Application of small cell lung cancer drug-resistant molecular marker CCDC80

Abstract

The invention discloses application of a small cell lung cancer chemotherapy drug resistance marker CCDC80, and belongs to the technical field of biomedicine. It is found for the first time that CCDC80 is remarkably increased in plasma of a small cell lung cancer drug-resistant patient, CCDC80 overexpression can reduce the sensitivity of SCLC cells to cis-platinum or / and etoposide, and CCDC80 knock-down can enhance the chemosensitivity of the SCLC cells. CCDC80 promotes chemotherapy drug resistance by inhibiting cell apoptosis, overexpression of CCDC80 can reduce cleavage of Caspase3, Caspase7 and PARP, and knock-down of CCDC80 can increase the cleavage level of Caspase3, Caspase7 and PARP. In-vivo experiments show that overexpression of the CCDC80 can accelerate tumor growth and reduce the chemotherapy effect, and knock-down of the CCDC80 can enhance the anti-tumor effect of chemotherapy, so that the reagent for inhibiting expression of the CCDC80 can be combined with chemotherapy drugs for use and can be used for treating small cell lung cancer. The invention provides a new thought for SCLC drug resistance treatment and research, and has important clinical application value and economic value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of a molecular marker CCDC80 for small cell lung cancer resistance. Background Art

[0002] Small cell lung cancer (SCLC) is a highly invasive pulmonary neuroendocrine tumor, which has high sensitivity to chemotherapy and radiotherapy and is prone to develop drug resistance in metastatic patients. It is the histological subtype with the highest malignancy in lung cancer. SCLC patients have a rapid disease progression, limited treatment options, and extremely poor prognosis. The median survival period is only 6 - 12 months, and the 5-year survival rate is only 5%. Approximately 70% of SCLC patients are initially sensitive to the etoposide-platinum treatment regimen. However, most of these patients will develop drug resistance about 5 months after chemotherapy with the EP regimen (etoposide combined with cisplatin), and then the disease progresses rapidly. Although immune checkpoint inhibitors have been used in the treatment of SCLC in the past, the overall survival period of patients has only increased by about 2 - 4 months, and the survival benefit is still very limited.

[0003] Regarding the chemotherapy resistance mechanism of SCLC, it is relatively complex. The classical resistance mechanisms include the following aspects: 1) Some membrane proteins with efflux pump functions (such as lung resistance protein LRP, P-glycoprotein P-gp, etc.) are overexpressed in vivo; 2) The anti-apoptotic ability of cells is enhanced, such as the overexpression of anti-apoptotic proteins MYC and BCL-2; 3) The cell repair system is strengthened. It has been found that DNA mismatch repair (MMR) plays an important role in the acquired drug resistance of SCLC, especially the decrease in the expression levels of MMR genes MSH2 and MLH1 has a great impact on the acquired drug resistance. These classical chemotherapy resistance mechanisms have been confirmed in SCLC, but these molecular targets are difficult to be developed into drugs or enter the tumor to play a role. Studying the molecular mechanism of early drug resistance in SCLC, finding possible molecular markers for predicting drug resistance and new targets for reversing drug resistance has very important clinical significance for overcoming early drug resistance in SCLC and prolonging the survival period of patients.

[0004] To further investigate the molecular mechanism of early drug resistance in SCLC and search for potential molecular markers for predicting drug resistance, the inventor team previously performed Olink proteomic sequencing on paired plasma samples from 22 SCLC patients before and after drug resistance. The results showed that the level of CCDC80 in the plasma of patients increased significantly after chemotherapy resistance compared with the samples at the first diagnosis. Data from the GEPIA database (http: / / gepia.cancer-pku.cn / ) showed that the expression of CCDC80 in lung adenocarcinoma and lung squamous cell carcinoma tissues was significantly lower than that in adjacent normal lung tissues. According to the relevant sequencing data in the research of Zhang Peng's team (GSA database: HRA003419), the expression level of CCDC80 RNA in primary small cell lung cancer tumor tissues was also lower than that in paired adjacent normal tissues (see Example 1 for details). In the Olink proteomic sequencing data of the inventor team, the level of CCDC80 in the plasma of SCLC patients increased significantly after drug resistance, suggesting that CCDC80 may be correlated with drug resistance in small cell lung cancer. However, CCDC80 has never been reported to be related to drug resistance in small cell lung cancer.

[0005] Coiled-coil domain-containing 80 (CCDC80) is a secreted protein mainly secreted by adipocytes and participates in processes such as lipid metabolism, inflammatory response, tumorigenesis, and cell adhesion through multiple mechanisms. Previous studies have shown that CCDC80 plays different functions in different tumors. CCDC80 has an anti-tumor effect in thyroid cancer, colorectal cancer, and ovarian cancer. However, there are literature reports that in colorectal cancer, CCDC80 may promote oxaliplatin resistance and affect the therapeutic effect of oxaliplatin on colon cancer. Colorectal cancer mainly occurs in the colon or rectum, with relatively slow progression, relatively low malignancy, good prognosis for early detection, mainly local lymph node metastasis, and the main treatment methods are surgery and chemotherapy regimens mainly based on fluorouracil combined with oxaliplatin. Small cell lung cancer mainly occurs in the lungs, often located in the central bronchi of the lungs, with rapid progression, easy early metastasis, high malignancy, strong invasiveness, poor prognosis, easy hematogenous or lymphatic metastasis in the early stage, and chemotherapy is the main treatment method, and the chemotherapy regimens are mostly cisplatin or carboplatin combined with etoposide. Based on the obvious differences in the site of occurrence, growth characteristics, malignancy, and treatment regimens between small cell lung cancer and colorectal cancer, as well as the expression differences of CCDC80 in normal tissues, cancer tissues, and cancer tissues after chemotherapy in cancer patients, the inventor team believes that the role of CCDC80 in chemotherapy resistance in small cell lung cancer is very valuable for research.

[0006] In view of the fact that there is no research report on CCDC80 in SCLC at present, and its function in promoting chemotherapy resistance in SCLC is still unclear, it is of great value to study whether CCDC80 can become a molecular marker of SCLC drug resistance and a new target for SCLC treatment. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the present invention provides an application of CCDC80, a chemotherapy resistance marker for small cell lung cancer.

[0008] The present invention protects the application of CCDC80 protein as a marker in the preparation of products for detecting chemotherapy resistance of small cell lung cancer or in the preparation of drugs for treating small cell lung cancer.

[0009] The present invention also protects the application of substances for detecting the expression level of CCDC80 protein in the preparation of products for detecting chemotherapy resistance of small cell lung cancer or in the preparation of drugs for treating small cell lung cancer.

[0010] Furthermore, the product for detecting chemotherapy resistance of small cell lung cancer includes a detection reagent for detecting the expression level of biomarker CCDC80 in a biological sample.

[0011] Furthermore, the biological sample is tumor tissue or blood of a small cell lung cancer patient.

[0012] The present invention protects the application of substances that inhibit the expression of CCDC80 protein in the preparation of products having at least one of the following functions: 1) Treating or assisting in the treatment of small cell lung cancer; 2) Promoting the treatment of small cell lung cancer with chemotherapy drugs; 3) Combining chemotherapy drugs to treat small cell lung cancer; 4) Reducing the sensitivity to chemotherapy with cisplatin, carboplatin or and etoposide.

[0013] Furthermore, the substance that inhibits the expression of CCDC80 protein is shRNA that inhibits the expression of CCDC80 protein.

[0014] The present invention protects a product having the function of treating or assisting in the treatment of small cell lung cancer as a substance that inhibits the expression of CCDC80 protein or a substance that inhibits the expression of CCDC80 protein and a chemotherapy drug.

[0015] Furthermore, the chemotherapy drug is cisplatin or / and carboplatin or / and etoposide; the substance that inhibits the expression of CCDC80 protein is shRNA that inhibits the expression of CCDC80 protein.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention firstly discovers that CCDC80 is significantly elevated in the plasma of patients with small cell lung cancer resistance. Overexpression of CCDC80 can reduce the sensitivity of small cell lung cancer cells to cisplatin or etoposide, while knockdown of CCDC80 can enhance its chemosensitivity. CCDC80 promotes chemotherapy resistance by inhibiting apoptosis. Overexpression of CCDC80 can reduce the cleavage of Caspase3, Caspase7, and PARP, while knockdown of CCDC80 increases the cleavage level. In vivo experiments show that overexpression of CCDC80 can accelerate tumor growth and reduce the chemotherapy effect, while knockdown of CCDC80 can enhance the anti-tumor effect of chemotherapy. The present invention provides new ideas for the treatment and research of small cell lung cancer resistance, and has important clinical application value and economic value.

[0017] The present invention proves that CCDC80 can be used as a biomarker for chemotherapy resistance in patients with small cell lung cancer, predict the response of patients to chemotherapy by detecting its expression level, and guide the individualized treatment decision-making of patients with small cell lung cancer and develop new chemotherapy resistance intervention strategies: Intervention targeting CCDC80, such as by inhibiting the expression of CCDC80, can be used as a new strategy for the treatment of small cell lung cancer resistance, improve the sensitivity of patients with small cell lung cancer to chemotherapy with cisplatin, carboplatin or etoposide, indicating that reagents that inhibit the expression of CCDC80 can be used in combination with chemotherapy drugs to treat small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a figure related to the results of Olink proteomics screening for proteins that change in SCLC-resistant plasma samples. Among them Figure 1 A is a volcano plot showing the upregulation and downregulation of each protein in the metabolic panel; red represents proteins with significant differences; Figure 1 B is a volcano plot of tumor panel 1; Figure 1 C is a volcano plot of tumor panel 2; Figure 1 D is a volcano plot of the inflammatory panel.

[0019] Figure 2 It is a figure comparing the expression levels of CCDC80 RNA in lung cancer tissues and adjacent normal tissues. Among them Figure 2 A is a figure comparing the expression levels of CCDC80 RNA in lung adenocarcinoma, lung squamous cell carcinoma and adjacent normal tissues in the GEPIA database; Figure 2 B is a figure comparing the expression levels of CCDC80 RNA in primary tumors of small cell lung cancer and 107 paired adjacent tissues in the relevant sequencing data (GSA database: HRA003419) of the Zhang Peng team's research.

[0020] Figure 3 It is a heat map showing the expression of CLMP and CCDC80 before and after chemotherapy resistance.

[0021] Figure 4 Results of the effects of overexpression and knockdown of CCDC80 on chemoresistance of SCLC cells. Among them, Figure 4 A shows the overexpression effect of CCDC80 detected by WB; Figure 4 B shows the cell viability after treating CCDC80-overexpressing or control cells with different concentration gradients of etoposide (VP-16) for 48 h; Figure 4 C shows the cell viability after treating CCDC80-overexpressing or control cells with different concentration gradients of cisplatin (DDP) for 48 h; Figure 4 D shows the knockdown effect of CCDC80 detected by WB; Figure 4 E shows the cell viability after treating CCDC80-knockdown or control cells with different concentration gradients of etoposide (VP-16) for 48 h; Figure 4 F shows the cell viability after treating CCDC80-knockdown or control cells with different concentration gradients of cisplatin (DDP) for 48 h. Data are shown as mean ± standard deviation.

[0022] Figure 5 Results of the effects of overexpression and knockdown of CCDC80 on apoptosis of SCLC cells. Figure 5 A shows the apoptosis rate of cells after treating CCDC80-overexpressing or control cells with cisplatin and etoposide for 48 h; Figure 5 B shows the apoptosis rate of cells after treating CCDC80-knockdown or control cells with cisplatin and etoposide for 48 h; Figure 5 C shows the expression of cleaved Caspase3, Caspase7, PARP and total protein detected by WB in CCDC80-overexpressing or control cells treated with cisplatin and etoposide; Figure 5 D shows the expression of cleaved Caspase3, Caspase7, PARP and total protein detected by WB in CCDC80-stably transfected knockdown or control cells treated with cisplatin or etoposide. Data are shown as mean ± standard deviation, ***p < 0.001.

[0023] Figure 6 Results of CCDC80 promoting chemoresistance to the EP regimen in small cell lung cancer in vivo. Small cell lung cancer DMS53 cells and SHP-77 cells stably overexpressing or knockdown of CCDC80 were inoculated subcutaneously into the axilla of BALB / c nude mice. One week after tumor-bearing, chemotherapy was performed using the EP regimen (cisplatin + etoposide). Figure 6 B and Figure 6 E are for harvesting tumor specimens from mice, Figure 6 A and Figure 6 D are for tumor growth curves, Figure 6 C and Figure 6F is a box plot of tumor weight. *p < 0.05, **p < 0.01, ***p < 0.001; data are shown as mean ± standard error ( Figure 6 A and Figure 6 D) and mean ± 5 - 95% percentile ( Figure 6 C and Figure 6 F). Detailed implementation manners

[0024] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0025] Example 1 Screening of chemoresistance markers for small cell lung cancer The inventor team collected paired plasma samples from 22 pairs of SCLC patients at the first diagnosis (Pre - chemo) and at the time of first - line EP - regimen chemotherapy resistance (Chemo - resistance) from the Second Department of Internal Medicine of the Third Affiliated Hospital of Kunming Medical University for Olink proteomics (devoted to the discovery of proteomes and protein markers in body fluids such as plasma) sequencing (4 Target 96 panel, including 2 tumor panels, 1 metabolic panel and 1 inflammatory panel), and detected the abundances of 384 proteins in plasma samples before and after resistance. The specific test methods are as follows: (1) Sample collection: After collecting whole blood with an EDTA anticoagulant tube, invert it 8 - 10 times to mix evenly, let it stand at room temperature for 30 min, and centrifuge at 1600 g at 4°C for 10 min. After centrifugation, the blood is divided into three layers. The upper layer is plasma, the middle layer is the white blood cell layer, and the lower layer is the red blood cell layer. Use a pipette to aspirate the plasma into a cryopreservation tube, store it at - 80°C in a refrigerator, and transport it with dry ice.

[0026] (2) Incubation: Prepare the incubation mixture according to the ratio (the volume required for each 96 - well plate): 280 µL of incubation solution, 40 µL of incubation stabilizer, 40 µL of A probe, 40 µL of B probe. Vortex and centrifuge. Take a PCR 8 - tube strip, and use reverse pipetting to add 47 µL of the incubation mixture to each test tube. Using reverse pipetting, transfer the incubation mixture taken from the 8 - tube strip to each well of a new 96 - well plate, and label this plate as the incubation plate. Use a multi - channel pipette to take 1 µL of the sample and add it to the bottom of each well of the incubation plate. Set up a negative control group (3 wells), an intra - plate control group (3 wells) and a sample control group of mixed plasma samples (2 wells) for each 96 - well plate. After adding the samples, seal the incubation plate with a thermal plastic seal, centrifuge at room temperature for 1 minute at a speed of 400×g. Incubate overnight at 4°C.

[0027] (3)Amplification: Prepare the extension mixture according to the ratio (volume required for each 96-well plate): 9385 µL of high-purity water, 1100 µL of PEA solution, 55 µL of PEA reaction enzyme, and 22 µL of PCR polymerase. Take out the overnight incubation plate from the previous step, let the temperature return to room temperature, centrifuge for 1 minute at a speed of 400×g. Preheat the PCR instrument. Vortex the extension mixture and pour it into the reagent trough. Set the timer for 5 minutes and start timing. Use reverse pipetting to add 96 µL of the extension mixture to each well of the incubation plate. The pipette tip should be vertical and above the sample surface in the incubation plate when dispensing the mixture. Seal the incubation plate with thermal plastic and vortex it thoroughly to ensure that the samples in each well are well mixed. Centrifuge at room temperature for 1 minute at a speed of 400×g. Place the incubation plate in the PCR instrument and start the PEA program: 50°C for 20 min, 95°C for 5 min (95°C for 30 s, 54°C for 1 min, 60°C for 1 min) × 17, 10°C hold.

[0028] (4)Detection: Prepare and pre-treat a 96.96 Dynamic Array TM Integrated Fluidic Circuit (IFC) chip. Inject control line fluid into the two valves of the chip respectively, and then load the chip into the IFC controller for pre-treatment for 20 minutes. Thaw the primer plate, vortex it and centrifuge. Prepare the detection mixture (volume required for each 96-well plate): 550.0 µL of detection solution, 230.0 µL of high-purity water, 7.8 µL of detection reaction enzyme, and 3.1 µL of PCR polymerase. Vortex the prepared detection mixture and centrifuge. Take a new PCR 8-tube strip, and add 95 µL of the detection mixture to each tube. Take a new 96-well plate, use reverse pipetting to add 7.2 µL of the detection mixture to each well, and label it as the sample plate. Take out the incubation plate from the PCR instrument, vortex it and centrifuge. Use forward pipetting to transfer 2.8 µL of the extended sample to the sample plate. When aspirating the liquid, it should be aspirated from the upper surface of the liquid, and the pipette tip should not be inserted into the bottom of the plate well. Seal the sample plate with thermal plastic and centrifuge at room temperature for 1 minute at a speed of 400×g. From each well of the primer plate, transfer 5 µL of the primer to the reagent cell on the left side of the pre-treated IFC chip. From each well of the sample plate, transfer 5 µL of the amplified sample to the reagent cell on the right side of the IFC chip. This operation should use reverse pipetting, and a new pipette tip should be used before each pipetting. There should be no empty reagent cells on the chip. If bubbles are generated during the sample addition process, carefully remove them with a needle. Load the IFC chip into the Fluidigm IFC Controller HX according to the instrument operation instructions. Put the loaded IFC chip into the Fluidigm Biomark according to the instrument operation instructions. TMIn the instrument, the detection program of Olink Protein Expression 96×96 was started (50°C for 120 s, 70°C for 1800 s, 25°C for 600 s, 95°C for 300 s (95°C for 15 s, 60°C for 60 s) × 40). The PCR reaction generates a fluorescence signal, and the number of labeled nucleic acid sequences is proportional to the number of proteins to be detected.

[0029] After analyzing the plasma samples of 22 pairs of SCLC patients before chemotherapy and after drug resistance by Olink proteomics sequencing technology, 7 differentially expressed proteins were screened out. When drug resistance occurred after the EP regimen treatment of the patients, the levels of CLMP, CCDC80, EPHA2, RET, and ADAM-TS 15 in the plasma increased ( Figure 1 A and Figure 1 B), and the levels of ICAM4 and IL33 decreased ( Figure 1 C and Figure 1 D).

[0030] Interestingly, according to the data in the GEPIA database (http: / / gepia.cancer-pku.cn / ), when 483 lung adenocarcinoma tissue samples were compared with 347 normal tissue samples, the expression level of CCDC80 RNA in lung adenocarcinoma was lower than that in normal tissues. When 486 lung squamous cell carcinoma samples were compared with 338 normal tissue samples, the expression level of CCDC80 RNA in lung squamous cell carcinoma was also lower than that in normal tissues, as shown in Figure 2 A. In addition, according to the relevant sequencing data in the research of Zhang Peng's team (GSA database: HRA003419), in 107 primary tumors of small cell lung cancer and 107 paired adjacent tissue samples, the expression level of CCDC80RNA was lower than that in normal tissues, and the results are shown in Figure 2 B.

[0031] According to the literature on the research of the above differentially expressed proteins and tumors, the inventors selected CCDC80 and CLMP, which had not been reported in SCLC drug resistance before, for a CCK8 pre-experiment of chemotherapy drug treatment after knocking down CCDC80 by transient transfection of si-CCDC80. The results showed that there was no significant phenotypic difference in CLMP, and finally CCDC80 was selected for subsequent experiments.

[0032] The levels of CCDC80 in the plasma before and after chemotherapy drug resistance are shown in the Figure 3 heat map. When chemotherapy drug resistance occurred, CCDC80 in the plasma of most patients increased significantly compared with that at the first diagnosis ( Figure 3 ). It is speculated that CCDC80 may be related to the drug resistance of small cell lung cancer and has the potential to become a marker for chemotherapy drug resistance in small cell lung cancer. Therefore, the inventors' team carried out subsequent experiments for further verification.

[0033] Example 2: CCDC80 promotes chemoresistance in small cell lung cancer cells The mRNA expression level of CCDC80 in SCLC cell lines was analyzed through the CCLE database, and SCLC cell lines with stable overexpression and knockdown of CCDC80 were constructed. The stable cell lines were constructed by lentivirus infection, and the specific method is as follows: (1) Lentiviruses carrying the CCDC80 overexpression plasmid and the CCDC80 knockdown plasmid were purchased from GenePharma.

[0034] (2) Prepare the cells to be infected, evenly inoculate the target cells in a 6-well plate at a density of (50 - 60%), and place them in the incubator overnight.

[0035] (3) Remove the cell culture medium, add 1 ml of normal cell culture medium and 1 ml of virus solution to each well, and 4 μg / ml polybrene can be used to promote the infection efficiency.

[0036] (4) Repeat step (2) after 24 hours. After 48 hours of infection, puromycin can be used to screen the stable cell lines. The screened stable cells can be used for subsequent experiments.

[0037] The sequences of the CCDC80 knockdown plasmids, where the sequences of NC, ShCCDC80 #1, and ShCCDC80 #2 are shown as SEQ ID NO: 1 - 3 respectively, and the sequence of the CCDC80 overexpression plasmid is shown as SEQ ID NO: 4.

[0038] In this invention, DMS53 and DMS114 cells were selected to construct cell lines with stable overexpression of CCDC80, and H446 and SHP-77 cells were selected to construct cell lines with stable knockdown of CCDC80. The effects after stable overexpression and knockdown of CCDC80 were verified by WB ( Figure 4 A and Figure 4 D). In CCDC80 overexpressing or control cells, cells overexpressing or knockdown of CCDC80 (DMS53, DMS114, H446, and SHP-77 cells) were treated with different concentration gradients of etoposide (VP-16) and cisplatin (DDP) for 48 hours, and the cell viability was detected by CCK8.

[0039] The results showed that overexpression of CCDC80 in small cell lung cancer DMS53 and DMS114 cells promoted chemoresistance of SCLC cells to cisplatin and etoposide ( Figure 4 B and Figure 4 C), and the cell viability detected by CCK8 increased significantly. Knockdown of CCDC80 in small cell lung cancer H446 and SHP-77 cells could increase the sensitivity of cells to cisplatin and etoposideFigure 4 E and Figure 4 F), and CCK8 assay showed a significant decrease in cell viability. The above in vitro experiments confirmed that CCDC80 could indeed promote in vitro chemoresistance in small cell lung cancer.

[0040] In vitro experiments showed that overexpression of CCDC80 significantly reduced the sensitivity of SCLC cells to cisplatin (Cisplatin, DDP) and etoposide (Etoposide, VP-16), while knockdown of CCDC80 enhanced their chemosensitivity.

[0041] Example 3 CCDC80 promotes chemoresistance by inhibiting apoptosis The above-mentioned DMS53, DMS114, H446 and SHP-77 cells with stable overexpression or knockdown of CCDC80 were treated with different concentration gradients of cisplatin or etoposide for 48 hours, and the apoptosis of cells in each group was detected by flow cytometry after Annexin V-PI staining. Further, the cleavage expression of apoptosis-related proteins such as Caspase3, Caspase7 and PARP in different cells was detected by WB.

[0042] Cells overexpressing CCDC80 had a stronger resistance to cisplatin-induced apoptosis, while knockdown of CCDC80 enhanced cisplatin-induced apoptosis. WB experiments showed that overexpression of CCDC80 could reduce the cleavage of Caspase3, Caspase7 and PARP, while knockdown of CCDC80 increased the cleavage of these apoptosis-related proteins.

[0043] The results showed that cisplatin or etoposide significantly induced apoptosis in small cell lung cancer cells, and overexpression of CCDC80 could partially reduce cisplatin-induced apoptosis ( Figure 5 A); on the contrary, in H446 and SHP-77 cells with knockdown of CCDC80, knockdown of CCDC80 could further increase cisplatin or etoposide-induced apoptosis ( Figure 5 B). Secondly, overexpression of CCDC80 could partially rescue cisplatin-induced apoptosis, with reduced cleavage of Caspase3, Caspase7 and PARP ( Figure 5 C); on the contrary, knockdown of CCDC80 further increased cisplatin-induced apoptosis, with increased cleavage of Caspase3, Caspase7 and PARP ( Figure 5 D). The above results indicated that CCDC80 could promote chemoresistance in small cell lung cancer by inhibiting apoptosis.

[0044] Example 4 CCDC80 promotes chemoresistance in small cell lung cancer in vivo An SCLC nude mouse xenograft model was established, and it was found that overexpression of CCDC80 could accelerate tumor growth and reduce the antitumor effect of EP regimen chemotherapy, while knockdown of CCDC80 enhanced the antitumor effect of chemotherapy.

[0045] Small cell lung cancer DMS53 cells and SHP-77 cells with stable overexpression or knockdown of CCDC80 were respectively inoculated subcutaneously into the axilla of BALB / c nude mice (1×10 7 cells / point). One week after tumor bearing, chemotherapy was performed using the EP regimen (cisplatin + etoposide). Etoposide was administered intraperitoneally at 4 mg / kg, twice a week, and cisplatin was administered intraperitoneally at 2 mg / kg, once every 8 days. The control group was injected with an equal volume of solvent, and the injection method and time were the same as those in the experimental group. The mice were sacrificed on the 17th day to harvest tumor specimens, the tumor size was measured, and a comparison chart of tumor volume and weight was drawn. The specific grouping is as follows: The xenograft experiment of the DMS53 CCDC80 stable overexpression cell line was divided into 4 groups: 2 control groups: NC + Vehicle (solvent); OE-CCDC80 + Vehicle (solvent); 2 experimental groups: NC + Drugs (cisplatin + etoposide); OE-CCDC80 + Drugs (cisplatin + etoposide).

[0046] The xenograft experiment of the SHP-77 CCDC80 stable knockdown cell line was divided into 6 groups: 3 control groups: NC + Vehicle (solvent); ShCCDC80 #1 + Vehicle (solvent); ShCCDC80 #2 + Vehicle (solvent); 3 experimental groups: NC + Drugs (cisplatin + etoposide); ShCCDC80 #1 + Drugs (cisplatin + etoposide); ShCCDC80 #2 + Drugs (cisplatin + etoposide).

[0047] The results showed that overexpression of CCDC80 significantly promoted chemoresistance in small cell lung cancer in vivo, and the growth of xenografts was significantly accelerated. Figure 6 A is the tumor growth curve of the xenograft drug resistance experiment of the DMS53 CCDC80 stable overexpression cell line. Figure 6 B is a comparison chart of tumor specimens after sacrificing the mice. Figure 6 C is a comparison chart of the weights of tumor samples. It can be seen that overexpression of CCDC80 promoted tumor growth and chemoresistance in small cell lung cancer in vivo. The volume and weight of CCDC80-overexpressing tumors were significantly higher than those of the NC group in both the control group and the experimental group. On the contrary, knockdown of CCDC80 significantly weakened chemoresistance in small cell lung cancer in vivo, and the growth of xenografts was significantly slowed down. Figure 6D shows the tumor growth curve of the transplantation tumor drug resistance experiment of the SHP-77 CCDC80 stable knockdown cell line, Figure 6 E shows the comparison diagram of tumor specimens after sacrificing the mice, Figure 6 F shows the comparison diagram of tumor sample weights. It can be seen that knockdown of CCDC80 inhibits tumor growth and chemoresistance of small cell lung cancer in vivo. Whether in the control group or the experimental group, the volume and weight of CCDC80-knockdown tumors are significantly lower than those of the NC group. The above experimental results fully confirm that CCDC80 has an important biological function of promoting chemoresistance of small cell lung cancer.

[0048] In summary, the present invention first discovers that CCDC80 is significantly elevated in the plasma of patients with small cell lung cancer (SCLC) drug resistance. Overexpression of CCDC80 can reduce the sensitivity of SCLC cells to cisplatin, carboplatin, or etoposide, while knockdown of CCDC80 can enhance its chemosensitivity. CCDC80 promotes chemoresistance by inhibiting apoptosis. Overexpression of CCDC80 can reduce the cleavage of Caspase3, Caspase7, and PARP, while knockdown of CCDC80 increases the cleavage level. In vivo experiments show that overexpression of CCDC80 can accelerate tumor growth and reduce the chemotherapy effect, while knockdown of CCDC80 can enhance the anti-tumor effect of chemotherapy. The present invention proves that CCDC80 can be used as a biomarker for chemoresistance in SCLC patients, predicting the response of patients to chemotherapy by detecting its expression level to guide individualized treatment decisions for SCLC patients; new chemoresistance intervention strategies can be developed: interventions targeting CCDC80, such as inhibiting CCDC80 expression, can be used as a new strategy for SCLC drug resistance treatment, and it is expected to improve the sensitivity of SCLC patients to chemotherapy with cisplatin, carboplatin, or etoposide, indicating that reagents that inhibit CCDC80 expression can be used in combination with chemotherapy drugs to treat SCLC. The present invention provides new ideas for SCLC drug resistance treatment and research, and has important clinical application value and economic value.

Claims

1. Use of CCDC80 protein as a biomarker in the preparation of a product for detecting chemoresistance of small cell lung cancer or in the preparation of a medicament for treating small cell lung cancer.

2. Use of a substance for detecting the expression level of CCDC80 protein in the preparation of a product for detecting chemoresistance of small cell lung cancer or in the preparation of a medicament for treating small cell lung cancer.

3. The use according to claim 1 or 2, characterized in that: The product for detecting chemoresistance of small cell lung cancer includes a detection reagent for detecting the expression level of biomarker CCDC80 in a biological sample.

4. The use according to claim 3, characterized in that: The biological sample is tumor tissue or blood of a small cell lung cancer patient.

5. Use of a substance that inhibits the expression of CCDC80 protein in the preparation of a product having at least one of the following functions: Use: 1) Treating or adjuvant-treating small cell lung cancer; 2) Promoting the treatment of small cell lung cancer with chemotherapeutic drugs; 3) Combining with chemotherapeutic drugs to treat small cell lung cancer; 4) Reducing the sensitivity to chemotherapy with cisplatin, carboplatin or etoposide.

6. The use according to claim 5, characterized in that: The substance for inhibiting CCDC80 protein expression is shRNA for inhibiting CCDC80 protein expression.

7. A product having the function of treating or adjuvant-treating small cell lung cancer is a substance that inhibits the expression of CCDC80 protein or a substance that inhibits the expression of CCDC80 protein and a chemotherapeutic drug.

8. The product according to claim 7, characterized in that: The chemotherapeutic drug is cisplatin or / and carboplatin or / and etoposide; the substance for inhibiting CCDC80 protein expression is shRNA for inhibiting CCDC80 protein expression.

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