CN110331203B

CN110331203B - Application of ubiquitin ligase CHAF1B as target site in preparation of lung adenocarcinoma cisplatin sensitization drug

Info

Publication number: CN110331203B
Application number: CN201910693697.8A
Authority: CN
Inventors: 曹科; 龚恋; 何东; 胡一; 朱煜星; 曾庆海; 张烨昱
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2019-07-30
Filing date: 2019-07-30
Publication date: 2023-04-18
Anticipated expiration: 2039-07-30
Also published as: CN110331203A

Abstract

The invention discloses application of ubiquitin ligase CHAF1B as a target site in preparation of a lung adenocarcinoma cisplatin sensitization medicine. The research of the invention shows that ubiquitin ligase CAHF1B in the lung adenocarcinoma cis-platinum drug-resistant cell strain is highly expressed, and the cis-platinum drug sensitivity of lung adenocarcinoma cells can be increased after the CAHF1B is knocked out. Therefore, CAHF1B can be used as a target site in the preparation of lung adenocarcinoma cisplatin sensitization medicines, and is also a molecular marker for clinically predicting cisplatin medicine sensitivity of lung adenocarcinoma patients. Mechanism research shows that high-expression CAHF1B can promote degradation of NCOR2 ubiquitination to cause lung adenocarcinoma cells to have cis-platinum resistance. Therefore, NCOR2 is also used as a target site in the preparation of lung adenocarcinoma cisplatin sensitizing drugs.

Description

Application of ubiquitin ligase CHAF1B as target site in preparation of lung adenocarcinoma cisplatin sensitization drug

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of ubiquitin ligase CHAF1B as a target site in preparation of a lung adenocarcinoma cisplatin sensitization medicine.

Background

The lung cancer is the most common malignant tumor worldwide, and the morbidity and mortality of the lung cancer are the first worldwide, and the morbidity and mortality of the lung cancer are also high in China. According to WHO lung cancer histological classification standard in 2015, the pathological types of the lung cancer mainly comprise adenocarcinoma, squamous carcinoma, large-cell carcinoma, adenosquamous carcinoma, small-cell carcinoma and other rare types of lung cancer, and the types except the small-cell carcinoma are collectively called non-small-cell lung cancer, and the proportion of the lung cancer in lung cancer patients is about 90%, wherein the proportion of the adenocarcinoma is about 40-55% at most. The existing treatment of the non-small cell lung cancer is comprehensive treatment combining various treatment means such as operation, chemotherapy, radiotherapy, molecular targeted therapy, immunotherapy and the like. Because the early symptoms of the non-small cell lung cancer patient are not obvious, the diagnosis is mostly in the middle and late stages, the operation opportunity is lost, the preoperative new auxiliary chemotherapy is needed or the postoperative chemotherapy anti-tumor treatment is needed, and the like, the chemotherapy is an important treatment means of the non-small cell lung cancer. The first-line chemotherapy regimen for lung cancer is based on platinum in combination with other chemotherapeutic drugs, with cisplatin being the most commonly used platinum chemotherapeutic drug. However, lung cancer patients typically become resistant to cisplatin as a result of initial treatment or gradually develop resistance during subsequent treatment, resulting in treatment failure. The lung adenocarcinoma accounts for the highest proportion of the non-small cell lung cancers, so a specific molecular mechanism of lung adenocarcinoma cis-platinum resistance is further discussed, and a new basis is provided for reversing the cis-platinum resistance of lung adenocarcinoma patients.

To date, lung adenocarcinoma patients lack information that would predict the susceptibility of individual patients to cisplatin drugs. Therefore, some patients do not obtain the expected therapeutic effect but suffer from the side effects of highly toxic drugs. More importantly, some patients who do not have the necessary treatment may lose additional treatment opportunities due to their worsening physical condition. Conventional histopathological parameters such as tumor stage or grade are generally considered prognostic factors for lung adenocarcinoma patients. However, even if tumors have similar histopathological characteristics, there may be a wide variety of molecular characteristics, belonging to a unique molecular subgroup, with different disease invasiveness and drug sensitivity. In order to perform effective accurate drug therapy aiming at the lung adenocarcinoma, a new target point for cisplatin drug sensitization of the lung adenocarcinoma needs to be explored. There is also a need to improve the ability of molecular markers to predict a patient's responsiveness to a particular treatment, or to assess the efficacy of a treatment. The multigene classification approach identified from gene expression analysis can predict cisplatin drug sensitivity, recurrence and survival in lung adenocarcinoma patients. There is therefore an urgent need for reliable markers that can predict cisplatin drug sensitivity, recurrence and prognosis in patients with lung adenocarcinoma to optimize treatment strategies and improve clinical outcome.

The cisplatin chemotherapy of lung cancer has a complex drug resistance mechanism, and the cisplatin drug resistance mechanism is mainly closely related to the platinum drugs in malignant tumor cells, such as little accumulation, increased DNA damage repair, apoptosis inactivation, epithelial-mesenchymal transition activation, tumor stem cell characteristics and the like. The related proteins and important molecules in their signal pathways are usually regulated by various factors such as genome, epigenetics and posttranslational modifications of proteins. The protein is the main performer and undertaker of life activity, and whether the function is normal or not determines whether the life activity can be performed orderly and efficiently. Genomics and epigenetics together direct the expression of genes unique to various cells at the genetic level. The post-translational modification of the protein directly influences the spatial conformation, activity, stability and the like of the protein at the protein level, and further regulates and controls various functions of the protein to regulate the life activities of organisms. Protein post-translational modification refers to chemical modification of a protein after an organism translates mRNA into protein, which can result in the formation of a mature protein product or regulate the level of protein expression. The posttranslational modification can be more than 400 types, and the main types comprise phosphorylation, acetylation, ubiquitination, methylation, glycosylation and the like.

The protein ubiquitination refers to a process of connecting ubiquitin molecules (highly conserved short peptides composed of 76 amino acids and widely expressed in eukaryotes) to a certain target protein molecule under the action of a series of enzymes to specifically modify the target protein. The process is catalyzed by three classes of enzymes: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). E1 activates ubiquitin and transfers it to E2, E3 recruits ubiquitinated E2, recognizes substrates and assists or directly assists the transfer of ubiquitin on E2 to protein substrates. In the ubiquitination process, E3 has an important function of identifying substrates, the human genome encodes more than 600 ubiquitin ligases, a plurality of ubiquitin ligases are proved to be closely related to the occurrence and development of tumors and malignant phenotypes, and researches prove that the ubiquitin ligases influence the cisplatin resistance of lung cancer by regulating the substrates.

Disclosure of Invention

The invention aims to provide a new target point for preparing lung adenocarcinoma cisplatin sensitization medicines.

In order to explore the reason of the cisplatin resistance of a lung adenocarcinoma patient, the lung adenocarcinoma cell strain A549 and the lung adenocarcinoma cisplatin resistant strain A549/DDP are screened by a protein group chip, a series of significantly changed proteins are identified, and the ubiquitin-dependent protein catabolic process in A549/DDP is found to be active compared with that in A549 by performing biochemical analysis on significantly up-regulated proteins. The invention shows that ubiquitin ligase CAHF1B in the lung adenocarcinoma cisplatin resistant cell strain is highly expressed, which prompts that the high expression is related to the lung adenocarcinoma cisplatin drug sensitivity. The research of the invention shows that ubiquitin ligase CAHF1B in the lung adenocarcinoma cis-platinum drug-resistant cell strain is highly expressed, and the cis-platinum drug sensitivity of the lung adenocarcinoma cells can be increased after the CAHF1B is knocked out. Mechanism research shows that high-expression CAHF1B can promote degradation of NCOR2 ubiquitination to cause lung adenocarcinoma cells to have cis-platinum resistance. Overall, the data indicate that high levels of CAHF1B and low expression of NCOR2 are associated with cisplatin resistance in lung adenocarcinoma. In addition, CAHF1B and NCOR2 are used as target sites in the preparation of lung adenocarcinoma cisplatin sensitizing drugs, are also molecular markers for clinically predicting the cisplatin drug sensitivity of lung adenocarcinoma patients, and can be a new strategy for improving the treatment and survival of lung adenocarcinoma patients.

The cisplatin resistance of a lung adenocarcinoma patient is one of the main reasons for poor tumor progression and prognosis, and in order to explore a lung adenocarcinoma cisplatin resistance mechanism, the subject screens a549/DDP and a549 cell lines by a whole protein group chip, quantifies and identifies 7475A 549A549/DDP cell line differential proteins, and 5758 proteins can be quantified. The P value is defined to be less than 0.05, 657 proteins with the A549/DDP up-regulated or down-regulated by 2 times and more than 2 times compared with the A549 are significant change proteins, wherein 312 proteins with the significant up-regulated and 345 proteins with the significant down-regulated, GO analysis is carried out on the significant up-regulated proteins through a public database David, and the ubiquitin-dependent protein catabolic process in the A549/DDP is found to be active compared with the A549. Therefore, the ubiquitination process is related to the cisplatin drug sensitivity of the lung adenocarcinoma.

The number of ubiquitination enzymes in the protein which is obviously up-regulated is more, and the total number is 46: 1 ubiquitin activating enzyme, 3 ubiquitin conjugating enzymes and 42 ubiquitin ligases. The protein ubiquitination process activates ubiquitin by E1 and transfers it to E2, and E3 recruits ubiquitinated E2, recognizes the substrate and assists or directly assists the transfer of ubiquitin on E2 to the protein substrate. E3 has an important function of identifying substrates in the ubiquitination process, and in 42E 3, the GEPIA analysis of a public database shows that the high expression of ubiquitin ligases WDHD1, ARPC1A, CDC, CHAF1B, PPP R13L, TRIP, AURKA, CDCA3 and FBXO22 is negatively correlated with the total life cycle of a patient with lung adenocarcinoma; the mRNA expression level of the first 6 cells was verified by PCR, and the results indicated that the expression levels of WDHD1, CHAF1B, PPP R13L and CDC20 in A549/DDP cell strain were up-regulated compared to A549 cell strain. The results of the MTT method show that after the 4 genes are knocked out respectively, the cisplatin drug sensitivity of A549/DDP cells can be increased, and CHAF1B with remarkable sensitization effect is selected for further research. These findings suggest that CHAF1B plays an important role in the mechanism of potential cisplatin resistance in lung adenocarcinoma cells.

The Ualcan database analysis shows that the expression level of the mRNA of the CHAF1B is higher than that of the paracarcinoma tissues in lung adenocarcinoma tissues, WB shows that the protein expression level of the CHAF1B in A549/DDP is higher than that of A549 cells, and the results are consistent with the chip results, which prove that the chip results are real and reliable, and suggest that the two are closely related to the lung adenocarcinoma cisplatin resistance. After CHAF1B is successfully knocked out, WB results show that CHAF1B expression is reduced, NCOR2 expression is up-regulated, and PPP5C expression is not obviously changed. The above results suggest that changes in CHAF1B can modulate changes in substrate NCOR2, respectively. MTT and flow cytometry results show that the cell activity is increased and the apoptosis rate is reduced under the action of cisplatin after NCOR2 is knocked out by the A549 cell strain. These data support the hypothesis that ubiquitin ligase CHAF1B and substrate NCOR2 affect important participants in cisplatin resistance in lung adenocarcinoma cells.

Further Co-IP results confirmed that CHAF1B and NCOR2 are interacting proteins, respectively; immunofluorescence double-color detection shows that the interaction position of CHAF1B and NCOR2 is mainly in the cell nucleus, and a protein stability experiment proves that CHAF1B promotes the ubiquitination and degradation of NCOR 2; the functional recovery experiment results show that: under the action of cisplatin, compared with a CHAF1B + knockout NCOR2 group which is knocked out independently in an A549/DDP group, the CHAF1B + knockout NCOR2 group has the advantages that the cell proliferation and migration capacity are increased, the apoptosis rate is reduced, the tumor body growth speed is increased and the tumor body size is obviously increased in an animal experiment, immunohistochemistry indicates that Ki-67 is increased, and the apoptosis related index is obviously reduced. Therefore, the invention suggests that the CHAF1B expression and the detection of the downstream target protein NCOR2 thereof can be used as an effective method for predicting the cis-platinum drug sensitivity of lung adenocarcinoma patients so as to guide clinical decision.

In summary, the present invention describes an altered expression model in lung adenocarcinoma and demonstrates its potential role in lung adenocarcinoma cisplatin drug sensitivity. In addition, further studies demonstrated that ubiquitin ligase CAHF1B can lead to cisplatin resistance in lung adenocarcinoma cells by promoting degradation of NCOR2 ubiquitination. Our findings support the concept of: high levels of CHAF1B may be a novel predictor of cisplatin resistance in lung adenocarcinoma, enabling clinicians to identify high-risk patients in need of more intensive treatment. Thus, targeting the CHAF1B pathway may be a new therapeutic strategy to improve the treatment and survival of patients with lung adenocarcinoma or other cancers.

Drawings

FIG. 1: A549/DDP in the protein chip screening chip is obviously changed compared with A549 protein. A: in the line A549/DDP and A549 full proteome chips, 5758 quantifiable differential proteins are identified. The P value is less than 0.05, and the protein with the A549/DDP up-regulated 2 times or down-regulated 2 times or more than the A549 is the significant change protein. B: protein posttranslational modification-related proteins are significantly upregulated. C: GO enrichment analysis is carried out on the significantly changed protein, and the ubiquitin-dependent protein catabolic process is found to be active. D: 657 proteins are obviously changed in the A549/DDP and A549 protein chips, 312 proteins are obviously up-regulated, 345 proteins are obviously down-regulated, and 46 ubiquitination enzymes are obviously up-regulated. E: of 46 upregulated significant ubiquitination enzymes, 1E 1, 3E 2 and 42E 3;

FIG. 2 is a schematic diagram: e3, which is significantly up-regulated in the protein chip, and E3, whose prognosis is negatively correlated. A: 46 proteins in A549/DDP and A549 significantly up-regulate the protein abundance and relative expression of ubiquitin-related enzyme, and the marked proteins are proteins which are suggested to be negatively related to prognosis by a database. B: according to the public database, 9E 3 high expressions (including: WDHD1, ARPC1A, AURKA, CDC20, CDCA3, CHAF1B, FBXO, PPP1R13L, TRIP) are negatively correlated with lung adenocarcinoma patient prognosis;

FIG. 3: the prognosis shows the negatively correlated and significantly up-regulated E3 expression level and the dose response line thereof. A: the result of the PCR method indicates that the mRNA expression of WDHD1, CHAF1B, PPP R13L and CDC20 in A549/DDP is obviously higher than that of A549.B: after WDHD1, CHAF1B, PPP R13L and CDC20 are knocked out, the proliferation capacity of A549/DDP cells is obviously reduced, and the IC50 is obviously reduced. * p <0.05, p < 0.01, p < 0.001;

FIG. 4 is a schematic view of: expression level of CHAF1B in lung adenocarcinoma and its substrate exploration. A: public database Ualcan suggested that CHAF1B expression was higher in lung cancer tissues than in paracarcinoma tissues. B: NCOR2 is up-regulated upon CHAF1B knock-out, whereas NCOR2 is not significantly altered. C: ubiquitination sites of NCOR2 and PPP 5C;

FIG. 5: downregulation of NCOR2 expression in a549 cells reduces cisplatin drug sensitivity. A-B: MTT method and flow cytometry result show that after NCOR2 is knocked out by A549 cells, the drug sensitivity of lung adenocarcinoma cells to cisplatin is reduced, cell proliferation is accelerated, and apoptosis is reduced. DDP: IC25 concentration of cisplatin to A549/DDP. * p is less than 0.05;

FIG. 6: CHAF1B in A549/DDP forms a complex with NCOR2 and promotes the ubiquitination and degradation of NCOR 2. A: the co-immunoprecipitation method showed that CHAF1B and NCOR2 are interacting proteins. B: immunofluorescence detection results show that NCOR2 (green) is mainly present in cell nucleus, CHAF1B (red) is present in cell nucleus and cell cytoplasm, and the two are co-localized in cell nucleus. C: protein stability experiments demonstrated a slower degradation rate of NCOR2 following CHAF1B knockout. * p is less than 0.05;

FIG. 7: CHAF1B in A549/DDP caused cisplatin resistance by increasing NCOR2 degradation. A-D: the functional recovery experiment carried out by respectively carrying out an MTT method, flow cytometry, a scratch experiment and a cell clone formation experiment proves that the degradation of the NCOR2 by the CHAF1B causes the cisplatin resistance;

FIG. 8: in vivo experiments further validated the function of CHAF 1B. A: the transplanted tumor experiment shows that the inhibiting effect of the cisplatin on the tumor volume after the CHAF1B is knocked out by the A549/DDP is obviously greater than that of a control group. B: WB demonstrated an upregulation of CHAF1B expression in tumor tissues of A549/DDP, knock-out of NCOR2 upregulation in tumor tissues of CHAF1B group. C-D: after CHAF1B is knocked out by A549/DDP, the Ki-67 expression is obviously less than that of a control group, and the apoptosis index is obviously higher than that of the control group. Bars indicates standard deviation, p <0.05, DDP treatment concentration is A549/DDP cell IC25 concentration.

Detailed description of the preferred embodiments

The invention is further explained and illustrated below with reference to the figures and experimental data

1. The material and the method are as follows: cell culture and transfection, qRT-PCR analysis, western blot analysis, immunohistochemical assay, MTT assay, flow cytometry, immunoprecipitation, immunofluorescence, cell scratch repair assay, colony formation assay, all of which are current methods and will not be described herein.

2. As a result: 2.1 Analyzing and verifying a full proteome chip;

2.1.1 The ubiquitin-dependent protein catabolism in the A549/DDP cell strain complete proteome chip is active, and the ubiquitination of the protein is related to cisplatin resistance; the cisplatin resistance of a lung adenocarcinoma patient is one of the main reasons for poor tumor progression and prognosis, and in order to explore a lung adenocarcinoma cisplatin resistance mechanism, the subject screens A549/DDP and A549 cell strains in a whole protein group chip. A stable isotope labeling and liquid chromatography mass spectrometry or mass spectrometry analysis integration combined method is used for quantifying A549 and A549/DDP cell line proteins, differential proteins of A549/DDP and A549 are analyzed, 7475 differential proteins are identified in total, and 5758 quantitative proteins are identified. We define that the P value is less than 0.05, and the protein with 2 times up-regulation or2 times down-regulation of A549/DDP is the significant change protein, and 657 significant change proteins are found, wherein 312 significant up-regulation proteins and 345 significant down-regulation proteins are shown (as the figure 1A). Analysis of the significantly upregulated proteins found post-translational modification-related protein upregulation (see figure 1B). GO analysis of the significantly upregulated proteins using public database https:// david. Ncifcrf. Gov/found that ubiquitin dependent protein catabolic processes were active in a549/DDP compared to a549, with statistical significance for the difference (p < 0.05) (see figure 1C). The ubiquitin-related websites Uniprot and ubibrown suggested that there were more, 46 in total, of proteins that were significantly upregulated (see fig. 1D): 1 ubiquitin activating enzyme (E1), 3 ubiquitin conjugating enzymes (E2) and 42 ubiquitin ligases (E3) (see fig. 1E).

2.1.2 multiple ubiquitin ligases can increase the drug sensitivity of lung adenocarcinoma cells cis-platinum; the protein chip of the subject has a total of 46 ubiquitination enzymes that are significantly up-regulated, including 1 ubiquitin activating enzyme, 3 ubiquitin conjugating enzymes and 42 ubiquitin ligases, and the protein abundance is shown in the heat map (as shown in fig. 2A). The protein ubiquitination process activates ubiquitin by E1 and transfers it to E2, and E3 recruits ubiquitinated E2, recognizes the substrate and assists or directly assists the transfer of ubiquitin on E2 to the protein substrate. Therefore, E3 plays an important role in substrate recognition in the ubiquitination process, and the research on E3 is mainly carried out by the subject group because the literature suggests that E3 is closely related to cisplatin resistance of malignant tumors. Of the 42E 3, to clarify the E3 function and explore whether it could affect lung adenocarcinoma cisplatin resistance, the public database http:// gepia. Cancer-pku. Cn/found up-regulated E3, 9 in total E3 that negatively correlated with prognosis (see FIG. 2B): WDHD1, ARPC1A, AURKA, CDC20, CDCA3, CHAF1B, FBXO, PPP1R13L, TRIP.

Among the 9E 3 which are remarkably up-regulated and negatively related to prognosis, partial up-regulation of ubiquitin ligase expression is related to cisplatin resistance in research reports, and the authenticity and reliability of the chip screened by the subject are confirmed. The subject selects proteins related to cisplatin resistance reported in no literature, and a few proteins related to cisplatin resistance reported in the literature are explored, including WDHD1, CHAF1B, ARPC1A, CDC and PPP1R13L, TRIP. The expression level of the protein is detected by a PCR method, and the result indicates that the expression levels of WDHD1, CHAF1B, PPP R13L and CDC20 in the A549/DDP cell strain are up-regulated compared with the expression level of the A549 cell strain, and the difference has statistical significance (P < 0.05), which is consistent with the result trend of a protein chip (as shown in figure 3A). Because the expression trends of ARPC1A and TRIP12 are not consistent with those of the chip, the proteins corresponding to the two genes are not studied subsequently. Further exploring the influence of WDHD1, CHAF1B, PPP R13L and CDC20 on the cisplatin drug sensitivity of A549/DDP cells, constructing corresponding si-RNA to knock out the 4 genes in A549/DDP, detecting the proliferation capacity of tumor cells by an MTT method, calculating the IC50 and the IC25 of the tumor cells, and finding that the proliferation capacity of A549/DDP cells is obviously reduced after the 4 genes are knocked out (for example, B) and the IC50 is obviously reduced. Since the PPP1R13L has been reported in the literature to be associated with cisplatin resistance, the IC50 drop of CDC20 is relatively small, and these two proteins are not subsequently studied. Therefore, the problem is to initially explore a mechanism of CHAF1B for regulating and controlling the cisplatin drug sensitivity of A549 lung adenocarcinoma cells.

2.2 ubiquitin ligase causes A549 lung adenocarcinoma cell cisplatin resistance by promoting substrate ubiquitination degradation; 2.2.1 CHAF1B promotes NCOR2 ubiquitination degradation to cause lung adenocarcinoma cisplatin resistance;

2.2.1.1 CHAF1B in A549/DDP can cause NCOR2 expression to be reduced; the results of the whole proteome chip show that the CHAF1B protein expression level of the A549/DDP cell strain is up-regulated by 2.01 times compared with that of the A549 cell strain, and the results of the unalkan of the public database show that the CHAF1B protein expression level is higher in the lung cancer tissues than in the paracarcinoma tissues (as shown in FIG. 4A). WB results confirmed that CHAF1B protein expression was up-regulated in A549/DDP compared to A549 (FIG. 4B). In combination with the whole proteome chip and public databases Biocuckoo, phosphosite and uaalcan, only NCOR2 and PPP5C (ubiquitination site as in fig. 4C) have an interactive relationship with CHAF1B, have ubiquitination sites and are low expressed in lung adenocarcinoma. After si-CHAF1B knockdown of CHAF1B in A549/DDP, the WB method was performed to detect the change of NCOR2 and PPP5C protein expression levels, and NCOR2 was up-regulated, while PPP5C was not significantly changed (see FIG. 4B).

2.2.1.2 Downregulation of NCOR2 expression in a549 may lead to cellular cisplatin resistance; to clarify the function of NCOR2, group A, B was the subject of a549, and used as the study target to transfect si-con and si-NCOR2, C, D was used to transfect si-con and si-NCOR2 in a549/DDP, MTT was used to detect cell viability (fig. 5A), and flow cytometry was used to detect apoptosis (fig. 5B), which showed that the cell viability was increased and apoptosis was decreased after knocking out NCOR2 by a549 cell strain, while si-NCOR2 was transfected or not transfected in a549/DDP, which indicated that NCOR2 down-regulation in lung adenocarcinoma cells is one of the causes of drug resistance.

Taken together, we speculate that NCOR2 might act as a downstream target protein for CHAF1B to modulate cisplatin drug sensitivity.

2.2.1.3 CHAF1B forms a complex with NCOR2 and promotes degradation of NCOR2 ubiquitination to cause cisplatin resistance; co-immunoprecipitation explored the interaction relationship between cha 1B and NCOR2, indicating that cha 1B and NCOR2 are interacting proteins (see fig. 6A). The two-color line IF assay revealed the interaction site of CHAF1B and NCOR2, indicating that CHAF1B (red) is present in the nucleus and cytoplasm and NCOR2 (green) is present in the nucleus, indicating that the interaction between CHAF1B and NCOR2 is mainly located in the nucleus (FIG. 6B). Protein stability results suggest that the degradation rate of NCOR2 is significantly slower following CHAF1B knockout (see figure 6C). The above experiments demonstrated that CHAF1B and NCOR2 are interacting proteins and that CHAF1B promotes the degradation of NCOR2 ubiquitination.

A function recovery experiment is carried out to explore that CHAF1B influences the function of cisplatin resistance by regulating NCOR 2. A549/DDP is taken as a research object to set 5 groups of A-E, A, B and Si-control and Si-CHAF1B respectively, and C, E and Si-CHAF1B, si-NCOR2 respectively, the group D simultaneously knocks out CHAF1B and NCOR2, and the three groups of C-E are transfected and then treated with DDP, and after the treatment, an MTT method (such as A), a flow cytometry (such as B), a scratch experiment (such as C) and a cell clone formation experiment (such as D) are respectively carried out to carry out a functional recovery experiment, and the experiment result shows that when CHAF1B in A549/DDP is knocked out during DDP treatment, the cell proliferation capacity is obviously reduced and the apoptosis is obviously increased. When the CHAF1B is knocked out and then the NCOR2 is knocked out, the cell proliferation capacity is recovered and the apoptosis is reduced.

2.2.1.4 Animal experiments prove that CHAF1B promotes NCOR2 degradation to increase lung adenocarcinoma cisplatin resistance; to further validate the function following interaction of CHAF1B and NCOR2, A549/DDP cells transfected with (1) si-con, (2) si-CHAF1B, (3) si-CHAF1B + si-NCOR2, respectively, were subcutaneously implanted in mice in animal experiments, and the growth rate and size of cisplatin-treated tumors after knockdown of CHAF1B was significantly less than in the control group (P <0.05, FIG. 8A-B). WB confirmed the upregulation of CHAF1B expression in the tissues of A549/DDP, and NCOR2 upregulation in tumor tissues after CHAF1B knockout. By detecting a tumor proliferation related index Ki-67 through immunohistochemistry, the positive rate of Ki67 in the CHAF1B knockout group is obviously lower than that in a control group (figure 8C), and the apoptosis index is obviously increased (figure 8D). In vivo experiments further demonstrate that CHAF1B knockout increases cisplatin sensitivity, decreases cell proliferation and increases apoptosis in lung adenocarcinoma, while CHAF1B knockout and NCOR2 knockout lead to decreased cisplatin drug sensitivity, increased cell proliferation and decreased apoptosis in lung adenocarcinoma.

In conclusion, cell experiments and animal experiments prove that CHAF1B promotes NCOR2 ubiquitination degradation to cause A549/DDP cell cisplatin resistance. By combining the up-regulation of CHAF1B expression and the down-regulation of NCOR2 expression in lung adenocarcinoma patient specimens, the negative relation of the two is very likely to provide new evidence for clinical prediction of cisplatin chemotherapy sensitivity of lung adenocarcinoma patients and provide an important molecular target for reversal of cisplatin resistance.

Claims

1. The application of the ubiquitin ligase CHAF1B inhibitor in preparing the lung adenocarcinoma cisplatin sensitization drugs.

2. Application of a preparation for detecting expression level of ubiquitin ligase CHAF1B in preparation of a preparation for predicting lung adenocarcinoma cisplatin sensitivity.

3. Application of a preparation for detecting NCOR2 expression level in preparing a preparation for predicting lung adenocarcinoma cisplatin sensitivity.