Biomarker for predicting risk of peritoneal recurrence of gastric cancer

Abstract

The present invention relates to a biomarker composition for predicting the risk of peritoneal recurrence of gastric cancer, the composition comprising circular RNA present in extracellular vesicles derived from cancer-associated fibroblasts (CAFs) as an active ingredient.

Description

Biomarkers for predicting the risk of peritoneal recurrence of gastric cancer

[0001] This study is about biomarkers for predicting the risk of peritoneal recurrence of gastric cancer.

[0002] Gastric cancer is one of the most common cancers worldwide, with a particularly high incidence in East Asia. Recurrence and metastasis are the main factors that worsen the prognosis of gastric cancer, with peritoneal recurrence being the most common.

[0003] Peritoneal recurrence occurs in approximately 30-40% of gastric cancer patients, with a higher incidence in patients with advanced gastric cancer. Patients with peritoneal recurrence have a very poor prognosis, with a significantly reduced 5-year survival rate. Therefore, early prediction and diagnosis of peritoneal recurrence are crucial for developing treatment strategies and improving survival rates in gastric cancer patients. However, reliable biomarkers for predicting peritoneal recurrence are currently lacking.

[0004] Therefore, there is an urgent need to develop a new biomarker that can accurately predict the risk of peritoneal recurrence in gastric cancer patients.

[0005] The technology that forms the background of this invention, Korean Patent No. 10-2253304, relates to a biomarker for predicting gastric cancer recurrence.

[0006] The present invention aims to solve the problems of the above-mentioned conventional technology and provides a biomarker composition for predicting the risk of peritoneal recurrence of gastric cancer.

[0007] In addition, a composition for predicting the risk of peritoneal recurrence of gastric cancer is provided.

[0008] In addition, a kit for predicting the risk of peritoneal recurrence of gastric cancer, including the composition for predicting the risk of peritoneal recurrence of gastric cancer, is provided.

[0009] Additionally, a method for predicting the risk of peritoneal recurrence of gastric cancer is provided.

[0010] However, the technical tasks to be achieved by the embodiments of the present invention are not limited to the technical tasks described above, and other technical tasks may exist.

[0011] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a biomarker composition for predicting the risk of peritoneal recurrence of gastric cancer, which comprises circular RNA in extracellular vesicles derived from cancer-associated fibroblasts (CAF) as an active ingredient.

[0012] According to one embodiment of the present invention, the circular RNA may include, but is not limited to, a member selected from the group consisting of circTSSC2 #2, circALG1, circALG1L2, and combinations thereof.

[0013] According to one embodiment of the present invention, if an upward regulation of the expression level of the circular RNA is observed, it may be determined that the rate of peritoneal recurrence of gastric cancer is high, but is not limited thereto.

[0014] In addition, the second aspect of the present invention provides a composition for predicting the risk of peritoneal recurrence of gastric cancer, comprising as an active ingredient a preparation capable of measuring the expression level of circular RNA in extracellular vesicles derived from cancer-related fibroblasts.

[0015] According to one embodiment of the present invention, the circular RNA may include, but is not limited to, a member selected from the group consisting of circTSSC2 #2, circALG1, circALG1L2, and combinations thereof.

[0016] According to one embodiment of the present invention, if an upward regulation of the expression level of the circular RNA is observed, it may be determined that the rate of peritoneal recurrence of gastric cancer is high, but is not limited thereto.

[0017] In addition, the third aspect of the present invention provides a kit for predicting the risk of peritoneal recurrence of gastric cancer, comprising a composition for predicting the risk of peritoneal recurrence of gastric cancer according to the second aspect of the present invention.

[0018] In addition, the fourth aspect of the present invention provides a method for predicting the risk of peritoneal recurrence of gastric cancer, comprising the steps of: measuring the expression level of circular RNA in extracellular vesicles derived from cancer-related fibroblasts from gastric cancer tissue; comparing the expression level of the circular RNA with that of a control group; and predicting the risk of peritoneal recurrence of gastric cancer based on the result of the comparison of the expression levels.

[0019] According to one embodiment of the present invention, the circular RNA may include, but is not limited to, a member selected from the group consisting of circTSSC2 #2, circALG1, circALG1L2, and combinations thereof.

[0020] According to one embodiment of the present invention, if an upward regulation of the expression level of the circular RNA is observed, it may be determined that the rate of peritoneal recurrence of gastric cancer is high, but is not limited thereto.

[0021] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be included in the drawings and detailed description of the invention.

[0022] The present invention proposes a novel biomarker for predicting the risk of peritoneal recurrence of gastric cancer using circular RNAs within cancer-associated fibroblast (CAF)-derived extracellular vesicles. Specifically, the circular RNAs (circTSSC2 #2, circALG1, circALG1L2) proposed in the present invention can be used as reliable indicators for early prediction of the risk of peritoneal recurrence in gastric cancer patients, thereby enabling the development of personalized treatment strategies. Furthermore, inhibition of these circular RNAs can be utilized as therapeutic targets to prevent peritoneal recurrence.

[0023] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0024] Figure 1 shows the results of analyzing circRNA expression between tissue groups after performing circRNA microarray on gastric cancer tissues between the peritoneal recurrence patient group (PR) and the non-recurrence patient group (NPR).

[0025] Figure 2 is a table showing candidate circRNAs whose expression is significantly upregulated in the tissues of patients with peritoneal recurrence selected through analysis of the circRNA microarray data of Figure 1.

[0026] Figure 3 is a graph showing the relative RNA expression levels of circTSSC2 #2, circALG1, and circALG1L2 in gastric cancer cell lines, normal mesothelial cell lines, and fibroblasts isolated from gastric cancer patients.

[0027] Figure 4 is a graph showing the relative RNA expression levels of circTSSC2 #2, circALG1, and circALG1L2 in extracellular vesicles (EVs) of various gastric cancer cell lines.

[0028] Figure 5 is a graph comparing the stability of circular RNA and linear RNA according to RNase R treatment.

[0029] Figure 6 shows the electrophoresis image and sequencing results showing the PCR results and the junction site base sequences in cDNA and gDNA of circTSSC2 and circALG1.

[0030] Figure 7 is a schematic diagram illustrating the process of attachment experiments between mesothelial cells and extracellular vesicles (EVs).

[0031] Figure 8 is a schematic diagram showing the schedule of an in vivo experiment using nude mice.

[0032] Figure 9 is a graph showing the fluorescence image and quantitative analysis results showing the degree of attachment of fluorescently labeled gastric cancer cells (SNU601) when various CAF-derived extracellular vesicles were treated on normal mesothelial cells (MeT-5A).

[0033] Figure 10 is a graph showing images of the control group (ctrl), the non-relapse group CAF culture medium-treated group (C98 CM), and the relapse group CAF culture medium-treated group (C104 CM) showing tumor nodule formation in the abdominal cavity of nude mice, and quantifying the number of tumor nodules.

[0034] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0035] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0036] Throughout this specification, when it is said that a member is located “on,” “above,” “upper,” “lower,” “lower” or “lower” another member, this includes not only cases where the member is in contact with the other member, but also cases where another member exists between the two members.

[0037] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0038] The terms "about," "substantially," and the like, as used herein, are used to mean at or near the numerical value when manufacturing and material tolerances inherent to the meanings referred to are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that contain precise or absolute numerical values ​​to aid understanding of the present disclosure. Furthermore, throughout the present disclosure, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0039] Throughout this specification, the term "combination thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0040] Throughout this specification, references to “A and / or B” mean “A, B, or A and B.”

[0041] Hereinafter, the biomarker composition for predicting the risk of peritoneal recurrence of gastric cancer of this hospital will be described in detail with reference to implementation examples, examples, and drawings. However, the present invention is not limited to these implementation examples, examples, and drawings.

[0042]

[0043] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a biomarker composition for predicting the risk of peritoneal recurrence of gastric cancer, which comprises circular RNA in extracellular vesicles derived from cancer-associated fibroblasts (CAF) as an active ingredient.

[0044] The present invention proposes a novel biomarker for predicting the risk of peritoneal recurrence of gastric cancer using circular RNA within extracellular vesicles derived from cancer-associated fibroblasts (CAFs). Specifically, the circular RNA proposed in the present invention can be utilized as a reliable indicator for early prediction of the risk of peritoneal recurrence in gastric cancer patients, thereby enabling the development of personalized treatment strategies. Furthermore, inhibition of the circular RNA can be utilized as a therapeutic target to prevent peritoneal recurrence.

[0045] According to one embodiment of the present invention, the circular RNA may include, but is not limited to, a member selected from the group consisting of circTSSC2 #2, circALG1, circALG1L2, and combinations thereof.

[0046] According to one embodiment of the present invention, if an upward regulation of the expression level of the circular RNA is observed, it may be determined that the rate of peritoneal recurrence of gastric cancer is high, but is not limited thereto.

[0047] If the expression of the above circular RNA is observed to be upregulated above normal levels, this suggests an increased risk of peritoneal recurrence of gastric cancer. These changes in expression levels can be measured quantitatively, and the risk of peritoneal recurrence can be assessed stepwise based on the degree of increase in expression levels.

[0048] In addition, the second aspect of the present invention provides a composition for predicting the risk of peritoneal recurrence of gastric cancer, comprising as an active ingredient a preparation capable of measuring the expression level of circular RNA in extracellular vesicles derived from cancer-related fibroblasts.

[0049] Regarding the composition for predicting the risk of peritoneal recurrence of gastric cancer according to the second aspect of the present invention, detailed descriptions of parts overlapping with the first aspect of the present invention have been omitted, but even if the descriptions have been omitted, the contents described in the first aspect of the present invention can be equally applied to the second aspect of the present invention.

[0050] According to one embodiment of the present invention, the circular RNA may include, but is not limited to, a member selected from the group consisting of circTSSC2 #2, circALG1, circALG1L2, and combinations thereof.

[0051] According to one embodiment of the present invention, if an upward regulation of the expression level of the circular RNA is observed, it may be determined that the rate of peritoneal recurrence of gastric cancer is high, but is not limited thereto.

[0052] In addition, the third aspect of the present invention provides a kit for predicting the risk of peritoneal recurrence of gastric cancer, comprising a composition for predicting the risk of peritoneal recurrence of gastric cancer according to the second aspect of the present invention.

[0053] Regarding the kit for predicting the risk of peritoneal recurrence of gastric cancer according to the third aspect of this application, detailed descriptions of parts overlapping with the first and second aspects of this application have been omitted. However, even if the descriptions have been omitted, the contents described in the first and second aspects of this application can be equally applied to the third aspect of this application.

[0054] In addition, the fourth aspect of the present invention provides a method for predicting the risk of peritoneal recurrence of gastric cancer, comprising the steps of: measuring the expression level of circular RNA in extracellular vesicles derived from cancer-related fibroblasts from gastric cancer tissue; comparing the expression level of the circular RNA with that of a control group; and predicting the risk of peritoneal recurrence of gastric cancer based on the result of the comparison of the expression levels.

[0055] Regarding the method for predicting the risk of peritoneal recurrence of gastric cancer according to the fourth aspect of this application, detailed descriptions of parts overlapping with the first to third aspects of this application have been omitted. However, even if the descriptions have been omitted, the contents described in the first to third aspects of this application can be equally applied to the fourth aspect of this application.

[0056] According to one embodiment of the present invention, the circular RNA may include, but is not limited to, a member selected from the group consisting of circTSSC2 #2, circALG1, circALG1L2, and combinations thereof.

[0057] According to one embodiment of the present invention, if an upward regulation of the expression level of the circular RNA is observed, it may be determined that the rate of peritoneal recurrence of gastric cancer is high, but is not limited thereto.

[0058] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0059]

[0060] [Example 1] Identification and expression analysis of circular RNA associated with peritoneal recurrence

[0061] CircRNA microarray was performed on gastric cancer tissues from patients with peritoneal recurrence (PR) and patients with non-recurrence (NPR). RNA was isolated from cell lines, and cDNA was synthesized. The expression of candidate circRNAs in each cell line was compared using qPCR.

[0062] Comparison of circular RNA expression between patients with peritoneal relapse and those without relapse

[0063] Figure 1 shows the results of analyzing circRNA expression between tissue groups after performing circRNA microarray on gastric cancer tissues between the peritoneal recurrence patient group (PR) and the non-recurrence patient group (NPR).

[0064] Statistical significance analysis of upregulated circular RNAs

[0065] Figure 2 is a table showing candidate circRNAs whose expression is significantly upregulated in the tissues of patients with peritoneal recurrence selected through analysis of the circRNA microarray data of Figure 1.

[0066] Analysis of circular RNA expression in gastric cancer cell lines

[0067] Figure 3 is a graph showing the relative RNA expression levels of circTSSC2 #2, circALG1, and circALG1L2 in gastric cancer cell lines, normal mesothelial cell lines, and fibroblasts isolated from gastric cancer patients.

[0068] Referring to Figure 3, RNA was isolated from gastric cancer cell lines, normal mesothelial cell lines, and fibroblasts isolated from gastric cancer patients, and the expression of candidate circRNAs was confirmed by qPCR. circTSSC2 #2, circALG1, and circALG1L2 were confirmed to be upregulated in fibroblasts obtained from gastric cancer patients.

[0069]

[0070] [Example 2] Analysis of expression characteristics of selected circular RNAs

[0071] The culture medium obtained by culturing patient fibroblasts was centrifuged at high speed to extract extracellular vesicles, and RNA was obtained and subjected to qPCR.

[0072] Analysis of circular RNA expression in extracellular vesicles

[0073] Figure 4 is a graph showing the relative RNA expression levels of circTSSC2 #2, circALG1, and circALG1L2 in extracellular vesicles (EVs) of various gastric cancer cell lines.

[0074] Referring to Fig. 4, a similar trend to Fig. 2 was also observed in the comparison of RNA expression contained in extracellular vesicles, and it was confirmed that candidate circular RNAs were abundant in the extracellular vesicles of fibroblasts.

[0075] RNase R resistance assessment

[0076] Figure 5 is a graph comparing the stability of circular RNA and linear RNA according to RNase R treatment.

[0077] Referring to Fig. 5, it was confirmed that circTSSC2, circALG1, and circALG1L are resistant to RNase R, which specifically destroys only linear RNA, and thus have a circular structure.

[0078] Confirmation of the structure of circular RNA

[0079] Figure 6 shows the electrophoresis image and sequencing results showing the PCR results and the junction site base sequences in cDNA and gDNA of circTSSC2 and circALG1.

[0080] Referring to Figure 6, it was confirmed that the junction sequence of the circular RNA formed by the back-splicing process during transcription was not synthesized in gDNA but only in cDNA, and thus had a circular structure.

[0081]

[0082] [Example 3] Analysis of the interaction between extracellular vesicles and mesothelial cells

[0083] Mesothelial cell adhesion experiment of extracellular vesicles

[0084] Figure 7 is a schematic diagram illustrating the process of attachment experiments between mesothelial cells and extracellular vesicles (EVs).

[0085] Referring to Figure 7, after treating mesothelial cells with extracellular vesicles obtained from cancer-associated fibroblasts and gastric cancer cells, they were cultured, and then fluorescently labeled gastric cancer cell lines were inoculated to compare the degree of adhesion between mesothelial cells and gastric cancer cells.

[0086] Establishment of an animal testing model

[0087] Figure 8 is a schematic diagram showing the schedule of an in vivo experiment using nude mice.

[0088] Referring to Figure 8, a culture medium of fibroblasts was administered into the abdominal cavity of nude mice, and a gastric cancer cell line was injected into the abdominal cavity one week later.

[0089] Evaluation of mesothelial cell-gastric cancer cell adhesion ability

[0090] Figure 9 is a graph showing the fluorescence image and quantitative analysis results showing the degree of attachment of fluorescently labeled gastric cancer cells (SNU601) when various CAF-derived extracellular vesicles were treated on normal mesothelial cells (MeT-5A).

[0091] Referring to Figure 9, it was confirmed that the adhesion ability to cancer cells increased in mesothelial cells treated with extracellular vesicles of fibroblasts.

[0092] In vivo tumorigenicity assessment

[0093] Figure 10 is a graph showing images of the control group (ctrl), the non-relapse group CAF culture medium-treated group (C98 CM), and the relapse group CAF culture medium-treated group (C104 CM) showing tumor nodule formation in the abdominal cavity of nude mice, and quantifying the number of tumor nodules.

[0094] Referring to Figure 10, it was confirmed that the number of intraperitoneal tumors increased in the group of mice administered a culture medium containing extracellular vesicles of fibroblasts.

[0095]

[0096] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0097] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. Containing circular RNA in extracellular vesicles derived from cancer-associated fibroblasts (CAF) as an active ingredient. Biomarker composition for predicting the risk of peritoneal recurrence of gastric cancer.

2. In paragraph 1, The above circular RNA comprises a nucleic acid selected from the group consisting of circTSSC2 #2, circALG1, circALG1L2 and combinations thereof. Biomarker composition for predicting the risk of peritoneal recurrence of gastric cancer.

3. In paragraph 2, If an upregulation in the expression level of the above circular RNA is observed, it is judged that the rate of peritoneal recurrence of gastric cancer is high. Biomarker composition for predicting the risk of peritoneal recurrence of gastric cancer.

4. A preparation comprising, as an active ingredient, a preparation capable of measuring the expression level of circular RNA in extracellular vesicles derived from cancer-related fibroblasts. A composition for predicting the risk of peritoneal recurrence of gastric cancer.

5. In paragraph 4, The above circular RNA comprises a nucleic acid selected from the group consisting of circTSSC2 #2, circALG1, circALG1L2 and combinations thereof. A composition for predicting the risk of peritoneal recurrence of gastric cancer.

6. In paragraph 5, If an upregulation in the expression level of the above circular RNA is observed, it is judged that the rate of peritoneal recurrence of gastric cancer is high. A composition for predicting the risk of peritoneal recurrence of gastric cancer.

7. A composition comprising a composition for predicting the risk of peritoneal recurrence of gastric cancer according to Article 4, Kit for predicting the risk of peritoneal recurrence of gastric cancer.

8. A step of measuring the expression level of circular RNA in extracellular vesicles derived from cancer-related fibroblasts from gastric cancer tissue; A step of comparing the expression level of the above circular RNA with the control group; and A step of predicting the risk of gastric cancer peritoneal recurrence through comparison of the above expression levels; including, Method for predicting the risk of peritoneal recurrence of gastric cancer.

9. In paragraph 8, The above circular RNA comprises a nucleic acid selected from the group consisting of circTSSC2 #2, circALG1, circALG1L2 and combinations thereof. Method for predicting the risk of peritoneal recurrence of gastric cancer.

10. In paragraph 9, If an upregulation in the expression level of the above circular RNA is observed, it is judged that the rate of peritoneal recurrence of gastric cancer is high. Method for predicting the risk of peritoneal recurrence of gastric cancer.

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