Application of ELOVL5 gene as biomarker and therapeutic target for adenocarcinoma of esophagogastric junction

Abstract

The application discloses application of an ELOVL5 gene as a biomarker and a therapeutic target of esophagogastric junction adenocarcinoma, and relates to the technical field of biological medicine.The application is proved by clinical sample detection that ELOVL5 is highly expressed in esophagogastric junction adenocarcinoma tissue, and is significantly different from normal tissue adjacent to the cancer, and the area under the diagnostic curve shows excellent diagnostic efficiency, providing a high-specificity biomarker for early screening and differential diagnosis of the disease.Metabolomics analysis shows that the fatty acid elongation process mediated by ELOVL5 is abnormally up-regulated in the patient, revealing the molecular mechanism of its participation in tumorigenesis.Cell experiments further confirm that the esophagogastric junction adenocarcinoma cell proliferation can be effectively inhibited by knocking down the expression of ELOVL5, and the feasibility of the ELOVL5 as a therapeutic target is clear.The application provides a new strategy for the diagnosis, efficacy evaluation and targeted treatment of esophagogastric junction adenocarcinoma, and has important scientific significance and clinical transformation value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the ELOVL5 gene as a biomarker and therapeutic target for adenocarcinoma of the esophagogastric junction. Background Technology

[0002] Esophagogastric junction adenocarcinoma is a malignant tumor originating at the junction of the esophagus and stomach, possessing biological characteristics of both esophageal and gastric cancer. Its incidence has been increasing in recent years. Early symptoms are often insidious, manifesting as mild acid reflux and a choking sensation when eating, which are easily overlooked, leading to most patients being diagnosed at an advanced stage. Despite advancements in comprehensive treatment methods such as surgery, chemotherapy, and radiotherapy, the tumor's aggressive nature and tendency to metastasize and recur result in a generally unsatisfactory prognosis. Therefore, in-depth analysis of the molecular mechanisms underlying the development and progression of esophagogastric junction adenocarcinoma, and the discovery of highly specific diagnostic biomarkers and effective therapeutic targets, are of significant clinical value and scientific importance for achieving early diagnosis and treatment and improving patient survival.

[0003] ELOVL5 (Elongation of Very Long Chain Fatty Acids Protein 5) is a member of the fatty acid elongase family (ELOVL) and plays a crucial role in the synthesis of polyunsaturated fatty acids (PUFAs). ELOVL5 primarily participates in the elongation of 18-carbon unsaturated fatty acids (such as linoleic acid and α-linolenic acid) into 20-carbon and 22-carbon unsaturated fatty acids (such as arachidonic acid and docosahexaenoic acid). ELOVL5 has important functions in various physiological activities in the human body. In the nervous system, ELOVL5 is expressed in both the central and peripheral nervous systems and is enriched in Purkinje cells of the cerebellum, brainstem, spinal cord, and hippocampus. It plays an important role in maintaining normal nervous system function, particularly in myelin formation and function. Regarding hepatic lipid metabolism, in a high-fat diet-induced obese mouse model, decreased ELOVL5 activity is associated with hepatic triglyceride (TG) accumulation and endoplasmic reticulum stress. Increasing the activity of hepatic ELOVL5 can reduce hepatic triglyceride (TG) levels, improve hyperglycemia and insulin resistance, and regulate the insulin signaling pathway. ELOVL5 plays a role in glycemic homeostasis by regulating hepatic fatty acid synthesis and affecting the mTORC2-Akt-FOXO1 signaling pathway.

[0004] ELOVL5 plays a dual role in different cancer types, with significant differences in its impact on tumorigenesis and development. In prostate cancer, overexpression of ELOVL5 is associated with the regulation of the androgen receptor (AR) signaling pathway and can promote the proliferation of prostate cancer cells. Furthermore, ELOVL5-mediated long-chain fatty acid elongation is considered one of the important mechanisms by which prostate cancer develops resistance to enzalutamide. Similarly, in renal cell carcinoma (RCC), high levels of ELOVL5 are positively correlated with poor clinical prognosis, and ELOVL5 can promote the proliferation and invasion of renal cancer cells. However, in breast cancer, low expression of ELOVL5 is associated with a poorer prognosis in patients with estrogen receptor-positive breast cancer. Downregulation of ELOVL5 leads to the accumulation of lipid droplets, which in turn mediates the induction of transforming growth factor-β (TGF-β) receptors, thereby promoting breast cancer metastasis. Furthermore, the expression of ELOVL5 and IGFBP6 genes affects the sensitivity of breast cancer cells to ferroptosis. Low expression of ELOVL5 may reduce the sensitivity of cells to ferroptosis, thereby promoting cancer cell survival. Currently, there are no reports of using ELOVL5 as a biomarker and therapeutic target for adenocarcinoma of the esophagogastric junction. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the ELOVL5 gene as a biomarker and therapeutic target for adenocarcinoma of the esophagogastric junction, thereby addressing the problems existing in the prior art. This invention has found that the ELOVL5 gene has high diagnostic value for adenocarcinoma of the esophagogastric junction and can be used as a biomarker for early screening and differential diagnosis of this disease.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of a reagent that inhibits the expression level of the ELOVL5 gene in the preparation of a drug for treating adenocarcinoma of the esophagogastric junction.

[0008] Furthermore, the reagent is siRNA.

[0009] Furthermore, the nucleotide sequence of the siRNA is shown in SEQ ID NO.2.

[0010] Furthermore, the nucleotide sequence of the siRNA is shown in SEQ ID NO.3.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0012] The present invention also provides the application of reagents for detecting the expression level of biomarkers in the preparation of diagnostic products for adenocarcinoma of the esophagogastric junction, wherein the biomarker is ELOVL5 protein or ELOVL5 gene.

[0013] Furthermore, the product is a chip.

[0014] Furthermore, the product is a testing reagent.

[0015] Furthermore, the product is a reagent kit.

[0016] The present invention discloses the following technical effects:

[0017] This invention clarifies the core role of the ELOVL5 gene in adenocarcinoma of the esophagogastric junction, possessing significant technical value and clinical application potential. Clinical sample testing confirmed that ELOVL5 is highly expressed in adenocarcinoma tissues of the esophagogastric junction, showing significant differences from adjacent normal tissues. Its area under the diagnostic curve demonstrates excellent diagnostic efficacy, providing a highly specific biomarker for early screening and differential diagnosis of this disease. Simultaneously, ELOVL5 expression levels are correlated with patient treatment efficacy, serving as a novel reference indicator for efficacy monitoring. Metabolomics analysis revealed that ELOVL5-mediated fatty acid elongation is abnormally upregulated in patients, elucidating its molecular mechanism of involvement in tumorigenesis. Cellular experiments further confirmed that knocking down ELOVL5 expression effectively inhibits the proliferation of adenocarcinoma cells in the esophagogastric junction, clarifying its feasibility as a therapeutic target. This invention provides a novel strategy for the diagnosis, efficacy evaluation, and targeted therapy of adenocarcinoma of the esophagogastric junction, possessing significant scientific importance and clinical translational value. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Heatmap of ELOVL family gene mRNA expression in gastric mucosa and adenocarcinoma of the esophagogastric junction;

[0020] Figure 2 A comparative diagram showing the differential expression of ELOVL family genes in gastric mucosa and adenocarcinoma of the esophagogastric junction; where N represents gastric mucosal tissue and T represents adenocarcinoma tumor tissue of the esophagogastric junction.

[0021] Figure 3 Immunohistochemical staining of adenocarcinoma and adjacent tissue at the esophagogastric junction;

[0022] Figure 4 A statistical graph showing the protein content of ELOVL5 in clinical tissue samples;

[0023] Figure 5For the subject operating characteristic curve;

[0024] Figure 6 Metabolomics heatmap of blood samples from patients with adenocarcinoma of the esophagogastric junction and patients with reflux esophagitis;

[0025] Figure 7 A heatmap showing the comparison of fatty acid content involved in the extension of ELOVL5;

[0026] Figure 8 The image shows the RT-PCR results of ELOVL5 mRNA expression level in SKGT4 cells after siRNA knockdown treatment.

[0027] Figure 9 The image shows the CCK-8 cell proliferation detection results of SKGT4 cells after siRNA knockdown treatment. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Example 1

[0034] The GSE74553 dataset from the GEO database was analyzed, containing 52 adenocarcinoma (AEG) samples from the esophagogastric junction and 5 gastric mucosal tissue samples. After standardization, the mRNA microarray data were used to generate mRNA expression heatmaps of ELOVL family genes in gastric mucosa and adenocarcinoma at the gastroesophageal junction using the "pheatmap" package in R software. The results are shown below. Figure 1 .

[0035] In GraphPad Prism 8 software, the t-test was used to analyze the differential expression of ELOVL family genes in normal gastric mucosa and adenocarcinoma of the esophagogastric junction. The results are shown in [Figure number missing]. Figure 2 The results showed that ELOVL2 / 5 / 7 were significantly overexpressed in AEG tissues, with ELOVL5 showing the most significant difference.

[0036] Tissue microarrays were prepared from tissue samples of 80 patients with adenocarcinoma of the esophagogastric junction, and immunohistochemical experiments were performed on ELOVL5. The results showed that ELOVL5 was also highly expressed in adenocarcinoma of the esophagogastric junction. Figure 3 ).

[0037] Example 2

[0038] Clinical tumor tissue samples (T group, n = 69) and adjacent normal tissue samples (N group, n = 60) were collected from patients with adenocarcinoma of the esophagogastric junction. Simultaneously, samples were collected from patients with adenocarcinoma of the esophagogastric junction who achieved complete remission after treatment (CR group, n = 21) and those who did not achieve complete remission (non-CR group, n = 35). Informed consent was obtained from all patients for all sample collection, and the process was approved by the ethics committee. The concentration of ELOVL5 protein (ng / mL) in each group of samples was detected using enzyme-linked immunosorbent assay (ELISA). Results are shown below. Figure 4 .

[0039] The results showed that the expression level of ELOVL5 in tumor tissue (T group) was significantly higher than that in adjacent normal tissue (N group), and the difference was statistically significant (P = 0.0073), suggesting that ELOVL5 can be used as a potential diagnostic marker for adenocarcinoma of the esophagogastric junction.

[0040] Analysis of the complete remission group (CR group) and the non-complete remission group (non-CR group) after treatment showed that the concentration of ELOVL5 in the non-CR group was significantly higher than that in the CR group (P = 0.0327), indicating that ELOVL5 can be used to evaluate the treatment efficacy of patients with adenocarcinoma of the esophagogastric junction and provides a new reference indicator for clinical efficacy monitoring.

[0041] Based on the ELOVL5 concentration data obtained from the aforementioned ELISA assay, this study used patients with adenocarcinoma of the esophagogastric junction (Group T) and adjacent normal tissue (Group N) as research subjects. Receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic efficacy of ELOVL5 for adenocarcinoma of the esophagogastric junction. The performance of ELOVL5 as a diagnostic biomarker was determined by calculating the area under the curve (AUC), sensitivity, and specificity. Figure 5 As shown, the AUC value of ELOVL5 for diagnosing adenocarcinoma of the esophagogastric junction was 0.715, indicating that it has high diagnostic value for adenocarcinoma of the esophagogastric junction and can be used as a biomarker for early screening and differential diagnosis of this disease.

[0042] Example 3

[0043] ELOVL5, as an ultra-long chain fatty acid elongation enzyme, is involved in the elongation of various fatty acids. Therefore, this invention collected blood samples from patients, including 13 cases of adenocarcinoma of the esophagogastric junction (AEG) and 35 cases of reflux esophagitis (GERD), for free fatty acid metabolomics analysis. Analysis results ( Figures 6-7 The results showed that multiple fatty acid elongation processes involved in the ELOVL5 response were upregulated to varying degrees (C18:2-C20:2, C18:3N6-C20:3N6, C20:4N6-C22:4N6, C20:5N3-C22:5N3). This indicates the presence of fatty acid differences in the blood of patients with esophagogastric junction adenocarcinoma and indirectly suggests differential expression of ELOVL5 in their bodies.

[0044] Example 4

[0045] This embodiment demonstrates through cell experiments that knocking down ELOVL5 can inhibit the proliferation of adenocarcinoma cells at the esophageal-gastric junction, as detailed below:

[0046] 1. Experimental Methods

[0047] 1.1 Small interfering RNA transfection of cells to knock down the target gene

[0048] (1) Design and synthesis of siRNA: Small interfering RNA was designed, and the specific sequences are shown in Table 1. All sequences were synthesized by Suzhou Jima Company. After the siRNA arrived, it was centrifuged at 1500g for 1 min. 125 μL of DEPC-treated water was pipetted into the tube and gently mixed by pipetting. It was then stored at -20℃.

[0049] (2) Seed preparation: Plant 2×10⁶ seeds in each well of a six-well plate. 5 Once SKGT4 cells have grown to more than 40%, siRNA transfection can be performed.

[0050] (3) Transfection: Prepare the siRNA transfection system as shown in Table 2. Mix the liquids in tubes 1 and 2 separately using a vortex mixer. Transfer the mixture from tube 2 to tube 1 and mix again using a vortex mixer. Let stand at room temperature for 15 minutes. Add the mixture dropwise to the culture medium in the wells. Replace the culture medium after one day, and digest the cells and collect the precipitate after another two days for subsequent experimental analysis. Verify the ELOVL5 knockdown efficiency using real-time quantitative PCR (primer sequences are shown in Table 3).

[0051] Table 1 siRNA sequences

[0052]

[0053] Table 2 siRNA transfection system

[0054]

[0055] Table 3 Primer Sequences

[0056]

[0057] 1.2 CCK-8 cell proliferation experiment

[0058] (1) Cell seeding: After digesting and centrifuging the cells, resuspend them in 1 mL of culture medium, mix well, and add 10 μL of the cell suspension to a counting chamber to count the number of cells. After dilution, add 1000 cells to each well of a 96-well plate. Prepare six replicates for each treatment group. After seeding all groups of cells, add another 100 μL of PBS around the outermost cells to help keep the cells moist and avoid affecting cell growth.

[0059] (2) CCK-8 treatment: 24 h after cell seeding, CCK-8 was added at a ratio of 10:1 (medium:CCK-8). After mixing, the cells were incubated for 1 h.

[0060] (3) Result determination: After the reaction is completed, the 96-well plate is wrapped with tin foil to protect it from light and the absorbance value at 450 nm is measured. Continue to perform the measurement every other day following this procedure.

[0061] 2. Experimental Results

[0062] ELOVL5 was knocked down using siRNA in SKGT4 cells, and the downregulation of ELOVL5 at the mRNA level was verified by RT-PCR. Figure 8 CCK8 proliferation assays showed that knocking down ELOVL5 inhibited SKGT4 cell proliferation. Figure 9 ).

[0063] In the development and progression of adenocarcinoma of the esophagogastric junction, ELOVL5 significantly promotes its proliferation by regulating key biological processes such as fatty acid metabolism. This embodiment demonstrates through cell experiments that knocking down ELOVL5 effectively inhibits the proliferative capacity of adenocarcinoma of the esophagogastric junction, clearly indicating that ELOVL5 plays a core driving role in the cell proliferation of this cancer. Based on this, ELOVL5 has the potential to become a therapeutic target for adenocarcinoma of the esophagogastric junction: by developing specific inhibitors of ELOVL5, interfering with its expression or function, the proliferation signaling pathway of adenocarcinoma of the esophagogastric junction can be blocked at the molecular level, providing a new strategy for the treatment of this cancer, potentially improving patient prognosis, and possessing significant clinical application value and scientific significance.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a reagent that inhibits ELOVL5 gene expression in the preparation of a drug for treating adenocarcinoma of the esophagogastric junction, characterized in that, The reagent is siRNA; The nucleotide sequence of the siRNA is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

3. The application of reagents for detecting biomarker expression levels in the preparation of diagnostic products for adenocarcinoma of the esophagogastric junction, characterized in that, The biomarker is the ELOVL5 protein or the ELOVL5 gene.

4. The application according to claim 3, characterized in that, The product in question is a chip.

5. The application according to claim 3, characterized in that, The product in question is a testing reagent.

6. The application according to claim 3, characterized in that, The product in question is a reagent kit.

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