Application of FBXO45 as prognostic marker of pancreatic cancer

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of FBXO45 in preparation of a prognostic marker for pancreatic cancer. According to the application of the FBXO45 as the prognostic marker of the pancreatic cancer, by analyzing the influence of the expression quantity of the FBXO45 on the prognosis of pancreatic cancer patients, the survival rate of patients in an FBXO45 high-expression group in one year is remarkably lower than that in an FBXO45 low-expression group; a single factor is combined with Cox multi-factor analysis to confirm that high expression of the FBXO45 is an independent risk factor influencing the survival rate of a pancreatic cancer patient in one year after radiotherapy and chemotherapy, and prompt that the FBXO45 is closely related to prognosis of the patient; rOC curve analysis shows that the FBXO45 has a high prediction value for evaluating whether the pancreatic cancer chemoradiotherapy patients die within one year after operation, and it is prompted that the FBXO45 is expected to become a good index for evaluating prognosis of the pancreatic cancer chemoradiotherapy patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to the application of FBXO45 in preparing a prognostic marker for pancreatic cancer. Background Art

[0002] Pancreatic cancer is one of the malignant tumors with the highest mortality rate. 90% of them are pancreatic ductal adenocarcinoma (PDAC), and about 80% of patients are diagnosed in the advanced stage and have no effective treatment. About 20% of cases have the opportunity to receive surgical treatment, and their 5-year survival rate is less than 30%. The introduction of combined chemotherapy has greatly improved the prognosis after resection, but the 5-year survival rate is still as low as 9%. The poor prognosis of pancreatic cancer is related to the lack of early specific symptoms, the lack of effective screening tests, and the inability of current therapies to prolong survival. Chemotherapy is suitable for patients with advanced disease, but it is ineffective due to acquired drug resistance. Gemcitabine, the first-line chemotherapy drug for pancreatic cancer patients, has been replaced by modified FOLFIRINOX (mFOLFIRINOX) (5-fluorouracil, leucine, oxaliplatin, and irinotecan) or nab-paclitaxel plus gemcitabine therapy, but these therapies can only provide moderate survival in the presence of high toxicity. So far, new targeted therapies have failed to bring better overall survival (OS). FBXO45 is an E3 ubiquitin ligase containing an F-box / SPRY domain and a member of the F-box protein family. The expression of FBXO45 in the peripheral blood of pancreatic cancer patients and its relationship with survival prognosis have not been reported. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a use of FBXO45 as a prognostic marker for pancreatic cancer.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides the use of FBXO45 as a marker in one or more of the following:

[0006] (1) Prepare products for predicting or assessing the prognosis of pancreatic cancer;

[0007] (2) Prepare products for predicting or evaluating the postoperative survival of pancreatic cancer patients.

[0008] Preferably, the marker also includes CA19-9.

[0009] The present invention provides the use of a reagent for detecting the expression amount of FBXO45 in one or more of the following:

[0010] (I) Preparation of products for predicting or assessing the prognosis of pancreatic cancer;

[0011] (II) Prepare products for predicting or evaluating the postoperative survival of pancreatic cancer patients.

[0012] Preferably, a reagent for detecting the expression level of CA19-9 is also included.

[0013] Preferably, the prediction or evaluation comprises: detecting the expression level of FBXO45 mRNA and / or the protein expression level of FBXO45 in the biological sample of the subject to predict the postoperative survival period or prognosis of the subject.

[0014] Preferably, the biological sample comprises a blood sample from a pancreatic cancer patient.

[0015] Preferably, the product includes a reagent, a chip, a test paper or a test kit.

[0016] The present invention provides a primer pair for detecting the expression level of pancreatic cancer prognosis marker FBXO45, including FBXO45-F and FBXO45-R; the nucleotide sequence of the FBXO45-F is shown in SEQ ID NO: 1, and the nucleotide sequence of the FBXO45-R is shown in SEQ ID NO: 2.

[0017] The present invention provides a product for evaluating the prognosis of pancreatic cancer, wherein the product comprises a primer pair for detecting the expression level of FBXO45 according to the above technical solution.

[0018] Preferably, the product includes reagents, chips, test strips or test kits; the test kits also include ELISA detection reagents.

[0019] Preferably, the ELISA detection reagent further comprises one or more of streptavidin-peroxidase, TMB substrate and stop solution.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides an application of FBXO45 as a prognostic marker for pancreatic cancer. The present invention analyzes the effect of FBXO45 expression on the prognosis of pancreatic cancer patients. The 1-year survival rate of patients in the FBXO45 high expression group is significantly lower than that in the FBXO45 low expression group. Single factor combined with Cox multivariate analysis confirms that high FBXO45 expression is an independent risk factor affecting the 1-year survival rate of pancreatic cancer patients after chemoradiotherapy, indicating that FBXO45 is closely related to the patient's prognosis. ROC curve analysis shows that FBXO45 has a high predictive value for assessing whether pancreatic cancer patients undergoing chemoradiotherapy will die within 1 year after surgery, indicating that FBXO45 is expected to become a good indicator for assessing the prognosis of pancreatic cancer patients undergoing chemoradiotherapy. Based on the biological specimen library system of the First Affiliated Hospital of Bengbu Medical University, the present invention analyzes the expression difference of FBXO45 in the peripheral blood of patients with pancreatic cancer without chemoradiotherapy and chemoradiotherapy, as well as its relationship with clinical pathological parameters and patient survival prognosis, to provide a new predictive indicator for assessing the survival of pancreatic cancer patients after chemoradiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 To compare the relative expression of FBXO45 in pancreatic cancer patients before and after chemoradiotherapy;

[0023] Figure 2 The KM survival curve was used to analyze the effect of FBXO45 expression on the 1-year survival rate of pancreatic cancer patients after chemoradiotherapy;

[0024] Figure 3 ROC curve analysis was performed to analyze the predictive value of FBXO45 relative expression and CA19-9 alone or in combination with FBXO45 for the 1-year survival of pancreatic cancer patients after chemoradiotherapy; Figure 3 The vertical axis is sensitivity. DETAILED DESCRIPTION

[0025] The present invention provides the use of FBXO45 as a marker in one or more of the following:

[0026] (1) Prepare products for predicting or assessing the prognosis of pancreatic cancer;

[0027] (2) Prepare products for predicting or evaluating the postoperative survival of pancreatic cancer patients.

[0028] As an embodiment, the marker may also include CA19-9. FBXO45 is an E3 ubiquitin ligase containing an F-box / SPRY domain, a member of the F-box protein family, and is expressed in pancreatic cancer tissue. The CA19-9 described in the present invention is cancer antigen 19-9. CA19-9 is a tumor marker related to pancreatic cancer, gallbladder cancer, colon cancer, gastric cancer, etc., also known as gastrointestinal tract-related antigen. Cancer antigen 19-9 has high sensitivity and good specificity for pancreatic cancer, with a positive rate between 85% and 95%, and decreases with surgery and improvement of the condition. FBXO45 and CA19-9 combined as biomarkers can more accurately predict the prognosis of pancreatic cancer and postoperative survival.

[0029] The present invention provides the use of a reagent for detecting the expression amount of FBXO45 in one or more of the following:

[0030] (I) Preparation of products for predicting or assessing the prognosis of pancreatic cancer;

[0031] (II) Prepare products for predicting or evaluating the postoperative survival of pancreatic cancer patients.

[0032] As an embodiment, the reagent also includes a preparation for detecting the expression level of CA19-9.

[0033] As an embodiment, the accession number of the amino acid sequence of FBXO45 in NCBI is GeneID:200933.

[0034] The research results of the present invention show that the 1-year survival rate of patients in the FBXO45 high expression group after surgery is significantly lower than that in the FBXO45 low expression group; univariate combined with Cox multivariate analysis confirms that high FBXO45 expression is an independent risk factor affecting the 1-year survival rate of pancreatic cancer patients after radiotherapy and chemotherapy, suggesting that FBXO45 is closely related to patient prognosis; ROC curve analysis shows that FBXO45 has a high predictive value for assessing whether pancreatic cancer patients undergoing radiotherapy and chemotherapy will die within 1 year after surgery, suggesting that FBXO45 is expected to become a good indicator for assessing the prognosis of pancreatic cancer patients undergoing radiotherapy and chemotherapy. The present invention shows that FBXO45 has a high predictive value for assessing whether pancreatic cancer patients undergoing radiotherapy and chemotherapy will die within 1 year after surgery through ROC curve analysis, suggesting that FBXO45 is expected to become a good indicator for assessing the prognosis of pancreatic cancer patients undergoing radiotherapy and chemotherapy.

[0035] As an embodiment, the type of the product may include a reagent, a chip, a test paper or a test kit.

[0036] As an embodiment, the prediction or evaluation of the present invention may include: detecting the expression level of FBXO45 mRNA and / or the protein expression level of FBXO45 in the biological sample of the subject to predict the postoperative survival or prognosis of the subject. As an embodiment, in the present invention, the specific method for determining the expression level of FBXO45 includes one or more of RT-qPCR, Western blot and immunohistochemistry. As an embodiment, the biological sample of the present invention may include a blood sample of a pancreatic cancer patient.

[0037] As an embodiment, the RT-qPCR method is used to detect the expression level of FBXO45 mRNA in the blood samples of pancreatic cancer patients, and the relative expression amount of FBXO45 mRNA is calculated by the 2^-(ΔΔCt) method using GAPDH as the internal reference gene.

[0038] When the mRNA expression level of FBXO45 is detected, the median (0.527774) of the expression of FBXO45 relative to the internal reference gene GAPDH is used as the boundary. When the expression of FBXO45 relative to the internal reference gene GAPDH is less than 0.527774, it means that the mRNA expression level of FBXO45 is low expression; when the mRNA expression level of FBXO45 is detected, when the expression of FBXO45 relative to the internal reference gene GAPDH is greater than 0.527774, it means that the mRNA expression level of FBXO45 is high expression. The results of the embodiment show that the 1-year survival rate of patients in the FBXO45 high expression group after radiotherapy and chemotherapy is significantly lower than that of patients in the FBXO45 low expression group (Log-rankχ 2 =25.003, P<0.001).

[0039] The present invention provides a primer pair for detecting the expression level of pancreatic cancer prognosis marker FBXO45, comprising FBXO45-F and FBXO45-R; the nucleotide sequence of the FBXO45-F is shown in SEQ ID NO: 1, and the nucleotide sequence of the FBXO45-R is shown in SEQ ID NO: 2.

[0040] The present invention provides a product for evaluating the prognosis of pancreatic cancer, wherein the product comprises a primer pair for detecting the expression level of FBXO45 as described in the above technical solution.

[0041] As an embodiment, the product of the present invention may include a reagent, a chip, a test paper or a kit; the kit also includes an ELISA detection reagent. As an embodiment, the ELISA detection reagent of the present invention also includes one or more of streptavidin-peroxidase, TMB substrate and stop solution.

[0042] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1

[0044] 1. Sources

[0045] Twenty-eight pancreatic cancer patients who did not receive chemotherapy and were admitted to the First Affiliated Hospital of Bengbu Medical University from March 2023 to April 2024 were collected as the pancreatic cancer non-chemoradiotherapy group, and 56 pancreatic cancer patients who received chemoradiotherapy were collected as the pancreatic cancer chemoradiotherapy group. The baseline data of the two groups were comparable (P>0.05).

[0046] 2. Inclusion and Exclusion Criteria

[0047] (1) Inclusion criteria

[0048] Patients were included if they met the following three conditions: 1) aged 18 years and above; 2) patients diagnosed with pancreatic cancer; 3) patients were willing to participate in the study and signed the informed consent.

[0049] (2) Exclusion criteria

[0050] Patients were excluded if at least one of the following conditions was met: 1) patients with other serious diseases or other malignant tumors; 2) patients with cognitive impairment or other inability to cooperate with the study requirements; 3) patients who lost the follow-up conditions or were unable to complete the scheduled follow-up.

[0051] Patients were enrolled according to the above inclusion and exclusion criteria and the following information was obtained:

[0052] 1) Basic information: Age, gender, carcinoembryonic antigen (CEA), carbohydrate antigen (CA19-9), tumor diameter, TNM stage and other data were obtained from the medical record system of our hospital.

[0053] 2) Survival data: The survival status of pancreatic cancer patients after chemoradiotherapy and adenocarcinoma patients without chemoradiotherapy was obtained through multiple channels such as telephone follow-up and outpatient review.

[0054] 3. Main Reagents

[0055] Whole blood total RNA kit (simgen), reverse transcription reagents (Takara PrimeScript RT Master Mix) and qPCR reagents (Vazyme ChamQ Universal SYBR qPCR Master Mix).

[0056] 4. Methods

[0057] (1) Total RNA extraction from whole blood

[0058] Blood samples collected from patients in the pancreatic cancer chemoradiotherapy group during the second hospitalization for chemotherapy 3 weeks after the first chemotherapy were used as samples for the pancreatic cancer chemoradiotherapy group. According to the instructions of the whole blood total RNA kit, 500 μL of peripheral blood was collected from each subject in the pancreatic cancer non-chemoradiotherapy group and the pancreatic cancer chemoradiotherapy group, and the blood was dissolved and precipitated with Buffer L9 to remove proteins. Ethanol was added to the supernatant containing RNA and then added to the nucleic acid purification column. RNA was bound to the nucleic acid purification column, and the residual protein and PCR inhibitors were filtered out. After RNA was washed with Buffer WA and Buffer WBR, it was eluted with Buffer TE and can be used for RT-qPCR.

[0059] (2) RT-qPCR detection of relative expression level of FBXO45 mRNA

[0060] 2 μg of peripheral blood total RNA obtained in step (1) was reverse transcribed into cDNA and amplified on ABI-7500. The RT-qPCR amplification program was as follows: 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, 60°C annealing and extension for 30 s, and repeated for 40 cycles. In this experiment, glyceraldehyde-3-phosphate dehydrogenase GAPDH was used as the internal reference gene, and the expression level of FBXO45 mRNA was evaluated by calculating the 2^-(ΔΔCt) value. The primers used in the experiment were synthesized by Qingke Biotechnology Co., Ltd., and the sequences are as follows: primers for amplifying the nucleotide sequence encoding FBXO45: upstream primer 5'-CAGCTACAAGGCCAAGATACG-3' (SEQ ID NO: 1), downstream primer 5'-TCTGAGCAATGGGGTTTCGAT-3' (SEQ ID NO: 2); primers for amplifying GAPDH: upstream primer 5'-CAGCCTCAAGATCATCAGCA-3' (SEQ ID NO: 3), downstream primer 5'-TGTGGTCATGAGTCCTTCCA-3' (SEQID NO: 4).

[0061] The expression levels of serum CEA and CA19-9 were quantitatively determined using the Abbott ARCHITECT i2000SR immunoassay analyzer and its supporting kit.

[0062] 5. Statistical Analysis

[0063] SPSS23.0 software and GraphPad prism 9.0 software were used for statistical analysis and graphics. The relative expression data of FBXO45 were calculated by 2^-(ΔΔCt), see Table 2 and Figure 1 .

[0064] If the measurement data obeyed the normal distribution, the mean ± standard deviation was used to express it, and the data between the two groups were compared with the independent sample t test; if the data did not obey the normal distribution, the median (quartile) and M (P25, P75) were used to express it, and the Mann-Whitney U test was used to compare the differences in the expression levels of indicators between the two groups, see Table 2, Table 4, Table 5. 2 The comparison of the test rate; Spearman correlation analysis, see Table 3; Kaplan-Meier (KM) curve for survival analysis, see Figure 2 ; The Cox proportional hazard regression model was used to analyze the independent risk factors affecting the 1-year survival rate of pancreatic cancer patients after the first chemoradiotherapy, see Table 6; The receiver operating characteristic curve (ROC) was used to analyze the evaluation value of FBXO45 in predicting the 1-year survival rate of pancreatic cancer patients after chemoradiotherapy, see Figure 3 and Table 7. P < 0.05 was considered statistically significant.

[0065] The general information of the pancreatic cancer non-chemoradiotherapy group and pancreatic cancer chemoradiotherapy group is shown in Table 1.

[0066] Table 1 Comparison of general data of patients in the non-chemoradiotherapy group and the chemoradiotherapy group of pancreatic cancer

[0067]

[0068] Table 2 Comparison of relative expression of FBXO45 in pancreatic cancer patients before and after chemoradiotherapy

[0069]

[0070] According to Table 2 and Figure 1 It can be seen that RT-qPCR detection showed that compared with the non-chemoradiotherapy group, the relative expression of FBXO45 mRNA in the peripheral blood of the chemoradiotherapy group was reduced, and the difference was statistically significant, Z=-2.002, P=0.045.

[0071] Table 3 Correlation between the relative expression of serum FBXO45 and the expression levels of serum CEA and CA19-9

[0072] Serum markers <![CDATA[r s ]]> P CEA -0.146 0.218 CA19-9 -0.045 0.705

[0073] Table 4 Relationship between serum FBXO45 expression level and clinical pathological parameters in pancreatic cancer patients without chemoradiotherapy

[0074]

[0075]

[0076] Note: Due to incomplete information in some medical records, the number of cases for the maximum tumor diameter, TNM stage, local invasion degree, lymph node metastasis, and distant metastasis is not 28.

[0077] Table 5 Relationship between serum FBXO45 expression level and clinical pathological parameters in patients with pancreatic cancer after chemoradiotherapy

[0078]

[0079]

[0080] Note: Due to incomplete information in some medical records, the number of cases for the maximum tumor diameter, TNM stage, local invasion degree, lymph node metastasis, and distant metastasis is not 56.

[0081] According to Tables 4 and 5, there was no statistically significant difference in the expression of FBXO45 mRNA and clinical pathological characteristics such as age, maximum tumor diameter, TNM stage, local invasion, lymph node metastasis, and distant metastasis between the pancreatic cancer non-chemotherapy group and the chemotherapy group (P>0.05).

[0082] Table 6 Univariate and Cox multivariate analysis of factors affecting the 1-year survival rate of pancreatic cancer patients after chemoradiotherapy

[0083]

[0084] According to Table 6, it can be seen that the expression of FBXO45 mRNA and CA19-9 are factors that affect the 1-year survival rate of pancreatic cancer patients after chemoradiotherapy.

[0085] The relative expression level of FBXO45 in patients with radiotherapy and chemotherapy was analyzed by RT-qPCR. The median of the relative expression level of FBXO45 in the radiotherapy and chemotherapy group (0.527774) was used as the boundary, with >0.527774 as the high expression group and <0.527774 as the low expression group.

[0086] KM survival curve was used to compare the effects of high and low FBXO45 expression on the 1-year survival rate of pancreatic cancer patients after radiotherapy and chemotherapy. The results showed that the 1-year survival rate of patients in the FBXO45 high expression group after radiotherapy and chemotherapy was significantly lower than that in the FBXO45 low expression group (Log-rankχ2=25.003, P<0.001). Figure 2 .

[0087] Table 7 ROC curve analysis of the predictive value of FBXO45 relative expression and CA19-9 alone or in combination for the 1-year survival of pancreatic cancer patients after chemoradiotherapy

[0088]

[0089] according to Figure 3 As shown in Table 7, ROC curve analysis showed that the sensitivity of evaluating the 1-year survival rate of pancreatic cancer patients after chemoradiotherapy with the relative expression of FBXO45 (0.445) as the cutoff value was 92.6%, the specificity was 88.2%, and the area under the curve was 0.926 (P<0.001). The sensitivity of evaluating the 1-year survival rate of pancreatic cancer patients after chemoradiotherapy with the CA19-9 detection value (26.410) as the cutoff value was 96.3%, the specificity was 58.8%, and the area under the curve was 0.810 (P=0.001). The combined sensitivity (96.3%), specificity (88.2%), and area under the curve (0.961) were higher (P<0.001). Taking all factors into consideration, the combined use of FBXO45 and CA19-9 for predicting the 1-year survival of pancreatic cancer after chemoradiotherapy is higher than that of FBXO45 alone.

[0090] In conclusion, FBXO45 alone or in combination with CA19-9 has predictive value for the 1-year survival of pancreatic cancer patients after chemoradiotherapy.

[0091] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of FBXO45 as a marker in one or more of the following: (1) Prepare products for predicting or assessing the prognosis of pancreatic cancer; (2) Prepare products for predicting or evaluating the postoperative survival of pancreatic cancer patients.

2. The use according to claim 1, characterized in that: The markers also include CA19-9.

3. Use of a reagent for detecting the expression of FBXO45 in one or more of the following: (I) Preparation of products for predicting or assessing the prognosis of pancreatic cancer; (II) Prepare products for predicting or evaluating the postoperative survival of pancreatic cancer patients.

4. The use according to claim 3, characterized in that: The reagents also include preparations for detecting the expression level of CA19-9.

5. The use according to claim 1 or 3, characterized in that: The prediction or evaluation includes: detecting the expression level of FBXO45 mRNA and / or the protein expression level of FBXO45 in the biological sample of the subject to predict the postoperative survival period or prognosis of the subject.

6. The use according to claim 5, characterized in that: The biological sample includes a blood sample from a pancreatic cancer patient.

7. The use according to claim 1 or 3, characterized in that: The products include reagents, chips, test strips or test kits.

8. A primer pair for detecting the expression level of pancreatic cancer prognostic marker FBXO45, characterized in that: It includes FBXO45-F and FBXO45-R; the nucleotide sequence of the FBXO45-F is shown in SEQ ID NO: 1, and the nucleotide sequence of the FBXO45-R is shown in SEQ ID NO:

2.

9. A product for evaluating the prognosis of pancreatic cancer, characterized in that: The product includes the primer pair for detecting the expression level of FBXO45 according to claim 8.

10. The product according to claim 7, characterized in that The product includes reagents, chips, test strips or test kits; the test kits also include ELISA detection reagents; the ELISA detection reagents also include one or more of streptavidin-peroxidase, TMB substrate and stop solution.

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