CN114480339B

CN114480339B - Application of PINK1 as marker for predicting sensitivity of lung adenocarcinoma to MAPK inhibitor and recurrence of lung adenocarcinoma

Info

Publication number: CN114480339B
Application number: CN202210037492.6A
Authority: CN
Inventors: 尹东; 李贇; 陈恒星
Original assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2024-05-14
Anticipated expiration: 2042-01-13
Also published as: CN114480339A

Abstract

The invention discloses an application of PINK1 as a marker for predicting susceptibility of lung adenocarcinoma to MAPK inhibitor and recurrence of lung adenocarcinoma. According to the invention, through experiments, the increase of the expression of the PINK1 in tumors indicates that a lung adenocarcinoma patient has higher tumor dormancy and recurrence risk, so that dormancy and recurrence of the lung adenocarcinoma can be predicted by detecting the expression level of the PINK1 in the lung adenocarcinoma patient, the blank of a biomarker for lung adenocarcinoma dormancy and recurrence in the prior art can be made up, and the expression level of the PINK1 can be used as a judgment standard for lung adenocarcinoma dormancy and recurrence according to the expression level of the PINK1, and can be used for developing a lung adenocarcinoma dormancy and recurrence prediction kit. In addition, for lung adenocarcinoma patients receiving MAPK pathway inhibitors, the increased expression of PINK1 prompts the increased risk of drug resistance and recurrence after treatment of the patients, which is favorable for clinically taking intervention measures in advance and preventing tumor recurrence, and provides a new strategy and thought for clinically treating lung adenocarcinoma.

Description

Application of PINK1 as marker for predicting sensitivity of lung adenocarcinoma to MAPK inhibitor and recurrence of lung adenocarcinoma

Technical Field

The invention relates to the fields of biomedical technology and biological medicine, in particular to application of PINK1 as a marker for predicting susceptibility of lung adenocarcinoma to MAPK inhibitor and recurrence of lung adenocarcinoma.

Background

Lung cancer is one of the common tumors that endanger human health, and the mortality rate of lung cancer is high and the first place of malignant tumor. Non-small cell lung cancer accounts for 80% -85% of all lung cancers, with lung adenocarcinoma being the most common pathological tissue type in non-small cell lung cancer. The treatment of lung cancer comprises surgical excision, radiotherapy and chemotherapy and targeted treatment. Even though the means of treatment continue to advance, the survival rate of lung adenocarcinoma patients for 5 years is still low. In patients with early lung adenocarcinoma with surgically complete tumor resection, about 30% experience tumor recurrence. In addition, most lung adenocarcinoma patients are advanced at diagnosis, and the treatment strategies of such patients are mainly chemotherapy and targeted therapy. 30% of patients with advanced lung adenocarcinoma develop tumor resistance and recurrence after receiving chemotherapy and targeted therapy. Therefore, tumor recurrence is an important cause of lung adenocarcinoma treatment failure and poor prognosis, and in order to overcome lung adenocarcinoma drug resistance and recurrence, it is highly desirable to develop accurate and specific biomarkers and therapeutic targets to aid in predicting the risk of lung adenocarcinoma drug resistance and recurrence.

Tumor dormancy is a major cause of recurrence. Tumor dormancy is the time that tumor cells enter a state resembling hibernation. When a tumor cell is in a dormant state, meaning that it is no longer dividing, can survive for years in vivo, is difficult to detect, and is no longer sensitive to conventional treatment methods. Under certain conditions, tumor cells "wake up" from a dormant state, resume proliferation, and cause tumor recurrence. Dormancy and reactivation of tumors are key factors in tumor resistance and recurrence. It is not clear how tumor cells go to dormancy and remain dormant. Some studies have shown that metabolic reprogramming activity is a major feature of dormant tumor cells. Since the 1952's warburg, mitochondrial respiratory function impairment in tumors is a prerequisite for malignant transformation of cells, with aerobic glycolysis and mitochondrial dysfunction being widely accepted as markers for tumors. However, recent studies have found that metabolic flexibility can meet different demands at various stages of tumor development, and mitochondrial function plays a vital role in a specific stage of tumorigenic development. In dormant tumor cells, mitochondrial function is particularly important. Many studies have demonstrated that mitochondria support survival of dormant cells from aspects of cellular energy metabolism, maintenance of redox homeostasis, regulation of apoptosis, etc. Healthy mitochondrial networks are the basis for tumor cell dormancy and survival.

There is a set of conserved mitochondrial quality control systems within cells, including mitochondrial synthesis, mitochondrial dynamics, mitochondrial autophagy, ROS monitoring, etc., where mitochondrial autophagy is the central link in mitochondrial quality control. When cells are subjected to harsh environments, such as starvation or chemotherapy, they can cause damage to the mitochondria, where they initiate autophagy, phagocytose and degrade the damaged mitochondria via the lysosomal pathway to maintain a functional mitochondrial network and cell homeostasis. A large number of researches show that under the treatment of anti-tumor drugs, the mitochondrial autophagy promotes the survival of tumor cells and enhances the drug resistance of the tumor cells. In mammals, multiple pathways have been found to be involved in mitochondrial autophagy, with mitochondrial autophagy mediated by PINK1 being the most classical. Pink1 is a mitochondrial outer membrane protein with serine/threonine protein kinase activity and can act as a molecular receptor for damaged mitochondria. Normally, PINK1 is transported to the inner mitochondrial membrane and immediately degraded; at mitochondrial dysfunction, PINK1 is stabilized on the mitochondrial outer membrane, recruiting effector proteins of other mitochondrial outer membranes to undergo a series of phosphorylation and ubiquitination events, ultimately leading to the encapsulation of mitochondria by acidic vesicles and transport to lysosomal degradation. The cell eliminates damaged mitochondria through PINK1 mediated mitochondrial autophagy, maintains mitochondrial quality, enhances oxidative phosphorylation, and is further beneficial to oxygen utilization and energy synthesis.

The MAPK pathway genes, including RAS, RAF, MEK, ERK, present mutations in about 40% of lung adenocarcinoma patients and drive progression of lung adenocarcinoma. Patients carrying such mutations have rapid tumor progression, poor survival prognosis, and are often insensitive to traditional chemotherapy. Currently, a number of MAPK pathway inhibitors are used to treat lung adenocarcinoma patients, such as AMG 510, a drug targeting KRAS G12C muteins, dabrafenib He Weimo, a RAF protein, and trimetinib, a MEK protein. These inhibitors of MAPK pathway proteins improve patient survival, but some patients relapse after treatment with MAPK pathway inhibitors, suggesting that MAPK pathway inhibitors may cause tumor dormancy. MAPK pathway inhibitor resistance caused by tumor dormancy has become a key factor limiting clinical application and poor patient survival prognosis, so in order to overcome and reverse the tolerance of lung adenocarcinoma to MAPK pathway inhibitors, accurate, sensitive and specific biomarkers are needed to assist in judging whether lung adenocarcinoma is resistant to MAPK pathway inhibitors as early as possible, guiding clinical medication so as to improve clinical efficacy and patient prognosis.

Currently, there is no reference standard for dormancy determination of lung adenocarcinoma, nor is there an indicator of specificity for lung adenocarcinoma dormancy and recurrence. Therefore, the dormancy of the lung adenocarcinoma is judged so as to select a proper treatment scheme, the dormant tumor cells are thoroughly killed, the recurrence risk of patients is obviously reduced, and the survival rate of the lung adenocarcinoma patients is improved. Furthermore, whether drug-induced tumor dormancy results in lung adenocarcinoma patients resistant to MAPK pathway inhibitors has not been studied. The method is deeply and comprehensively researched to play an important guiding role in reversing the drug resistance of the lung adenocarcinoma MAPK pathway inhibitor.

In order to judge dormancy and recurrence of lung adenocarcinoma as early as possible, and overcome and reverse tolerance of lung adenocarcinoma therapeutic drugs, accurate, sensitive and highly specific biomarkers are needed to assist in predicting risk of lung adenocarcinoma dormancy and recurrence so as to select an optimal therapeutic scheme and improve survival rate of patients.

Disclosure of Invention

The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide the application of PINK1 in preparing a product for regulating and controlling the sensitivity of lung adenocarcinoma to MAPK inhibitors.

It is another object of the present invention to provide the use of an agent for detecting the level of PINK1 expression in the preparation of a product for predicting the susceptibility of lung adenocarcinoma to MAPK inhibitors.

It is still another object of the present invention to provide the use of an agent for detecting the level of Pink1 expression in the preparation of a product for predicting tumor dormancy and recurrence in a patient suffering from lung adenocarcinoma.

The aim of the invention is achieved by the following technical scheme:

application of PINK1 in preparation of products for regulating and controlling sensitivity of lung adenocarcinoma to MAPK inhibitors.

The PINK1 is a mitochondrial outer membrane protein with serine/threonine protein kinase activity, and the amino acid sequence of the mitochondrial outer membrane protein is shown in SEQ ID NO. 1:

MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVGLGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAGPCGRAVFLAFGLGLGLIEEKQAESRRAVSACQEIQAIFTQKSKPGPDPLDTRRLQGFRLEEYLIGQSIGKGCSAAVYEATMPTLPQNLEVTKSTGLLPGRGPGTSAPGEGQERAPGAPAFPLAIKMMWNISAGSSSEAILNTMSQELVPASRVALAGEYGAVTYRKSKRGPKQLAPHPNIIRVLRAFTSSVPLLPGALVDYPDVLPSRLHPEGLGHGRTLFLVMKNYPCTLRQYLCVNTPSPRLAAMMLLQLLEGVDHLVQQGIAHRDLKSDNILVELDPDGCPWLVIADFGCCLADESIGLQLPFSSWYVDRGGNGCLMAPEVSTARPGPRAVIDYSKADAWAVGAIAYEIFGLVNPFYGQGKAHLESRSYQEAQLPALPESVPPDVRQLVRALLQREASKRPSARVAANVLHLSLWGEHILALKNLKLDKMVGWLLQQSAATLLANRLTEKCCVETKMKMLFLANLECETLCQAALLLCSWRAAL.

The regulation and control are realized by the following modes:

(1) Reducing sensitivity of lung adenocarcinoma to MAPK inhibitors by overexpressing the PINK1 gene;

(2) Inhibiting the expression of the PINK1 gene increases the susceptibility of lung adenocarcinoma to MAPK inhibitors.

The over-expression of the PINK1 gene in the mode (1) is realized by the following steps: connecting the PINK1 gene to an over-expression framework vector, and constructing to obtain a plasmid over-expressing the PINK1 gene; and then, carrying out lentivirus packaging on the plasmid over-expressing the PINK1 by using a lentivirus packaging system, and screening to obtain a cell strain over-expressing the PINK 1.

The over-expression framework vector is preferably pLVX-DsRed-Monomer-N1 vector.

The plasmids used for the lentiviral packaging are pMD2.G plasmid and psPAX. Mu.m 2 plasmid.

The method of inhibiting the expression of the PINK1 gene described in the above-mentioned mode (2) is carried out by using an agent for inhibiting PINK1 or knocking down the PINK1 gene.

The target sequence of the knockdown PINK1 gene is any one of the following sequences:

shPINK1#1:5′-TTTCCACACAACACTTCTCTGT-3′；

shPINK1#2:5′-TTCCACACAACACTTCTCTGTG-3′。

The agent for inhibiting PINK1 comprises an agent for reducing the expression or activity of PINK1, such as antisense nucleic acid combined with PINK1mRNA, a substance combined with PINK1 protein, and an agent for inhibiting autophagy of downstream mitochondria of PINK1, such as a medicament for inhibiting lysosomal acidification; preferably an agent that inhibits PINK 1-mediated mitochondrial autophagy; more preferably hydroxychloroquine.

The PINK1 gene knockdown is realized through the following steps: connecting the sequence of knocking down the PINK1 to an expression vector, and constructing a plasmid for knocking down the PINK 1; and then, carrying out lentivirus packaging on the PINK1 knockdown plasmid by using a lentivirus packaging system, and screening to obtain the PINK1 knockdown cell strain.

The expression vector is preferably a plko.1 vector.

The MAPK inhibitor comprises at least one of KRAS G12C inhibitor, RAF inhibitor and MEK inhibitor; preferably at least one of AMG 510 (KRAS G12C inhibitor), dapafinib (RAF inhibitor), vitamin Mo Feini (RAF inhibitor) and trametinib (MEK inhibitor); more preferably, it is trimetinib.

The application of the reagent for detecting the expression level of the PINK1 in preparing the product for predicting the sensitivity of the lung adenocarcinoma to the MAPK inhibitor can be used for preparing the biomarker for predicting the sensitivity of the lung adenocarcinoma to the MAPK inhibitor by the PINK 1.

The reagent for detecting the PINK1 expression level comprises a reagent capable of quantitatively detecting PINK1 protein in a sample, such as a specific antibody combined with the PINK1 protein, goat serum, horseradish peroxidase-labeled goat anti-mouse/rabbit IgG polymer, 3-diaminobenzidine and the like.

The detection sample can be a tissue slice prepared by fixing, embedding and slicing tumor tissues of a lung adenocarcinoma patient.

The specific antibody binding to the PINK1 protein can be obtained by methods well known to those skilled in the art, for example, preparing as an antigen a mammalian cell expression vector retaining the polypeptide of the whole or part of the target protein or integrating the polynucleotide encoding them; after immunizing an animal with an antigen, obtaining immune cells from the immunized animal and fusing myeloma cells to obtain hybridomas, then collecting antibodies from the hybridoma culture, and finally obtaining monoclonal antibodies against the molecular marker protein by subjecting the obtained antibodies to antigen-specific purification using the molecular marker protein or a portion thereof used as an antigen.

Furthermore, the specific antibody combined with the PINK1 protein can be a polyclonal antibody, and the monoclonal antibody is prepared by the following method: the antibody fragment can be obtained by immunizing an animal with the same antigen as described above, collecting a blood sample from the immunized animal, separating serum from the blood, and then performing antigen-specific purification on the serum using the above antigen, by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.

Still further, binding of the marker to the antibody or fragment thereof may be carried out by methods commonly known in the art; for example, proteins or peptides may be fluorescently labeled as follows: washing protein or peptide with phosphate buffer, adding dye prepared with DMSO, buffer, etc., mixing the solutions, and standing at room temperature for 10 min; in addition, the label may be a commercial peroxidase labeling kit such as peroxidase labeling kit-NH 2; for proper labeling, a suitable instrument may be used to detect the labeled antibody or fragment thereof.

The PINK1 is highly expressed, and lung adenocarcinoma is insensitive to MAPK inhibitors, namely lung adenocarcinoma cells highly expressing the PINK1 are more resistant to MAPK inhibitors; accordingly, PINK1 is under-expressed and lung adenocarcinoma is sensitive to MAPK inhibitors.

The products include kits and the like.

Application of PINK1 in preparing products for regulating and controlling tumor dormancy and recurrence of lung adenocarcinoma patients.

The regulation and control are realized by the following modes:

(I) Through over-expression of the PINK1 gene, PINK1 promotes lung adenocarcinoma cells to enter dormancy by promoting mitochondrial function;

(II) inhibiting recurrence of lung adenocarcinoma following MAPK inhibitor treatment by knocking down the PINK1 gene.

The application of the reagent for detecting the expression level of the PINK1 in preparing a product for predicting the occurrence of tumor dormancy and recurrence of a lung adenocarcinoma patient can be used as a judgment standard of the lung adenocarcinoma dormancy and recurrence, and the expression level of the PINK1 can be used as a biomarker for preparing a prognosis preparation for predicting the lung adenocarcinoma patient and for predicting the risk of the lung adenocarcinoma dormancy and recurrence; therefore, the judgment can be made according to the expression level of PINK1 in lung adenocarcinoma tissues, and when the expression of PINK1 is obviously increased, the patient is prompted to have high risk of dormant tumor cells in the body and larger recurrence risk after MAPK inhibitor is used.

The products include kits and the like.

Use of MAPK inhibitor and PINK1 mediated mitophagy inhibitor in combination for the manufacture of a medicament for the prevention and treatment of lung adenocarcinoma.

The PINK1 mediated mitochondrial autophagy inhibitor is preferably hydroxychloroquine.

The application of the combination of the MAPK inhibitor and the PINK1 mediated mitochondrial autophagy inhibitor in preparing the product for preventing tumor dormancy and recurrence of lung adenocarcinoma patients can prevent the lung adenocarcinoma patients from tumor dormancy and recurrence after receiving the treatment of the MAPK inhibitor by using the PINK1 mediated mitochondrial autophagy inhibitor, so that the treatment effect of the MAPK inhibitor is improved.

Application of an agent for inhibiting PINK1 in preparing a medicament (sensitizer) for increasing sensitivity of lung adenocarcinoma to MAPK inhibitors, wherein the agent for inhibiting PINK1 can improve the curative effect of MAPK channel inhibitor treatment.

The agent for inhibiting PINK1 comprises an agent for reducing the expression or activity of PINK1, such as antisense nucleic acid combined with PINK1mRNA, a substance combined with PINK1 protein, and an agent for inhibiting autophagy of downstream mitochondria of PINK1, such as a medicament for inhibiting lysosomal acidification; preferably an agent that inhibits PINK 1-mediated mitochondrial autophagy; more preferably hydroxychloroquine, has good safety and effectiveness.

The medicament of the present invention may be prepared into various dosage forms as required, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.

The dosage of the drug of the present invention is not limited as long as the desired effect is obtained, and can be appropriately determined depending on symptoms, sex, age, and the like.

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

1. The invention discloses application of PINK1 as a biomarker for predicting dormancy and recurrence of lung adenocarcinoma, and experiments show that the increase of the expression of PINK1 in tumors prompts a lung adenocarcinoma patient to have higher tumor dormancy and recurrence risk, so that the dormancy and recurrence of the lung adenocarcinoma can be predicted by detecting the expression level of PINK1 in the lung adenocarcinoma patient, so as to make up for the blank of the biomarker for the dormancy and recurrence of the lung adenocarcinoma in the prior art, and the biomarker can be used as a judgment standard of the dormancy and recurrence of the lung adenocarcinoma according to the expression level of PINK1 to develop a lung adenocarcinoma dormancy and recurrence prediction kit; in addition, for lung adenocarcinoma patients receiving MAPK pathway inhibitors, the increased expression of PINK1 prompts the increased risk of drug resistance and recurrence after treatment of the patients, which is favorable for clinically taking intervention measures in advance and preventing tumor recurrence, and provides a new strategy and thought for clinically treating lung adenocarcinoma.

2. For a lung adenocarcinoma patient applicable to the MAPK pathway inhibitor, by detecting the expression level of PINK1 in lung adenocarcinoma tissues, when the expression of PINK1 is obviously increased, the patient is prompted to have larger risk of drug resistance and recurrence after using the MAPK inhibitor, and drug resistance and tumor recurrence after treatment can be effectively avoided after using the preparation for inhibiting PINK1 in combination.

3. The invention provides a drug combination therapy combining MAPK pathway inhibitor and PINK1 inhibitor, and the preparation for inhibiting PINK1 can thoroughly kill dormant tumor cells, is beneficial to improving the problems of tumor dormancy and recurrence after a patient uses the MAPK pathway inhibitor, and has profound clinical significance and popularization in improving the treatment effect of the MAPK pathway inhibitor.

4. The invention provides a method for improving the curative effect of MAPK pathway inhibitors on lung adenocarcinoma, which is characterized in that PINK1 promotes the survival of tumor cells entering dormancy and dormant cells by regulating and controlling the mitochondrial network and steady state of dormant tumor cells, and can inhibit the tumor cells from entering dormancy by inhibiting PINK 1-mediated mitochondrial autophagy, so that tumor recurrence after the MAPK pathway inhibitor treatment is avoided, and the curative effect of the MAPK pathway inhibitor is improved.

5. According to the invention, the PINK1 can be used as a biomarker for predicting the sensitivity of lung adenocarcinoma to MAPK pathway inhibitor treatment, so that a reagent for detecting the expression level of the PINK1 can be used for preparing a preparation for predicting the sensitivity of lung adenocarcinoma patients to MAPK pathway inhibitor, and an effective way is provided for developing a lung adenocarcinoma MAPK pathway inhibitor sensitivity prediction kit easy to popularize and apply.

Drawings

FIG. 1 is a graph showing the relationship between the expression of PINK1 and recurrence and prognosis of 90 cases of lung adenocarcinoma patients; wherein A: in tumor tissues of patients with recurrent lung adenocarcinoma, the expression level of PINK1 was higher (p=0.0111); b: lung adenocarcinoma patients with high PINK1 expression had a higher rate of recurrence (p=0.0111); c: patients with lung adenocarcinoma with high PINK1 expression survived worse and had no recurrence.

FIG. 2 is a graph of PINK1 promoting lung adenocarcinoma cells to dormancy by promoting mitochondrial function; wherein A: in 503 lung adenocarcinoma patients, the expression of the PINK1 and the expression of the negative regulation gene of the cell cycle show a remarkable positive correlation, and the expression of the PINK1 and the expression of the positive regulation gene of the cell cycle show a remarkable negative correlation; b: overexpression of PINK1 in lung adenocarcinoma cells significantly promotes mitochondrial oxidative phosphorylation; c: after treatment of MAPK pathway inhibitor-trametinib, the mitochondrial membrane potential of lung adenocarcinoma cells is obviously reduced, while the mitochondrial membrane potential of lung adenocarcinoma cells over-expressing PINK1 is not affected after treatment of trametinib, which indicates that the cell over-expressing PINK1 has stronger mitochondrial function; d: in a lung adenocarcinoma cell line, the over-expression of the PINK1 obviously inhibits cell division, and after the treatment of the mitochondrial oxidative phosphorylation inhibitor, the over-expression of the PINK1 can not inhibit cell division any more, which indicates that the PINK1 promotes tumor cell dormancy by promoting mitochondrial oxidative phosphorylation; e: and a Western blot result diagram of the PINK1 over-expression cells is shown.

FIG. 3 is a graph of PINK1 promoting resistance to MAPK pathway inhibitors resulting in tumor recurrence following treatment with MAPK pathway inhibitors; wherein A: for lung adenocarcinoma cells with high PINK1 expression, the median lethal concentration of MAPK pathway inhibitor is higher; b: after PINK1 is knocked down in lung adenocarcinoma cells, the effect of trametinib on inhibiting tumor cell proliferation is obviously enhanced; c: after PINK1 is knocked down in lung adenocarcinoma cells, the effect of trametinib in promoting tumor cell apoptosis is obviously enhanced; d: a Western blot result diagram of PINK1 knockdown cells is displayed; e: in a nude mouse subcutaneously transplanted lung adenocarcinoma model, a doxycycline induced shPINK1 system is used, and tumor recurrence caused by MAPK inhibitor use is obviously inhibited after PINK1 is knocked down.

FIG. 4 is a graph showing the effect of PINK1 mediated mitochondrial autophagy inhibitor in combination with MAPK pathway inhibitor drug combination therapies; wherein A: is the result of lung adenocarcinoma cell line and lung adenocarcinoma patient source organoid; b: is the result of lung adenocarcinoma subcutaneous transplantation tumor model.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.

The test method of the present invention, in which specific experimental conditions are not specified in the examples, is generally performed according to conventional conditions, such as those described in the "molecular cloning experiment guide" published in 2002 by the scientific press of j.

The results of 3 independent experiments in each example were analyzed using statistical methods, and "mean ± standard deviation" was calculated, and a single factor analysis of variance was used for the significance analysis (in the figure, "" represents p <0.05, "" represents p <0.01, "" represents p <0.001, "" represents p < 0.0001).

EXAMPLE 1 study of the correlation of the expression of PINK1 with tumor recurrence in patients with pulmonary adenocarcinoma

To verify the relationship between the expression of PINK1 and the recurrence and survival prognosis of lung adenocarcinoma patients, 90 lung adenocarcinoma patient tissue chips (Shanghai core super biosystems) were subjected to immunohistochemical staining with PINK1 antibody (23274-1-AP, proteintect) in this study, and the expression classification of PINK1 was interpreted according to the staining intensity and positive rate of PINK 1. The method is divided into 4 stages: grade 1 represents no staining, grade 2 represents weak positive, grade 3 represents positive, and grade 4 represents strong positive. Grouping patients according to recurrence, and counting the expression of PINK1 in tumor tissues of two groups of patients.

Statistical results showed that the expression of PINK1 protein was higher in lung adenocarcinoma tissues of recurrent patients (p=0.0111), and that the results were statistically significant by chi-square test (fig. 1A). In addition, among lung adenocarcinoma patients with high tumor tissue PINK1 expression scores, the proportion of recurrent patients is higher, and the results show statistical significance after chi-square test (figure 1B). The lung adenocarcinoma patients are divided into two groups according to the expression of the tumor tissue PINK1, the PINK1 score is 1 grade and 2 grade are low-expression PINK1 groups, and the PINK1 score is 3 grade and 4 grade are high-expression PINK1 groups. The survival analysis test was performed using the log-rank method and the risk Ratio (Hazard Ratio, HR) was calculated. Survival analysis results show that the recurrence-free survival time and total survival time of the patient with the lung adenocarcinoma in the high-expression group of the PINK1 are shorter than those of the patient with the lung adenocarcinoma in the low-expression group of the PINK1 (p <0.0001, HR=3.896), the recurrence rate of the patient with the high-expression of the PINK1 protein is obviously higher than that of the patient with the low-expression group (p=0.0004, HR= 4.479), and the two groups have obvious statistical differences (figure 1C), which shows that the high-expression of the PINK1 is an independent risk factor for predicting the recurrence risk of the patient with the lung adenocarcinoma.

EXAMPLE 2 cell level study of the Effect of PINK1 expression on lung adenocarcinoma dormancy

1. Correlation analysis of PINK1 expression and cell cycle regulatory gene expression

Data 503 lung adenocarcinoma RNA sequencing data from TCGA database, obtained expression of the PINK1 gene and expression of cell cycle regulatory genes, including genes PCNA, MKi67, CDK1, CDK2 that positively regulate the cell cycle, and genes such as CDKN1A, CDKN C that negatively regulate the cell cycle.

The results show that the expression of the PINK1 gene and the expression of the negative regulation gene of the cell cycle show a remarkable positive correlation, and the expression of the positive regulation gene of the cell cycle shows a remarkable negative correlation (figure 2A), which shows that the PINK1 is a negative regulation factor of the cell cycle, and indicates that the PINK1 expression and the tumor dormancy have correlation in tumors, and the PINK1 possibly promotes the tumor dormancy.

2. Cell culture

A549 and H460 cells (both purchased from the chinese sciences Shanghai cell bank) were cultured in DMEM medium [ 10% (v/v) fetal bovine serum added ]. The 6cm cell culture plate is passaged and expanded every other day, and the cells in the logarithmic growth phase are taken for subsequent experiments.

3. Construction of PINK1 gene over-expression cell line

(1) First, a plasmid over-expressing PINK1 is constructed: RNA was extracted after A549 cells were lysed, and cDNA was obtained using the Siemens reverse transcription kit (M1632, siemens). Designing a PCR primer of the PINK1 coding region according to the PINK1 coding region sequence (CCDS 211.1) recorded in the NCBI database, carrying out PCR amplification on the PINK1 coding region, and connecting the PINK1 coding region to an over-expression skeleton vector through enzyme digestion to construct pLVX-PINK1 plasmid; wherein the used over-expression framework Vector is pLVX-DsRed-Monomer-N1 Vector (632152, takara); the PCR primer sequences of the PINK1 coding region are as follows:

F1：5′-CCGCTCGAGACCATGGCGGTGCGACAGGCGCTGGG-3′；

R1：5′-CGCGGATCCTCACAGGGCTGCCCTCCATGAGC-3′。

(2) Lentiviral packaging of plasmids overexpressing Pink1 using a lentiviral packaging system: the pLVX-DsRed-Monomer-N1 Vector plasmid and the pLVX-PINK1 plasmid constructed in step (1) were mixed with pMD2.G plasmids (12259, adedge ne), psPAX plasmids (12260, adedge ne), respectively, and 293T cells (purchased from the Shandong sea cell Bank of the national institute) were transfected with PEI transfection reagent. Supernatants from 293T cells 3 days post transfection were collected. Adding PEG8000 and sodium chloride solution into cell supernatant, precipitating, and centrifuging to obtain viral precipitate, which is pLVX-DsRed-Monomer-N1 Vector virus and pLVX-PINK1 virus respectively.

(3) Infection with virus: a549 and H460 cells were each divided into two groups of about 1x 10 ⁶ cells each, each 100 μl of pLVX-DsRed-Monomer-N1 Vector virus pellet and pLVX-PINK1 virus pellet were added, respectively, with 10mL of DMEM complete medium containing polybrene [10% (v/v) fetal bovine serum, 1% (v/v) diabody (penicillin+streptomycin), 8 μg/mL polybrene, 89% (v/v) DMEM medium ].

(4) And (3) screening: after 48H of infection of A549 and H460 cells with virus, 10mL of medium containing puromycin [10% (v/v) fetal bovine serum, 1% (v/v) diab, 10 μg/mL puromycin, 89% (v/v) DMEM medium ] was added to the cells after infection with virus, and when no cells infected with virus all died, normal DMEM complete medium [10% (v/v) fetal bovine serum, 1% (v/v) diab, 89% (v/v) DMEM medium ] was exchanged to obtain a control cell strain over-expressing empty vector and a cell strain over-expressing PINK 1.

(5) Western blot verification: the control cell strain over-expressing the empty vector and the cell strain over-expressing the PINK1 are subjected to expansion culture, cells after expansion culture are digested, a small amount of cells are left for continuous passage, western blot is carried out on the rest cells, the expression condition of the PINK1 is detected, and the over-expression efficiency is verified (figure 2E).

4. Mitochondrial oxidative phosphorylation level detection

To verify that PINK1 regulates mitochondrial function, the study used a real-time energy metabolizer of the agilent hippocampus (Seahorse XFe analyzer) to conduct mitochondrial pressure testing on a549 and H460 cells. Mitochondrial oxidative phosphorylation level changes of a549 and H460 cells overexpressing PINK1 were compared with a control group of a549 and H460 cells overexpressing empty vector, respectively. The mitochondrial pressure test procedure operates strictly according to the instructions for mitochondrial pressure testing provided by Agilent corporation.

The required consumables and part of the reagents, including XF 96 well cell culture plates, XF 96 well tool plates, XF 96 well probe plates and XF calibration solutions, are derived from Seahorse XFe FluxPak kit (102601-100, agilent). The required reagents include 1M glucose solution (103577-100, agilent), 100mM pyruvic acid solution (103578-100, agilent), 200mM glutamine (103579-100, agilent) and Seahorse XF basal medium (103335-100, agilent). The required drugs, including the mixture of oligomycin (Oligomycin, oligo), decoupling agent FCCP, antimycin a and rotenone (ANTIMYCIN A/Rotenone, AA/Rot), were derived from mitochondrial pressure test kit (103015-100, agilent).

The test procedure was performed in two days, with the following brief descriptions of the steps (where step (1), step (2) and step (3) were completed on the first day and the remaining steps were completed on the second day):

(1) Cell plating: the cultured cells were collected, after calculating the cell density, a cell suspension was prepared with DMEM complete medium [10% (v/v) fetal bovine serum, 1% (v/v) diab, 89% (v/v) DMEM medium ], 80 μl of a cell suspension containing 2 ten thousand cells per well was inoculated in an XF 96-well cell culture plate, and the cell culture plate was placed in a 5% (v/v) CO ₂ cell culture box at 37 ℃ to allow the cells to adhere to the wall and cultured overnight.

(2) Hydration probe card: 200 mu L of XF calibration solution per well is added to an XF 96-well tool plate, an XF 96 Kong Tan needle plate is stacked on the 96-well tool plate, the probe is completely immersed in the XF calibration solution, and the probe is placed in a cell incubator without CO ₂ at 37 ℃ for incubation overnight.

(3) The Agilent hippocampus real-time energy metabolizer was turned on overnight pre-heating instrument.

(4) Washing the cells: the culture medium in the XF 96-well cell culture plate is sucked and removed; 200. Mu.L of XF assay (10 mM glucose, 1mM pyruvic acid, 2mM glutamine, seahorse XF basal medium, pH 7.4) was added and then aspirated, and the procedure was repeated 1 time; finally, after 180 μl of XF detection solution was added to each well, XF 96 well cell culture plates were loaded onto the machine.

(5) Dispensing and loading the drugs into a probe card drug adding bin: the mixed powder of the oligomycin powder, the FCCP powder, the antimycin A and the rotenone provided by the mitochondrial pressure test kit is respectively prepared into 15 mu M oligomycin solution, 5 mu M FCCP solution and 5 mu M antimycin A and rotenone mixed solution by using XF detection solution. 20 mu L of oligomycin solution per hole, 22 mu L of FCCP solution per hole and 25 mu L of antimycin A and rotenone mixed solution per hole are added into a sample adding bin A, a sample adding bin B and a sample adding bin C of an XF 96-hole probe plate. After the dosing is completed, the XF 96 well probe plate is loaded onto the machine. The instrument automatically stacks the XF 96 Kong Tan needle plate on the XF 96 well cell culture plate to fully submerge the probes in the cell culture medium.

(6) Detecting the oxygen consumption rate: mitochondrial pressure test experiments the oxygen consumption rate (Oxygen consumption rate, OCR) of cells was measured by sequential addition of targeted drugs for mitochondrial electron transport chains (mixture of oligomycin, FCCP, antimycin a and rotenone) to obtain key parameters reflecting mitochondrial function such as maximum respiration rate (Maximal respiration), sparing respiration capacity (Spare respiratory capacity). Where the maximum respiration rate is the maximum oxygen consumption of the cell obtained after addition of FCCP, which operates at maximum capacity by stimulating the cell's respiratory chain, represents the maximum respiration rate that the cell can achieve. The reserve respiratory capacity is the maximum respiratory minus the basal respiratory oxygen consumption, representing the potential response capacity of the cell to energy demand and the gap between the basal and theoretical respiratory maxima, and the capacity of the cell to respond to demand can be an indicator of cell fitness or flexibility.

(7) Normalization of results: after the mitochondrial pressure test was completed, XF 96-well cell culture plates were unloaded from the agilent hippocampus real-time energy metabolizer, and after pipetting away XF assay, 40 μl of RIPA protein lysate (R0278, sigma) was added to each well. After 1 hour of on-ice lysis, protein lysates were taken and detected with BCA protein quantification kit (23227, sameid) to obtain the protein amount per well of XF 96 well cell culture plates. And (3) normalizing the maximum respiratory rate and the standby respiratory capacity detected in the step (6) according to the protein per pore amount. A normalized maximum respiration rate and normalized reserve respiration capacity are obtained, which together reflect the mitochondrial oxidative respiration level of the cell.

The results show that over-expression of PINK1 significantly enhanced mitochondrial oxidative phosphorylation levels in lung adenocarcinoma cells (fig. 2B).

5. Mitochondrial membrane potential detection

To further verify that PINK1 maintained a mitochondrial health network, the present study used mitochondrial membrane potential probes JC-10 (FXP 134-100, four cypress organisms) to detect mitochondrial membrane potential in lung adenocarcinoma cells that overexpressed PINK 1. A549 and H460 cells over-expressing empty vector were used as control groups, a549 and H460 cells over-expressing PINK1 were used as experimental groups, and the change of mitochondrial membrane potential of cells after the cells were cultured in a complete medium [10% (v/v) fetal bovine serum, 1% (v/v) diab, 0.1% (v/v) DMSO,89% (v/v) DMEM medium ] and a complete medium [10% (v/v) fetal bovine serum, 1% (v/v) diab, 100nM trimetinib, 89% (v/v) DMEM medium ], 5% (v/v) CO ₂ cell incubator at 37 ℃ for 72 hours, respectively, was compared.

The results showed that the proportion of cells with reduced mitochondrial membrane potential was significantly lower in the a549 and H460 cells overexpressing PINK1 compared to the control group of a549 and H460 cells overexpressing empty vector after treatment with MAPK pathway inhibitor trametinib (fig. 2C), suggesting that the mitochondrial function network was healthier and less susceptible to mitochondrial damage by trametinib in lung adenocarcinoma cells overexpressing PINK 1.

6. Cell proliferation assay

In order to prove that the PINK1 promotes the dormancy of lung adenocarcinoma cells by regulating mitochondrial function, cell proliferation dye CFDA (M5117, aumera organism) is used, A549 and H460 cells which over express empty vector are used as a control group to detect the proliferation rate of the lung adenocarcinoma cells which over express the PINK1, and meanwhile, the influence of mitochondrial respiration inhibitor oligomycin on the proliferation rate of the lung adenocarcinoma cells is explored.

CFSE is a protein dye that can be taken up by living cells and stain them with green fluorescence. CFSE-labeled proteins are equally distributed among daughter cells at the time of cell division, so that the staining intensity of CFSE is diluted with cell division, whereby the proliferation rate of cells is detected. To control A549 and H460 cells and A549 and H460 cells overexpressing PINK1, complete medium [10% (v/v) fetal bovine serum, 1% (v/v) diab, 10. Mu.M CFSE,89% (v/v) DMEM medium ] containing CFSE dye was added, and the cells were incubated at 37℃for 20 minutes in A5% (v/v) CO ₂ cell incubator to label the cells with the CFSE dye. After labeling, the complete medium containing CFSE dye was aspirated from each cell group. The stained control group and the over-expressed PINK1 groups A549 and H460 cells were each divided into two groups, one group was supplemented with DMSO-containing complete medium [10% (v/v) fetal bovine serum, 1% (v/v) diabody, 0.01% (v/v), 89% (v/v) DMEM medium ], and the other group was supplemented with oligomycin-containing complete medium [10% (v/v) fetal bovine serum, 1% (v/v) diabody, 100nM oligomycin, 89% (v/v) DMEM medium ],37℃and 5% (v/v) CO ₂ cell incubator. Fluorescence intensity of CFSE was detected after labeling for 24 hours, 72 hours, 120 hours.

The results show that: compared with the control group, the proliferation speed of the cells which over-express the PINK1 groups A549 and H460 is obviously slower; in the a549 and H460 cells of the control group, there was little change in the cell proliferation rate after oligomycin treatment; however, in the cells of the groups A549 and H460 which overexpress PINK1, the proliferation rate of the cells is increased after the oligomycin treatment. This suggests that PINK1 promotes lung adenocarcinoma cells to sleep by promoting mitochondrial function (fig. 2D).

EXAMPLE 3 cellular level study of the Effect of PINK1 expression on the efficacy of MAPK pathway inhibitors in treating lung adenocarcinoma

1. The analysis data of the correlation between the PINK1 expression and the sensitivity of the lung adenocarcinoma cell line to the MAPK pathway inhibitor is from a cancer drug sensitivity Genome Database (GDSC) database, and the expression of the PINK1 gene in the lung adenocarcinoma cell line is obtained according to the detection data of the gene chip recorded in the database. Lung adenocarcinoma cell lines were divided into two groups according to PINK1 expression: cells that express 20% of the primary PINK1, i.e., cells that highly express PINK1, and cells that express 20% of the secondary PINK1, i.e., cells that lowly express PINK 1. Half-lethal concentration data (IC 50) of 11 MAPK pathway inhibitors on lung adenocarcinoma cell lines were obtained from this database. Comparing the difference in sensitivity of high-expression and low-expression PINK1 cells to MAPK pathway inhibitors, the results show that the half-lethal concentration of lung adenocarcinoma cells of high-expression PINK1 is higher, suggesting that the lung adenocarcinoma cells of high-expression PINK1 are less sensitive to MAPK pathway inhibitors (figure 3A), i.e. the lung adenocarcinoma cells of high-expression PINK1 are generally more resistant to MAPK pathway inhibitors.

2. PINK1 knockdown cell line construction

(1) Designing shPINK sequences, searching and knocking down sequences of PINK1 by using a Sigma website, designing two targets shPINK in total, wherein the sequences are respectively (2 complementary paired single-stranded DNA primers are respectively synthesized by the targets of each shPINK1, the sequence of one primer is listed below, and the other primer is complementary paired with the sequence):

shPINK1#1:5′-TTTCCACACAACACTTCTCTGT-3′；

shPINK1#2:5′-TTCCACACAACACTTCTCTGTG-3′。

(2) shPINK1 plasmid construction, annealing the synthesized shPINK1 primer to form double-stranded DNA.

(3) The vector plko.1puro (8453, addgene) was digested with agoi and EcoRI. And (3) connecting the digested vector with annealed shPINK double-stranded DNA by using T4 DNA ligase, and finally transforming, screening positive bacteria and extracting plasmids to obtain pLKO.1-shPINK1#1 and pLKO.1-shPINK1#2 plasmids respectively.

(4) Lentiviral packaging was performed on the PINK1 knockdown plasmid using a lentiviral packaging system. The plasmids pLKO.1-scrambled (136035, addgene), pLKO.1-shPINK1#1, and pLKO.1-shPINK1#2 were mixed with pMD2.G and psPAX2, respectively, and 293T cells were transfected with PEI transfection reagent. Supernatants from 293T cells 3 days post transfection were collected. Adding PEG8000 and sodium chloride solution into cell supernatant, precipitating, and centrifuging to obtain viral precipitate, which is pLKO.1-scrambled virus, pLKO.1-shPINK #1 virus and pLKO.1-shPINK #2 virus respectively.

(5) Infection with virus: a549 and H460 cells were each divided into three groups of about 1 x 10 ⁶ cells each, each 100 μl of each of plko.1-scrambled viral pellet, plko.1-shPINK #1 viral pellet, and plko.1-shPINK # 1#2 viral pellet. Simultaneously, 10mL of complete DMEM medium containing polybrene [10% (v/v) fetal bovine serum, 1% (v/v) diabody, 8. Mu.g/mL polybrene, 89% (v/v) DMEM medium ] was added.

(6) After infection of A549 and H460 cells with virus for 48H, 10mL of puromycin-containing medium [10% (v/v) fetal bovine serum, 1% (v/v) diabody, 10. Mu.g/mL puromycin, 89% (v/v) DMEM medium ] was added to the virus-infected cells. When all the cells not infected by the virus die, the cells can be replaced by normal DMEM complete medium [10% (v/v) fetal bovine serum, 1% (v/v) double antibody and 89% (v/v) DMEM medium ], so as to obtain the cell strain of the knockdown control group, the cell strain of the knockdown PINK1 (target point 1) and the cell strain of the knockdown PINK1 (target point 2).

(7) Western blot verification: the cell lines of the knockdown control group, the cell lines of the knockdown PINK1 (target point 1) and the cell lines of the knockdown PINK1 (target point 2) are subjected to expansion culture, cells after expansion culture are digested, a small amount of cells are left for continuous passage, and the expression condition of the PINK1 is detected by the residual cells through Western blot, so that the knockdown efficiency is verified (figure 3D).

3. Detecting the effect of trimetinib on lung adenocarcinoma cell proliferation

(1) 3 Compound holes of each group, inoculating lung adenocarcinoma cells A549 and H460 into a 12-hole plate according to 1000 cells of each hole, and culturing at 37 ℃ in A5% (v/v) CO ₂ cell incubator;

(2) After cell attachment, different concentrations (final concentrations of 5nM, 10nM, 25 nM) of trimetinib were given, and the control group was added with an equal volume of dimethyl sulfoxide (DMSO) and cultured continuously;

(3) After one week of culture, the original medium was aspirated, cells were fixed with 4% paraformaldehyde for 20min, followed by 0.1% crystal violet for 20min;

(4) The experimental results are analyzed after photographing, and the results show that the effect of the trimetinib on inhibiting the proliferation of lung adenocarcinoma cells is remarkably enhanced after knocking down the PINK1 (figure 3B).

4. Detecting the effect of trimetinib on apoptosis of lung adenocarcinoma cells

(1) Inoculating lung adenocarcinoma cells A549 and H460 into 12-hole plates according to 50000 cells per hole respectively, and culturing in incubator;

(2) After the cells are attached, trimetinib with the final concentration of 100nM is given, DMSO is added to a control group, and the culture is continued;

(3) After 72h of culture, supernatant floating cells and cell adhesion at the bottom of the dish were collected and subjected to apoptosis staining using an annexin v-FITC/PI apoptosis double staining kit (556547, bd);

(4) Flow cytometry analysis results show that the inhibition effect of MAPK pathway inhibitor Qu Meiti Ni on tumor cell proliferation is obviously enhanced after PINK1 is knocked down, and meanwhile, the effect of trimetinib on promoting lung adenocarcinoma cell apoptosis is obviously enhanced after PINK1 is knockdown (figure 3C).

EXAMPLE 4 animal level study of the Effect of PINK1 expression on the therapeutic efficacy of MAPK pathway inhibitors

1. Construction of doxycycline (doxycycline) induced PINK1 knock-down lung adenocarcinoma cell line

(1) Using shPINK #1 target, the sequence was: 5'-TTTCCACACAACACTTCTCTGT-3'.

(2) ShPINK1 plasmid construction, annealing the synthesized shPINK1 primer (shPINK 1#1) to form double-stranded DNA;

(3) The vector EZ-Tet-pLKO-Puro (85966, adedge) was digested with AgeI and EcoRI. Connecting the digested vector with annealed primers by using T4 DNA ligase, and finally converting, screening positive bacteria and extracting plasmids to obtain shPINK-tet-on plasmids;

(4) Lentiviral packaging was performed on the PINK1 knockdown plasmid using a lentiviral packaging system. shPINK1-tet-on obtained in step (3) was mixed with pMD2.G, psPAX, respectively, and 293T cells were transfected with PEI transfection reagent. Supernatants from 293T cells 3 days post transfection were collected. Adding PEG8000 and sodium chloride solution into cell supernatant for precipitation, and centrifuging to obtain shPINK-tet-on virus precipitate;

(5) Infection with virus: 1.10 ⁶ A549 cells were added to 100. Mu. L shPINK1-tet-on viral pellet, respectively. Simultaneously, 10mL of complete DMEM medium containing polybrene [10% (v/v) fetal bovine serum, 1% (v/v) diabody, 8. Mu.g/mL polybrene, 89% (v/v) DMEM medium ] was added.

(6) And (3) screening: after 48h of infection of A549 cell virus, 10mL of puromycin-containing medium [10% (v/v) fetal bovine serum, 1% (v/v) diabody, 10. Mu.g/mL puromycin, 89% (v/v) DMEM medium ] was added to the virus-infected cells. When the cells without the infection virus are all dead, the cells can be replaced by normal DMEM complete medium [10% (v/v) fetal bovine serum, 1% (v/v) double antibody and 89% (v/v) DMEM medium ], so as to obtain the cell strain of the doxycycline-induced knockdown PINK 1.

2. Construction of doxycycline-induced knockdown PINK 1A 549 cell subcutaneous transplantation lung adenocarcinoma model

And (3) performing expanded culture on the doxycycline-induced knockdown PINK1A549 cells to a certain quantity, wherein doxycycline is not added during culture. Balb/c male nude mice (purchased from university of middle mountain animal laboratory center) weighing about 20g were purchased 4-6 weeks in advance, for a total of 36 nude mice, each nude mouse was inoculated with one subcutaneous graft on one side. Cells were collected, resuspended with 50 μl of PBS every two million cells, and after mixing the cell suspension with matrigel (354231, corning) 1:1 (v/v), tumor cells were inoculated subcutaneously into the back of nude mice with a1 mL-sized insulin syringe, and two million cells were inoculated per tumor spot.

3. Effect of knockdown PINK1 on MAPK pathway inhibitor-trametinib treatment effect

After the tumor growth reached 100mm ³, as shown in fig. 3E, the tumor-bearing mice were divided into 2 groups, and the tumor-bearing mice induced by the doxycycline was not administered as a control group, and the tumor-bearing mice induced by the doxycycline was administered as a knock-down PINK1 group. Two groups of tumor-bearing mice were treated with trimetinib, respectively, and the treatment effect of trimetinib on tumors of the two groups of tumor-bearing mice was compared. In addition, after 6 weeks of treatment with trimetinib, treatment with trimetinib was stopped for two weeks, and the difference in tumor recurrence in the two groups of tumor-bearing mice was compared. The administration mode was that doxycycline (dox, 20mg/kg, continuous administration before killing tumor-bearing mice, 2 times a week) was given by intraperitoneal injection, trametin (tra, 2ppm, continuous administration in drinking water of mice before killing tumor-bearing mice) was given by drinking water, while the state of nude mice and tumor growth were observed, and the longest and shortest diameters were measured according to the formula: v=0.5 (longest diameter. Shortest diameter) calculate tumor volume. Tumor weights and volumes were measured by sacrificed nude mice of the corresponding groups at weeks 4, 7, and 9, respectively.

The results showed that tumors began to relapse 6 weeks after treatment with the MAPK pathway inhibitor trimetinib. Tumor recurrence rate was faster after cessation of treatment. Whereas knocking down PINK1 in tumors significantly inhibited tumor recurrence following trimetinib treatment (fig. 3E). Indicating that PINK1 is a risk factor for drug resistance recurrence after MAPK pathway inhibitor-trametinib treatment.

EXAMPLE 5 cellular level study of the efficacy of MAPK pathway inhibitor and PINK1 mediated combination therapies of mitochondrial autophagy inhibitors

The MAPK pathway inhibitor selected in this example was trimetinib and the PINK 1-mediated autophagy inhibitor was hydroxychloroquine.

1. Cell proliferation assay after combination of trimetinib and hydroxychloroquine

(1) After A549 cells and H460 cells grow to a certain number, digesting the cells, inoculating 1000 cells per well into a 96-well plate, and culturing in a incubator;

(2) After cell attachment, different concentrations of trimetinib (Trametinib, tra) (final concentration of trimetinib 0.001 μm, 0.005 μm, 0.01 μm,0.1 μm, 0.5 μm) and hydroxychloroquine (Hydroxychloroquine, HCQ) (final concentration of hydroxychloroquine 5 μm, 10 μm, 25 μm, 50 μm, 100 μm, 250 μm) were given, and the control group was added DMSO for further culture;

(3) After 72 hours, the original culture medium is sucked, 100 mu L of CCK-8 detection liquid is added into each hole, and the mixture is continuously put back into the incubator for 2 hours of incubation;

(4) The cells were removed and absorbance at 450nm was measured using an enzyme-labeled instrument.

2. Analysis of combined effects of trametinib and hydroxychloroquine

The effect of the combination of trametinib and hydroxychloroquine was analyzed using Combenefit software based on the results of CCK8 assays. The results show that trametinib and hydroxychloroquine combined have a synergistic effect (figure 4A).

Example 6 evaluation of the level of organoids derived from patients with lung adenocarcinoma effective combination therapies of MAPK pathway inhibitors and PINK1 mediated mitophagy inhibitors

1. Construction of lung adenocarcinoma patient-derived organoids

(1) Tumor tissues resected by the lung adenocarcinoma patients after the operation are cut into about 1mm ³ pieces by a disposable operation blade;

(2) Digesting the tissue fragments into single cells by collagenase II (17101-015, gibco) and collagenase IV (17104-019, gibco), centrifuging, and then re-suspending the single cells in matrigel (356255, corning) with concentration of more than 80%, and uniformly mixing;

(3) Inoculating the matrigel containing single cells into a 48-well plate, and continuously putting the matrigel into an incubator with 20 mu L of matrigel per well;

(4) Preparing an organoid culture medium, using DMEM/F-12 as a basal medium, adding 100ng/mL of epidermal cell growth factor (PHG 0315, gibco), 20ng/mL of fibroblast growth factor (PHG 0266, sieimer's fly), 50X B27 (12587010, gibco), 100X N2 (17502048, gibco), 10 mu M Y27632 (HY-10071, MCE), and filtering by a 0.22 mu m filter head for later use;

(5) After the matrigel was solidified, the culture medium was added to the matrigel containing single cells, 500. Mu.L per well, and the mixture was returned to the incubator, and after about 20 days of culture, the culture effect was observed, during which the culture medium was changed every 3 days.

2. Organoid activity assay after combination of trimetinib and hydroxychloroquine

(1) Taking the patient-derived organoids cultured until 3 rd generation, inoculating about 20 organoids per well to a 96-well plate, and culturing in a incubator at a concentration of 5 mu L per well;

(2) After the matrigel was coagulated, various concentrations of trimetinib (Trametinib, tra) (final concentration of trimetinib 0.001 μm, 0.005 μm, 0.01 μm, 0.1 μm, 0.5 μm) and hydroxychloroquine (Hydroxychloroquine, HCQ) (final concentration of hydroxychloroquine 5 μm, 10 μm, 25 μm, 50 μm, 100 μm, 250 μm) were given, and DMSO was added to the control group to continue the culture;

(3) After 72h, organoid viability was detected using 3D cultured cell viability detection kit (G9681, promega).

2. Analysis of combined effects of trametinib and hydroxychloroquine

And using Combenefit software to analyze the effect of the combination of the trametinib and hydroxychloroquine according to the result of the organoid activity detection. The results show that trametinib and hydroxychloroquine combined have a synergistic effect (figure 4A).

EXAMPLE 7 animal level study of the effectiveness of MAPK pathway inhibitor and PINK1 mediated combination therapies of mitochondrial autophagy inhibitors

1. Construction of A549 cell nude mice subcutaneously transplanted lung adenocarcinoma model

A549 cells were expanded to a number. Balb/c male nude mice (purchased from university of Zhongshan animal laboratory center) weighing about 20g were purchased 4-6 weeks in advance. A total of 24 nude mice, each nude mouse was vaccinated on one side with one subcutaneous graft tumor. Cells were collected, resuspended in 50 μl PBS for every two million cells, and after mixing the cell suspension with matrigel (354231, corning) 1:1 (v/v), tumor cells were inoculated subcutaneously into the back of nude mice with a 1mL insulin syringe, and two million cells were inoculated per tumor spot.

2. Effect of combination use of PINK1 mediated mitophagy inhibitors on MAPK pathway inhibitor-trametinib treatment effect

After the tumor growth reached 100mm ³, the tumor-bearing mice were divided into 4 groups, and tumor growth of the tumor-bearing mice administered with hydroxychloroquine, the tumor-bearing mice administered with trimetinib, and the tumor-bearing mice administered with hydroxychloroquine and trimetinib simultaneously were compared with each other. The administration mode is that hydroxychloroquine is injected intraperitoneally (50 mg/kg, continuous administration is carried out 2 times a week before tumor-bearing mice are killed), trimetinib is administered orally (2 ppm, continuous administration is carried out in drinking water of mice before tumor-bearing mice are killed), the state and tumor growth of nude mice are observed, the longest diameter and the shortest diameter are measured, and the formula is as follows: v=0.5 x (longest diameter x shortest diameter) calculate tumor volume; when the tumor diameter reached 1.8cm, nude mice were sacrificed and tumor weight and volume were measured.

The results showed that the combination of trimetinib and hydroxychloroquine had a synergistic effect in a549 cell nude mice subcutaneously transplanted lung adenocarcinoma model (fig. 4B).

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

<110> University of Zhongshan Sun Yixian commemorative Hospital

<120> Application of PINK1 as marker for predicting sensitivity of lung adenocarcinoma to MAPK inhibitor and recurrence of lung adenocarcinoma

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 581

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> PINK1

<400> 1

Met Ala Val Arg Gln Ala Leu Gly Arg Gly Leu Gln Leu Gly Arg Ala

1 5 10 15

Leu Leu Leu Arg Phe Thr Gly Lys Pro Gly Arg Ala Tyr Gly Leu Gly

20 25 30

Arg Pro Gly Pro Ala Ala Gly Cys Val Arg Gly Glu Arg Pro Gly Trp

35 40 45

Ala Ala Gly Pro Gly Ala Glu Pro Arg Arg Val Gly Leu Gly Leu Pro

50 55 60

Asn Arg Leu Arg Phe Phe Arg Gln Ser Val Ala Gly Leu Ala Ala Arg

65 70 75 80

Leu Gln Arg Gln Phe Val Val Arg Ala Trp Gly Cys Ala Gly Pro Cys

85 90 95

Gly Arg Ala Val Phe Leu Ala Phe Gly Leu Gly Leu Gly Leu Ile Glu

100 105 110

Glu Lys Gln Ala Glu Ser Arg Arg Ala Val Ser Ala Cys Gln Glu Ile

115 120 125

Gln Ala Ile Phe Thr Gln Lys Ser Lys Pro Gly Pro Asp Pro Leu Asp

130 135 140

Thr Arg Arg Leu Gln Gly Phe Arg Leu Glu Glu Tyr Leu Ile Gly Gln

145 150 155 160

Ser Ile Gly Lys Gly Cys Ser Ala Ala Val Tyr Glu Ala Thr Met Pro

165 170 175

Thr Leu Pro Gln Asn Leu Glu Val Thr Lys Ser Thr Gly Leu Leu Pro

180 185 190

Gly Arg Gly Pro Gly Thr Ser Ala Pro Gly Glu Gly Gln Glu Arg Ala

195 200 205

Pro Gly Ala Pro Ala Phe Pro Leu Ala Ile Lys Met Met Trp Asn Ile

210 215 220

Ser Ala Gly Ser Ser Ser Glu Ala Ile Leu Asn Thr Met Ser Gln Glu

225 230 235 240

Leu Val Pro Ala Ser Arg Val Ala Leu Ala Gly Glu Tyr Gly Ala Val

245 250 255

Thr Tyr Arg Lys Ser Lys Arg Gly Pro Lys Gln Leu Ala Pro His Pro

260 265 270

Asn Ile Ile Arg Val Leu Arg Ala Phe Thr Ser Ser Val Pro Leu Leu

275 280 285

Pro Gly Ala Leu Val Asp Tyr Pro Asp Val Leu Pro Ser Arg Leu His

290 295 300

Pro Glu Gly Leu Gly His Gly Arg Thr Leu Phe Leu Val Met Lys Asn

305 310 315 320

Tyr Pro Cys Thr Leu Arg Gln Tyr Leu Cys Val Asn Thr Pro Ser Pro

325 330 335

Arg Leu Ala Ala Met Met Leu Leu Gln Leu Leu Glu Gly Val Asp His

340 345 350

Leu Val Gln Gln Gly Ile Ala His Arg Asp Leu Lys Ser Asp Asn Ile

355 360 365

Leu Val Glu Leu Asp Pro Asp Gly Cys Pro Trp Leu Val Ile Ala Asp

370 375 380

Phe Gly Cys Cys Leu Ala Asp Glu Ser Ile Gly Leu Gln Leu Pro Phe

385 390 395 400

Ser Ser Trp Tyr Val Asp Arg Gly Gly Asn Gly Cys Leu Met Ala Pro

405 410 415

Glu Val Ser Thr Ala Arg Pro Gly Pro Arg Ala Val Ile Asp Tyr Ser

420 425 430

Lys Ala Asp Ala Trp Ala Val Gly Ala Ile Ala Tyr Glu Ile Phe Gly

435 440 445

Leu Val Asn Pro Phe Tyr Gly Gln Gly Lys Ala His Leu Glu Ser Arg

450 455 460

Ser Tyr Gln Glu Ala Gln Leu Pro Ala Leu Pro Glu Ser Val Pro Pro

465 470 475 480

Asp Val Arg Gln Leu Val Arg Ala Leu Leu Gln Arg Glu Ala Ser Lys

485 490 495

Arg Pro Ser Ala Arg Val Ala Ala Asn Val Leu His Leu Ser Leu Trp

500 505 510

Gly Glu His Ile Leu Ala Leu Lys Asn Leu Lys Leu Asp Lys Met Val

515 520 525

Gly Trp Leu Leu Gln Gln Ser Ala Ala Thr Leu Leu Ala Asn Arg Leu

530 535 540

Thr Glu Lys Cys Cys Val Glu Thr Lys Met Lys Met Leu Phe Leu Ala

545 550 555 560

Asn Leu Glu Cys Glu Thr Leu Cys Gln Ala Ala Leu Leu Leu Cys Ser

565 570 575

Trp Arg Ala Ala Leu

580

<210> 2

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> shPINK1 #1

<400> 2

tttccacaca acacttctct gt 22

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> shPINK1 #2

<400> 3

ttccacacaa cacttctctg tg 22

<210> 4

<211> 35

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> F1

<400> 4

ccgctcgaga ccatggcggt gcgacaggcg ctggg 35

<210> 5

<211> 32

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> R1

<400> 5

cgcggatcct cacagggctg ccctccatga gc 32

Claims

Use of pink1 for the preparation of a product for modulating susceptibility of lung adenocarcinoma to MAPK inhibitors, characterized in that said modulation is effected by:

(1) Reducing sensitivity of lung adenocarcinoma to MAPK inhibitors by overexpressing the PINK1 gene;

(2) Inhibiting the expression of the PINK1 gene increases the sensitivity of lung adenocarcinoma to MAPK inhibitors;

The MAPK inhibitor is trametinib.
2. The use according to claim 1, characterized in that:

The over-expression of the PINK1 gene in the mode (1) is realized by the following steps: connecting the PINK1 gene to an over-expression framework vector, and constructing to obtain a plasmid over-expressing the PINK1 gene; then, carrying out slow virus packaging on the plasmid over-expressing the PINK1 by using a slow virus packaging system, and screening to obtain a cell strain over-expressing the PINK 1;

the inhibition of the expression of the PINK1 gene described in the mode (2) is achieved by knocking down the PINK1 gene;

The target sequence of the knockdown PINK1 gene is any one of the following sequences:

shPINK1 #1: 5′-TTTCCACACAACACTTCTCTGT-3′；

shPINK1 #2: 5′-TTCCACACAACACTTCTCTGTG-3′。
3. Use of an agent for detecting the level of PINK1 expression in the manufacture of a product for predicting susceptibility of lung adenocarcinoma to MAPK inhibitors, characterized in that: wherein, high PINK1 expression makes lung adenocarcinoma insensitive to MAPK inhibitors; PINK1 low expression makes lung adenocarcinoma susceptible to MAPK inhibitors; the MAPK inhibitor is trametinib.
4. The application of an agent for inhibiting PINK1 in preparing a medicament for increasing the sensitivity of lung adenocarcinoma to MAPK inhibitors is characterized in that:

the PINK1 is inhibited by knocking down the PINK1 gene;

The target sequence of the knockdown PINK1 gene is any one of the following sequences:

shPINK1 #1: 5′-TTTCCACACAACACTTCTCTGT-3′；

shPINK1 #2: 5′-TTCCACACAACACTTCTCTGTG-3′；

The MAPK inhibitor is trametinib.