Application of clu gene as a marker in screening or treating cervical gastric-type adenocarcinoma

Abstract

The application discloses application of a CLU gene as a marker in screening or treatment of cervical gastric-type adenocarcinoma and belongs to the technical field of molecular biology. The application finds that, compared with cervical ordinary-type adenocarcinoma, CLU is highly expressed in the tumor microenvironment of cervical gastric-type adenocarcinoma through single-cell transcriptome sequencing technology, and determines the potential role of the CLU gene in diagnosis or differentiation of cervical gastric-type adenocarcinoma and cervical ordinary-type adenocarcinoma. Further, through cervical gastric-type adenocarcinoma organoids, it is verified that a CLU inhibitor (OGX-011) can effectively inhibit the growth of the cervical gastric-type adenocarcinoma organoids, and the role of the CLU gene as a treatment target in treatment of cervical gastric-type adenocarcinoma is determined, thereby providing a new idea for clinical treatment and drug development of cervical gastric-type adenocarcinoma.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the application of the CLU gene as a biomarker in the screening or treatment of cervical gastric adenocarcinoma. Background Technology

[0002] Cervical cancer (CC) is one of the most common malignant tumors in women, accounting for 7.5% of all female cancer deaths. The vast majority of cervical cancers are associated with persistent infection with high-risk human papillomavirus (HPV); however, a small number, such as cervical adenocarcinoma, are unrelated to HPV infection. Therefore, the International Endocervical Adenocarcinoma Criteria and Classification (IECC) classifies cervical adenocarcinoma into two main categories: HPV-related and non-HPV-related. With the widespread adoption of primary prevention (HPV vaccination and public education) and secondary prevention (cervical cancer screening), the incidence of HPV-related cervical cancer will inevitably decrease significantly, while the proportion of non-HPV-related cervical cancer will gradually increase. The latter has unique epidemiological, clinical, pathological, and molecular genetic characteristics; it is highly malignant, has a poor prognosis, and is easily missed or misdiagnosed, posing a significant threat that must be taken seriously.

[0003] Gastric-type endocervical adenocarcinoma (G-EAC) is the most common type of non-HPV-related cervical cancer, ranking second only to ordinary-type endocervical adenocarcinoma (UEA) as the second most common primary adenocarcinoma of the cervix. It is a mucinous adenocarcinoma differentiated from gastric-type adenocarcinoma, with morphological features resembling pyloric gland epithelium. G-EAC is not associated with high-risk HPV infection, and its clinical presentation is highly atypical. The occult lesions make biopsy difficult, resulting in low screening and biopsy positivity rates. G-EAC exhibits some tolerance to radiotherapy and chemotherapy, and treatment remains primarily surgical. Unlike ordinary cervical adenocarcinoma, which is confined to the pelvis, G-EAC is highly invasive, with a tendency to spread to the peritoneum, omentum, and adnexa, leading to a poor prognosis. The diagnosis and treatment of G-EAC currently face significant challenges. Therefore, there is a need to further develop new treatment strategies and in vitro models that can be used to verify individualized efficacy, in order to address the problem of poor efficacy of traditional treatments for G-EAC, thereby improving treatment response rates and prognostic outcomes for G-EAC patients.

[0004] G-EAC has a complex pathogenesis, with rare cases reported in conjunction with Peutz-Jeghers syndrome (PJS) or Lynch syndrome. Both PJS and Lynch syndrome are autosomal dominant inherited diseases caused by gene mutations, specifically mutations in the serine-threonine kinase gene STK11 and the MSH mismatch repair gene, respectively. G-EAC may also involve some of the same gene mutations. Furthermore, studies have proposed that G-EAC possesses a complex tumor microenvironment composed of various components, including cancer epithelial cells, stromal cells, chemokines, and cytokines. These components contribute to G-EAC's high invasiveness and limited response to current treatments. Therefore, identifying specific biomarkers and therapeutic targets for cervical gastric adenocarcinoma is a crucial topic in therapeutic research. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the CLU gene as a biomarker in the screening or treatment of cervical gastric adenocarcinoma, thereby addressing the problems existing in the prior art. This invention utilizes single-cell transcriptome sequencing technology to discover that CLU is highly expressed in the tumor microenvironment of cervical gastric adenocarcinoma compared to common cervical adenocarcinoma, thus identifying the potential role of the CLU gene in diagnosing or differentiating cervical gastric adenocarcinoma from common cervical adenocarcinoma. Furthermore, through cervical gastric adenocarcinoma organoids, the invention validates that the CLU inhibitor (OGX-011) can effectively inhibit the growth of cervical gastric adenocarcinoma organoids, confirming the role of the CLU gene as a therapeutic target in the treatment of cervical gastric adenocarcinoma, and providing new insights for the clinical treatment and drug development of cervical gastric adenocarcinoma.

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

[0007] This invention provides the application of a reagent for detecting CLU gene expression levels in the preparation of products for screening cervical gastric adenocarcinoma.

[0008] The present invention also provides the application of a reagent for detecting CLU gene expression levels in the preparation of products that differentiate between cervical gastric adenocarcinoma and cervical common adenocarcinoma, wherein the expression level of the CLU gene in cervical gastric adenocarcinoma is higher than that in cervical common adenocarcinoma.

[0009] This invention also provides the use of reagents that inhibit CLU gene expression in the preparation of drugs that inhibit the growth of cervical gastric adenocarcinoma cells.

[0010] Optionally, the reagent for inhibiting CLU gene expression includes OGX-011.

[0011] This invention also provides the use of reagents that inhibit CLU gene expression in the preparation of drugs for treating cervical gastric adenocarcinoma.

[0012] Optionally, the reagent for inhibiting CLU gene expression includes OGX-011.

[0013] The present invention discloses the following technical effects:

[0014] This invention uses single-cell transcriptome sequencing technology to identify CLU molecules highly expressed in key structural cell groups of the cervical gastric adenocarcinoma tumor microenvironment (genomically unstable tumor epithelial cells and fibroblasts enriched in specific gastric adenocarcinoma). Immunohistochemical verification of clinical specimens confirmed that CLU is highly expressed in the cervical gastric adenocarcinoma tumor microenvironment compared to cervical common adenocarcinoma, thus establishing the potential role of the CLU gene in diagnosing or differentiating cervical gastric adenocarcinoma from cervical common adenocarcinoma.

[0015] This invention constructs organoids of cervical gastric adenocarcinoma and, based on these organoids, verifies that the CLU inhibitor (OGX-011) can effectively inhibit the growth of cervical gastric adenocarcinoma organoids, thus determining the role of the CLU gene as a therapeutic target in the treatment of cervical gastric adenocarcinoma. This provides new insights for the clinical treatment and drug development of cervical gastric adenocarcinoma. Attached Figure Description

[0016] 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.

[0017] Figure 1 This is the cell type distribution after clustering of 3 cases of cervical gastric adenocarcinoma and 2 cases of ordinary adenocarcinoma tissues using single-cell transcriptome sequencing technology in Example 1;

[0018] Figure 2 This is a labeling of the cell and tissue origins after clustering three cases of cervical gastric adenocarcinoma and two cases of common adenocarcinoma using single-cell transcriptome sequencing technology; among them, GAS represents cervical gastric adenocarcinoma and UEA represents cervical common adenocarcinoma.

[0019] Figure 3 Violin plots of copy number variations (CNVs) in epithelial cells of common cervical adenocarcinoma and gastric cervical adenocarcinoma, **** p<0.0001;

[0020] Figure 4 This is a bar chart showing the functional enrichment pathways of genomically unstable tumor epithelial cells in cervical gastric adenocarcinoma; the longer the bar, the smaller the p-value, and the more statistically significant it is.

[0021] Figure 5 This is a bar graph showing the enrichment of functional pathways in fibroblasts specific to gastric adenocarcinoma of the cervical gastric type; the longer the bar, the smaller the p-value, and the more statistically significant it is.

[0022] Figure 6 Differences in CLU gene expression between genomically unstable tumor epithelial cells and other epithelial cells in cervical gastric adenocarcinoma; * p < 0.05;

[0023] Figure 7 The expression differences of CLU gene between fibroblasts enriched in cervical gastric adenocarcinoma and other fibroblasts; *** p<0.001;

[0024] Figure 8 Immunohistochemical staining of clinical specimens of cervical gastric adenocarcinoma to show the expression of CLU molecules in the tumor microenvironment of cervical gastric adenocarcinoma;

[0025] Figure 9 Immunohistochemical staining of clinical specimens of common cervical adenocarcinoma to show the expression of CLU molecules in the tumor microenvironment of common cervical adenocarcinoma;

[0026] Figure 10 ROC curves for diagnosing cervical gastric adenocarcinoma and common cervical adenocarcinoma based on CLU molecule expression levels;

[0027] Figure 11 Survival curves based on CLU expression levels were plotted using cervical cancer transcriptomics data and survival prognostic information from the TCGA public database.

[0028] Figure 12 The results of immunofluorescence identification of human cervical gastric adenocarcinoma organoids constructed in Example 2 are shown below. Among them, A represents the expression of MUC6, a characteristic marker of cervical gastric adenocarcinoma tissue; B represents the expression of HIK1083, a characteristic marker of cervical gastric adenocarcinoma tissue; C represents the expression of PAX8, a characteristic marker of cervical gastric adenocarcinoma tissue; and D represents the expression of TFF2, a characteristic marker of cervical gastric adenocarcinoma tissue.

[0029] Figure 13 Bright field images of the cervical gastric adenocarcinoma control group (Control) and intervention group (OGX-011) constructed in Example 2. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0036] The sources of some of the raw materials involved in the following embodiments are as follows:

[0037] The basal culture medium, Advanced DMEM / F12, was sourced from Shanghai, China (Thermo Fisher Scientific 1263401); HEPES buffer was sourced from Shanghai, China (Thermo Fisher Scientific 15630130); glutamine was sourced from Shanghai, China (Thermo Fisher Scientific A1286001); penicillin / streptomycin / amphotericidal B was sourced from Shanghai, China (New Semmel C100C8); recombinant human glycoprotein (Nuojin) was sourced from Shanghai, China (R&D Company 6057-NG-100); fibroblast growth factor 7 (FGF-7) was sourced from Shanghai, China (Peptec 100-19); fibroblast growth factor 10 (FGF-10) was sourced from Shanghai, China (Peptec 100-26); recombinant human Wnt3a factor was sourced from Shanghai, China (Peptec 315-20); hepatocyte growth factor was sourced from Shanghai, China (MCE, HY-P7121); 1 μM SB202190 (signaling pathway inhibitor) is from Shanghai, China, Sigma S7067; Nicotinamide (vitamin B3 derivative) is from Shanghai, China, Sigma N0636; Acetylcysteine ​​(antioxidant) is from Shanghai, China, Sigma A9165-5g; Forskolin (cell proliferation agent) is from Shanghai, China, Sigma F6886; B-27 (cell culture additive) is from Shanghai, China, Gibico, 17504-44; Y-27632 (ROCK inhibitor) is from Shanghai, China, Abmole, M1817; A83-01 (transforming growth factor-β inhibitor) is from Shanghai, China, Sigma SML0788-5MG; CHIR (glycogen synthesis kinase-3β inhibitor) is from Shanghai, China, Sigma SML1046; Prostaglandin E2 is from Shanghai, China, MCE, HY-101952.

[0038] Example 1

[0039] This embodiment uses single-cell sequencing technology to identify key structural cell groups (genomically unstable tumor epithelial cells and fibroblasts enriched in gastric adenocarcinoma) and their characteristic genes in the tumor microenvironment of cervical gastric adenocarcinoma. Immunohistochemical verification using clinical specimens revealed that, compared to conventional cervical adenocarcinoma, the tumor microenvironment of cervical gastric adenocarcinoma highly expresses CLU molecules. The specific process is as follows:

[0040] Three samples of cervical gastric adenocarcinoma and two samples of cervical common adenocarcinoma (samples from five cervical adenocarcinoma patients who underwent surgical treatment at the Obstetrics and Gynecology Hospital Affiliated to Fudan University, with informed consent signed and approved by the ethics committee) were prepared into single-cell suspensions, constructed into libraries, and subjected to single-cell transcriptome sequencing.

[0041] Single-cell sequencing identified key structural cell populations in the tumor microenvironment of cervical gastric adenocarcinoma (genomically unstable tumor epithelial cells and fibroblasts enriched in gastric adenocarcinoma), elucidating their functional characteristics and differentially expressed genes. Principal component analysis was used to reduce the dimensionality of cells in the microenvironment of cervical gastric adenocarcinoma and common adenocarcinoma, and cluster analysis based on the Unified Manifold Approximation and Projection (UMAP) method yielded a total of 8 cell subpopulations (see...). Figure 1 Among them, significant heterogeneity exists in the epithelial cells and fibroblasts of cervical gastric adenocarcinoma and common adenocarcinoma (see...). Figure 2 ).

[0042] Further in-depth research on epithelial cells revealed that the mean copy number variation (CNV) of epithelial cells in cervical gastric adenocarcinoma was significantly higher than that in ordinary adenocarcinoma (see...). Figure 3 This indicates that genomic instability exists in cervical gastric adenocarcinoma epithelial cells. Further functional enrichment and differential gene analysis of genomically unstable tumor epithelial cells and other epithelial cells in cervical gastric adenocarcinoma revealed that significantly enriched functional pathways in genomically unstable tumor epithelial cells of cervical gastric adenocarcinoma included the heat shock protein binding pathway (see...). Figure 4 ), the characteristic gene of genomically unstable tumor epithelial cells of cervical gastric adenocarcinoma includes CLU (see Figure 6 ).

[0043] Further in-depth research on fibroblasts revealed that functional enrichment analysis and differential gene analysis of fibroblasts enriched in cervical gastric adenocarcinoma-specific gastric adenocarcinoma and other fibroblasts showed that the significantly enriched functional pathway in fibroblasts enriched in cervical gastric adenocarcinoma-specific gastric adenocarcinoma also included the heat shock protein binding pathway (see...). Figure 5 CLU is also a characteristic gene of cervical gastric adenocarcinoma-specific gastric adenocarcinoma enriched in fibroblasts (see CLU). Figure 7 ).

[0044] Based on the above research results, this embodiment collected 25 clinical specimens of cervical gastric adenocarcinoma and 25 clinical specimens of cervical common adenocarcinoma that underwent surgical treatment at the Obstetrics and Gynecology Hospital Affiliated to Fudan University. Immunohistochemical validation was further performed on the clinical specimens of cervical gastric adenocarcinoma and common adenocarcinoma to detect CLU expression levels in different patients. Receiver operating characteristic (ROC) curve analysis was used to evaluate the sensitivity and specificity of CLU expression levels in distinguishing between patients with cervical gastric adenocarcinoma and those with common adenocarcinoma. The results showed that, compared to common adenocarcinoma, the tumor microenvironment of cervical gastric adenocarcinoma specifically showed high expression of CLU molecules (see...). Figure 8 , Figure 9 and Figure 10 Finally, analysis using the TCGA database revealed that high expression of CLU molecules was associated with poor prognosis in cervical cancer. Figure 11Based on this preliminary analysis, it is believed that high expression of CLU molecules is a specific biomarker and potential target for cervical gastric adenocarcinoma, which is considered to have a higher degree of malignancy than cervical common adenocarcinoma.

[0045] Example 2

[0046] To verify the practical application of the CLU gene in the clinical treatment of cervical gastric adenocarcinoma, this embodiment constructs cervical gastric adenocarcinoma organoids and inhibits the CLU gene in these organoids. It was found that inhibiting the CLU gene can suppress the growth of cervical gastric adenocarcinoma cells. The specific process is as follows:

[0047] 1. Preparation of organoids from cervical gastric adenocarcinoma:

[0048] (1) After lysing red blood cells with red blood cell lysis buffer, the cervical cancer tissue specimens were placed in pre-cooled (4℃) DMEM / F12 medium, cut into small pieces with scissors, and then digested in a solution containing 1 mg / mL collagenase I. After shaking on a shaker at 37℃ for 1.5 h, mechanical shearing was performed to further process the suspension. The cell suspension was filtered through a 70 μm nylon cell filter and centrifuged at 1200 rpm for 5 min to collect primary cells. The cells were then washed with phosphate-buffered saline for later use.

[0049] (2) The extract obtained in step (1) was resuspended in organoid culture medium. 30 μL of cell suspension was added to each well of a 24-well culture plate containing BME (basement membrane extract) preheated at 37°C. After standing for 30 min, the non-adherent cells were removed to obtain cervical gastric adenocarcinoma organoids.

[0050] The above-mentioned organoid culture medium is prepared from the following raw materials:

[0051] The basal medium was Advanced DMEM / F12 medium supplemented with 0.1 mg / mL HEPES buffer, 0.1 mg / mL glutamine, 0.1 mg / mL penicillin / streptomycin / amphoteric B, 100 ng / mL recombinant human glycoprotein, 200 ng / mL fibroblast growth factor 7, 100 ng / mL fibroblast growth factor 10, 100 ng / mL recombinant human Wnt3a, 10 ng / mL hepatocyte growth factor, 1 μM signaling pathway inhibitor, 2.5 mM nicotinamide, 1.25 mM acetylcysteine, 10 μM cell proliferation agent, cell culture additive at twice the volume of the basal medium, 10 μM ROCK inhibitor, 500 nM transforming growth factor-β inhibitor, 300 nM glycogen synthesis kinase-3β inhibitor, and 1 μM prostaglandin E2.

[0052] Among them, HEPES buffer, HEPES, namely 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, is an amphoteric N-substituted aminosulfonic acid buffer that exhibits excellent buffering capacity in the pH range of 6.8-8.2, with a pKa of 7.48 at 25℃; the penicillin / streptomycin / amphotericidal B solution contains 10 kU / mL of penicillin, 10 mg / mL of streptomycin, and 25 μg / mL of amphotericin B, prepared with 0.9% sodium chloride; hepatocyte growth factor (HGF) is a multifunctional factor that regulates various cell growth, movement, and morphogenesis; the signaling pathway inhibitor SB202190 is a selective p38 MAPK inhibitor; the cell culture additive B-27 is a serum-free additive with defined components containing antioxidants, proteins, vitamins, and fatty acids, which, when mixed in optimal proportions, support the culture of nerve cells; the ROCK inhibitor Y-27632, ROCK (Rho Related protein kinases (PKA / PKG / PKC) are kinases belonging to the serine-threonine kinase AGC (PKA / PKG / PKC) family.

[0053] 2. Immunofluorescence staining identification of cervical cancer organoids

[0054] (1) Fix the sample: remove the culture medium from the 24-well plate, wash once with phosphate buffer; add 4% paraformaldehyde, fix for 10 min, and then wash 3 times with phosphate buffer for 5 min each time;

[0055] (2) Blocking and staining: Add 500 μL of immunostaining blocking solution and block at room temperature for 20 min; remove the immunostaining blocking solution and add 100 μL of primary antibody (anti-MUC6 1:200, anti-HIK1083 1:50, anti-TFF2 1:200, anti-PAX8 1:200) and incubate at 4℃ overnight.

[0056] (3) Secondary antibody incubation and staining: Wash 3 times with PBS for 5 min each time; add 100 μL of diluted secondary antibody (horseradish peroxidase-labeled antibody targeting the primary antibody) and incubate at room temperature for 1 h; wash 2 times with phosphate buffer for 10 min each time; add 100 μL of DAPI and stain at room temperature for 5 min; remove DAPI and wash 3 times with phosphate buffer for 5 min each time; add an appropriate amount of anti-fluorescence quenching mounting solution, cover with a coverslip and observe under a fluorescence microscope.

[0057] like Figure 12 As shown, the immunofluorescence staining results indicate that the organoid model constructed by the above process has the characteristics of cervical gastric adenocarcinoma tissue, indicating that the cervical gastric adenocarcinoma organoid was successfully constructed.

[0058] 3. Practical application verification of CLU gene in the clinical treatment of cervical gastric adenocarcinoma

[0059] Cervical gastric adenocarcinoma organoids were seeded in cell culture plates with a low adhesion surface. After three passages, the control group (Control) was given only 500 μL of the above-mentioned cervical gastric adenocarcinoma organoid culture medium, while the intervention group (OGX-011) was given 500 μL of the above-mentioned cervical gastric adenocarcinoma organoid culture medium and 300 nM of OGX-011 (CLU gene inhibitor). Bright-field images of cervical gastric adenocarcinoma organoids from the control group and the intervention group were collected at 0 h (D0), 24 h (D1), 48 h (D2), and 72 h (D3) after intervention.

[0060] like Figure 13 As shown, inhibiting the CLU gene with a CLU gene inhibitor significantly reduced the number of cancer cells in cervical gastric adenocarcinoma organoids. This indicates that the CLU gene can serve as a therapeutic target for cervical gastric adenocarcinoma, and therapeutic drugs for cervical gastric adenocarcinoma can be developed based on the CLU gene.

[0061] 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. Use of an agent that inhibits the expression of a CLU gene for the manufacture of a medicament for the treatment of cervical gastric-type adenocarcinoma, characterized in that, The agent that inhibits expression of the CLU gene is OGX-011. The agent that inhibits expression of the CLU gene is OGX-011.

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