Application of CEACAM1 gene or protein as marker in screening medicine for preventing or treating small cell lung cancer

Abstract

The invention discloses an application of a CEACAM1 gene or protein as a marker in screening a medicine for preventing or treating small cell lung cancer, and belongs to the technical field of molecular biology. The invention provides application of a CEACAM1 gene or protein as a marker in SCLC chemotherapy curative effect monitoring, chemotherapy drug resistance, disease recurrence, small cell lung cancer treatment and improvement of the sensitivity of small cell lung cancer to chemotherapy drugs. The invention discovers that the molecular marker CEACAM1 participates in SCLC and is resistant to EP combined chemotherapy for the first time, and VP-16, DDP or combination of VP-16 and DDP or two drugs (EP) can increase the molecular expression level of CEACAM1 in SCLC cells. The CEACAM1 can be used as a marker for SCLC chemotherapy curative effect monitoring, chemotherapy drug resistance and disease recurrence. The CEACAM1 is taken as a target spot, and a drug for reversing SCLC EP combined chemotherapy drug resistance can be developed.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and specifically relates to CEACAM1 The use of genes or proteins as markers in screening drugs for preventing or treating small cell lung cancer, and as markers for monitoring SCLC chemotherapy efficacy, chemotherapy resistance and disease recurrence. Background Art

[0002] Small cell lung cancer (SCLC) is a highly aggressive, poorly differentiated neuroendocrine cancer with a poor prognosis and a high malignancy among lung cancers. It accounts for approximately 15% of lung cancers and ranks first in mortality among malignant tumors worldwide. SCLC cells have a short doubling time, high heterogeneity, and are prone to metastasis at an early stage, making patients highly susceptible to drug resistance. From the time of diagnosis, the five-year survival rates for patients with limited-stage (LS-) and extensive-stage (ES-) SCLC are only approximately 10%-13% and 1%-2%, respectively.

[0003] Chemotherapy is the cornerstone of SCLC treatment. According to the "2024 Guidelines for the Diagnosis and Treatment of Small Cell Lung Cancer" of the Chinese Society of Clinical Oncology (CSCO), first-line chemotherapy for both LS- and ES-SCLC patients includes a combination of a topoisomerase inhibitor and a platinum compound, including the EP combination of etoposide (VP-16) and cisplatin (DDP). In recent years, immunotherapy has achieved significant breakthroughs in SCLC. With the release of positive results from the CASPIAN and IMpower133 studies, PD-1 / PD-L1 inhibitors (serotonin, atezolizumab, and durvalumab) combined with chemotherapy have become the new standard of care for ES-SCLC. However, multiple studies have shown that the benefits of combination immunotherapy for SCLC patients have been less than expected, increasing the economic burden on SCLC patients. Cell cycle dysregulation plays a key role in the development of SCLC, and cell cycle regulation is a potential treatment strategy for SCLC. Trilaciclib, a cyclin-dependent kinase (CDK) 4 / 6 inhibitor, is clinically used to mitigate myelosuppression induced by first-line chemotherapy in SCLC. Analysis of clinical research data suggests that while the use of trilaciclib prior to chemotherapy combined with immunotherapy can improve the prognosis of ES-SCLC patients to a certain extent, treatment-related complications significantly increase the medical burden for Chinese SCLC patients. TP53 and RB1 genes are almost universally deleted in SCLC, resulting in abrogation of G1 / S checkpoint activity and reliance on DNA repair during the G2 / M checkpoint. However, G2 / M checkpoint inhibitors currently in clinical trials, such as WEE1 protein kinase inhibitors and Aurora kinase inhibitors, also suffer from limited patient benefit.

[0004] Multidrug resistance to EP combined with chemotherapy, leading to treatment failure, is the leading cause of death in SCLC patients. PD-1 / PD-L1 inhibitors, CDK inhibitors, and G2 / M checkpoint inhibitors have yet to achieve significant clinical benefits in overcoming drug resistance in relapsed SCLC. Therefore, identifying specific biomarkers or therapeutic targets that can predict drug response in SCLC patients and exploring strategies to reverse SCLC resistance or enhance sensitivity are of great clinical value. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a CEACAM1 The use of genes or proteins as markers in screening drugs for the prevention or treatment of small cell lung cancer, and the first discovery of molecular markers CEACAM1 It is related to the in vitro chemotherapy resistance of SCLC cells and can affect the apoptosis level of SCLC cells. VP-16 and DDP can increase the expression of VP-16 in SCLC cells. CEACAM1 Molecular expression levels.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is: CEACAM1 Use of genes or proteins as markers in screening drugs for preventing or treating small cell lung cancer.

[0007] CEACAM1 Use of a gene or protein expression inhibitor in the preparation of a drug for preventing or treating small cell lung cancer.

[0008] Furthermore, CEACAM1 The gene or protein expression inhibitor is at least one of a nucleic acid molecule, a small molecule compound, a polypeptide, a protein, a gene editing vector, a lentivirus or an adeno-associated virus.

[0009] Furthermore, CEACAM1 Gene or protein expression inhibitors are CEACAM1 siRNA designed as a target for a gene or protein, or targeted knockout CEACAM1 sgRNA targeting a gene or protein.

[0010] Furthermore, the sequence of the siRNA is: CEACAM1 siRNA sense: 5'-CACCUAACAAGAUGAAUGATT-3'; (SEQ ID NO.1) CEACAM1 siRNA anti-sense: 5'-UCAUUCAUCUUGUUAGGUGTT-3'. (SEQ ID NO.2) CEACAM1Use of gene or protein expression inhibitors in the preparation of drugs for enhancing the chemotherapy sensitivity of small cell lung cancer.

[0011] A combination drug comprising the above CEACAM1 Gene or protein expression inhibitors, and chemotherapy drugs.

[0012] Furthermore, the chemotherapy drug is VP-16 and / or DDP.

[0013] For detection CEACAM1 Use of a substance that increases gene expression in any of the following: A1) Preparation of products for evaluating the efficacy of treatment for small cell lung cancer, or for evaluating the efficacy, chemotherapy resistance, or recurrence of small cell lung cancer; A2) Preparing a product for evaluating apoptosis in small cell lung cancer cells, or evaluating apoptosis in small cell lung cancer cells.

[0014] Further, for detection CEACAM1 Gene expression markers include specific amplification CEACAM1 The primer sequence of the gene is: CEACAM1 -F: 5'-AGCCCTCCATCTCCAGCAA-3'; (SEQ ID NO.3) CEACAM1 -R:5'-GGGTCCTGTGTCATTCCTTGT-3'. (SEQ ID NO.4) One for detection CEACAM1 Gene expression methods, such as Western Blot, ELISA, digital PCR, IHC, etc.

[0015] Beneficial effects of the present invention: The present invention discovered molecular markers for the first time CEACAM1 Involved in SCLC resistance to EP combined chemotherapy, and VP-16, DDP or the combination of the two drugs can increase the expression of cytokines in SCLC cells. CEACAM1 Molecular expression levels. CEACAM1 It can be used as a molecular marker for monitoring SCLC chemotherapy efficacy, chemotherapy resistance and disease recurrence. CEACAM1 This target can be used to develop drugs that can reverse the resistance of SCLC to EP combined chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The construction process of SCLC drug-resistant cell lines and identification of drug-resistant characteristics; Figure 2 In SCLC drug-resistant cells CEACAM1 Abnormally high expression; Figure 3for CEACAM1 Participates in the resistance of SCLC cells to EP combined with chemotherapy; Figure 4 VP-16, DDP and the combination of the two drugs (EP) can increase the expression of VP-16 in SCLC cells. CEACAM1 Molecular expression levels. DETAILED DESCRIPTION

[0017] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0018] Example 1 Construction of SCLC multidrug-resistant cell lines Drug-resistant cell lines were established using increasing concentrations of VP-16 (MedChemExpress, 33419-42-0) and DDP (MedChemExpress, 15663-27-1). NCI-H446 cells were stably cultured in culture flasks. When the cell density reached 80% at passage 3, they were treated with VP-16, DDP, or a combination of VP-16 and DDP (EP) at a final concentration of 0.2 μM for 24 hours. The cells were then passaged at a 1:2 ratio and cultured in drug-free complete medium. When the cell density reached 80% again, the cells were treated with the next higher drug concentration for 24 hours, and the above procedure was repeated. The drug concentration gradient was 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 μM, respectively. The development period was 13 months. Finally, the SCLC VP-16 single-drug resistant cell line H446 / VP-R, the DDP single-drug resistant cell line H446 / DP-R, and the VP-16 and DDP dual-drug resistant cell line H446 / EP-R were constructed. The results are shown in Figure 1 .

[0019] Example 2 Identification of drug resistance characteristics of the SCLC multidrug-resistant cell line H446 / EP 1. Cell culture Human SCLC cell lines NCI-H446, H446 / VP-R, H446 / DP-R, and H446 / EP-R were cultured in RPMI Medium 1640 Basic (Gibco, 11875119) supplemented with 10% FBS (Procell, 164210). HEK-293T cells were cultured in DMEM Basic (1×) (Gibco, 6124145) supplemented with 10% FBS (DEME complete medium). These cells were cultured in a 37°C, 5% CO2 incubator.

[0020] 2. Cell viability assay Take cells in the logarithmic growth phase or cells after experimental treatment, digest them and resuspend them in complete medium to adjust the cell density to 6×10 per well. 3 100 μL of single-cell suspension was plated per well in a 96-well culture plate (LABSELECT, 11510). After overnight cell attachment, the original culture medium was discarded. SCLC cells were treated with different final concentrations of VP-16 and DDP, with triplicate wells set up for each group. After 48 hours of drug treatment, CCK8 solution (Life-ilab, AC11L054) was mixed with RPMI Medium 1640 Basic medium at a ratio of 1:10. The original culture medium in the 96-well plate was discarded, and 100 μL of the prepared CCK8 solution was added to each well. Cultures were continued for 1–4 hours, and the absorbance (OD) of each well was measured at 450 nm using a microplate reader. Cell viability = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group); drug resistance index (RI) = IC value of drug-resistant cells. 50 Value / parental cell drug IC 50 The drug resistance standard was RI ≥ 2. The data were plotted and analyzed using GraphPad Prism 10.0 and IBM SPSS Statistics 27.0 software. Figure 1 B.

[0021] The present invention uses CCK8 method to detect the cell viability of parental cells NCI-H446, drug-resistant cells H446 / VP-R, H446 / DP-R and H446 / EP-R cells after 48 h of VP-16 and DDP treatment ( Figure 1 B) and calculated the RI of drug-resistant cells. The experimental results showed that the RIs of H446 / VP-R cells to VP-16, H446 / DP-R cells to DDP, and H446 / EP-R cells to VP-16 and DDP were all greater than 2, indicating that the H446 / VP-R, H446 / DP-R, and H446 / EP-R cell lines were successfully established.

[0022] 3. Cell proliferation curve detection Cell proliferation was measured using the Incucyte cell proliferation (label-free) assay. After digestion, cells were resuspended in complete culture medium and the cell density was adjusted to 2.5 × 10 cells per ml. 3 SCLC single-cell suspensions were seeded into 96-well culture plates (CORNING, 3599) at a rate of 100 μL per well. Two groups, untreated and treated, were set up, with triplicate wells per group. After allowing cells to adhere overnight, the treated groups were treated with VP-16, DDP, or a combination of the two drugs (EP). The cell culture plates were then placed in an Incucyte S3 live cell analyzer, programmed, and cell proliferation in each group was measured over a 120-hour period.

[0023] The present invention uses the Incucyte cell proliferation (label-free) method to detect cell proliferation and finds that the proliferation ability of drug-resistant cells H446 / VP-R, H446 / DP-R and H446 / EP-R is stronger than that of their parent cell NCI-H446. Moreover, under the action of VP-16, DDP or VP-16 combined with DDP (EP), the proliferation ability of drug-resistant cells is stronger than that of NCI-H446. The results are shown in FIG. Figure 1 C.

[0024] 4. Flow cytometry detection of apoptosis SCLC cells in the logarithmic growth phase were plated in 6-well cell culture plates. After the cells stabilized, RPMI 1640 complete medium was replaced. VP-16, DDP, or the combination of VP-16 and DDP (EP) were added to the drug-treated group, while DMSO was added to the control group. After 48 hours of drug treatment, the cells were digested into single cells using EDTA-free trypsin. The cell pellet was collected by centrifugation at 1000 rpm for 5 minutes and washed three times with 4°C pre-cooled PBS, each time at 1000 rpm for 5 minutes. After washing, the cells were resuspended in 500 μL of pre-cooled PBS and counted to 1×10 cells. 6 Cells were centrifuged and the supernatant was discarded. 195 μL of Annexin V-FITC binding solution was added to resuspend the cells; 5 μL of Annexin V-FITC was added and gently pipetted to mix; 10 μL of propidium iodide (PI) staining solution was added and gently pipetted to mix; incubated at room temperature (20-25°C) in the dark for 10-20 min, then placed on ice, and immediately detected by flow cytometry (Annexin V-FITC is green fluorescence, PI is red fluorescence). ModFit and Graphpad 10.0 were used to analyze the distribution of cell apoptosis in each treatment group. Results are shown in Figure 2. Figure 1 D, 1E.

[0025] Flow cytometry confirmed that under the action of VP-16, DDP and EP drugs, the apoptosis rates of H446 / VP-R, H446 / DP-R and H446 / EP-R were lower than those of the parental cell line NCI-H446.

[0026] Through the above studies, the present invention demonstrates that the SCLC VP-16 single-drug resistant cell line H446 / VP-R, the DDP single-drug resistant cell line H446 / DP-R, and the VP-16 and DDP dual-drug resistant cell line H446 / EP-R were successfully constructed.

[0027] Example 3 Drug-resistant cells CEACAM1 Expression detection 1. RNA extraction and real-time quantitative PCR (RT-qPCR) Total RNA was extracted from the parental cells NCI-H446, drug-resistant cells H446 / VP-R, H446 / DP-R, and H446 / EP-R using RNA extraction reagent RNAiso Plus reagent (TaKaRa, 9109), chloroform, isopropanol, and anhydrous ethanol. The first-strand cDNA was then synthesized using PrimeScript™ FAST RT reagent Kit with gDNA Eraser (TaKaRa, RR092A) according to the instructions. RT-qPCR was performed using TB Green ® Premix Ex Taq™ II (Tli RNaseHPlus) (TaKaRa, RR820A) was used with the CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA). ^ (-ΔΔCt) method to calculate target mRNA CEACAM1 The relative expression levels of Figure 2 The target gene amplification primers are as follows: CEACAM1 Forward primer: 5′-AGCCCTCCATCTCCAGCAA-3′; CEACAM1 Reverse primer: 5′-GGGTCCTGTGTCATTCCTTGT-3′.

[0028] β-Actin was used as the internal control. The β-Actin forward primer was 5'-TGGCACCCAGCACAATGAA-3'; the β-Actin reverse primer was 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3'.

[0029] 2. Western blot detection of cell protein expression levels When the cells reached the logarithmic growth phase or after drug treatment, the cells in each group were collected and rinsed twice with pre-chilled PBS, the supernatant was discarded, and RIPA lysis buffer (Beyotime, P0013B) containing protease and phosphatase inhibitors was added to extract total cell protein. The protein concentration was determined by BCA protein quantification method, and then SDS-PAGE electrophoresis was performed, transferred to the membrane, blocked with 5% skim milk powder for 2 h at room temperature, incubated with primary antibody at 4°C overnight, incubated with secondary antibody at room temperature for 2 h, and developed with ELC chemiluminescence.

[0030] like Figure 2 As shown in A and 2B, the present invention found through RT-qPCR and WB experiments that CEACAM1 It is abnormally highly expressed in SCLC drug-resistant cells H446 / VP-R, H446 / DP-R and H446 / EP-R.

[0031] Example 4: Stable overexpression of lentivirus CEACAM1 and siRNA knockdown CEACAM1 1. Overexpression CEACAM1 CEACAM1 After dissolving the (NM_001184813.2) and its control empty vector dry powder plasmid (Vector) in 20 μL sterile water, take 2 μL and add it to 100 μL DH5α competent cells, incubate on ice for 30 minutes, then heat shock at 42°C for 60 seconds, incubate on ice for 2 minutes, add it to 900 μL of LB medium without resistance, shake and culture at 180 rpm for 2 hours, take 10 μL and spread it on the corresponding resistance LB plate and culture it upside down overnight. Then pick a single colony and culture it in the corresponding resistance LB liquid medium with shaking overnight, and use the endotoxin-free plasmid miniprep kit (CW2106S) to extract. CEACAM1 Plasmids. pSPAX2, pMD2G, CEACAM1 / Vector plasmid was transfected into HEK-293T cells, viral supernatant was collected and infected into NCI-H446 cells. After infection, drug screening was performed to determine stable overexpression CEACAM1 / Vector cells. Virus infection efficiency was verified by WB experiment. The results are shown in Figure 3 A.

[0032] 2. siRNA knockdown CEACAM1 CEACAM1 (Gene ID: 634, NCBI) siRNA and NC siRNA were synthesized by Shanghai GeneGene. CEACAM1The siRNA sense sequence is: 5′-CACCUAACAAGAUGAAUGATT-3′; CEACAM1 The siRNA anti-sense sequence is: 5'-UCAUUCAUCUUGUUAGGUGTT-3'. The NC siRNA sense sequence is: 5'-UUCUCCGAACGUGUCACGUTT-3'; the NC siRNA anti-sense sequence is: 5'-ACGUGACACGUUCGGAGAATT-3'. After dissolving the siRNA in sterile water, use Lipofectamine ® RNAiMAX transfection reagent (Invitrogen, 13778-150) was used for transfection according to its instructions. 48 h after transfection, the transfection efficiency was verified by Western blotting. Figure 3 BD.

[0033] The present invention constructs a stable overexpression CEACAM1 The NCI-H446 cell line was used and NC siRNA was used to CEACAM1 siRNA was transfected into H446 / VP-R, H446 / DP-R, and H446 / EP-R cells, and the transfection was successful verified by WB experiment ( Figure 3 AD).

[0034] 3. CEACAM1 Participates in the resistance of SCLC cells to EP combined with chemotherapy CCK8 experiments found that it was overexpressed in the SCLC cell parent cell NCI-H446 CEACAM1 Genes can reduce cell sensitivity to VP-16 and DDP chemotherapy ( Figure 3 A); Knockdown in drug-resistant cells H446 / VP-R, H446 / DP-R and H446 / EP-R CEACAM1 Genes can increase cell sensitivity to VP-16 and DDP chemotherapy ( Figure 3 BD).

[0035] 4. If Figure 3 As shown, flow cytometry analysis of cell apoptosis revealed that the gene was overexpressed in the SCLC parent cell line NCI-H446. CEACAM1 Gene can reduce the level of cell apoptosis caused by VP-16, DDP, and EP ( Figure 3 E, F); Knockdown in drug-resistant cells H446 / VP-R, H446 / DP-R and H446 / EP-R CEACAM1 Genes can increase the level of apoptosis caused by VP-16, DDP, and EP ( Figure 3 G).

[0036] Example 5 VP-16, DDP or VP-16 combined with DDP (EP) can increase the expression of SCLC cells CEACAM1 Molecular expression levels.

[0037] By RT-qPCR ( Figure 4 AD, IL), WB ( Figure 4 EH, MO) experiments found that treatment of NCI-H446 and drug-resistant cells H446 / VP-R, H446 / DP-R and H446 / EP-R with VP-16, DDP or EP could increase the expression of SCLC cells in a time- and concentration-dependent manner. CEACAM1 Molecular expression level. CEACAM1 It is a drug response molecule for VP-16, DDP and EP. Since EP combined with chemotherapy is the first-line treatment for SCLC, it is further confirmed CEACAM1 It is a molecule that mediates SCLC resistance to VP-16, DDP and EP chemotherapy, indicating that CEACAM1 It can be used as a marker for monitoring SCLC chemotherapy efficacy, chemotherapy resistance and disease recurrence. CEACAM1 This target can be used to develop drugs that can reverse the resistance of SCLC to EP combined chemotherapy.

[0038] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. CEACAM1 Use of a gene or protein as a marker in any of the following: A1) Use in screening drugs for preventing or treating small cell lung cancer; A2) Use in the preparation of preparations for monitoring SCLC chemotherapy efficacy, chemotherapy resistance and disease recurrence.

2. CEACAM1 Use of a gene or protein expression inhibitor in the preparation of a drug for preventing or treating small cell lung cancer.

3. The use according to claim 2, characterized in that described CEACAM1 The gene or protein expression inhibitor is at least one of a nucleic acid molecule, a small molecule compound, a polypeptide, a protein, a gene editing vector, a lentivirus or an adeno-associated virus.

4. The use according to claim 3, characterized in that described CEACAM1 Gene or protein expression inhibitors are CEACAM1 siRNA designed as a target for a gene or protein, or targeted knockout CEACAM1 sgRNA targeting a gene or protein.

5. The use according to claim 4, characterized in that The sequence of the siRNA is: CEACAM1 siRNA sense:5’-CACCUAACAAGAUGAAUGATT-3’; CEACAM1 siRNA anti-sense: 5'-UCAUUCAUCUUGUUAGGUGTT-3'.

6. CEACAM1 Use of gene or protein expression inhibitors in the preparation of drugs for enhancing the chemotherapy sensitivity of small cell lung cancer.

7. A combined drug, characterized in that: include CEACAM1 Gene or protein expression inhibitors, and chemotherapy drugs.

8. The combined drug according to claim 7, characterized in that The chemotherapy drug is VP-16 and / or DDP.

9. For detection CEACAM1 Use of a substance for regulating gene expression in any of the following applications, characterized in that: A1) Preparation of products for evaluating the efficacy of treatment for small cell lung cancer, or for evaluating the efficacy, chemotherapy resistance, or recurrence of small cell lung cancer; A2) Preparing a product for evaluating apoptosis in small cell lung cancer cells, or evaluating apoptosis in small cell lung cancer cells.

10. The use according to claim 9, characterized in that For detection CEACAM1 Gene expression markers include specific amplification CEACAM1 The primer sequence of the gene is: CEACAM1 -F:5’-AGCCCTCCATCTCCAGCAA-3’; CEACAM1 -R:5’-GGGTCCTGTGTCATTCCTTGT-3’。

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