Application of CCDC80, a molecular marker of drug resistance in small cell lung cancer

Abstract

The present invention discloses an application of CCDC80, a small cell lung cancer chemotherapy resistance marker, and belongs to the field of biomedical technology. The present invention discovered for the first time that CCDC80 is significantly elevated in the plasma of patients with small cell lung cancer resistance. Overexpression of CCDC80 can reduce the sensitivity of SCLC cells to cisplatin and / or etoposide, while knocking down CCDC80 can enhance their chemotherapy sensitivity. CCDC80 promotes chemotherapy resistance by inhibiting cell apoptosis. Overexpression of CCDC80 can reduce the cleavage of Caspase3, Caspase7, and PARP, while knocking down CCDC80 can increase their cleavage levels. In vivo experiments showed that overexpression of CCDC80 can accelerate tumor growth and reduce the effect of chemotherapy, while knocking down CCDC80 can enhance the anti-tumor effect of chemotherapy, indicating that agents that inhibit CCDC80 expression can be used in combination with chemotherapy drugs to treat small cell lung cancer. The present invention provides new ideas for the treatment and research of SCLC resistance and has important clinical application value and economic value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of CCDC80, a molecular marker for drug resistance of small cell lung cancer. Background Art

[0002] Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine lung tumor that is highly sensitive to chemotherapy and radiotherapy and prone to developing drug resistance in metastatic patients. It is the most aggressive histological subtype of lung cancer. SCLC patients experience rapid disease progression, limited treatment options, and a dismal prognosis, with a median survival of only 6-12 months and a 5-year survival rate of only 5%. Approximately 70% of SCLC patients are initially sensitive to etoposide-platinum therapy, but most develop resistance to first-line chemotherapy with EP (etoposide plus cisplatin) within approximately 5 months, leading to rapid disease progression. Although immune checkpoint inhibitors have been used in the past to treat SCLC, the overall survival benefit has only improved by approximately 2-4 months, representing a very limited survival benefit.

[0003] The mechanisms of chemoresistance in SCLC are relatively complex. Classic mechanisms include the following: 1) overexpression of membrane proteins that function as efflux pumps (such as lung resistance protein (LRP) and P-glycoprotein (P-gp); 2) enhanced cellular resistance to apoptosis, such as overexpression of the anti-apoptotic proteins MYC and BCL-2; and 3) enhanced cellular repair systems. Studies have shown that DNA mismatch repair (MMR) plays a crucial role in acquired chemoresistance in SCLC, with decreased expression of the MMR genes MSH2 and MLH1 having a significant impact. These classic mechanisms of chemoresistance have been demonstrated in SCLC, but these molecular targets remain difficult to drug or to target in tumors. Studying the molecular mechanisms of early SCLC chemoresistance, identifying potential molecular markers for predicting resistance and novel targets for reversing resistance, is of great clinical significance for overcoming early SCLC chemoresistance and prolonging patient survival.

[0004] To further investigate the molecular mechanisms of early SCLC drug resistance and identify potential molecular markers for predicting drug resistance, the inventors' team previously performed Olink proteomic sequencing on paired plasma samples collected from 22 SCLC patients before and after chemotherapy resistance. The results showed that CCDC80 levels in plasma significantly increased after chemotherapy resistance developed, compared to samples collected at the initial visit. Data from the GEPIA database (http: / / gepia.cancer-pku.cn / ) indicate that CCDC80 expression in lung adenocarcinoma and squamous cell lung carcinoma tissues is significantly lower than in adjacent normal lung tissue. Sequencing data from Zhang Peng's team (GSA database: HRA003419) also showed that CCDC80 RNA expression levels in primary small cell lung cancer tumor tissues were lower than in paired adjacent normal tissues (see Example 1 for details). Furthermore, the inventors' team's Olink proteomic sequencing data showed that CCDC80 levels in the plasma of SCLC patients after chemotherapy resistance developed, suggesting a possible correlation between CCDC80 and drug resistance in small cell lung cancer. However, CCDC80 has never been reported to be associated with drug resistance in small cell lung cancer.

[0005] Coiled-coil domain-containing protein 80 (CCDC80) is a secreted protein primarily produced by adipocytes. It participates in lipid metabolism, inflammatory responses, tumorigenesis, and cell adhesion through multiple mechanisms. Previous studies have shown that CCDC80 functions differently in different tumors, including a tumor suppressor in thyroid, colorectal, and ovarian cancers. However, some studies have reported that CCDC80 may promote oxaliplatin resistance in colorectal cancer, potentially impacting the therapeutic efficacy of oxaliplatin in colorectal cancer. Colorectal cancer primarily arises in the colon or rectum, progresses relatively slowly, and is less malignant. Early detection offers a better prognosis, with regional lymph node metastasis being the primary pathology. Treatment is primarily surgical, with chemotherapy typically consisting of fluorouracil combined with oxaliplatin. Small cell lung cancer primarily develops in the lungs, often located in the central bronchus. It progresses rapidly, is prone to early metastasis, is highly malignant, and is highly invasive. It has a poor prognosis and is prone to early hematogenous or lymphatic metastasis. Chemotherapy is the primary treatment, often with cisplatin or carboplatin combined with etoposide. Given the significant differences between small cell lung cancer and colorectal cancer in terms of location, growth characteristics, malignancy, and treatment options, and the differential expression of CCDC80 in normal tissues, cancerous tissues, and cancerous tissues of cancer patients after chemotherapy, the inventors believe that the role of CCDC80 in chemotherapy resistance in small cell lung cancer is of great research value.

[0006] Given that no research reports on CCDC80 in SCLC have been reported so far, 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 resistance and a new target for SCLC treatment. Summary of the Invention

[0007] In view of the deficiencies in 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 use of CCDC80 protein as a marker in preparing a product for detecting chemotherapy resistance of small cell lung cancer or in preparing a medicine for treating small cell lung cancer.

[0009] The present invention also protects the use of a substance for detecting the expression level of CCDC80 protein in preparing a product for detecting chemotherapy resistance of small cell lung cancer or in preparing a medicine 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 the biomarker CCDC80 in a biological sample.

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

[0012] The present invention provides for the use of a substance for inhibiting CCDC80 protein expression in the preparation of a product having at least one of the following functions:

[0013] 1) Treatment or adjuvant treatment of small cell lung cancer;

[0014] 2) Promote chemotherapy drugs to treat small cell lung cancer;

[0015] 3) Combined chemotherapy for the treatment of small cell lung cancer;

[0016] 4) Reduce sensitivity to cisplatin, carboplatin or etoposide chemotherapy.

[0017] Furthermore, the substance that inhibits the expression of CCDC80 protein is shRNA that inhibits the expression of CCDC80 protein.

[0018] The present invention protects a product for treating or assisting in the treatment of small cell lung cancer, which is a substance that inhibits the expression of CCDC80 protein, or a substance that inhibits the expression of CCDC80 protein and a chemotherapy drug.

[0019] Furthermore, the chemotherapy drug is cisplatin and / or carboplatin and / or etoposide; and the substance that inhibits the expression of CCDC80 protein is shRNA that inhibits the expression of CCDC80 protein.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention discovered for the first time that CCDC80 is significantly elevated in the plasma of patients with drug-resistant small cell lung cancer. Overexpression of CCDC80 can reduce the sensitivity of small cell lung cancer cells to cisplatin and etoposide, while knocking down CCDC80 can enhance their chemotherapy sensitivity. CCDC80 promotes chemotherapy resistance by inhibiting cell apoptosis. Overexpression of CCDC80 can reduce the cleavage of Caspase3, Caspase7, and PARP, while knocking down CCDC80 can increase their cleavage levels. In vivo experiments have shown that overexpression of CCDC80 can accelerate tumor growth and reduce the effectiveness of chemotherapy, while knocking down CCDC80 can enhance the anti-tumor effect of chemotherapy. This invention provides new ideas for the treatment and research of drug-resistant small cell lung cancer and has important clinical application value and economic value.

[0022] The present invention demonstrates that CCDC80 can be used as a biomarker for chemotherapy resistance in patients with small cell lung cancer. By detecting its expression level, the patient's response to chemotherapy can be predicted, thereby guiding individualized treatment decisions for patients with small cell lung cancer and developing new chemotherapy resistance intervention strategies: intervention targeting CCDC80, such as by inhibiting CCDC80 expression, can be used as a new strategy for the treatment of small cell lung cancer resistance, thereby increasing the sensitivity of small cell lung cancer patients to cisplatin, carboplatin or etoposide chemotherapy. This indicates that agents 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

[0023] Figure 1 This is the result of Olink proteomics screening of proteins in SCLC drug-resistant plasma samples. Figure 1 A is a volcano plot showing the up- and down-regulation of proteins in the metabolic panel; red represents proteins with significant differences; Figure 1 B is the volcano map of tumor panel 1; Figure 1 C is the volcano map of tumor panel 2; Figure 1 D is the volcano plot of the inflammatory panel.

[0024] Figure 2 The figure shows the comparison of CCDC80 RNA expression levels in lung cancer tissues and adjacent normal tissues. Figure 2 A is a comparison of CCDC80 RNA expression levels in lung adenocarcinoma, lung squamous cell carcinoma and adjacent normal tissues in the GEPIA database; Figure 2 B is a comparison of the CCDC80 RNA expression levels in primary small cell lung cancer tumors and 107 paired adjacent cancer tissues in the relevant sequencing data (GSA database: HRA003419) from Zhang Peng's team's study.

[0025] Figure 3The heat map shows the expression of CLMP and CCDC80 before and after chemotherapy resistance.

[0026] Figure 4 The results of the effects of overexpression and knockdown of CCDC80 on chemotherapy resistance of SCLC cells are shown in Figure 2. Figure 4 A is the overexpression effect of CCDC80 detected by WB; Figure 4 B shows the cell survival of CCDC80 overexpressing or control cells after treatment with different concentration gradients of etoposide (VP-16) for 48 h; Figure 4 C shows the cell survival of CCDC80 overexpressing or control cells after treatment with different concentration gradients of cisplatin (DDP) for 48 h; Figure 4 D is the effect of knocking down CCDC80 detected by WB; Figure 4 E shows the cell survival of CCDC80 knockdown or control cells after treatment with different concentration gradients of etoposide (VP-16) for 48 h; Figure 4 F shows the cell survival of CCDC80 knockdown or control cells after treatment with different concentration gradients of cisplatin (DDP) for 48 h. The data are shown as mean ± standard deviation.

[0027] Figure 5 The results show the effects of overexpression and knockdown of CCDC80 on apoptosis of SCLC cells. Figure 5 A is the apoptosis rate of CCDC80 overexpressing or control cells after 48 h of cisplatin and etoposide treatment; Figure 5 B is the apoptosis rate of CCDC80 knockdown or control cells after 48 h of cisplatin and etoposide treatment; Figure 5 C is WB detection of the expression of Caspase3, Caspase7, PARP cleavage protein and total protein in CCDC80 overexpression or control cells treated with cisplatin and etoposide; Figure 5 D shows Western blotting analysis of the expression of Caspase 3, Caspase 7, and PARP cleavage proteins and total proteins in CCDC80 stably knocked-down or control cells treated with cisplatin or etoposide. Data are shown as mean ± SD, ***p < 0.001.

[0028] Figure 6 Figure 2 shows the results of CCDC80 promoting chemoresistance to the EP regimen in small cell lung cancer in vivo. Small cell lung cancer DMS53 and SHP-77 cells stably overexpressing or knocking down CCDC80 were inoculated subcutaneously in the axilla of BALB / c nude mice. One week after tumor growth, EP (cisplatin + etoposide) chemotherapy was initiated. Figure 6 B and Figure 6 E is the tumor specimen harvested from mice. Figure 6 A and Figure 6 D is the tumor growth curve, 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 DESCRIPTION

[0029] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0030] Example 1 Screening of chemotherapy resistance markers for small cell lung cancer

[0031] The inventors' team collected 22 pairs of paired plasma samples from SCLC patients at the time of their first visit (pre-chemo) and when they were resistant to first-line EP chemotherapy (chemo-resistance) from the Second Department of Internal Medicine of the Third Affiliated Hospital of Kunming Medical University. They performed Olink proteomics sequencing (dedicated to the discovery of proteomes and protein markers in body fluids such as plasma) (four Target 96 panels, including two tumor panels, one metabolic panel, and one inflammatory panel). A total of 384 proteins were detected in plasma samples before and after resistance. The specific experimental methods are as follows:

[0032] (1) Sample collection: Collect whole blood in an EDTA anticoagulant tube and mix thoroughly by inverting it 8-10 times. Let it stand at room temperature for 30 minutes and then centrifuge it at 1600g at 4°C for 10 minutes. After centrifugation, the blood will separate 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 draw the plasma into a cryopreservation tube, store it in a -80°C refrigerator, and transport it on dry ice.

[0033] (2) Incubation: Prepare the incubation mixture in proportion (the required volume for each 96-well plate): 280µL incubation solution, 40µL incubation stabilization solution, 40µL probe A, and 40µL probe B. Vortex and centrifuge. Take a PCR 8-tube strip and add 47µL of incubation mixture to each test tube using the reverse pipetting method. Use the reverse pipetting method to add the incubation mixture transferred from the 8-tube strip to each well of a new 96-well plate and mark this plate as the incubation plate. Use a multichannel pipette to take 1µL of sample and add it to the bottom of each well of the incubation plate. Set up a negative control group (3 wells), an on-plate control group (3 wells), and a sample control group for mixed plasma samples (2 wells) for each 96-well plate. After the sample addition is completed, seal the incubation plate with a heat seal and centrifuge at room temperature for 1 minute at a speed of 400×g. Incubate overnight at 4°C.

[0034] (3) Amplification: Prepare the extension mixture in the following proportions (the required volume for each 96-well plate): 9385µL of high-purity water, 1100µL of PEA solution, 55µL of PEA reaction enzyme, and 22µL of PCR polymerase. Remove the plate from the previous step, return it to room temperature, and centrifuge it for 1 minute at 400×g. Preheat the PCR instrument. Vortex the extension mixture and pour it into the reagent tank. Set the timer for 5 minutes and start the timer. Use the reverse pipetting method to add 96µL of extension mixture to each well of the incubation plate. When dispensing the mixture, the tip of the pipette should be vertical and above the sample surface in the incubation plate. Seal the incubation plate with a heat seal and vortex it thoroughly to ensure that the samples in each well are evenly mixed. Centrifuge it for 1 minute at room temperature at 400×g. Place the incubation plate in a 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.

[0035] (4) Detection: Prepare and pre-process a 96.96 Dynamic Array TM Integrated FluidicCircuit (IFC) chip. Inject control line fluid into each of the two valves on the chip, then load the chip into the IFC controller for pretreatment for 20 minutes. Thaw the primer plate, vortex, and centrifuge. Prepare the detection mix (required volume for each 96-well plate): 550.0µL detection solution, 230.0µL high-purity water, 7.8µL detection enzyme, and 3.1µL PCR polymerase. Vortex the prepared detection mix and centrifuge. Take a new 8-tube PCR strip and add 95µL of detection mix to each tube. Take a new 96-well plate and add 7.2µL of detection mix to each well using reverse pipetting, and label it as the sample plate. Remove the incubation plate from the PCR instrument, vortex, and centrifuge. Use forward pipetting to transfer 2.8µL of the extended sample to the sample plate. When aspirating, pipette from the top surface of the liquid; do not insert the pipette tip into the bottom of the plate well. Seal the sample plate with heat plastic and centrifuge at room temperature for 1 minute at 400×g. From each well of the primer plate, transfer 5µL of primer to the reagent compartment on the left side of the pre-treated IFC chip. From each well of the sample plate, transfer 5µL of amplified sample to the reagent compartment on the right side of the IFC chip. This step should use the reverse pipetting method, and the pipette tip should be updated before each pipetting. There should be no empty reagent compartments 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 instructions. Place the loaded IFC chip into the Fluidigm Biomark according to the instrument instructions. TMStart the Olink Protein Expression 96×96 assay (50°C for 120 seconds, 70°C for 1800 seconds, 25°C for 600 seconds, 95°C for 300 seconds (95°C for 15 seconds, 60°C for 60 seconds) × 40). The PCR reaction generates a fluorescent signal, and the amount of labeled nucleic acid sequence is proportional to the amount of protein being detected.

[0036] After analyzing 22 pairs of plasma samples from SCLC patients before and after chemotherapy and drug resistance using Olink proteomics sequencing technology, 7 differentially expressed proteins were screened. When patients developed drug resistance after EP regimen treatment, the levels of CLMP, CCDC80, EPHA2, RET, and ADAM-TS 15 in plasma increased ( Figure 1 A and Figure 1 B), ICAM4 and IL33 levels decreased ( Figure 1 C and Figure 1 D).

[0037] Interestingly, the inventors, based on data from the GEPIA database (http: / / gepia.cancer-pku.cn / ), found that the expression level of CCDC80 RNA in lung adenocarcinoma tissue samples was lower than that in normal tissue samples. In a comparison of 486 lung squamous cell carcinoma tissue samples with 338 normal tissue samples, the expression level of CCDC80 RNA in lung squamous cell carcinoma tissue samples was also lower than that in normal tissue samples. Figure 2 As shown in A. In addition, according to the relevant sequencing data from Zhang Peng's team's study (GSA database: HRA003419), the expression level of CCDC80 RNA was lower in 107 primary tumors of small cell lung cancer and 107 paired adjacent tissue samples than in normal tissues. Figure 2 As shown in B.

[0038] Based on the above-mentioned literature related to differentially expressed proteins and tumor research, the inventors selected CCDC80 and CLMP, which had not been previously reported in SCLC resistance, and conducted a preliminary CCK8 experiment in which transient transfection of si-CCDC80 knocked down CCDC80 followed by chemotherapy drug treatment. The results showed that there was no significant phenotypic difference in CLMP, and CCDC80 was ultimately selected for subsequent experiments.

[0039] The levels of CCDC80 in plasma before and after chemotherapy resistance were as follows Figure 3 As shown in the heat map, CCDC80 levels in the plasma of most patients were significantly higher than those at the time of initial diagnosis when they were resistant to chemotherapy ( 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 resistance of small cell lung cancer. Therefore, the inventor team conducted follow-up experiments to further verify it.

[0040] Example 2 CCDC80 promotes chemotherapy resistance in small cell lung cancer cells

[0041] The mRNA expression levels of CCDC80 in SCLC cell lines were analyzed using the CCLE database, and SCLC cell lines with stable overexpression and knockdown of CCDC80 were constructed. Stable cell lines were constructed by lentiviral infection as follows:

[0042] (1) Purchase lentivirus carrying CCDC80 overexpression plasmid and CCDC80 knockdown plasmid from Genema.

[0043] (2) Prepare the cells to be infected. Evenly inoculate the target cells into a 6-well plate (50-60%) and place them in an incubator overnight.

[0044] (3) Remove the cell culture medium and add 1 ml of ordinary cell culture medium and 1 ml of virus solution to each well. 4 μg / ml polybrene can be used to promote infection efficiency.

[0045] (4) Repeat step (2) 24 hours later. 48 hours after infection, puromycin can be used to screen for stable transfected cell lines. The screened stable transfected cells can be used for subsequent experiments.

[0046] The CCDC80 knockdown plasmid sequence, wherein the sequences of NC, ShCCDC80 #1, and ShCCDC80 #2 are shown as SEQ ID NOs: 1-3, respectively, and the CCDC80 overexpression plasmid sequence is shown as SEQ ID NO: 4.

[0047] In the present invention, DMS53 and DMS114 cells were selected to construct cell lines stably overexpressing CCDC80, and H446 and SHP-77 cells were selected to construct cell lines stably knocking down CCDC80. The effects of stably overexpressing and knocking down CCDC80 were verified by WB ( Figure 4 A and Figure 4 D). CCDC80-overexpressing or knockdown cells (DMS53, DMS114, H446, and SHP-77 cells) were treated with different concentration gradients of etoposide (VP-16) and cisplatin (DDP) for 48 hours, and cell survival was detected by CCK8.

[0048] The results showed that overexpression of CCDC80 in small cell lung cancer DMS53 and DMS114 cells promoted SCLC cell resistance to cisplatin and etoposide. Figure 4 B and Figure 4C), CCK8 detection significantly increased cell survival. Knockdown of CCDC80 in small cell lung cancer H446 and SHP-77 cells can increase the sensitivity of cells to cisplatin and etoposide ( Figure 4 E and Figure 4 F) CCK8 assay showed a significant decrease in cell survival. The above in vitro experiments confirmed that CCDC80 can indeed promote in vitro chemotherapy resistance in small cell lung cancer.

[0049] In vitro experiments showed that overexpression of CCDC80 significantly reduced the sensitivity of SCLC cells to cisplatin (DDP) and etoposide (VP-16), while knockdown of CCDC80 enhanced their chemotherapy sensitivity.

[0050] Example 3 CCDC80 promotes chemotherapy resistance by inhibiting cell apoptosis

[0051] DMS53, DMS114, H446, and SHP-77 cells stably overexpressing or knocking down CCDC80 were treated with varying concentrations of cisplatin or etoposide for 48 hours. Cell apoptosis in each group was assessed by flow cytometry after Annexin V-PI staining. Western blotting was also used to examine the expression of apoptosis-related proteins, such as Caspase 3, Caspase 7, and PARP cleavage, in the different cells.

[0052] Cells overexpressing CCDC80 were more resistant to cisplatin-induced apoptosis, while knockdown of CCDC80 enhanced cisplatin-induced apoptosis. Western blot experiments showed that overexpression of CCDC80 reduced the cleavage of Caspase3, Caspase7, and PARP, while knockdown of CCDC80 increased the cleavage of these apoptosis-related proteins.

[0053] 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); In contrast, in H446 and SHP-77 cells with CCDC80 knockdown, knockdown of CCDC80 further increased cisplatin- or etoposide-induced apoptosis ( Figure 5 B). Secondly, overexpression of CCDC80 can partially rescue cisplatin-induced apoptosis, and the cleavage of Caspase3, Caspase7, and PARP is reduced ( Figure 5 C) In contrast, knockdown of CCDC80 further enhanced cisplatin-induced apoptosis, with increased cleavage of Caspase3, Caspase7, and PARP ( Figure 5 D). The above results indicate that CCDC80 can promote chemotherapy resistance in small cell lung cancer by inhibiting cell apoptosis.

[0054] Example 4 CCDC80 promotes chemotherapy resistance in small cell lung cancer in vivo

[0055] A SCLC nude mouse xenograft tumor model was established, and it was found that overexpression of CCDC80 accelerated tumor growth and reduced the tumor inhibitory effect of EP chemotherapy, while knockdown of CCDC80 enhanced the anti-tumor effect of chemotherapy.

[0056] Small cell lung cancer DMS53 cells and SHP-77 cells that stably overexpress or knockdown CCDC80 were inoculated subcutaneously in the axilla of BALB / c nude mice (1×10 7 Cells / site) were injected into the EP regimen (cisplatin + etoposide) for one week after tumor loading. Etoposide was administered intraperitoneally at 4 mg / kg twice weekly, and cisplatin was administered intraperitoneally at 2 mg / kg every 8 days. The control group received an equal volume of solvent, using the same injection method and schedule as the experimental group. On day 17, mice were sacrificed, tumor specimens were harvested, tumor size was measured, and a comparison of tumor volume and weight was plotted. The specific groupings are as follows:

[0057] The DMS53 CCDC80 stable overexpression cell line transplanted tumor experiment was divided into 4 groups:

[0058] Control group 2: NC + Vehicle (solvent); OE-CCDC80 + Vehicle (solvent);

[0059] There were 2 experimental groups: NC + Drugs (cisplatin + etoposide); OE-CCDC80 + Drugs (cisplatin + etoposide).

[0060] The SHP-77 CCDC80 stable knockdown cell line transplanted tumor experiment was divided into 6 groups:

[0061] 3 control groups: NC + Vehicle (solvent); ShCCDC80 #1 + Vehicle (solvent); ShCCDC80 #2 + Vehicle (solvent);

[0062] There were three control groups: NC + Drugs (cisplatin + etoposide); ShCCDC80 #1 + Drugs (cisplatin + etoposide); ShCCDC80 #2 + Drugs (cisplatin + etoposide).

[0063] The results showed that overexpression of CCDC80 significantly promoted chemotherapy resistance in small cell lung cancer and significantly accelerated the growth of transplanted tumors. Figure 6 A is the tumor growth curve of the drug resistance experiment of transplanted tumors of DMS53 CCDC80 stably overexpressing cell lines. Figure 6 B is a comparison of tumor specimens after killing mice. Figure 6C is a comparison chart of tumor sample weights, showing that CCDC80 overexpression promoted tumor growth and chemotherapy resistance in small cell lung cancer. The volume and weight of CCDC80 overexpressing tumors in both the control and experimental groups were significantly higher than those in the NC group. Conversely, knocking down CCDC80 significantly weakened chemotherapy resistance in small cell lung cancer, and the growth of transplanted tumors was significantly slowed down. Figure 6 D is the tumor growth curve of the drug resistance experiment of transplanted tumors of SHP-77 CCDC80 stable knockdown cell line. Figure 6 E is a comparison of tumor specimens after mice were killed. Figure 6 Figure F shows a comparison of tumor sample weights, demonstrating that CCDC80 knockdown inhibits tumor growth and chemoresistance in small cell lung cancer. In both the control and experimental groups, the volume and weight of CCDC80 knockdown tumors were significantly lower than those in the NC group. These experimental results fully demonstrate the important biological function of CCDC80 in promoting chemoresistance in small cell lung cancer.

[0064] In summary, this study demonstrates for the first time that CCDC80 is significantly elevated in the plasma of patients with drug-resistant small cell lung cancer (SCLC). Overexpression of CCDC80 reduces the sensitivity of SCLC cells to cisplatin, carboplatin, and etoposide, while knockdown of CCDC80 enhances their chemotherapy sensitivity. CCDC80 promotes chemotherapy resistance by inhibiting apoptosis. Overexpression of CCDC80 reduces the cleavage of Caspase3, Caspase7, and PARP, while knockdown of CCDC80 increases their cleavage levels. In vivo experiments have shown that overexpression of CCDC80 accelerates tumor growth and reduces the efficacy of chemotherapy, while knockdown of CCDC80 enhances the anti-tumor effect of chemotherapy. This study demonstrates that CCDC80 can serve as a biomarker for chemotherapy resistance in SCLC patients. By detecting its expression level, patients' responses to chemotherapy can be predicted, guiding personalized treatment decisions for SCLC patients. This study also explores the potential for developing new strategies for chemotherapeutic resistance intervention. Intervention targeting CCDC80, such as inhibiting CCDC80 expression, could serve as a novel strategy for treating SCLC resistance, potentially increasing SCLC patients' sensitivity to cisplatin, carboplatin, or etoposide chemotherapy. This suggests that agents that inhibit CCDC80 expression could be used in combination with chemotherapy drugs to treat SCLC. This study provides new insights into the treatment and research of SCLC resistance and has significant clinical and economic value.

Claims

1. Application of CCDC80 protein as a marker in the preparation of products for detecting chemotherapy resistance of small cell lung cancer treated with cisplatin and etoposide.

2. Application of a substance for detecting the expression level of CCDC80 protein in the preparation of a product for detecting chemotherapy resistance of small cell lung cancer treated with cisplatin and etoposide.

3. The use according to claim 1 or 2, characterized in that: The product for detecting chemotherapy resistance of small cell lung cancer treated with cisplatin and etoposide includes a detection reagent for detecting the expression amount of the 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 patient with small cell lung cancer.

5. Use of a substance that inhibits CCDC80 protein expression in the preparation of a product for treating small cell lung cancer, characterized in that: The substance that inhibits the expression of CCDC80 protein is shRNA that inhibits the expression of CCDC80 protein. The sequences of the shRNA are shown in SEQ ID NO: 2 and SEQ ID NO:

3.

6. The use according to claim 5, characterized in that: The product can increase the sensitivity of small cell lung cancer to cisplatin and etoposide chemotherapy.

7. The use according to claim 5, characterized in that: The product can be used in combination with chemotherapy drugs to treat small cell lung cancer.

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