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

Abstract

The invention discloses the application of the ubiquitin ligase CHAF1B as a target site in the preparation of cisplatin-sensitizing drugs for lung adenocarcinoma. The research of the present invention shows that the ubiquitin ligase CAHF1B is highly expressed in the cisplatin-resistant cell line of lung adenocarcinoma, and the cisplatin drug sensitivity of lung adenocarcinoma cells can be increased after knocking out CAHF1B. Therefore, CAHF1B can be used as a target site in the preparation of cisplatin-sensitizing drugs for lung adenocarcinoma, and it is also a molecular marker for clinically predicting cisplatin drug sensitivity in patients with lung adenocarcinoma. Mechanistic studies have shown that highly expressed CAHF1B can lead to cisplatin resistance in lung adenocarcinoma cells by promoting the ubiquitination and degradation of NCOR2. Therefore, NCOR2 is also used as a target site in the preparation of cisplatin sensitizers for lung adenocarcinoma.

Description

Application of ubiquitin ligase CHAF1B as a target site in the preparation of cisplatin-sensitizing drugs for lung adenocarcinoma

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to the application of ubiquitin ligase CHAF1B as a target site in the preparation of cisplatin-sensitizing drugs for lung adenocarcinoma.

Background technique

Lung cancer is the most common malignant tumor in the world, and its morbidity and mortality rate rank first in the world, and the morbidity and mortality rate in China are also high. According to the 2015 WHO lung cancer histological classification standard, the pathological types of lung cancer mainly include adenocarcinoma, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma, small cell carcinoma and other rare types of lung cancer, and other types except small cell carcinoma are collectively referred to as non-small cell carcinoma. Cell lung cancer accounts for about 90% of lung cancer patients, and the highest proportion of adenocarcinoma is about 40% to 55%. At present, the treatment of non-small cell lung cancer is a comprehensive treatment including surgery, chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy. Chemotherapy is an important treatment for non-small cell lung cancer because the early symptoms of non-small cell lung cancer patients are not obvious, and most of them are in the middle and late stages when they are diagnosed. . The first-line chemotherapy for lung cancer is based on platinum-based chemotherapy combined with other chemotherapy drugs, among which cisplatin is the most commonly used platinum-based chemotherapy drug. However, in general, lung cancer patients are resistant to cisplatin after initial treatment or gradually develop drug resistance during subsequent treatment, which leads to treatment failure. Lung adenocarcinoma accounts for the highest proportion of non-small cell lung cancer. Therefore, it is urgent to further explore the specific molecular mechanism of cisplatin resistance in lung adenocarcinoma and provide new evidence for the reversal of cisplatin resistance in lung adenocarcinoma patients.

To date, there is a lack of information in patients with lung adenocarcinoma to predict sensitivity to cisplatin in individual patients. Therefore, some patients did not obtain the expected therapeutic effect but suffered side effects of highly toxic drugs. What's more, some unnecessarily treated patients may lose the opportunity for additional treatment due to their deteriorating physical condition. Traditional histopathological parameters such as tumor stage or grade are generally considered to be prognostic factors in patients with lung adenocarcinoma. However, even tumors that share similar histopathologic features may similarly have widely divergent molecular features, belong to distinct molecular subgroups, and have distinct disease aggressiveness and drug sensitivity. In order to implement effective precision drug therapy for lung adenocarcinoma, it is necessary to explore new targets for cisplatin drug sensitization in lung adenocarcinoma. There is also a need to improve the ability of molecular markers to predict a patient's response to a particular treatment, or to assess the effect of a treatment. A multigene classification method identified from gene expression analysis predicts cisplatin sensitivity, recurrence, and survival in patients with lung adenocarcinoma. Therefore, there is an urgent need for reliable markers that can predict cisplatin drug sensitivity, recurrence, and prognosis in patients with lung adenocarcinoma in order to optimize treatment strategies and improve clinical outcomes.

The mechanism of cisplatin chemotherapy resistance in lung cancer is complex. So far, the mechanism of cisplatin resistance is mainly related to less accumulation of platinum drugs in malignant tumor cells, increased DNA damage repair, apoptosis inactivation, activation of epithelial-mesenchymal transition, and tumor stem cell characteristics. relevant. The related proteins and important molecules in the signaling pathway are usually regulated by multiple factors such as genome, epigenetics and protein post-translational modification. Protein is the main executor and bearer of life activities, and whether its function is normal or not determines whether life activities can be carried out in an orderly and efficient manner. Genomics and epigenetics work together at the genetic level to direct the unique gene expression of various cells. The protein post-translational modification directly affects the spatial conformation, activity and stability of the protein at the protein level, and then regulates various functions of the protein to regulate the life activities of the body. Protein post-translational modification refers to the chemical modification of protein after the organism translates mRNA into protein. This process can form mature protein products or regulate protein expression levels. There are more than 400 types of post-translational modifications, and the main types include phosphorylation, acetylation, ubiquitination, ubiquitination, methylation, and glycosylation.

Protein ubiquitination refers to that ubiquitin molecules (a highly conserved short peptide composed of 76 amino acids widely expressed in eukaryotes) are connected to a target protein molecule under the action of a series of enzymes. The process of protein specific modification. This process is catalyzed by three classes of enzymes: ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). E1 activates ubiquitin and transfers it to E2, E3 recruits ubiquitinated E2, recognizes the substrate and assists or directly assists the transfer of ubiquitin on E2 to the protein substrate. E3 plays an important role in identifying substrates in the ubiquitination process. The human genome encodes more than 600 ubiquitin ligases, many of which have been proven to be closely related to tumor development and malignant phenotypes, and studies have confirmed Ubiquitin ligases affect cisplatin resistance in lung cancer by modulating substrates.

Contents of the invention

The present invention aims to provide a new target that can be used to prepare cisplatin-sensitizing drugs for lung adenocarcinoma.

In order to explore the cause of cisplatin resistance in patients with lung adenocarcinoma, the present invention carried out proteome chip screening of lung adenocarcinoma cell line A549 and lung adenocarcinoma cisplatin-resistant strain A549/DDP, and identified a series of significantly changed proteins, which were significantly up-regulated Bioinformatics analysis of the protein revealed that compared with A549, the ubiquitin-dependent protein catabolism process was active in A549/DDP. The present invention shows that the ubiquitin ligase CAHF1B is highly expressed in lung adenocarcinoma cisplatin drug-resistant cell lines, suggesting that its high expression is related to lung adenocarcinoma cisplatin drug sensitivity. The research of the present invention shows that the ubiquitin ligase CAHF1B is highly expressed in the cisplatin-resistant cell line of lung adenocarcinoma, and the cisplatin drug sensitivity of lung adenocarcinoma cells can be increased after knocking out CAHF1B. Mechanistic studies have shown that highly expressed CAHF1B can lead to cisplatin resistance in lung adenocarcinoma cells by promoting the ubiquitination and degradation of NCOR2. Collectively, the data suggest 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 cisplatin sensitizers for lung adenocarcinoma, and they are also molecular markers for clinically predicting cisplatin drug sensitivity in patients with lung adenocarcinoma, which may be a new method to improve lung adenocarcinoma Strategies for patient care and survival.

Cisplatin resistance in patients with lung adenocarcinoma is one of the main reasons for tumor progression and poor prognosis. In order to explore the mechanism of cisplatin resistance in lung adenocarcinoma, this project screened A549/DDP and A549 cell lines with a whole proteome chip , A total of 7475 differential proteins were quantified and identified in the A549A549/DDP cell line, and 5758 proteins could be quantified. Defined as P value <0.05, A549/DDP up-regulated 2 times or down-regulated 2 times or more than A549 as significant changes. There are 657 proteins, including 312 significantly up-regulated proteins and 345 significantly down-regulated proteins. Through the public database David Significantly up-regulated proteins were analyzed by GO, and it was found that the ubiquitin-dependent protein catabolism process was active in A549/DDP compared with A549. It can be seen that the ubiquitination process is related to the sensitivity of lung adenocarcinoma to cisplatin.

Among the significantly up-regulated proteins, there were more ubiquitinases, 46 in total: 1 ubiquitin activating enzyme, 3 ubiquitin conjugating enzymes and 42 ubiquitin ligases. The process of protein ubiquitination is that E1 activates ubiquitin 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 plays an important role in identifying substrates in the process of ubiquitination. Among the 42 E3s, the public database GEPIA analysis showed that the high expression of ubiquitin ligases WDHD1, ARPC1A, CDC20, CHAF1B, PPP1R13L, TRIP12, AURKA, CDCA3, and FBXO22 were associated with pulmonary The overall survival of adenocarcinoma patients was negatively correlated; the mRNA expression levels of the first 6 patients were verified by PCR, and the results indicated that the expression levels of WDHD1, CHAF1B, PPP1R13L and CDC20 in A549/DDP cell lines were up-regulated compared with A549 cell lines. The results of MTT method showed that the knockout of the above four genes could increase the drug sensitivity of A549/DDP cells to cisplatin, and CHAF1B with a significant sensitization effect was selected for further research. These findings suggest that CHAF1B plays an important role in the underlying mechanism of cisplatin resistance in lung adenocarcinoma cells.

Ualcan database analysis showed that the mRNA expression level of CHAF1B in lung adenocarcinoma tissue was higher than that in adjacent tissues. WB showed that the protein expression level of CHAF1B in A549/DDP was higher than that in A549 cells, which was consistent with the chip results, confirming that the chip results were true and reliable, and It is suggested that the two are closely related to cisplatin resistance in lung adenocarcinoma. After successfully knocking out CHAF1B, WB results showed that the expression of CHAF1B decreased, the expression of NCOR2 was up-regulated, and the expression of PPP5C had no significant change. The above results suggest that the changes of CHAF1B can regulate the changes of the substrate NCOR2 respectively. The results of MTT and flow cytometry showed that after knocking out NCOR2 in the A549 cell line, the cell viability increased and the apoptosis rate decreased under the action of cisplatin. These data support the hypothesis that the ubiquitin ligase CHAF1B and substrate NCOR2 affect important players in cisplatin resistance in lung adenocarcinoma cells.

Further Co-IP results confirmed that CHAF1B and NCOR2 are interacting proteins; immunofluorescence double-color detection showed that the interaction between CHAF1B and NCOR2 is mainly in the nucleus, and protein stability experiments confirmed that CHAF1B promotes the ubiquitination and degradation of NCOR2; the results of functional recovery experiments It shows that under the action of cisplatin, the knockout CHAF1B + knockout NCOR2 group in A549/DDP has increased cell proliferation and migration ability in cell experiments, decreased apoptosis rate, and increased tumor growth rate in animal experiments compared with CHAF1B knockout group alone. Immunohistochemistry showed that Ki-67 increased significantly, while apoptosis-related indicators decreased significantly. Therefore, the present invention suggests that the detection of CHAF1B expression and its downstream target protein NCOR2 can be used as an effective method for predicting cisplatin drug sensitivity in patients with lung adenocarcinoma, so as to guide clinical decision-making.

In conclusion, 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 confirmed that the ubiquitin ligase CAHF1B can lead to cisplatin resistance in lung adenocarcinoma cells by promoting the ubiquitination and degradation of NCOR2. Our findings support the concept that high levels of CHAF1B may be a novel predictor of cisplatin resistance in lung adenocarcinoma, allowing clinicians to identify high-risk patients who require more intensive therapy. Therefore, targeting the CHAF1B pathway may become a new therapeutic strategy to improve the treatment and survival of patients with lung adenocarcinoma or other cancers.

Description of drawings

Figure 1: A549/DDP significantly changed protein compared with A549 in the protein chip screening chip. A: Run A549/DDP and A549 whole proteome chip, identify and quantify 5758 differential proteins. Define the P value <0.05, and the protein that A549/DDP is up-regulated by 2 times or down-regulated by 2 times or more than A549 is a significantly changed protein. B: Protein post-translational modification related proteins are significantly upregulated. C: GO enrichment analysis of significantly changed proteins revealed that ubiquitin-dependent protein catabolism was active. D: In A549/DDP and A549 protein chips, there are 657 significantly changed proteins, 312 significantly up-regulated proteins, 345 significantly down-regulated proteins, and 46 significantly up-regulated ubiquitinases. E: Among the 46 significantly up-regulated ubiquitinases, there is 1 E1, 3 E2 and 42 E3;

Figure 2: Significantly upregulated E3 and E3 with negative prognosis in protein microarray. A: The abundance and relative expression of 46 ubiquitin-related enzymes significantly upregulated in A549/DDP and A549. The proteins marked by * are proteins that the database suggests are negatively correlated with prognosis. B: According to the public database, the high expression of 9 E3s (including: WDHD1, ARPC1A, AURKA, CDC20, CDCA3, CHAF1B, FBXO22, PPP1R13L, TRIP12) is negatively correlated with the prognosis of lung adenocarcinoma patients;

Figure 3: Significantly upregulated E3 expression levels and their dose-response lines negatively correlated with prognosis. A: The results of PCR showed that the mRNA expressions of WDHD1, CHAF1B, PPP1R13L and CDC20 in A549/DDP were significantly higher than those in A549. B: After knocking out WDHD1, CHAF1B, PPP1R13L and CDC20, the proliferation ability of A549/DDP cells was significantly decreased, and IC50 was significantly down-regulated. *p<0.05, **p<0.01, ***p<0.001;

Figure 4: Expression level of CHAF1B in lung adenocarcinoma and its substrate exploration. A: The public database Ualcan suggests that the expression of CHAF1B in lung cancer tissues is higher than that in paracancerous tissues. B: NCOR2 is upregulated after knocking out CHAF1B, while NCOR2 has no significant change. C: ubiquitination sites of NCOR2 and PPP5C;

Figure 5: Down-regulation of NCOR2 expression in A549 cells reduces cisplatin drug sensitivity. A-B: The results of MTT assay and flow cytometry showed that after knocking out NCOR2 in A549 cells, the drug sensitivity of lung adenocarcinoma cells to cisplatin decreased, cell proliferation accelerated and apoptosis decreased. DDP: IC25 concentration of cisplatin on A549/DDP. *p<0.05;

Figure 6: CHAF1B forms a complex with NCOR2 in A549/DDP and promotes ubiquitination and degradation of NCOR2. A: Co-immunoprecipitation method shows that CHAF1B and NCOR2 are interacting proteins. B: The results of immunofluorescence detection show that NCOR2 (green) mainly exists in the nucleus, CHAF1B (red) exists in both the nucleus and the cytoplasm, and both co-localize in the nucleus. C: Protein stability experiments confirmed that the degradation rate of NCOR2 slowed down after knocking out CHAF1B. *p<0.05;

Figure 7: CHAF1B contributes to cisplatin resistance by increasing NCOR2 degradation in A549/DDP. A-D: MTT method, flow cytometry, scratch test and cell clone formation test were performed to perform functional recovery experiments to confirm that CHAF1B leads to cisplatin resistance by increasing the degradation of NCOR2;

Figure 8: In vivo experiments further validate the function of CHAF1B. A: The transplanted tumor experiment found that the inhibitory effect of cisplatin on the tumor volume after A549/DDP knocked out CHAF1B was significantly greater than that of the control group. B: WB confirmed that the expression of CHAF1B was up-regulated in the tumor tissue of A549/DDP, and the expression of NCOR2 was up-regulated in the tumor tissue of the knockout CHAF1B group. C-D: After A549/DDP knockout CHAF1B, the expression of Ki-67 was significantly less than that of the control group, and the apoptosis index was significantly higher than that of the control group. Bars represent the standard deviation, *p<0.05, the DDP treatment concentration is the IC25 concentration of A549/DDP cells.

Implementation

Below in conjunction with accompanying drawing and experimental data, the present invention is further explained and illustrated

1. Materials and methods: cell culture and transfection, qRT-PCR analysis, western blot analysis, immunohistochemical assay, MTT assay, flow cytometry, immunoprecipitation, immunofluorescence, cell scratch repair experiment, colony formation assay, The above methods are all existing methods and will not be repeated here.

2. Results: 2.1 Analysis and verification of the whole proteome chip;

2.1.1 Active ubiquitin-dependent protein catabolism in the whole proteome chip of A549/DDP cell line, suggesting that protein ubiquitination is related to cisplatin resistance; cisplatin resistance in lung adenocarcinoma patients is the cause of tumor progression and poor prognosis One of the main reasons is that in order to explore the mechanism of cisplatin resistance in lung adenocarcinoma, this project screened A549/DDP and A549 cell lines with a whole proteome microarray. Using stable isotope labeling combined with liquid chromatography mass spectrometry or mass spectrometry analysis to quantify A549 and A549/DDP cell line proteins, analyze the differential proteins of A549/DDP and A549, a total of 7475 differential proteins were identified, and 5758 proteins could be quantified indivual. We defined P value < 0.05, A549/DDP up-regulated 2 times or down-regulated 2 times or more than A549 as significantly changed proteins, and found a total of 657 significantly changed proteins, including 312 significantly up-regulated proteins and 345 significantly down-regulated proteins (Figure 1A). Analysis of significantly up-regulated proteins found that post-translational modification-related proteins were up-regulated (Figure 1 B). Using the public database https://david.ncifcrf.gov/ to perform GO analysis on the significantly up-regulated proteins, it was found that the ubiquitin-dependent protein catabolism process was active in A549/DDP compared with A549, and the difference was statistically significant (p<0.05 ) (as shown in Figure 1 C). The ubiquitinase-related websites Uniprot and Ubibrowser suggest that there are more ubiquitinases in significantly up-regulated proteins, a total of 46 (as shown in Figure 1 D): 1 ubiquitin activating enzyme (E1), 3 ubiquitin conjugating enzymes (E2 ) and 42 ubiquitin ligases (E3) (Figure 1 E).

2.1.2 A variety of ubiquitin ligases can increase the drug sensitivity of lung adenocarcinoma cells to cisplatin; a total of 46 ubiquitinases were significantly up-regulated in the protein chip of this project, including 1 ubiquitin activating enzyme and 3 ubiquitin binding enzymes enzymes and 42 ubiquitin ligases, and their protein abundances are shown in the heatmap (Fig. 2A). The process of protein ubiquitination is that E1 activates ubiquitin 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. It can be seen that E3 plays an important role in identifying substrates in the ubiquitination process, and the literature suggests that E3 is closely related to cisplatin resistance in malignant tumors. Therefore, the research group mainly studies E3. Among the 42 E3, in order to clarify the function of E3 and To explore whether it can affect cisplatin resistance in lung adenocarcinoma, consult the public database http://gepia.cancer-pku.cn/ and find that among the up-regulated E3s, there are 9 E3s that are negatively correlated with prognosis (as shown in Figure 2B): WDHD1 , ARPC1A, AURKA, CDC20, CDCA3, CHAF1B, FBXO22, PPP1R13L, TRIP12.

Among the nine E3s that were significantly up-regulated and negatively correlated with prognosis, some studies have reported that the up-regulation of ubiquitin ligase expression is related to cisplatin resistance, which confirms the authenticity and reliability of the chip screening results in this project. In this project, the proteins that have not been reported to be associated with cisplatin resistance and the few proteins that have been reported to be associated with cisplatin resistance are selected for exploration, including WDHD1, CHAF1B, ARPC1A, CDC20, PPP1R13L, and TRIP12. The expression levels were detected by PCR, and the results indicated that the expression levels of WDHD1, CHAF1B, PPP1R13L and CDC20 in the A549/DDP cell line were up-regulated compared with those in the A549 cell line, and the difference was statistically significant (P<0.05), which was consistent with the trend of the protein chip results ( Figure 3 A). Because the expression trends of ARPC1A and TRIP12 were not consistent with the microarray, the corresponding proteins of these two genes were not studied in the follow-up. To further explore the effects of WDHD1, CHAF1B, PPP1R13L and CDC20 on the sensitivity of A549/DDP cells to cisplatin, construct the corresponding si-RNA to knock down the above four genes in A549/DDP, and perform MTT assay to detect the proliferation ability of tumor cells and calculate For IC50 and IC25, it was found that the proliferation ability of A549/DDP cells was significantly reduced after knocking out the expression of the above four genes (as shown in Figure 3 B), and IC50 was significantly down-regulated. Since it has been reported in the literature that PPP1R13L is associated with cisplatin resistance, and the IC50 drop of CDC20 is relatively small, these two proteins will not be studied in the future. Therefore, this topic explores the mechanism of CHAF1B regulation of cisplatin drug sensitivity in A549 lung adenocarcinoma cells.

2.2 Ubiquitin ligase leads to cisplatin resistance in A549 lung adenocarcinoma cells by promoting substrate ubiquitination and degradation; 2.2.1 CHAF1B promotes NCOR2 ubiquitination and degradation to lead to cisplatin resistance in lung adenocarcinoma cells;

2.2.1.1 CHAF1B in A549/DDP can reduce the expression of NCOR2; the whole proteome microarray results show that the expression of CHAF1B protein in A549/DDP cell line is 2.01 times higher than that in A549 cell line, and the results of public database Ualcan suggest that the expression of CHAF1B in lung cancer tissue is higher than that of Paracancerous tissue (Figure 4A). WB results confirmed that the expression of CHAF1B protein in A549/DDP was up-regulated compared with A549 (Figure 4 B). Combined with the whole proteome chip and public databases Biocuckoo, Phosphosite and Ualcan, only NCOR2 and PPP5C (ubiquitination site as shown in Figure 4 C) have an interaction relationship with CHAF1B, have ubiquitination sites and are low expressed in lung adenocarcinoma . After knocking out CHAF1B in A549/DDP with si-CHAF1B, the protein expression levels of NCOR2 and PPP5C were detected by WB method, and NCOR2 was up-regulated, while PPP5C had no significant change (as shown in Figure 4 B).

2.2.1.2 Down-regulating the expression of NCOR2 in A549 can lead to cisplatin-resistant cells; in order to clarify the function of NCOR2, A549 was used as the research object in groups A and B, which were transfected with si-con and si-NCOR2 respectively. DDP was transfected with si-con and si-NCOR2 respectively, and cell viability was detected by MTT method (as shown in Figure 5 A), and cell apoptosis was detected by flow cytometry (as shown in Figure 5 B). Cell viability increased and apoptosis decreased, while A549/DDP transfected or not transfected with si-NCOR2 had high cell viability and less apoptosis, suggesting that down-regulation of NCOR2 in lung adenocarcinoma cells is one of the reasons for cisplatin resistance .

In summary, we speculate that NCOR2 may act as a downstream target protein of CHAF1B to regulate cisplatin drug sensitivity.

2.2.1.3 CHAF1B forms a complex with NCOR2 and promotes the ubiquitination and degradation of NCOR2 to cause cisplatin resistance; co-immunoprecipitation explores the interaction relationship between CHAF1B and NCOR2, and the results show that CHAF1B and NCOR2 are interacting proteins (as shown in Figure 6 A) . IF dual-color detection was performed to clarify the joint interaction between CHAF1B and NCOR2. The results showed that CHAF1B (red) exists in the nucleus and cytoplasm, and NCOR2 (green) exists in the nucleus, suggesting that the interaction between CHAF1B and NCOR2 is mainly located in the nucleus (Figure 6 B) . The results of protein stability experiments suggested that the degradation rate of NCOR2 was significantly slowed down after knocking out CHAF1B (Figure 6C). The above experiments confirmed that CHAF1B and NCOR2 are interacting proteins, and CHAF1B promotes the ubiquitination and degradation of NCOR2.

Functional recovery experiments were performed to explore the function of CHAF1B affecting cisplatin resistance by regulating NCOR2. A549/DDP was used as the research object to set up 5 groups A-E, groups A and B were transfected with si-control and si-CHAF1B respectively, groups C and E were transfected with si-CHAF1B and si-NCOR2 respectively, and group D was knocked out at the same time CHAF1B and NCOR2, and three groups C-E were treated with DDP after transfection. After the above treatment, MTT method (as shown in Figure 7 A), flow cytometry (as shown in Figure 7 B), and scratch test (as shown in Figure 7 C) were performed respectively. And the cell clone formation experiment (as shown in Figure 7 D) was used for functional recovery experiments. The experimental results showed that when CHAF1B in A549/DDP was knocked out when DDP was treated, the cell proliferation ability was significantly decreased and apoptosis was significantly increased. After knocking out CHAF1B and then knocking out NCOR2, the cell proliferation ability was restored and apoptosis was reduced.

2.2.1.4 Animal experiments confirmed that CHAF1B promotes the degradation of NCOR2 and increases lung adenocarcinoma cisplatin resistance; in order to further verify the function of the interaction between CHAF1B and NCOR2, in animal experiments, transfect (1) si-con, (2) si-CHAF1B, (3) A549/DDP cells of si-CHAF1B+si-NCOR2 were used to subcutaneously implant tumors in mice, and the growth rate and size of the tumors treated with cisplatin after knocking out CHAF1B were significantly smaller than those in the control group (P<0.05, Figure 8A-B). WB confirmed that the expression of CHAF1B was up-regulated in A549/DDP tissues, and NCOR2 was up-regulated in tumor tissues after knocking out CHAF1B. By immunohistochemical detection of Ki-67, an indicator related to tumor proliferation, it can be found that the positive rate of Ki67 in the CHAF1B knockout group was significantly lower than that in the control group (Figure 8C), and the apoptosis indicators were significantly up-regulated (Figure 8D). In vivo experiments further proved that knocking out CHAF1B can increase cisplatin sensitivity, decrease cell proliferation and increase apoptosis in lung adenocarcinoma, while knocking out CHAF1B and NCOR2 can lead to decreased cisplatin sensitivity, increased cell proliferation and increased apoptosis in lung adenocarcinoma reduce.

In summary, cell experiments and animal experiments have confirmed that CHAF1B promotes the ubiquitination and degradation of NCOR2, which can lead to cisplatin resistance in A549/DDP cells. Combined with the up-regulation of CHAF1B expression and the down-regulation of NCOR2 expression in lung adenocarcinoma patient specimens, the negative relationship between the two is likely to provide new evidence for the prediction of cisplatin chemotherapy sensitivity in clinical lung adenocarcinoma patients, and provide important evidence for the reversal of cisplatin resistance. molecular target.

Claims (3)

1. Application of an inhibitor of ubiquitin ligase CHAF1B in the preparation of cisplatin-sensitizing drugs for lung adenocarcinoma. 2. Application of a preparation for detecting the expression level of ubiquitin ligase CHAF1B in preparing a preparation for predicting cisplatin sensitivity of lung adenocarcinoma. 3. The application of the preparation for detecting the expression level of NCOR2 in the preparation of the preparation for predicting the cisplatin sensitivity of lung adenocarcinoma.

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