Application of hsacirc0078269 in pancreatic cancer diagnosis, prognosis and nerve cleaning operation

Abstract

The invention relates to the technical field of biological medicine, in particular to application of hsacirc0078269 in diagnosis and prognosis of pancreatic cancer and nerve cleaning operation. Researches show that the hsacirc0078269 is highly expressed in the blood exosome of a pancreatic cancer patient, the total lifetime of a subject with high expression of the hsacirc0078269 is shorter than that of a subject with low expression of the hsacirc0078269, and the disease-free lifetime of the subject with high expression of the hsacirc0078269 is shorter than that of the subject with low expression of the hsacirc0078269; in addition, experiments also find that the hsacirc0078269 can be used for remarkably promoting the nerve infiltration of the pancreatic cancer; therefore, the hsacirc0078269, which is further used as a molecular marker for diagnosis and prognosis of pancreatic cancer, has an important application value for guiding treatment of nerve cleaning in pancreatic cancer operation.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly to an application of hsa_circ_0078269 in the diagnosis, prognosis, and nerve clearance surgery of pancreatic cancer. Background Art

[0002] Pancreatic cancer is one of the most malignant solid tumors, characterized by insidious onset, low surgical resectability, strong local invasiveness, high risk of early metastasis, high recurrence after surgery, and an overall poor prognosis. In recent years, with changes in dietary patterns and lifestyles, as well as increased clinical detection rates, the incidence of pancreatic cancer has steadily increased, posing a serious threat to public health and life. It is estimated that in 2020, the number of new cases of pancreatic cancer worldwide reached 420,000, and the number of deaths reached 410,000. Pancreatic cancer is highly invasive, metastatic, and recurrent, with perineural invasion being one of the most important factors affecting prognosis. Perineural invasion (PNI) is a prominent biological hallmark of pancreatic cancer, primarily characterized by tumor cell infiltration into neural tissue, followed by neural dissemination and metastasis. Pancreatic cancer has the highest incidence of PNI among all solid tumors, and the severity of PNI is associated with worse survival and significant disease-related pain. We have previously demonstrated that nerve clearance can effectively improve the prognosis of patients with CA199 levels less than 200, but specific biomarkers associated with neural invasion are still lacking. Therefore, exploring biomarkers and therapeutic targets for pancreatic cancer neural invasion has become an urgent clinical challenge. While a growing body of basic research has provided us with a deeper understanding of the molecular mechanisms of pancreatic cancer neural invasion, effective biomarkers and clinical therapeutic targets for the diagnosis and treatment of pancreatic cancer patients with neural invasion remain lacking.

[0003] Exosomes are microscopic vesicles secreted by various cells, each containing a phospholipid bilayer membrane and an average diameter of 100 nm. They are primarily composed of RNA, DNA, liposomes, proteins, and miRNAs. Research has shown that exosomes, by carrying bioactive substances, participate in many important physiological and pathological processes, particularly in tumor invasion and metastasis. For example, exosomes facilitate cellular communication within the tumor microenvironment by transferring functional nucleic acids and proteins. This communication promotes abnormal tumor growth, invasion, and metastasis by interacting with immune cells, fibroblasts, endothelial cells, and cancer cells. Therefore, blocking or inhibiting the production of tumor-derived exosomes is a potential therapeutic target for pancreatic cancer. Furthermore, because exosomes are widely present in various bodily fluid samples and are easily accessible, exosome detection could become a truly noninvasive diagnostic method, thus holding great potential for application in tumor diagnosis.

[0004] Circular RNAs (circRNAs) are a unique class of non-coding RNA molecules with a circular structure and no 5' to 3' polarity. This makes them more stable than linear RNAs, less susceptible to degradation by RNA-degrading enzymes, and able to persist in cells for extended periods. CircRNAs have diverse functions, including regulating gene expression by acting as microRNA sponges, binding to proteins to modulate their function or localization, mediating translation to produce peptides via IRES or m6A modifications, and regulating parent gene expression. Numerous studies have demonstrated that under pathological conditions, particularly tumors, the expression levels of many exosomal circRNAs differ significantly from those in normal controls. This suggests that exosomes can selectively package, secrete, and transfer specific circRNAs, exerting their corresponding biological functions. Therefore, tumor-derived exosomal circRNAs hold promise as novel biomarkers and potential targets for early diagnosis and treatment of cancer. Exploring their diagnostic efficacy and the efficacy of targeted nerve clearance in patients with pancreatic cancer with neural invasion is of great clinical significance, potentially improving the prognosis of pancreatic cancer patients. Summary of the Invention

[0005] In order to overcome at least one technical problem existing in the prior art, the present invention provides an application of hsa_circ_0078269 in the diagnosis, prognosis and nerve clearance surgery of pancreatic cancer.

[0006] The present invention first provides the use of hsa_circ_0078269 as a molecular marker in the preparation of a product for diagnosing pancreatic cancer.

[0007] Preferably, the subject with high expression of hsa_circ_0078269 suffers from pancreatic cancer; and the subject with low expression of hsa_circ_0078269 is a normal person.

[0008] The present invention also provides the use of hsa_circ_0078269 as a molecular marker in the preparation of a product for predicting the prognosis of pancreatic cancer.

[0009] Preferably, the prognosis of the subject with high expression of hsa_circ_0078269 is relatively poor, and the prognosis of the subject with low expression of hsa_circ_0078269 is relatively good.

[0010] Preferably, the subject with high expression of hsa_circ_0078269 has a shorter overall survival than the subject with low expression of hsa_circ_0078269; the subject with high expression of hsa_circ_0078269 has a shorter disease-free survival than the subject with low expression of hsa_circ_0078269.

[0011] The present invention also provides the use of hsa_circ_0078269 as a molecular marker in the preparation of a product for guiding whether to perform nerve clearance during surgery.

[0012] Preferably, the subject with high hsa_circ_0078269 expression suffers from pancreatic cancer and requires nerve dissection during surgery.

[0013] The present invention also provides a reagent for detecting the expression level of hsa_circ_0078269, wherein the reagent includes an hsa_circ_0078269 probe for detecting the expression level of hsa_circ_0078269.

[0014] The present invention also provides a kit comprising an hsa_circ_0078269 expression level detection system, wherein the hsa_circ_0078269 expression level detection system comprises the reagent for detecting the hsa_circ_0078269 expression level according to claim 8; A label or instructions indicating that the kit is used for diagnosing pancreatic cancer and / or detecting prognostic effects, and for evaluating whether nerve clearance is performed during pancreatic cancer surgery.

[0015] Preferably, the kit further comprises a CA19-9 detection system, which comprises a reagent for detecting the CA19-9 content in serum.

[0016] Beneficial effects: The inventors surprisingly discovered in their study that hsa_circ_0078269 was highly expressed in the blood exosomes of pancreatic cancer patients. The subjects with high hsa_circ_0078269 expression had a shorter overall survival than those with low hsa_circ_0078269 expression, and the subjects with high hsa_circ_0078269 expression had a shorter disease-free survival than those with low hsa_circ_0078269 expression. In addition, experiments also found that hsa_circ_0078269 can significantly promote the nerve infiltration of pancreatic cancer. Therefore, further application of hsa_circ_0078269 as a molecular marker for the diagnosis and prognosis of pancreatic cancer and for guiding the treatment of intraoperative nerve clearance for pancreatic cancer has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Scanning electron microscopy images of exosomes.

[0018] Figure 2 The expression of exosome hsa_circ_0078269 in NF, CAF, and pancreatic cancer cells was detected by qRT-PCR.

[0019] Figure 3 Figure 2 is the relationship between the expression level of serum exosome hsa_circ_0078269 and the overall survival of patients.

[0020] Figure 4 This is a diagram showing the diagnostic efficacy of serum exosome hsa_circ_0078269 for pancreatic cancer.

[0021] Figure 5 This is a representative image of the transwell taken under a microscope after tumor cells were treated with CAF exosomes.

[0022] Figure 6 This figure is a statistical analysis of the differences in the number of tumor cell penetrating cells in transwell experiments of different groups.

[0023] Figure 7 Representative images of tumor cells and dorsal root ganglion (DRG) neural infiltration after treatment with CAF exosomes, taken under a microscope.

[0024] Figure 8 Figure 2 is a statistical analysis of the differences in neural infiltration of tumor cells and DRG in different groups after treatment with CAF exosomes.

[0025] Figure 9 Statistical analysis of the number of nude mice with sciatic nerve dysfunction in different groups. DETAILED DESCRIPTION

[0026] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] Example 1 hsa_circ_0078269 is highly expressed in serum exosomes of pancreatic cancer patients The inventors first collected serum samples from five pancreatic cancer patients and five healthy volunteers. They extracted and purified exosomes from the serum by ultracentrifugation (2000g × 20 min, transfer the supernatant to a new centrifuge tube, then 10000g × 40 min, transfer the supernatant to a new centrifuge tube, and then centrifuge at 120000g × 70 min, finally discard the supernatant and resuspend the exosomes at the bottom of the tube with PBS). They then used high-throughput sequencing to screen hsa_circ_0078269, which was highly expressed in the serum exosomes of pancreatic cancer patients.

[0028] The inventors expanded the clinical samples, collected serum specimens from 122 pancreatic cancer patients and 88 healthy volunteers, extracted serum exosomes, and identified the extracted vesicles as exosomes by scanning electron microscopy, NTA particle size analysis, and protein electrophoresis analysis ( Figure 1 : Scanning electron microscopy image); The expression of hsa_circ_0078269 was detected by qRT-PCR, and it was found that the expression level of hsa_circ_0078269 in serum exosomes of pancreatic cancer patients was significantly higher than that in normal healthy volunteers.

[0029] Furthermore, by collecting the culture supernatants of normal fibroblasts (NFs), cancer-associated fibroblasts (CAFs), pancreatic cancer cells PANC-1, Capan-2, MiaCaPa-2, and normal pancreatic cells (HPDE) and extracting the total exosome RNA, the expression of hsa_circ_0078269 was detected by qRT-PCR. It was found that the expression of hsa_circ_0078269 in exosomes of CAFs and pancreatic cancer cells was significantly higher than that in NFs and normal pancreatic cells ( Figure 2 ).

[0030] The above results indicate that serum exosome hsa_circ_0078269 can be used as a molecular marker to distinguish pancreatic cancer patients from normal subjects.

[0031] Example 2 Relationship between the expression level of exosome hsa_circ_0078269 and disease-free survival and overall survival of pancreatic cancer patients Clinical information of all pancreatic cancer patients was collected and statistically analyzed in combination with the above qRT-PCR test results. It was found that the expression of hsa_circ_0078269 in serum exosomes of pancreatic cancer patients with high degree of neural invasion was significantly higher than that of patients without neural invasion. In addition, patients with high expression of hsa_circ_0078269 in serum exosomes had shorter disease-free survival and overall survival ( Figure 3 , Analysis method: Kaplan–Meier survival curve analysis, exosomal hsa_circ_0078269 expression above the median was defined as exosomal hsa_circ_0078269 high expression, and below was defined as exosomal hsa_circ_0078269 low expression).

[0032] The above results indicate that the subjects with high expression of exosomal hsa_circ_0078269 have a shorter overall survival than the subjects with low expression of exosomal hsa_circ_0078269; the subjects with high expression of exosomal hsa_circ_0078269 have a shorter disease-free survival than the subjects with low expression of exosomal hsa_circ_0078269, and exosomal hsa_circ_0078269 can promote nerve infiltration in pancreatic patients.

[0033] The expression level of exosomal hsa_circ_0078269 can be used as a molecular marker for diagnosing pancreatic cancer and judging pancreatic cancer progression and prognosis.

[0034] Example 3 Diagnostic efficacy of exosome hsa_circ_0078269 for pancreatic cancer By detecting the expression of hsa_circ_0078269 in serum exosomes and combining it with receiver operating characteristic (ROC) analysis, we found that: Serum exosome hsa_circ_0078269 has good diagnostic efficacy for pancreatic cancer, with an area under the curve (AUC) of 0.78 (95% confidence interval: 0.70-0.87). Figure 4 .

[0035] Example 4 Exosome hsa_circ_0078269 can significantly promote the invasion, metastasis and neural infiltration of pancreatic cancer The expression of hsa_circ_0078269 in CAFs was upregulated by transfection with hsa_circ_0078269 overexpression plasmid, and the cell culture supernatant was collected and co-cultured with tumor cells. For the transwell experiment, the treated cells were resuspended in fresh serum-free medium and counted. 1×10 5 The cells were diluted to a total volume of 300 μl with serum-free culture medium. The cell suspension was then added to the upper chamber of a transwell chamber, while 700 μl of culture medium containing 10% serum was added to the lower chamber. The entire system was incubated in an incubator for 8 hours. The chamber was then removed and the cells were fixed with 4% paraformaldehyde for 15 minutes. The cells were gently washed three times with PBS and then stained with crystal violet for 15 minutes. The excess crystal violet stain was then washed away with PBS. The cells inside the chamber were gently wiped clean with a cotton swab and observed and photographed under a microscope. Random fields of view were counted using Image J, and statistical analysis was performed to analyze the differences in cell migration ability between the different exosome-induced groups. The results showed that overexpression of the exosome hsa_circ_0078269 significantly enhanced the migration ability of pancreatic cancer cells compared to the control group.

[0036] For the neural invasion assay, resuspend the cells in normal complete medium containing 5% serum and count them. 5 cells / well, gently add matrigel, and inoculate in a 6-well plate. DRG is inoculated with matrigel 2μm away from the tumor cells. After the matrigel solidifies, complete culture medium containing 10% serum is added, and photos are taken. Then, the cells are placed in a cell culture incubator for culture. After 7 days, the tube formation of the cells is observed again, photos are taken, and the length and area are measured using Image J software. Through statistical analysis, the neural infiltration between different groups is compared. It is found that compared with the control group, the neural infiltration ability of pancreatic cancer cells is significantly enhanced after overexpression of exosomes hsa_circ_0078269. In vitro transwell experiments (indicating that exosomes hsa_circ_0078269 promote the migration of tumor cells) Figure 5 This is a representative image of a tumor cell transwell taken under a microscope. Figure 6 The differences in tumor cell migration ability among different groups were statistically analyzed using one-way ANOVA followed by Dunnett's tests. Figure 7 It's a nerve infiltration experiment. Figure 8 The differences in tumor cell neural infiltration among different groups were statistically analyzed using one-way ANOVA followed by Dunnett's tests; two The P value of statistical analysis for comparison between the two groups was less than 0.001.

[0037] In vivo experiments confirmed that exosome hsa_circ_0078269 can significantly promote the neural invasion of pancreatic cancer. A mouse sciatic nerve invasion model was established using 4-week-old BALB / c nude mice (n=25). The mice were anesthetized, the sciatic nerve was surgically exposed, and tumor cells (1×10 4 PANC-1 cells) were injected into the right sciatic nerve strip, while the left sciatic nerve strip was injected with 3 μL of PBS as a control group. Subsequently, CAF-derived EVs (10 μg) overexpressing hsa_circ_0078269 were intravenously injected weekly, while the control group was injected with 20 μL of PBS. The sciatic nerve index was monitored by measuring the distance between the first and fifth toes of the hind limbs of the mice, ranging from 4 (normal) to 1 (complete paralysis of the paw). Statistical analysis was performed every two weeks using a two-tailed Student's t test. Figure 9 : The number of nude mice with right leg dysfunction in different groups was analyzed using statistical tables, and the chi-square test was used to analyze the significance of the difference. It means the statistical P value is less than 0.001.

[0038] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0039] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Application of hsa_circ_0078269 as a molecular marker in the preparation of products for the diagnosis of pancreatic cancer.

2. The use according to claim 1, characterized in that The subject with high expression of hsa_circ_0078269 suffers from pancreatic cancer; the subject with low expression of hsa_circ_0078269 is a normal person.

3. Application of hsa_circ_0078269 as a molecular marker in the preparation of products for predicting the prognosis of pancreatic cancer.

4. The use according to claim 3, characterized in that The prognosis of the subjects with high expression of hsa_circ_0078269 is relatively poor, and the prognosis of the subjects with low expression of hsa_circ_0078269 is relatively good.

5. The use according to claim 3, characterized in that The subjects with high hsa_circ_0078269 expression had a shorter overall survival than the subjects with low hsa_circ_0078269 expression; the subjects with high hsa_circ_0078269 expression had a shorter disease-free survival than the subjects with low hsa_circ_0078269 expression.

6. Application of hsa_circ_0078269 as a molecular marker in the preparation of products for guiding whether to perform nerve dissection during surgery.

7. The use according to claim 6, characterized in that The subject with high expression of hsa_circ_0078269 suffers from pancreatic cancer and requires nerve dissection during surgery.

8. A reagent for detecting the expression level of hsa_circ_0078269, characterized in that The reagent includes an hsa_circ_0078269 probe, which is used to detect the expression level of hsa_circ_0078269.

9. A kit, characterized in that It includes an hsa_circ_0078269 expression detection system, wherein the hsa_circ_0078269 expression detection system includes the reagent for detecting the hsa_circ_0078269 expression level according to claim 8; A label or instructions indicating that the kit is used for diagnosing pancreatic cancer and / or detecting prognostic effects, and for evaluating whether nerve clearance is performed during pancreatic cancer surgery.

10. The kit according to claim 9, characterized in that The kit also includes a CA19-9 detection system, which includes reagents for detecting the CA19-9 content in serum.

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