Biomarkers for the diagnosis and prognosis of small cell lung cancer and their applications

Abstract

This invention discloses a biomarker for the diagnosis and prognosis of small cell lung cancer (SCLC) and its applications. The biomarker, DEPDC1, is present in the tissues of SCLC patients, and its high expression is associated with poor prognosis. The invention also discloses reagents for detecting DEPDC1 expression levels, kits containing these reagents, and the applications of these reagents and kits in the preparation of SCLC diagnostic or prognostic products. The reagents include primers and probes for detecting DEPDC1. Furthermore, the application of DEPDC1 inhibitors in the preparation of SCLC therapeutic products is also disclosed. This invention provides novel molecular biomarkers and potential targets for the diagnosis, prognostic assessment, and treatment of SCLC, contributing to precision diagnosis and treatment of SCLC.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a biomarker for the diagnosis and prognosis of small cell lung cancer and its application. Background Technology

[0002] Lung cancer is classified into two main categories based on histopathology: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). SCLC, the most aggressive histological form of lung cancer and the most common primary neuroendocrine tumor of the lung, accounts for approximately 15%–20% of all lung cancers. It is characterized by high heterogeneity, early metastasis, and difficulty in treatment. Nearly 70% of SCLC patients already have metastatic disease at diagnosis. Furthermore, while SCLC patients are sensitive to chemotherapy, they rapidly develop resistance. Even after the approval of immune checkpoint inhibitors in combination with chemotherapy as first-line treatment, the additional survival benefits for patients remain very limited. Early metastasis and treatment resistance lead to poor long-term prognosis; the 5-year survival rate for limited-stage disease is 15%–30%, and the 5-year survival rate for extensive-stage disease is less than 1%, causing more than 200,000 deaths annually. Therefore, there is an urgent need to find new therapeutic targets and strategies for SCLC. Although some progress has been made in SCLC immunotherapy, the lack of specific therapeutic targets has limited the advancement of SCLC treatment. Therefore, early diagnosis and the identification of specific therapeutic targets are crucial for improving the prognosis of SCLC patients.

[0003] While NSE and ProGRP have demonstrated excellent sensitivity and specificity in diagnosing SCLC, they have failed to show superior efficacy in predicting disease prognosis. Therefore, identifying novel biomarkers with both high diagnostic efficacy and the ability to assess treatment response and prognosis is crucial. To further investigate the molecular mechanisms of SCLC development and progression and to identify potential tumor biomarkers, the inventors' team previously conducted bioinformatics analysis using publicly available online databases. The results showed that DEPDC1 was significantly overexpressed in SCLC tumor tissues compared to normal tissues from SCLC patients, and survival analysis revealed that high DEPDC1 expression was associated with poor prognosis in SCLC patients, suggesting a possible correlation between DEPDC1 and the development of SCLC. However, DEPDC1 has never been previously reported to be associated with SCLC.

[0004] DEPDC1 (DEP domain-containing protein 1), named after the Disheveled, EGL-10, and pleckstrin (DEP) domains, interacts with phospholipids and membrane receptors, recruiting proteins to the plasma membrane for signal transduction. Previous studies have identified DEPDC1 as a key factor in the development and progression of various cancers. This protein is almost undetectable in normal tissues but is highly expressed in various tumor types, playing a crucial role in cell mitosis, proliferation, migration, invasion, angiogenesis, autophagy, and apoptosis. Furthermore, DEPDC1 is involved in several key signaling pathways, such as the NF-κB, PI3K / Akt, Wnt / β-catenin, and Hippo pathways, which are essential for cell proliferation and survival. DEPDC1 expression is associated with poor prognosis and survival in various cancers, including hepatocellular carcinoma, lung adenocarcinoma, colorectal cancer, and breast cancer. Therefore, DEPDC1 has the potential to serve as a diagnostic and prognostic biomarker and a therapeutic target. Furthermore, clinical trials have shown that DEPDC1-based peptide vaccines exhibit good safety profiles and potential efficacy in cancer treatment. The inventors believe that the role of DEPDC1 in the diagnosis and prognosis of SCLC is of great research value.

[0005] Given that no studies have been reported on DEPDC1 in SCLC, its function in SCLC is still unclear. Therefore, it is of great value to study whether DEPDC1 can become a molecular marker for the diagnosis and prognosis of SCLC and a new target for the treatment of SCLC. Summary of the Invention

[0006] Based on the aforementioned research and development objectives, this invention relates to applications of DEPDC1 in SCLC, including using DEPDC1 as a marker for SCLC, and using the study of DEPDC1 expression changes in SCLC to prepare products related to the detection and treatment of SCLC. Specifically, these applications include:

[0007] The first objective of this invention is to provide a biomarker for the diagnosis or prognosis of small cell lung cancer, wherein the biomarker is DEPDC1, which is present in biological samples of patients with small cell lung cancer (SCLC).

[0008] A second objective of this invention is to provide a kit for detecting the expression level of DEPDC1 in patients with small cell lung cancer.

[0009] Furthermore, the kit includes primers and / or probes for detecting the expression level of DEPDC1, a biomarker for the diagnosis or prognosis of small cell lung cancer.

[0010] Furthermore, the kit includes siRNA and shRNA for knocking down the expression of the biomarker DEPDC1.

[0011] Furthermore, the kit is used in the preparation of products for detecting small cell lung cancer or for assessing the prognosis of small cell lung cancer.

[0012] A fourth objective of this invention is to provide an application of a DEPDC1 inhibitor in the preparation of a product for treating small cell lung cancer, wherein the DEPDC1 inhibitor comprises siRNA or shRNA.

[0013] The working principle of this invention: This invention is the first to discover that DEPDC1 expression is significantly elevated in small cell lung cancer (SCLC) patient tissues. Knockdown of DEPDC1 can inhibit SCLC cell proliferation, promote apoptosis, regulate the cell cycle, and inhibit migration and invasion. Knockdown of DEPDC1 inhibits proliferation by promoting apoptosis, increases Caspase 3 cleavage, and reduces the expression of the anti-apoptotic protein Bcl-2. Simultaneously, knockdown of DEPDC1 inhibits proliferation by mediating G0 / G1 phase arrest in small cell lung cancer cells, and increases p21 expression and reduces Cyclin D1 expression. In vivo experiments show that knockdown of DEPDC1 can slow tumor growth. This provides a new approach for targeted therapy and research in small cell lung cancer.

[0014] This invention demonstrates through experiments that DEPDC1 can serve as a therapeutic target for patients with small cell lung cancer and as a diagnostic and prognostic biomarker. By detecting its expression level, it can guide diagnosis and predict patient prognosis, providing new ideas for individualized treatment decisions for patients with small cell lung cancer and developing new therapeutic intervention strategies: interventions targeting DEPDC1, such as inhibiting DEPDC1 expression, can serve as a new strategy for the treatment of small cell lung cancer and inhibit tumor progression.

[0015] Compared with existing technologies, the beneficial effects of this invention are as follows: by using the method of "multi-database differential analysis + Venn diagram intersection screening", DEPDC1 is quickly and accurately screened as a core oncogene in small cell lung cancer (SCLC), filling the gap in the functional study of DEPDC1 in SCLC, providing a new target direction for the study of molecular mechanisms of SCLC, and this method can also be extended to the screening of key genes in other tumors, improving the efficiency of tumor molecular target research. Attached Figure Description

[0016] Figure 1 The Venn diagram shows the intersection of genes that are significantly overexpressed in tumor tissues from three databases.

[0017] Figure 2 This shows the expression of DEPDC1 in the GSE30219 database.

[0018] Figure 3 This shows the expression of DEPDC1 in the GSE60052 database.

[0019] Figure 4 The expression of DEPDC1 in paired array cancer and adjacent normal tissues.

[0020] Figure 5 This is a survival analysis diagram.

[0021] Figure 6 The expression of DEPDC1 mRNA in normal human lung epithelial cells and human small cell lung cancer cells;

[0022] Figure 7 The protein expression level of DEPDC1 in normal human lung epithelial cells and human small cell lung cancer cells is shown.

[0023] Figure 8 To test the knockdown effect of siDEPDC1 using WB.

[0024] Figure 9 The proliferation of cells in the DEPDC1 knockdown group and the control group was detected by CCK8 from day 0 to 4.

[0025] Figure 10 To test the knockdown effect of shDEPDC1 in WB.

[0026] Figure 11 To detect cell proliferation after DEPDC1 knockdown in a plate colony assay, the left side shows the plate colony formation, and the right side shows the relative quantitative statistics.

[0027] Figure 12 The total apoptosis rate of DEPDC1 knockdown and control cells is shown in the graph. The left side is a flow cytometry plot of apoptosis, and the right side is a quantitative statistical graph of total apoptosis rate.

[0028] Figure 13 Western blot diagram showing the expression of anti-apoptotic proteins Bcl-2, Caspase-3 cleaved proteins, and total protein in DEPDC1 knockdown and control cells.

[0029] Figure 14 The cell cycle regulation of DEPDC1 knockdown and control cells is shown, with the left side being a cell cycle flow cytometry plot and the right side being a quantitative statistical graph of cell cycle distribution ratio.

[0030] Figure 15 Western blot analysis was performed to detect the expression of p21 and Cyclin D1 proteins in DEPDC1 knockdown and control cells.

[0031] Figure 16To inhibit SCLC growth in vivo by knocking down DEPDC1, SCLC cells DMS53 with stable DEPDC1 knockdown and control cells were subcutaneously seeded in the axillary region of BALB / c nude mice and incubated for 32 days. A. Sacrifice the mouse to collect tumor specimens; B represents the tumor growth curve; C is a scatter plot of tumor weight. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0033] Example 1: Screening of biomarkers

[0034] Transcriptome data of small cell lung cancer (SCLC) were collected from three databases: TU-SCLC cohort (SCLC sample cohort from Tongji University, published in Cell), GSE30219 (SCLC sample from a public database), and GSE60052 (SCLC sample from a public database).

[0035] Differential analysis was performed on the tumor tissue vs. normal / adjacent tissue in each database, with the selection criteria being log2FC≥3.0 and adjusted p < 0.05.

[0036] By screening for significantly overexpressed genes shared by three databases using Venn diagrams (Figure 1), DEPDC1 was identified as a key candidate gene. Subsequent expression validation was performed, and the results are as follows: Figure 2-4 As shown, in GSE30219, the expression level of DEPDC1 in tumor tissue was significantly higher than that in normal tissue (p<0.0001) (Figure 2); the expression level of DEPDC1 in GSE60052 tumor tissue was also significantly higher than that in normal tissue (p<0.0001) (Figure 3); similarly, in TU-SCLC tumor tissue, the expression level of DEPDC1 was significantly higher than that in paired adjacent normal tissue (p<0.0001) (Figure 4).

[0037] Then, the expression level of DEPDC1 in TU-SCLC was grouped (high expression / low expression), and the association between DEPDC1 expression level and overall survival (OS) of SCLC patients was analyzed. Figure 5 ):

[0038] SCLC patients with high DEPDC1 expression had significantly shorter overall survival than those with low expression (Log-rank P=0.0244), indicating that DEPDC1 is a poor prognostic biomarker for SCLC.

[0039] Normal human lung epithelial cells BEAS-2B and SCLC cells (including DMS53, DMS114, SHP-77, SW1271, NCI-H196, NCI-H446, NCI-H69, NCI-H82, NCI-H146, NCI-H209, NCI-H526, and DMS153) were collected. RNA and protein were extracted and analyzed by RT-qPCR. Figure 6 ) and Western Blot ( Figure 7 The experiment detected the mRNA and protein expression levels of DEPDC1.

[0040] Total RNA was extracted using Magen's Cell RNA Rapid Extraction Kit, and cDNA was obtained by reverse transcription using the FastKing RT Kit. A qPCR system was prepared (1 μL each of the forward and reverse primers for either DEPDC1 or ACTB genes, 5 μL of PerfectStartGreen qPCR SuperMix fluorescent dye, 0.2 μL of cDNA, and 2.8 μL of RNase-Free ddH2O). Real-time quantitative PCR was performed to detect the relative expression level of DEPDC1, according to Formula 2. -ΔΔCt Calculations and statistical analysis using GraphPad Prism 9.5.1 software showed that the expression level of DEPDC1 in SCLC cells was higher than that in BEAS-2B cells.

[0041] Cells were lysed on ice for 20 minutes using RIPA lysis buffer, pre-cooled at 4°C, and centrifuged at 13,000 rpm for 20 minutes. The supernatant was collected and protein was quantified using a BCA protein quantification kit to homogenize the protein concentration. After adding 5×SDS protein loading buffer, the protein was boiled at 95°C for 10 minutes. Prepare SDS-PAGE gels, load 20 μg protein samples uniformly, and run electrophoresis at 80V for approximately 30 minutes until the separating gel is reached. Then adjust the voltage to 120V until bromophenol blue reaches the bottom of the gel and stop electrophoresis. After electrophoresis, perform membrane transfer. The PVDF membrane needs to be activated with methanol beforehand. Assemble it according to the "sandwich" structure (sponge-filter paper-gel-membrane-filter paper-sponge). Transfer the membrane in the transfer tank at 300mA for 90 minutes. After the transfer, block the membrane with 5% milk (prepared with TBST) and incubate at room temperature for 1 hour. Then cut the membrane according to the protein molecular weight. Incubate overnight at 4°C with primary antibodies DEPDC1 Polyclonal Antibody (Invitogen, catalog number PA5-70206) and β-Actin Rabbit mAb (ABclonal, catalog number AC026). After the transfer, wash the membrane three times with TBST for 10 minutes each time, and then use the secondary antibody HRP Goat Anti-Rabbit. IgG (H+L) (Invitogen, catalog number 31460, requires dilution with antibody diluent before use) was incubated at room temperature for 1 hour. After incubation, the membrane was washed three times with TBST for 10 minutes each time. ECL developing solution was prepared by mixing 1 mL of solution A and solution B thoroughly. The membrane was developed using a multi-functional image workstation, and the image was saved. ImageJ software was used to analyze the gray values ​​of protein bands and perform quantitative analysis. Western blotting results showed that the protein expression level of DEPDC1 in most SCLC cells was higher than that in BEAS-2B cells.

[0042] Both RT-qPCR and WB results showed that DEPDC1 expression was increased in SCLC cells. Figure 6 , Figure 7 ).

[0043] Example 2: Cellular level functional experiments

[0044] (1) The role of DEPDC1 in SCLC

[0045] Based on the overall basal expression levels (mRNA and protein expression), DMS53 and NCI-H446 cells, which had high basal expression levels of DEPDC1, were selected. siRNA (synthesized by Gemma Gene) was transfected using the siRNA-Mate plus transfection kit. Western blotting confirmed that both siRNA and shRNA effectively reduced DEPDC1 protein expression in DMS53 and NCI-H446 cells (Figures 8 and 10). Figure 8The results showed that compared with the NC group, the DEPDC1 protein band (~100 kDa) in the siDEPDC1 #1 and #2 groups was significantly lighter, while the internal control β-actin (~40 kDa) band was uniform. This indicates that both siRNAs can effectively knock down DEPDC1 protein expression, providing a reliable model for subsequent functional experiments. Figure 10 The results showed that, compared with the shNC group, the expression of DEPDC1 protein in both shDEPDC1 #1 and #2 groups was significantly reduced, and the internal control β-actin band was uniform. This indicates that a stable knockdown cell model of DEPDC1 was successfully constructed and can be used for long-term functional experiments (such as colony formation). After confirming the knockdown, the cells were seeded in 96-well plates, and the proliferation of cells in the NC group and the siDEPDC1 group was detected by CCK8 assay for 5 consecutive days.

[0046] Table 1 lists the siRNA sequences used in this application.

[0047]

[0048] Table 2 lists the shRNA sequences used in this application.

[0049]

[0050] The results are as follows Figure 9 As shown, the proliferation curve of the control group (NC) continued to rise; while the proliferation rate of siDEPDC1 #1 and #2 groups was significantly slower than that of the NC group (the curve slope was lower), and the cell proliferation ability was significantly reduced after knocking down DEPDC1 (Figure 9, p<0.01); furthermore, the plate colony experiment showed that the colony formation rate of the knockdown group was reduced by about 50% compared with the control group ( Figure 11 (p<0.01), indicating that knockdown of DEPDC1 significantly inhibited the long-term proliferation / clonal formation ability of SCLC cells.

[0051] Through the experimental combination of "knockdown verification (A, C) + short-term proliferation (B) + long-term cloning (D)," it was determined that DEPDC1 is a key promoting factor for the proliferation of SCLC cells. Knockdown of DEPDC1 can significantly inhibit the proliferation ability of SCLC cells. After knockdown of DEPDC1, the proliferation rate of SCLC cells slows down, and it is preliminarily concluded that DEPDC1 plays a pro-cancer role in SCLC.

[0052] (2) The effect of long-term silencing of DEPDC1 on SCLC

[0053] DEPDC1 stably knocked-down cell lines were constructed using DMS53 and NCI-H446 cells. The interfering lentivirus was purchased from GKG (viral titer 1×10⁻⁶). 8The viral load was measured (TU / mL), and HitransGP was used as the auxiliary viral infection reagent. DMS53 and NCI-H446 cells were prepared, plated in 6-well plates with 500,000 cells per well, and cultured overnight. Afterward, 1 mL of culture medium was mixed with 5 μL of viral solution and 40 μL of HitransGP viral infection reagent. The mixture was then used to infect the cells. The medium was changed 24 hours after infection, and after 72 hours, puromycin selection medium was added to screen for positive cells. Cells were harvested 5 days later to extract protein, and Western blotting was used to detect the knockdown efficiency. After confirming successful DEPDC1 knockdown, 6-well plates were plated for colony formation assays at 1000 cells / well for 2 weeks, with the medium changed every 3 days. Once the majority of individual clones reached 50 cells, they were fixed, stained, and photographed. The number of individual clones was counted, revealing a weakened colony-forming ability in the stably knocked-down DEPDC1 group. Therefore, it was concluded that DEPDC1 knockdown inhibits the growth of small cell lung cancer cells.

[0054] (3) The association between the pro-cancer effect of DEPDC1 and apoptosis in SCLC

[0055] DMS53 and NCI-H446 cells with DEPDC1 knocked down were collected, washed once with PBS, centrifuged, and the cell pellet was resuspended in 1 mL PBS. 100,000 cells were counted, centrifuged again, the supernatant was discarded, and 195 μL Annexin V-FITC binding buffer, 5 μL Annexin V-FITC, and 10 μL PI staining solution were added. The apoptosis rate of cells in the knockdown group and control group was detected by flow cytometry. FlowJo software was used to plot the data and analyze the differences between groups. It was found that the proportion of apoptotic cells increased after DEPDC1 knockdown. Figure 12 To confirm the promoting effect of DEPDC1 on apoptosis, we also used Western blotting to detect the expression of the anti-apoptotic protein Bcl-2, the important biomarker of apoptosis Cleaved Caspase-3, and total Caspase-3. The results are as follows: Figure 13 As shown, knocking down DEODC1 downregulated the expression of the anti-apoptotic protein Bcl-2 and upregulated the expression of the apoptosis marker Cleaved Caspase-3. These experiments demonstrate that knocking down DEODC1 inhibits apoptosis in SCLC cells, and that DEODC1 can promote SCLC survival by inhibiting the apoptosis pathway.

[0056] Given the role of DEPDC1 in cell cycle progression, and the potential for tumorigenesis due to abnormal cell cycle regulation, this study aimed to further understand the role of DEPDC1 in the development and progression of SCLC. Cell cycle changes in DMS53 and NCI-H446 cells (shNC and shDEPDC1 groups) were detected by flow cytometry. Cells were collected and fixed with pre-chilled anhydrous ethanol, ensuring constant mixing to prevent clumping. After fixation, cells were washed once with PBS, the supernatant was discarded, and 500 μL of PI staining solution was added for staining. After incubation at room temperature for 30 minutes, the cells were analyzed, and the results were recorded. ModFit LT software was used for plotting and analyzing the proportion of cells in different cell cycle stages. The results showed that knocking down DEPDC1 resulted in cell cycle arrest at the G0 / G1 phase, and the proportion of cells in the G0 / G1 phase increased. Figure 14 Western blot analysis of cell cycle-related proteins (p21 and Cyclin D1) showed that the expression of the cell cycle repressor p21 was increased, while the expression of the cell cycle promoter Cyclin D1 was decreased (Figure 15), indicating that DEPDC1 promotes cell cycle progression by regulating the G1 / S phase transition.

[0057] Example 3: Animal Experiment – ​​Knockdown of DEPDC1 Inhibits SCLC Tumor Growth in Vivo

[0058] Animal model: DMS53 cells with stable DEPDC1 knockdown (shDEPDC1 #1, #2) and negative control cells (shNC) were used at 1×10⁻⁶. 7 Tumors were inoculated subcutaneously in the axillae of BALB / c nude mice at a density of 6 mice per group. Tumor volume was measured every 4 days after tumor implantation (formula: volume = (length × width)). 2 ) / 2, in mm 3 Growth curves were plotted, and nude mice were sacrificed 32 days after tumor implantation. Tumor tissue was dissected, weighed, and the tumor weight was recorded.

[0059] In vivo tumor-bearing experiments showed that the tumor growth rate in the DEPDC1 knockdown group was significantly slower than that in the control group (Figure 16B, p<0.01); after 32 days of tumor bearing, the tumor weight in the knockdown group was reduced by approximately 60% compared to the control group. Figure 16 (C, p<0.01), which verified the pro-cancer effect of DEPDC1 in vivo.

[0060] In summary, DEPDC1 is a significantly overexpressed oncogene in SCLC, driving the development and progression of SCLC through mechanisms that promote cell proliferation, inhibit apoptosis, and accelerate cell cycle progression. Furthermore, high DEPDC1 expression is associated with poor prognosis in SCLC patients, making it a potential therapeutic target. Therefore, DEPDC1, present in biosamples from SCLC patients, can serve as a biomarker for the diagnosis or prognosis of SCLC. It can also be used to develop kits for detecting DEPDC1 expression levels. By using primers and / or probes capable of detecting DEPDC1 expression levels, biological agents or diagnostic kits for the diagnosis or prognosis of SCLC can be prepared.

Claims

1. A biomarker for the diagnosis or prognosis of small cell lung cancer, characterized in that, The biomarker is DEPDC1, which is present in biological samples from patients with small cell lung cancer (SCLC).

2. A kit for detecting the expression level of the biomarker of claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit includes primers and / or probes for detecting the expression level of DEPDC1, a biomarker for the diagnosis or prognosis of small cell lung cancer.

4. The reagent kit according to claim 3, characterized in that, The kit includes siRNA and shRNA for knocking down the expression of the biomarker DEPDC1.

5. The use of the kit according to claim 2 in the preparation of products for detecting small cell lung cancer or for prognostic assessment of small cell lung cancer.

6. The use of DEPDC1 inhibitors in the preparation of products for the treatment of small cell lung cancer, wherein the DEPDC1 inhibitors include siRNA or shRNA.

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