of two single-stranded DNAs shown as SEQ ID No.6 and SEQ ID No. 7.

In a fifth aspect, the invention claims a kit comprising a primer pair or primer pair set as described in the fourth aspect above.

The kit can be, for example, an RT-qPCR kit.

Further, the kit may further comprise all or part of the following: internal reference primers for homogenization, positive control templates, negative control templates, cDNA synthesis reagents and PCR reaction reagents.

In a specific embodiment of the invention, the internal reference primer for homogenization is a primer taking Beta-action as an internal reference, and the nucleotide sequence of the internal reference primer can be shown as SEQ ID No.8 and SEQ ID No. 9.

The positive control template is a MUC22 gene PCR amplified product verified by sequencing, or a MUC22 gene cloning plasmid verified by sequencing; preferably, the MUC22 gene cloning plasmid comprises the consensus sequence of the MUC22 gene, as shown in SEQ ID No. 1.

The negative control template may be deionized water.

In the above aspects, the diagnosis of non-small cell lung cancer includes not only distinguishing lung squamous cell carcinoma from lung adenocarcinoma in non-small cell lung cancer, but also determining whether or not to switch from lung adenocarcinoma in non-small cell lung cancer to lung squamous cell carcinoma (if monitored, if the MUC22 gene expression level of a patient suffering from lung adenocarcinoma is continuously decreased, it is determined that it is highly likely to switch from lung adenocarcinoma to lung squamous cell carcinoma).

In the above aspects, the targeted therapy monitoring of non-small cell lung cancer may be as follows: an increasing number of cases report a new NSCLC phenotype, namely "EGFR mutant lung adenocarcinoma transformed to lung squamous carcinoma" that develops acquired resistance to treatment with an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), i.e., there is a transformation of lung adenocarcinoma to lung squamous carcinoma during treatment, which suggests resistance. Dynamic monitoring is needed by a simple and easy method. The present invention helps to solve such problems.

In a sixth aspect, the invention claims a data processing device or system for distinguishing or aiding in distinguishing between a lung squamous carcinoma patient and a lung adenocarcinoma patient in non-small cell lung cancer patients.

The system consists of the data processing device and the primer pair or primer pair set described in the fourth aspect or the kit described in the fifth aspect.

The data processing device is a data processing device A or a data processing device B.

The data processing device A comprises the following modules:

(A1) A data receiving module; the data receiving module is configured to receive the expression quantity value of MUC22 genes in lung cancer focus tissues and paracancerous tissues of a non-small cell lung cancer patient to be detected;

(A2) A data comparison module; the data comparison module is configured to receive the expression quantity value of the MUC22 gene in the lung cancer focus tissue and the paracancerous tissue of the non-small cell lung cancer patient to be detected from the data receiving module, and compare the expression quantity value of the MUC22 gene in the lung cancer focus tissue and the paracancerous tissue of the non-small cell lung cancer patient to be detected;

(A3) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition and output a judging result;

The predetermined determination condition is: if the expression quantity value of the MUC22 gene in the lung cancer focus tissue is smaller than the expression quantity value of the MUC22 gene in the near-cancer tissue, the non-small cell lung cancer patient to be detected is or is candidate to be a lung squamous cell lung cancer patient; if the expression quantity value of the MUC22 gene in the lung cancer focus tissue is larger than that of the MUC22 gene in the cancer-nearby tissue, the non-small cell lung cancer patient to be detected is or is candidate to be a lung adenocarcinoma patient.

The data processing device B includes the following modules:

(B1) A data receiving module; the data receiving module is configured to receive the expression quantity value of MUC22 genes in a test sample from a patient with non-small cell lung cancer to be tested;

(B2) A data storage module; the data storage module is configured to store a judgment threshold;

(B3) A data comparison module; the data comparison module is configured to receive the value of the expression level of the MUC22 gene in the test sample of the non-small cell lung cancer patient to be tested from the data receiving module, and call the judgment threshold value from the data storage module to compare with the value of the expression level of the MUC22 gene in the test sample of the non-small cell lung cancer patient to be tested;

(B4) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition and output a judging result;

the predetermined determination condition is: if the value of the expression quantity of the MUC22 gene in the test sample from the non-small cell lung cancer patient to be tested is smaller than the judgment threshold value, the non-small cell lung cancer patient to be tested is or is candidate to be a lung squamous cell carcinoma patient; if the value of the expression quantity of the MUC22 gene in the test sample from the non-small cell lung cancer patient to be tested is higher than the judgment threshold value, the non-small cell lung cancer patient to be tested is or is candidate to be a lung adenocarcinoma patient.

Further, the judgment threshold is the expression level value of the MUC22 gene in the test sample of the same species of the normal healthy control.

In a seventh aspect, the present invention claims a data processing apparatus or system of the type:

First kind: a data processing device or system for assessing the prognostic risk of a non-staged squamous lung carcinoma patient in a non-small cell lung carcinoma patient.

The system consists of the data processing device and the primer pair or primer pair set described in the fourth aspect or the kit described in the fifth aspect; the data processing device comprises the following modules:

(C1) A data receiving module; the data receiving module is configured to receive the expression quantity value of MUC22 genes in lung cancer focus tissues of non-stage lung squamous carcinoma patients in non-small cell lung cancer patients to be detected;

(C2) A data storage module; the data storage module is configured to store a judgment threshold;

(C3) A data comparison module; the data comparison module is configured to receive the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the non-stage lung squamous cell carcinoma patient in the non-stage lung cancer patient to be detected from the data receiving module, and call the judgment threshold value from the data storage module to compare with the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the non-stage lung squamous cell carcinoma patient in the non-stage lung cancer patient to be detected;

(C4) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition and output a judging result;

The predetermined determination condition is: if the expression quantity value of the MUC22 gene in the lung cancer focus tissue is smaller than the judgment threshold value, the prognosis risk of the non-stage lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is low and/or the survival period is short); if the expression quantity value of the MUC22 gene in the lung cancer focus tissue is larger than the judgment threshold value, the prognosis risk of the non-stage lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer).

Further, the judgment threshold value is 0.02FPKM. FPKM is FRAGMENTS PER Kilobase of exon model per Million MAPPED FRAGMENTS abbreviation, representing a fragment read per million maps per kilobase of transcription.

Second kind: a data processing device or system for assessing the prognostic risk of stage I lung squamous cancer patients in non-small cell lung cancer patients.

(D1) A data receiving module; the data receiving module is configured to receive the expression quantity value of MUC22 genes in lung cancer focus tissues of a patient suffering from stage I lung squamous carcinoma in a non-small cell lung cancer patient to be detected;

(D2) A data storage module; the data storage module is configured to store a judgment threshold;

(D3) A data comparison module; the data comparison module is configured to receive the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the stage I lung squamous carcinoma patient in the non-small cell lung cancer patient to be detected from the data receiving module, and call the judgment threshold value from the data storage module to compare with the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the stage I lung squamous carcinoma patient in the non-small cell lung cancer patient to be detected;

(D4) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition and output a judging result;

The predetermined determination condition is: if the expression level value of the MUC22 gene in the lung cancer focus tissue is smaller than the judgment threshold value, the prognosis risk of the stage I lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is low and/or the survival period is short); if the expression quantity value of the MUC22 gene in the lung cancer focus tissue is larger than the judgment threshold value, the prognosis risk of the stage I lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer).

Further, the judgment threshold value is 0.02FPKM.

Third kind: a data processing device or system for assessing the prognostic risk of a stage III lung squamous carcinoma patient in a non-small cell lung carcinoma patient; the system consists of the data processing device and the primer pair or primer pair set described in the fourth aspect or the kit described in the fifth aspect; the data processing device comprises the following modules:

(E1) A data receiving module; the data receiving module is configured to receive the expression quantity value of MUC22 genes in lung cancer focus tissues of a stage III lung squamous carcinoma patient in a non-small cell lung cancer patient to be detected;

(E2) A data storage module; the data storage module is configured to store a judgment threshold;

(E3) A data comparison module; the data comparison module is configured to receive the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the stage III lung squamous carcinoma patient in the non-small cell lung cancer patient to be detected from the data receiving module, and call the judgment threshold value from the data storage module to compare with the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the stage III lung squamous carcinoma patient in the non-small cell lung cancer patient to be detected;

(E4) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition and output a judging result;

The predetermined determination condition is: if the expression level value of the MUC22 gene in the lung cancer focus tissue is smaller than the judgment threshold value, the prognosis risk of the stage III lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is low and/or the survival period is short); if the expression level value of the MUC22 gene in the lung cancer focus tissue is larger than the judgment threshold value, the prognosis risk of the stage III lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer).

Further, the judgment threshold value is 0.02FPKM.

Fourth kind: a data processing device or system for assessing the prognostic risk of a non-staged lung adenocarcinoma patient in a non-small cell lung cancer patient.

(F1) A data receiving module; the data receiving module is configured to receive the expression quantity value of MUC22 genes in lung cancer focus tissues of non-stage lung adenocarcinoma patients in non-small cell lung cancer patients to be detected;

(F2) A data storage module; the data storage module is configured to store a judgment threshold;

(F3) A data comparison module; the data comparison module is configured to receive the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the non-stage lung adenocarcinoma patients in the non-small cell lung cancer patients to be detected from the data receiving module, and call the judgment threshold value from the data storage module to compare with the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the non-stage lung adenocarcinoma patients in the non-small cell lung cancer patients to be detected;

(F4) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition and output a judging result;

The predetermined determination condition is: if the expression level value of the MUC22 gene in the lung cancer focus tissue is smaller than the judgment threshold value, the prognosis risk of the non-stage lung adenocarcinoma patient in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer); if the expression quantity value of the MUC22 gene in the lung cancer focus tissue is larger than the judgment threshold value, the prognosis risk of the non-stage lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is low and/or the survival period is short).

Further, the judgment threshold value is 0.02FPKM.

Fifth: a data processing device or system for assessing the prognostic risk of a patient with stage I lung adenocarcinoma in a patient with non-small cell lung cancer.

(G1) A data receiving module; the data receiving module is configured to receive the expression quantity value of MUC22 genes in lung cancer focus tissues of a patient suffering from the I-stage lung adenocarcinoma among the non-small cell lung cancer patients to be detected;

(G2) A data storage module; the data storage module is configured to store a judgment threshold;

(G3) A data comparison module; the data comparison module is configured to receive the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the lung adenocarcinoma patient in the I phase of lung cancer patient in the non-small cell lung cancer patient to be detected from the data receiving module, and call the judgment threshold value from the data storage module to compare with the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the lung adenocarcinoma patient in the I phase of lung cancer patient in the non-small cell lung cancer patient to be detected;

(G4) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition and output a judging result;

The predetermined determination condition is: if the expression level value of the MUC22 gene in the lung cancer focus tissue is smaller than the judgment threshold value, the prognosis risk of the patient with the lung adenocarcinoma of the I stage in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer); if the expression level value of the MUC22 gene in the lung cancer focus tissue is larger than the judgment threshold value, the prognosis risk of the stage I lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is low and/or the survival period is short).

Further, the judgment threshold value is 0.02FPKM.

Sixth: a data processing device or system for assessing the prognostic risk of a stage II lung adenocarcinoma patient in a non-small cell lung cancer patient; the system consists of the data processing device and the primer pair or primer pair set described in the fourth aspect or the kit described in the fifth aspect; the data processing device comprises the following modules:

(H1) A data receiving module; the data receiving module is configured to receive the expression quantity value of MUC22 genes in lung cancer focus tissues of a phase II lung adenocarcinoma patient in a non-small cell lung cancer patient to be detected;

(H2) A data storage module; the data storage module is configured to store a judgment threshold;

(H3) A data comparison module; the data comparison module is configured to receive the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the lung adenocarcinoma patient in phase II in the non-small cell lung cancer patient to be detected from the data receiving module, and call the judgment threshold value from the data storage module to compare with the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the lung adenocarcinoma patient in phase II in the non-small cell lung cancer patient to be detected;

(H4) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition and output a judging result;

The predetermined determination condition is: if the expression level value of the MUC22 gene in the lung cancer focus tissue is smaller than the judgment threshold value, the prognosis risk of the phase II lung adenocarcinoma patient in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer); if the expression level value of the MUC22 gene in the lung cancer focus tissue is larger than the judgment threshold value, the prognosis risk of the stage II lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is low and/or the survival period is short).

Further, the judgment threshold value is 0.02FPKM.

Seventh: a data processing device or system for assessing the prognostic risk of a stage III lung adenocarcinoma patient in a non-small cell lung cancer patient.

(I1) A data receiving module; the data receiving module is configured to receive the expression quantity value of MUC22 genes in lung cancer focus tissues of a lung cancer patient in a phase III lung adenocarcinoma patient in a non-small cell lung cancer patient to be detected;

(I2) A data storage module; the data storage module is configured to store a judgment threshold;

(I3) A data comparison module; the data comparison module is configured to receive the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the lung adenocarcinoma patient in stage III in the non-small cell lung cancer patient to be detected from the data receiving module, and call the judgment threshold value from the data storage module to compare with the expression quantity value of the MUC22 gene in the lung cancer focus tissue of the lung adenocarcinoma patient in stage III in the non-small cell lung cancer patient to be detected;

(I4) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition and output a judging result;

The predetermined determination condition is: if the expression level value of the MUC22 gene in the lung cancer focus tissue is smaller than the judgment threshold value, the prognosis risk of the patient with the III-stage lung adenocarcinoma in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is low and/or the survival period is short); if the expression level value of the MUC22 gene in the lung cancer focus tissue is larger than the judgment threshold value, the prognosis risk of the stage III lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer).

Further, the judgment threshold value is 0.02FPKM.

Eighth: a data processing device or system for assessing the prognostic risk of a stage IV lung adenocarcinoma patient in a non-small cell lung cancer patient.

(J1) A data receiving module; the data receiving module is configured to receive the expression quantity value of MUC22 genes in lung cancer focus tissues of IV-phase lung adenocarcinoma patients in non-small cell lung cancer patients to be detected;

(J2) A data storage module; the data storage module is configured to store a judgment threshold;

(J3) A data comparison module; the data comparison module is configured to receive the expression quantity value of the MUC22 gene in lung cancer focus tissues of the IV-phase lung adenocarcinoma patients in the non-small cell lung cancer patients to be detected from the data receiving module, and call the judgment threshold value from the data storage module to compare with the expression quantity value of the MUC22 gene in lung cancer focus tissues of the IV-phase lung adenocarcinoma patients in the non-small cell lung cancer patients to be detected;

(J4) A judging module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result according to a preset judging condition and output a judging result;

the predetermined determination condition is: if the expression level value of the MUC22 gene in the lung cancer focus tissue is smaller than the judgment threshold value, the prognosis risk of the IV-stage lung adenocarcinoma patient in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer); if the expression level value of the MUC22 gene in the lung cancer focus tissue is larger than the judgment threshold value, the prognosis risk of the stage IV lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is low and/or the survival period is short).

Further, the judgment threshold value is 0.02FPKM.

In an eighth aspect, the invention claims a method of distinguishing or aiding in distinguishing between a lung squamous carcinoma patient and a lung adenocarcinoma patient in a non-small cell lung carcinoma patient.

The method for distinguishing or assisting in distinguishing lung squamous cancer patients from lung adenocarcinoma patients in non-small cell lung cancer patients claimed by the invention can be a method A or a method B:

The method a may comprise the steps of: the expression level of MUC22 gene in lung cancer focus tissue and paracancerous tissue (i.e., normal control tissue paired with lung cancer focus tissue) from a non-small cell lung cancer patient to be tested was detected, respectively, and then whether the non-small cell lung cancer patient to be tested was a lung squamous cell carcinoma patient or a lung adenocarcinoma patient was determined as follows: if the expression level of MUC22 gene in lung cancer focus tissue is lower than that of cancer tissue, the non-small cell lung cancer patient to be detected is or is candidate to be lung squamous cancer patient; if the expression level of MUC22 gene in lung cancer focus tissue is higher than that of cancer tissue, the non-small cell lung cancer patient to be detected is or is candidate to be lung adenocarcinoma patient.

The method B may include the steps of: detecting the expression level of MUC22 gene in the homogeneous sample (i.e. normal control tissue paired with lung cancer focus tissue) from the non-small cell lung cancer patient to be detected and normal healthy control patient, respectively, and determining whether the non-small cell lung cancer patient to be detected is a lung squamous cell carcinoma patient or a lung adenocarcinoma patient according to the following steps: if the expression level of MUC22 gene in the test sample from the non-small cell lung cancer patient to be tested is lower than the expression level of MUC22 gene in the test sample from the normal healthy control, the non-small cell lung cancer patient to be tested is or is candidate to be a lung squamous cell carcinoma patient; if the expression level of MUC22 gene in the test sample from the non-small cell lung cancer patient to be tested is higher than the expression level of MUC22 gene in the test sample from the normal healthy control, the non-small cell lung cancer patient to be tested is or is candidate to be a lung adenocarcinoma patient.

Further, the sample may be cells, tissues, body fluids, secretions (e.g., sputum, broncholavage, etc.), and the like.

In a ninth aspect, the invention claims any one of the following methods:

first kind: a method of assessing the prognostic risk in a non-staged lung squamous carcinoma patient in a non-small cell lung carcinoma patient.

The method may comprise the steps of: detecting the expression level of MUC22 gene in lung cancer focus tissues of non-stage lung squamous cell carcinoma patients among non-small cell lung cancer patients to be detected, and then evaluating the prognosis risk of the non-stage lung squamous cell carcinoma patients among the non-small cell lung cancer patients to be detected according to the following steps: if the expression level of MUC22 gene in the lung cancer focus tissue is lower than 0.02FPKM, the prognosis risk of the non-stage lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is lower and/or the survival period is shorter); if the expression level of MUC22 gene in the focus tissue of lung cancer is higher than 0.02FPKM, the prognosis risk of the patients without stage lung squamous cancer in the patients with non-small cell lung cancer to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer).

Second kind: a method of assessing the prognostic risk in a stage I lung squamous carcinoma patient in a non-small cell lung carcinoma patient.

The method may comprise the steps of: detecting the expression level of MUC22 gene in lung cancer focus tissues of a patient suffering from stage I lung squamous carcinoma in a patient suffering from non-small cell lung cancer to be tested, and then evaluating the prognosis risk of the patient suffering from stage I lung squamous carcinoma in the patient suffering from non-small cell lung cancer to be tested as follows: if the expression level of MUC22 gene in the lung cancer focus tissue is lower than 0.02FPKM, the prognosis risk of the stage I lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is lower and/or the survival period is shorter); if the expression level of MUC22 gene in the focus tissue of lung cancer is higher than 0.02FPKM, the prognosis risk of stage I lung squamous cancer patient in the non-small cell lung cancer patient to be tested is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer).

Third kind: a method of assessing the prognostic risk in a stage III lung squamous carcinoma patient in a non-small cell lung carcinoma patient.

The method may comprise the steps of: detecting the expression level of MUC22 gene in lung cancer focus tissues of stage III lung squamous carcinoma patients in a non-small cell lung cancer patient to be detected, and then evaluating the prognosis risk of the stage III lung squamous carcinoma patients in the non-small cell lung cancer patient to be detected according to the following steps: if the expression level of MUC22 gene in the lung cancer focus tissue is lower than 0.02FPKM, the prognosis risk of the stage III lung squamous cancer patient in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is lower and/or the survival period is shorter); if the expression level of MUC22 gene in the focus tissue of lung cancer is higher than 0.02FPKM, the prognosis risk of the stage III lung squamous carcinoma patient in the non-small cell lung cancer patient to be tested is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer).

Fourth kind: a method of assessing the prognostic risk of a non-staged lung adenocarcinoma patient in a non-small cell lung cancer patient.

The method may comprise the steps of: detecting the expression level of MUC22 gene in lung cancer focus tissue of non-stage lung adenocarcinoma patients among non-small cell lung cancer patients to be tested, and then evaluating the prognosis risk of the non-stage lung adenocarcinoma patients among the non-small cell lung cancer patients to be tested according to the following steps: if the expression level of MUC22 gene in lung cancer focus tissue is lower than 0.02FPKM, the prognosis risk of non-stage lung adenocarcinoma patients in the non-small cell lung cancer patients to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer); if the expression level of MUC22 gene in the focus tissue of lung cancer is higher than 0.02FPKM, the prognosis risk of the patients without stage lung squamous cancer in the patients with non-small cell lung cancer to be detected is relatively high (specifically, the overall survival rate is low and/or the survival period is short).

Fifth: a method of assessing the prognostic risk in a patient with stage I lung adenocarcinoma in a patient with non-small cell lung cancer.

The method may comprise the steps of: detecting the expression level of MUC22 gene in lung cancer focus tissue of a patient suffering from phase I lung adenocarcinoma in a patient suffering from non-small cell lung cancer, and then evaluating the prognostic risk of the patient suffering from phase I lung adenocarcinoma in the patient suffering from non-small cell lung cancer as follows: if the expression level of MUC22 gene in the lung cancer focus tissue is lower than 0.02FPKM, the prognosis risk of the patient with the stage I lung adenocarcinoma in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer); if the expression level of MUC22 gene in the focus tissue of lung cancer is higher than 0.02FPKM, the prognosis risk of stage I lung squamous cancer patient in the non-small cell lung cancer patient to be tested is relatively high (specifically, the overall survival rate is low and/or the survival period is short).

Sixth: a method of assessing the prognostic risk in a stage II lung adenocarcinoma patient in a non-small cell lung cancer patient.

The method may comprise the steps of: detecting the expression level of MUC22 gene in lung cancer focus tissue of phase II lung adenocarcinoma patients in non-small cell lung cancer patients to be detected, and then evaluating the prognosis risk of phase II lung adenocarcinoma patients in the non-small cell lung cancer patients as follows: if the expression level of MUC22 gene in the lung cancer focus tissue is lower than 0.02FPKM, the prognosis risk of the patient with the phase II lung adenocarcinoma in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer); if the expression level of MUC22 gene in the focus tissue of lung cancer is higher than 0.02FPKM, the prognosis risk of stage II lung squamous carcinoma patient in the non-small cell lung cancer patient to be tested is relatively high (specifically, the overall survival rate is low and/or the survival period is short).

Seventh: a method of assessing the prognostic risk in a patient with stage III lung adenocarcinoma in a patient with non-small cell lung cancer.

The method may comprise the steps of: detecting the expression level of MUC22 gene in lung cancer focus tissue of a patient suffering from phase III lung adenocarcinoma among non-small cell lung cancer patients to be tested, and then evaluating the prognosis risk of the patient suffering from phase III lung adenocarcinoma among the non-small cell lung cancer patients as follows: if the expression level of MUC22 gene in the lung cancer focus tissue is lower than 0.02FPKM, the prognosis risk of the patient with the III-stage lung adenocarcinoma in the non-small cell lung cancer patient to be detected is relatively high (specifically, the overall survival rate is lower and/or the survival period is shorter); if the expression level of MUC22 gene in the focus tissue of lung cancer is higher than 0.02FPKM, the prognosis risk of the stage III lung squamous carcinoma patient in the non-small cell lung cancer patient to be tested is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer).

Eighth: a method of assessing the prognostic risk of a stage IV lung adenocarcinoma patient in a non-small cell lung cancer patient.

The method may comprise the steps of: detecting the expression level of MUC22 gene in lung cancer focus tissue of a patient suffering from IV-stage lung adenocarcinoma in a non-small cell lung cancer patient to be tested, and then evaluating the prognosis risk of the patient suffering from IV-stage lung adenocarcinoma in the non-small cell lung cancer patient as follows: if the expression level of MUC22 gene in the lung cancer focus tissue is lower than 0.02FPKM, the prognosis risk of the IV-stage lung adenocarcinoma patient in the non-small cell lung cancer patient to be detected is relatively low (specifically, the overall survival rate is higher and/or the survival period is longer); if the expression level of MUC22 gene in the focus tissue of lung cancer is higher than 0.02FPKM, the prognosis risk of stage IV lung squamous carcinoma patient in the non-small cell lung cancer patient to be tested is relatively high (specifically, the overall survival rate is low and/or the survival period is short).

In the method, the detection of the expression level of the MUC22 gene can be specifically performed by the primer set or the primer set according to the fourth aspect or the kit according to the fifth aspect.

The detection of the expression level of MUC22 gene by RT-qPCR using the primer set or primer set according to the fourth aspect or the kit according to the fifth aspect may be specifically performed as follows:

1) Reverse transcription is carried out to synthesize cDNA of a sample to be detected by adopting the primer pair or the primer pair group in the fourth aspect or the kit in the fifth aspect and simultaneously using Beta-action as a homogenizing reference primer pair, and qPCR amplification is carried out to obtain an amplification product;

2) Interpretation of the results:

observing the specificity of the PCR amplification product through the dissolution profile of qPCR, obtaining Ct value < or=33 through the amplification profile, indicating the presence of MUC22 mRNA expression in the sample; no lysis curve was observed by qPCR or a non-specific lysis curve, no amplification curve, or Ct value > 33 by amplification curve was observed, indicating that MUC22 mRNA was not expressed in the sample. After determining that MUC22 mRNA was expressed in the sample, the amount of MUC22 mRNA expressed in the sample was calculated using the 2 ^-ΔΔCt method.

In a tenth aspect, the invention claims the use of the MUC22 gene or a substance capable of promoting expression of the MUC22 gene in any of the following:

Q1, preparing a product for inhibiting proliferation and/or migration of non-small cell lung cancer cells, or inhibiting proliferation and/or migration of non-small cell lung cancer cells;

q2, preparing a product for inhibiting the growth and/or metastasis of non-small cell lung cancer tumor, or inhibiting the growth and/or metastasis of non-small cell lung cancer tumor.

In an eleventh aspect, the invention claims the use of an agent capable of inhibiting the expression of the MUC22 gene in any of the following:

m1, preparing a non-small cell lung cancer cell model with enhanced proliferation and/or migration capacity;

M2, preparing a non-small cell lung cancer animal model with enhanced tumor growth and/or metastasis capability.

Wherein the non-small cell lung cancer can be lung squamous carcinoma or lung adenocarcinoma.

In a specific embodiment of the present invention, the substance capable of inhibiting the expression of the MUC22 gene is an siRNA for inhibiting the expression of the MUC22 gene, specifically 5'-AAGAGAAGCAGCCACAGGAACCAGA-3' or 5'-UAUAGUAGUCCCUGAGUCUGUGGUG-3'.

The research of the invention finds that MUC22 expression shows difference in non-small cell lung cancer, the expression is abnormally decreased in cells and tissues of lung squamous cell carcinoma, and the expression is abnormally increased in lung adenocarcinoma; the MUC22 gene can inhibit the growth and metastasis of lung cancer cells, is related to tumorigenesis and development, and has important effect; differential expression of MUC22 in lung squamous carcinoma and lung adenocarcinoma patients is predictive of prognosis.

The primer or the kit for detecting the differential expression of the MUC22 gene can specifically detect the transcription level of the MUC22 gene in samples such as cells, tissues, body fluid, secretion and the like, thereby carrying out molecular typing and therapeutic drug evaluation on lung squamous cell carcinoma and lung adenocarcinoma in non-small cell lung cancer. The detection result can provide a basis for the formulation of a treatment scheme of a tumor patient and also provides an evaluation method for the prognosis of the drug treatment of the tumor patient. In addition, the detection method is simple and convenient to operate, accurate and stable, and has clinical application significance.

The invention provides a molecular basis for effectively identifying the function of MUC22 in tumors, and is beneficial to the transformation application of MUC22 genes in the molecular diagnosis and treatment of tumors. The researches find that not only provides a new mechanism for the occurrence and development of tumors, but also provides a new clue for the clinical application of MUC22 in tumor personalized diagnosis and treatment, thus having important scientific value and clinical significance.

Drawings

FIG. 1 shows the RT-qPCR assay of MUC22 gene expression. A is the analysis of MUC22 mRNA expression in non-small cell lung carcinoma cell lines (lung squamous carcinoma: NCI-H1703, NCI-H2170, SK-MES-1 and NCI-H226; lung adenocarcinoma: NCI-H1975, HCC-827, NCI-H1395 and NCI-H522; immortalized human bronchial epithelial cell line BEAS-2B as control). MUC22 mRNA relative expression levels were normalized to Beta-Aactin and presented as mean.+ -. Standard deviation from triplicate experimental data of fold ratio BEAS-2B. B and C are comparisons of MUC22 mRNA in tumor tissue with its paired normal control tissue. B is lung squamous carcinoma (n=24); c is lung adenocarcinoma (n=24). * P <0.01, compared to control group (unpaired t test).

FIG. 2 shows that knocking down MUC22 gene can promote proliferation and migration of lung cancer cells by activating NF- κB pathway. A is RT-qPCR analysis of RNA interference efficiency. SK-MES-1, NCI-H522 and BEAS-2B cells were transfected with siRNA oligonucleotides (siMUC-1 and-2) or RNAi negative control (siNC). The expression of MUC22mRNA was then detected. B is the growth and proliferation of SK-MES-1, NCI-H522 and BEAS-2B cells transfected siMUC s or siNC by MTT method detection. Data are expressed as mean ± standard deviation, < P <0.05, < P <0.01, compared to control. C is the migration ability of the cells tested by a Transwell migration assay. The transfer viability after transfection of SK-MES-1, NCI-H522 and bees-2B cells was represented as mean ± standard deviation in a quantification graph (right graph) by representative images (left graph) or by summarizing three experimental data. D is protein level of key protein molecules I kappa B-alpha, phosphorylated I kappa B-alpha and NF-kappa B p subunit of the NF-kappa B channel by Western blot analysis. Whole cell extracts (top panel), nuclear extracts (middle panel) and cytoplasmic extracts (bottom panel) of SK-MES-1 cells after MUC22 knockout. Beta-actin serves as a cytoplasmic, laminA as a nuclear protein load control.

FIG. 3 is a graph showing the predictive value of MUC22 expression for prognosis of lung squamous carcinoma and lung adenocarcinoma in non-small cell lung carcinoma. The Kaplan-Meier method performed survival analysis on patients with lung squamous carcinoma (left panel) and lung adenocarcinoma (right panel) at different stages. The results were stratified according to the expression pattern of MUC22 in stage (A) free or stage I-IV (stages I, II, III and IV in this order) cancer patients. * P <0.05, < P <0.01, MUC22 low expression compared to MUC22 high expression.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 preparation of MUC22 Gene detection primer and kit

1. Primer design

The MUC22 gene mRNA sequence (SEQ ID No. 1) presented from NCBI was artificially designed with upstream, intermediate and downstream primer pairs, respectively. The target sequence of the target sequence covers +217bp to +5349bp of the mRNA sequence of the MUC22 gene.

Primer pair 1: MUC22 mRNA upstream primer (PCR amplified fragment is 342-580 bit of SEQ ID No.1, size 239 bp)

An upstream primer: 5'-TGGCCTCTACTTCGGCCTTA-3' (SEQ ID No. 2);

a downstream primer: 5'-GGTGGAGGCCACGATAGTTT-3' (SEQ ID No. 3).

Primer pair 2: MUC22 mRNA intermediate primer (PCR amplified fragment is position 217-286 of SEQ ID No.1, size 70 bp)

An upstream primer: 5'-GTTCCTGTGGCTGCTTCTCT-3' (SEQ ID No. 4);

A downstream primer: 5'-GCCTTTTGTGAAGGCTGTGG-3' (SEQ ID No. 5).

Primer pair 3: MUC22 gene downstream primer (PCR amplified fragment is 5178-5349 bit of SEQ ID No.1, size is 172 bp)

An upstream primer: 5'-TCATTTCCCTGGCTGCAGTT-3' (SEQ ID No. 6);

a downstream primer: 5'-CTGTGGAATGTCCCAGAGCC-3' (SEQ ID No. 7).

2. Kit composition

The kit comprises a MUC22 gene splice variant RT-qPCR primer (primer pair 1, primer pair 2 or primer pair 3), an internal reference primer for homogenization, a positive control template, deionized water as a negative control template, a cDNA synthesis reagent and a PCR reaction reagent, wherein the primer pair 1, the primer pair 2 or the primer pair 3 are designed in the step one;

wherein the internal reference primer for homogenization is a primer taking Beta-Aactin as an internal reference,

An upstream primer: 5'-TTAGTTGCGTTACACCCTTTC-3' (SEQ ID No. 8);

A downstream primer: 5'-ACCTTCACCGTTCCAGTTT-3' (SEQ ID No. 9).

The negative control template is deionized water.

EXAMPLE 2 detection of the expression and biological Functions of the MUC22 Gene in lung cancer tissue cells

1. Experimental materials

Lung squamous carcinoma cells tested: NCl-H1703, NCl-H2170, SK-MES-1 and NCI-H226.

Lung adenocarcinoma cells tested: NCI-H1975, HCC-827, NCI-H1395, and NCI-H522.

Test immortalized human bronchial epithelial cell line: BEAS-2B.

All the cells are purchased from a national biomedical experiment cell resource library and are cultured and passaged under normal conditions in a laboratory of an applicant unit.

Test frozen tissue (lung squamous carcinoma and lung adenocarcinoma clinical tissue specimens and corresponding paired paracancerous tissue): specific information is shown in table 1.

TABLE 1 clinical information on non-small cell lung cancer samples

2. Experimental method

1. Cellular RNA extraction and reverse transcription process

(1) The procedure of cell RNA extraction was to select cells in good growth, and Trizol reagent (Invitrogen, inc. USA, cat# 15596026) was added to 1mL/10 ⁶ cells. The tissue RNA extraction procedure is to take out frozen tissue from a liquid nitrogen tank, cut about 200mg, and grind the tissue specimen as a cold mortar. 1mLTrizol reagent was added per 100mg of tissue specimen.

(2) After 10min at room temperature, chloroform extraction was performed after sufficient lysis, and 0.2mL chloroform was added per 1mL Trizol. Shaking vigorously for 15s, standing at room temperature for 5min, centrifuging at 12000g at 4℃for 15min.

(3) The upper colorless liquid after delamination was transferred to a new centrifuge tube, precipitated with pre-chilled isopropanol, 0.5mL isopropanol was added per 1mL Trizol, and left on ice for 20min.12000g, centrifuging at 4 ℃ for 10min, and discarding the supernatant.

(4) The precipitate was washed with pre-chilled 75% ethanol, 1mL Trizol was added to 1mL 75% ethanol, 7500g, centrifuged at 4℃for 5min, and the supernatant discarded. After drying, an appropriate amount of DEPC-H ₂ O was added for dissolution, and 0.8% agarose gel electrophoresis was performed to confirm that the RNA concentration was measured by Nanodrop, and the sample was stored at-80 ℃.

(5) 1.0. Mu.g of RNA was reverse transcribed into cDNA using TRANSSCRIPT II FIRST-STRAND CDNA SYNTHESIS Supermix kit (cat# AH 301-02) manufactured by Beijing full gold Biotechnology Co., ltd.): mu. L Anchored Oligo (dT) 20, 10. Mu.L of 2 XTS Reaction Mix and 1. Mu.L of RT/RI Enzyme Mix were added to 20. Mu.L of DEPC-H ₂ O. The reaction conditions, 42℃30min,85℃5min, and the cDNA obtained by reverse transcription were allowed to stand at-20 ℃.

2、qPCR

(1) QPCR primer sequence:

The RT-qPCR primer set was primer set 1 of example 1.

(2) Reaction system for qPCR amplification

As shown in table 2.

TABLE 2 reaction System for qPCR amplification

Note that: 2 XSYBR-Green: zymo Research, usa, cat No. E2004.

(3) The PCR conditions were as follows:

2min at 50 ℃,10 min at 95 ℃ and 15s at 95 ℃;60 ℃ for 1min; collecting fluorescence in 40 cycles; a dissolution profile was prepared at 95℃for 15s,60℃for 1min,95℃for 15s, and 60℃for 15 s. The experimental result adopts 2 ^-ΔΔCt method to analyze data, and the dissolution curve ensures the specificity of the product.

3. Results and analysis

The results show that:

In non-small cell lung cancer cells, MUC22 mRNA exhibited higher expression levels in lung squamous carcinoma cells and lower expression levels in lung adenocarcinoma cells compared to the immortalized human bronchial epithelial cell line BEAS-2B (FIG. 1A).

In non-small cell lung cancer sample tissues, MUC22 mRNA expression was lower in lung squamous carcinoma than in the paracancerous control (B in FIG. 1) and higher in lung adenocarcinoma than in the paracancerous control (C in FIG. 1).

Example 3 biological function verification of MUC22 as tumor marker

1. Interfering with MUC22 Gene expression in SK-MES-1, NCI-H522 and BEAS-2B cells

Universal negative control, available from guangzhou sharp biotechnology limited, cat No.: siN0000002-1-5.

MUC22 interference sequence 1:5'-AAGAGAAGCAGCCACAGGAACCAGA-3';

MUC22 interference sequence 2:5'-UAUAGUAGUCCCUGAGUCUGUGGUG-3'.

Non-small cell lung cancer cell lines SK-MES-1, NCI-H522 and BEAS-2B were selected for transient transfection. Cells were inoculated into 6-well cell culture plates, and when the cells were grown to 60% confluence, 200. Mu.L of DMEM was added to 20nM of interfering siRNA and empty siRNA (i.e., the general negative control described above), and the mixture was left at room temperature for 5min after homogenization. 200. Mu.L of DMEM was added to 6. Mu.L of Lipofectamine 2000, and the mixture was left at room temperature for 5min after mixing. 200mL Lipofectamine 2000 (Thermo Fisher, inc. of America, cat. No.: 11668019) was added to 200. Mu.L of the interference fragment, and the mixture was left at room temperature for 20 minutes after mixing. After adding 400. Mu.L of the mixture to the cell culture plate and culturing at 37℃for 8 hours in the absence of serum, 10% FBS DMEM was replaced, and after incubation at 37℃for 24 hours, detection was performed by RT-qPCR (the reference gene was β -actin and the detection primer for the objective gene MUC22 was primer set 1 in example 1).

2. Cell biological function experiment

1. MTT (3- [4,5-dimethylthiazol-2-yl ] -2,5-diphenyl-tetrazolium bromide) assay

Performing digestion counting on SK-MES-1, NCI-H522 and BEAS-2B cell lines containing no-load and interference MUC22 constructed in the first step of growing in the logarithmic phase and good growing condition, adjusting the concentration of cell suspension, adding 100 mu L of the cell suspension into 96-well cell culture plates, paving 2000 cells in each well, arranging 8 compound wells, and continuously observing for 4 days; mu.L of MTT (product number: M2128 of sigma Co., USA) was added to each well at a fixed time every day to give a final concentration of 5. Mu.g/mL, and after co-culturing for 4 hours, the medium was discarded. After 4 days, 150. Mu.L of dimethyl sulfoxide (dimethyl sulfoxide, DMSO, sigma Co., USA, cat# D2650) was added to each well, and after shaking for 10min in the dark, the absorbance of each well was measured at OD 490nm of the ELISA, and the data obtained was analyzed.

2. Migration experiment

The SK-MES-1, NCI-H522 and BEAS-2B cell lines containing the interference MUC22 constructed in the first step are respectively digested and counted, and are respectively added into an upper chamber of a Transwell cell (product number: CLS3422 of the company of corning U.S.A.) with a pore size of 8.0 mu m, 200 mu L of cell suspension without serum is added into each upper chamber, 2X 10 ⁴ cells are added into each hole, and 600 mu L of complete culture medium is added into the lower chamber; culturing in a 37 ℃ cell culture box, culturing cells for 24 hours, and carefully cleaning the cells with 1 XPBS for several times; fixing 4% paraformaldehyde for 30min, and cleaning with 1×PBS three times; 0.5% crystal violet staining for 30min, rinsing to colorless, carefully removing upper chamber cells with cotton swab, and counting the number of migrating cells under microscope (100×).

3. Statistical analysis

The experiment was repeated at least three times. The experimental results were all tested using a two-sided t-test, the results being expressed as mean ± standard deviation, p <0.05 representing significant statistical differences and p <0.01 representing very significant statistical differences.

4. Western blot experiment

1. Extraction of cytoplasmic protein

Specific procedures were performed with reference to the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher, U.S. Pat. No. 78833) as follows: when cells were 70% long, cells were collected and washed 2 times with PBS. The cell volume was estimated, 50. Mu.l of cells were added to 250. Mu.l of CEB-A and shaken for 30s and incubated on ice for 10min. Add 15. Mu.l CEB-B and shake for 10s and incubate on ice for 1min. Centrifuge at 1000rpm at 4℃for 15min. The supernatant was transferred to a fresh EP tube and centrifuged at 12000rpm at 4℃for 5min. The supernatant was then transferred to a new EP tube, which was the cytoplasmic protein. After washing the pellet, it was centrifuged at 1000rpm for 5min with 100. Mu.l CEB-A and the supernatant was discarded. 70 μl NEB was added, shaken and incubated on ice for 30min. Centrifuge at 12000rpm for 5min. Transferring the supernatant to a new tube to obtain the nucleoprotein. After 20% glycerol was added to the cytoplasmic protein, the protein was stored at-80 ℃. The cytokinin is preserved at-20deg.C.

2. Western blot analysis

Cells with good growth status were inoculated into 60mm ² dishes. When the cell fusion degree reached 80%, 100. Mu. L RIPA LYSIS Buffer (China Genstar company, cat# E112-01) was added after washing with 1 XPBS. After the cells were sufficiently lysed on ice, the cells were scraped from the culture dish with a cell scraper, transferred to a 1.5ml centrifuge tube, and 1 μl PMSF was added. Centrifuge at 12000rpm for 30min at 4℃and transfer the supernatant to a new 1.5ml centrifuge tube. The product concentration was determined by NanoDrop. Preserving at-20 ℃.

The quantitative sample was subjected to SDS-PAGE electrophoresis at a voltage of 120V by taking 30. Mu.g/well and calculating the amount of the sample, adding the sample to a 5 XSDS gel sample-loading buffer (product number: E153-05 of Genstar Co., ltd., china) and heating at 100℃for 10 min. After electrophoresis, the membrane is put into a wet type electrotransfer instrument for membrane transfer, the current is 350mA, and the membrane transfer time is adjusted according to the molecular weight of the protein. After the transfer was completed, PVDF (Millipore Co., U.S.A., cat# IPVH 00010) membrane was removed. After blocking for 1h with 5% skimmed milk, the primary antibody was incubated: beta-actin (U.S. sigma, cat# A4551), anti-NF-kappa B p (Santa Cruz, cat# sc-8008), anti-IκB-alpha (Ab 32518, cat# ab 32518), anti-p-IκB-alpha (Ab 133462, cat# ab 133462), and Lamin A (Santa Cruz, cat# sc-71481), overnight at 4 ℃. Anti-rabbit horseradish peroxidase-labeled secondary antibody (Ab 6721, cat# of abcam, UK) was added and incubated for 1h, and ECL chemiluminescent kit (Thermo Fisher, USA; cat# 32132) was added for development and developed with Smart Gel ^TM IMAGE ANALYSIS SYSTEM.

3. Results and analysis

1. Knocking down MUC22 gene

The results show that: after transfection with siRNA fragments interfering with MUC22, the expression of MUC22 in SK-MES-1, NCI-H522 and BEAS-2B cells was significantly reduced (FIG. 2A).

2. Effects of knock-down MUC22 on cell growth ability (proliferation ability)

MTT experiments were used to examine the effect of inhibiting MUC22 gene expression on the growth capacity (proliferation capacity) of cell lines. The results show (B in fig. 2) that knockdown MUC22 significantly inhibited the growth capacity (proliferation capacity) of the cells (P < 0.01) compared to the control cell line.

3. Effect of knockdown MUC22 on cell line migration ability

The ability of the cell lines to migrate was further verified by transwell cell migration experiments. The results show (C in fig. 2) that knockdown MUC22 significantly inhibited the migration capacity of the cells (P < 0.01) compared to the control cell line.

The biological function verification experiment of the MUC22 shows that the knock-down MUC22 can obviously inhibit the cell proliferation capacity and migration in the cell lines SK-MES-1 and NCI-H522, shows that the MUC22 gene plays an important role in lung cancer cells, and lays a theoretical foundation for the application of the differential expression of the MUC22 gene to molecular markers for tumor or inflammatory infection diagnosis and prognosis detection.

4. Western blot analysis of protein levels of the NF- κB pathway key protein molecules IκB- α, phosphorylated IκB- α and NF- κ B p65 subunits

As shown in fig. 2D, whole cell extracts (upper panel), nuclear extracts (middle panel) and cytoplasmic extracts (lower panel) of SK-MES-1 cells after MUC22 knockout. Beta-actin serves as a cytoplasmic, laminA as a nuclear protein load control.

Example 4, MUC22 expression predictive value for prognosis of lung squamous carcinoma and lung adenocarcinoma patients in non-small cell lung carcinoma.

In this example, survival analysis was performed on patients with lung squamous carcinoma (left panel) and lung adenocarcinoma (right panel) at different stages using the Kaplan-Meier method.

Raw data were from human protein maps (https:// www.proteinatlas.org). The TCGA RNA sequencing raw data is downloaded, samples are analyzed and divided into MUC22 low expression and MUC22 high expression groups (taking the median as a cutoff value, the cutoff value is 0.02 FPKM) in different disease stages, and the low expression and high expression groups are further compared with clinical data (see https:// www.proteinatlas.org in detail) related to tumor patients (the sample capacity is lung squamous carcinoma n=494 and lung adenocarcinoma n=499).

The Kaplan-Meier method performed survival analysis on cancer patients with different stages of lung squamous carcinoma (left panel in fig. 3) and lung adenocarcinoma (right panel in fig. 3). The results were stratified according to the expression pattern of MUC22 in stage-free (A in FIG. 3) or stage I-IV (B-E in FIG. 3) cancer patients. Calculating a risk Ratio (HR), wherein HR is the Ratio of the risk ratios of the MUC22 high expression group to the risk Ratio of the MUC22 low expression group, and if the P value is obviously different, and the HR value is <1, the prognosis of the patient with MUC22 high expression is good; if there is a significant difference in P values and HR values >1, a patient with high MUC22 expression is indicated to have a poor prognosis.

The results show that:

For non-stage lung squamous carcinoma patients among non-small cell lung carcinoma patients, the overall survival rate of the MUC22 high-expression group in lung cancer lesion tissue was significantly higher than that of the low-expression group (P value <0.01, risk ratio (HR) = 0.6039, 95% confidence interval= 0.3828-0.9527).

For stage I lung squamous carcinoma patients in non-small cell lung carcinoma patients, the overall survival rate of the MUC22 high-expression group in lung cancer lesion tissue was significantly higher than that of the low-expression group (P value <0.05, risk ratio (HR) = 0.5794, 95% confidence interval= 0.2739-1.226).

For stage II lung squamous carcinoma patients in non-small cell lung carcinoma patients, the overall survival of MUC22 high-expression groups in lung cancer lesion tissues was not significantly different from the low-expression groups (P-value >0.05, risk ratio (HR) = 0.9459, 95% confidence interval= 0.4421-2.024).

For stage III lung squamous carcinoma patients in non-small cell lung carcinoma patients, the overall survival rate of the MUC22 high-expression group in lung cancer lesion tissue was significantly higher than that of the low-expression group (P value <0.05, risk ratio (HR) = 0.4136, 95% confidence interval= 0.1157-1.478).

For stage IV lung squamous carcinoma patients in non-small cell lung carcinoma patients, the overall survival of MUC22 high-expression groups in lung cancer lesion tissues was not significantly different from the low-expression groups (P-value >0.05, risk ratio (HR) = 0.4778, 95% confidence interval= 0.02852-8.005).

For non-staged lung adenocarcinoma patients in non-small cell lung cancer patients, the overall survival rate of the MUC22 high-expression group in lung cancer lesion tissue was significantly lower than that of the low-expression group (P value <0.05, risk ratio (HR) =1.160, 95% confidence interval= 0.8676-1.551).

For stage I lung adenocarcinoma patients in non-small cell lung cancer patients, the overall survival rate of the MUC22 high-expression group in lung cancer lesion tissue was significantly lower than that of the low-expression group (P value <0.05, risk ratio (HR) =1.367, 95% confidence interval= 0.8431-2.217).

For phase II lung adenocarcinoma patients in non-small cell lung carcinoma patients, the overall survival rate of the MUC22 high-expression group in lung cancer lesion tissue was significantly lower than that of the low-expression group (P value <0.05, risk ratio (HR) =1.409, 95% confidence interval= 0.8115-2.446).

For stage III lung adenocarcinoma patients in non-small cell lung carcinoma patients, the overall survival rate of the MUC22 high-expression group in lung cancer lesion tissue was significantly higher than that of the low-expression group (P value <0.01, risk ratio (HR) = 0.5811, 95% confidence interval= 0.3167-1.066).

For stage IV lung adenocarcinoma patients in non-small cell lung carcinoma patients, the overall survival rate of the MUC22 high-expression group in lung cancer lesion tissue was significantly lower than that of the low-expression group (P value <0.05, risk ratio (HR) =1.198, 95% confidence interval= 0.4239-3.383).

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

<110> Beijing chest hospital affiliated to university of capital medical science; beijing city tuberculosis chest tumor institute

<120> A molecular marker for tumor molecular typing and therapeutic drug evaluation, and detection primer and kit therefor

<130> GNCLN210086

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 5970

<212> DNA

<213> Artificial sequence

<400> 1

acaaaaacta gctggcgtgg tggcaggcac ctgtagtccc agctacgtgg gaagctgagg 60

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agagatgaga agaggaaata tctctcctgc tttctggttc ctgtggctgc ttctctttgg 240

acttctggga cccagctctg agaataccac agccttcaca aaaggctccg acaccaccac 300

agcctccatc acaggctctg agaccaccat ggcctccacc atggcctcta cttcggcctt 360

aactacaggc tctaagatca ccacagactc taccacaggc tctgagacaa cctcagcctc 420

caccatggct tctactgcag ccttcaccac aggctctgag accaacacgg cctccaccac 480

agactcaggg actactatag cctccactag gaccttcacc acaggctctg acacaaccac 540

aggctccact gcaggctctg aaactatcgt ggcctccacc acagtctctg ggaccacaac 600

aacctttact atagcctcca ctacagtccc tgagactacc atggcctcca gcacaacctc 660

cactgcaggc tctgagaaaa cgatggcctc ctccataatt tctgagacca ccatggcctc 720

caccacaggc tctgagactg ccacagtctc taccacaggc tctgagacca ccaccacctc 780

cactgcaagc tctgaggcca ctaaagtctc taccacaggc tctgaaacca ccacagcatc 840

tactgcaggt tctgagacca ccactacctc cacctccatg gcaggctctg aggccaccac 900

aacctcaact gcagactcca aggtgatcac ggcatccagc atgagctctg agaccactgt 960

ggcccccgct gcaggctcta acaccaccac agcctctacc acaggctctg agaccactac 1020

aatcctgatt aaagcctctg agaccaccac agcctctaca gcaggttctg agaccaccac 1080

cccctccccc acaggctctc agaccaccat agtctctatt tcaggttctg agatcaccac 1140

cacctctacg gcaggatccg agaacaccac agtctctagt gcaggctctg ggaccaccac 1200

agcttctatg gcaggctctg agaccaccgt ctccactgca ggctctgaga ccactacagt 1260

ctctatcaca ggcactgaga ccaccatggt ctctgccatg ggctcagaga ccaccacaaa 1320

ctctactaca agctctgaga ccaccgtcac ctctactgca ggctctgaga ccaccacagt 1380

ctccaccgtg ggctctgaga ccaccacagc ctatactgca gattctgaga ccactgcagc 1440

ctctaccaca ggctctgaga tgaccacagt cttcactgca ggctcggaaa ccatcacacc 1500

ctctactgca ggctcagaga ccaccacagt ctctactgca ggctctgaga ccactacagt 1560

ctccaccaca ggctctgaga ccacaacagc ctctactgca cattctgaga cgactgcagc 1620

ctccaccatg ggctctgaga ccaccaaagt ctcaactgca ggctctgaga ccacagtctc 1680

cactgcaggc tctgagacca ctgcagcctc tactgaagat tctgaaacca acacagcatt 1740

tactgaagat tctaagacta ccacagcctc tactacaggg tttgagacaa ccgcagcctc 1800

tactacaggc tctgagccta ccatggcatc caccatgggc tctgagacca ctatggcctc 1860

taccataggc cctgagacca ccaaggtctc cactgcaagc tctgaggtga ccacagtctt 1920

tgctgcaggc tctgagacaa tcagagcctc taccgtaggc tctgagacca ccacagtctc 1980

taccacaggc tctgagacca ccacagcctc catcatgggc tctgagacca gcacagattc 2040

taccacaggc tctgagacca ccacagcctc tactgaaggc tctgagacca ccacagcttc 2100

cactgaaggc tctgaggcca ctacagtctc caccacaggc tctgagacca ctacagtttc 2160

tatcacagac tcagagacca ccaccacctg tactgaaggc tctgagatga ctgcagtctc 2220

caccacagtc tttgagacca ctacagcctc tactgaaggc tctgagatca caatagcctc 2280

tacttcagac tctgagacca ccacagcttc tactgaaggt tctgagacca ctacagtcac 2340

taccgcaggc tctgagacca aaacagccta tactacaggc tctgagacca ccacagcctc 2400

taatacaggc ttggagacca ccacagtctt taccataggc tctgacacca ccacagcctc 2460

tactgaaggc tctgagacca ctgcagtctc tgccacaggc tctgagatga ccacagtctc 2520

tactgaaggc tctgagaaca ctacagtctc caccacaggc tctgagacca ctacagtttc 2580

caccacaggc ttggagacca ccaccacttc cactgaaggc tctgagatga ctacagtctc 2640

caccacaggt gctgagacca ccacagactc tactgaaggc tctgggacca ctgcagcctc 2700

cactgcaggc tctgagacca ccacagtctc tactgcagat tctgagaaca ccacagcatc 2760

tactgcagat tctgagacca cctcagcctc tactacaggc tctgagacca ccacagcctc 2820

tactacaagc tctgagacca ccacagcctc tactgaaggc tctgagacca ctacagtctc 2880

caccacagac tctgagacca ccatggtctc taccacaggc tctgagagga ccatcacctc 2940

tactgaaggc tctgagacca ctacagtatc tgccacaggc tctgagacca cagtctctac 3000

tgaaggctct gggaccacta cagtctccat cacaggctct gagaccacta aagtttctac 3060

cacaggttca gagaccacca ccacttctac tgaaggctct gagattacta cagcctccat 3120

cacaggctct gagaccacca cagcctctac tgaaggctcc gagaccacca cagcctctac 3180

tgaaggctcc gagaccacct cagcctctac tacaggctct gagaccacca cagcctctac 3240

tacaagctct gagaccacca tggcatccat catgggctct gagaccacta tggcctctac 3300

cataggctct gagaccacca aggtctccac tgcaagctct aaaatgacca cagtcttcac 3360

tgaaaactct gagaccacca tagcctctac cacagcctct gagaccacca cagtctccac 3420

tgcaggctct gagaccatcc cagcctctac agcaggctct gagaccacca ccaccacctc 3480

tactgaaggc tctgagacca ctacagcctc tactgaaggc tctgagacca ccacagcctc 3540

tactgaaagc tctgagacca ctacagccac taccataggc tctgagacca ccacagcctc 3600

tactgaaggc tctgagacta ccaccacctc tactgaaggc tctgagacca ccacagcctc 3660

tactgaaggc tctgagatca ctacagtttc taccacaggc tctgagacca ccacagcctc 3720

tactgaaggc tctgagacca ccacagcctc tactgaaggc tctgagctca ctacagtttc 3780

taccacaggc tctgagacca tcacagtctc tgctgaaggc tctgagacca ctacagtcac 3840

tactatgggc tctgagacca ccacggcctc tactgcaggc tcagagacca ccacagtctc 3900

tactgcaggc tctgagacca ccacagcctc tattgaaggc tctgagacca ctacagtctc 3960

ctccacaggc tctgagacca ccacagtctc taccacaggc actgagacta ccatcacctc 4020

tactgaaggt tcagagacca ctacagtcac tactgcaggt tctgagacca cagcagtcta 4080

taccacaggc tctgagacta ccaccacctc tactgaaggc tctgagacaa ccacagtctc 4140

taccacgggc tctgagacca ccacagcctc taccgcagat ttggagacca ccacagtctc 4200

cacctcaggc tctgggacca ccacagcctc taccgcaggc tctgagacca caacagtcta 4260

tatcacaggc tctaagacta ccaccgcctc tactgaaggc tctgaggcca ctacagtttc 4320

taccactagc tctgagacca ccacagcctc taccacaggc tctgagatga ctacagtctt 4380

taccacagtc tctgagacca ccacagtctc taccataggc tctgaggcca ccacatcctc 4440

tgctgcaggc tctgaggcca ccaccacctc tactgaaggc tctgagacca ccacagcctc 4500

cactgcaggc tctgagacca ccacagcctc cactgcaggc tctgagacca ccacagcctc 4560

cacttcaggc tctgagacca acacagcctg taccacaggt tctgagacct ccacaccctc 4620

cagtgcaggc tctgagacca acactgcctt catcataggc tctgagagca ccatagcttc 4680

cactgcaagc ttggagccca ctgcaacttc cctcacaggc tctgagacca ccacagtctc 4740

tatcacagct tctggggcca ctgcagcctc caccactgtc tcttccacca cgtttgtact 4800

caccaaggcc actgacgttt ctatccagcc catcaccaac acacctatgt caggcaccag 4860

aaccactgga accagactca ctgcctccag ctctgtcacc atggcccctg gaatggactt 4920

cacggcctct gctgccagcc atactgtgcc aggaatagtc ttaaacacct ctggcctggg 4980

tacatccact atgggagcat catctaccac ctcagcccac ggcgtcagga ccaccacagg 5040

atccacccgt gagccaacca gcagcacctt ccaggaaaca ggcccggtgt ccatgggcac 5100

aaacacagtt agcatgagcc acacacccac aaacgtgatc aaaccaagtg gatatttaca 5160

gccctgggct atcatcctca tttccctggc tgcagttgtg gctgctgttg gattgtcagt 5220

aggactgagt ttttgtctga gaaacctttt cttccccctg agatattgtg gtatttatta 5280

cccccatggc cacagccaca gccttggtct ggacctgaac ttgggcctgg gctctgggac 5340

attccacagc ctgggaaatg cactggttca tggaggagaa cttgaaatgg gacatggagg 5400

aacacacggc tttggatatg gagtgggcca tggactgagc cacatccatg gagatggcta 5460

cggagtgaat catggcgggc attatggaca tggaggaggc cactgaggac accatggagt 5520

ggatcacaga gggagccacc aaggaggcca cggcaggaca agatggctgt ggccatagat 5580

tgggtatcaa aacatattat gggtgggagg gggtcatgga ggagaaaaaa ataatgatca 5640

tgaaataatt aaaatggagc ataggaagct tcccaggatg tgatccatgg agatggacat 5700

ggactaggtc aagaaaagaa ccagcaaaag gacctcagag actttgactg gcttggaggg 5760

gacttcaagt caaagcttct gtgagttttt cctgagtctc agcctctgtt gtggggagtc 5820

acgacaacca ccctcaggac atcttctctc ccatttcccg ccacatcagg gtcaacgttt 5880

ctcatccctg tgtttcctca tggtgctata aatattacca agacatgtct aagaaacaaa 5940

agcacataat gaatgtatta tcagggccac 5970

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

tggcctctac ttcggcctta 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<400> 3

ggtggaggcc acgatagttt 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

gttcctgtgg ctgcttctct 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

gccttttgtg aaggctgtgg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

tcatttccct ggctgcagtt 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

ctgtggaatg tcccagagcc 20

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<400> 8

ttagttgcgt tacacccttt c 21

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence

<400> 9

accttcaccg ttccagttt 19

Claims

1. Application of primer pair for detecting MUC22 gene expression amount in preparation of products for molecular typing of lung squamous carcinoma and lung adenocarcinoma in non-small cell lung cancer.

2. Application of primer pair for detecting MUC22 gene expression quantity in preparation of products for diagnosing non-small cell lung cancer; the non-small cell lung cancer is lung squamous carcinoma or lung adenocarcinoma.

3. Use according to claim 1 or 2, characterized in that: the primer pair is a primer pair A, a primer pair B or a primer pair C; the primer pair A consists of two single-stranded DNA shown in SEQ ID No.2 and SEQ ID No. 3; the primer pair B consists of two single-stranded DNA shown in SEQ ID No.4 and SEQ ID No. 5; the primer pair C consists of two single-stranded DNAs shown as SEQ ID No.6 and SEQ ID No. 7.