Marker, detection reagent or kit and system for diagnosis, grading or prognosis of intrahepatic cholangiocarcinoma and application of marker, detection reagent or kit and system

Abstract

The invention relates to the technical field of medical diagnosis, in particular to an intrahepatic cholangiocarcinoma diagnosis, grading or prognosis marker, a detection reagent or kit, a system and application thereof. According to the present invention, the expression of the exosome source circUBR5 in the serum in the serum exosome of the intrahepatic cholangiocarcinoma patient is significantly up-regulated for the first time, the subject working characteristic curve analysis result shows that the under-curve area of the circUBR5 in the intrahepatic cholangiocarcinoma diagnosis reaches 0.833, and is significantly superior to the traditional tumor markers CA19-9 and CEA, and the sensitivity and the specificity of the circUBR5 have significant advantages; and when the circUBR5 and the CA19-9 are used in a combined manner, the diagnosis AUC is further improved to 0.858. In addition, the invention also finds that the expression level of circUBR5 is closely related to the TNM staging, vascular invasion, lymph node metastasis and distant metastasis of the patient, and the high expression of circUBR5 is positively related to the obvious shortening of the overall lifetime of the patient.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical diagnosis, in particular to an intrahepatic cholangiocarcinoma diagnosis, grading or prognosis marker, detection reagent or kit, system and application thereof. BACKGROUND

[0002] Intrahepatic cholangiocarcinoma (iCCA) is a malignant tumor originating from intrahepatic bile duct epithelial cells, which is the second most common malignant tumor of the liver after hepatocellular carcinoma, accounting for about 10-15% of primary liver cancer, and its incidence has been increasing globally in recent years. Its 5-year survival rate is about 5-15%, and the average survival period without surgery is only 9 months. The main reasons for the poor prognosis of intrahepatic cholangiocarcinoma are as follows: first, the clinical features of intrahepatic cholangiocarcinoma are not obvious, early diagnosis is difficult, and lymph node and distant metastasis easily occurs, so that patients may have missed the best surgical opportunity after diagnosis, resulting in low and unstable radical resection rate; second, the pathogenesis of intrahepatic cholangiocarcinoma is unclear, and there is a lack of effective targeted therapeutic drugs. Therefore, studying the mechanism of intrahepatic cholangiocarcinoma development and developing more effective diagnostic and therapeutic methods are key factors for prolonging the survival period of patients.

[0003] Circular RNA (circRNA) is a closed loop non-coding RNA molecule generated by reverse splicing of precursor mRNA. With the latest progress of RNA deep sequencing technology and bioinformatics, a large number of circRNAs have been found to be expressed abundantly in mammalian cells. CircRNAs have characteristics such as conservation of different species, high stability, and specific expression in tissues and developmental stages in eukaryotes. Current studies have shown that circRNAs can act as miRNA sponges, have functions such as protein binding, protein translation, and regulation of parent gene transcription, and can be transported to body fluids, thus having great potential as biomarkers.

[0004] Exosomes are a class of extracellular vesicles with a diameter of 30-100 nm, which are secreted by various cells and exist in almost all body fluids. They play a role in cell-to-cell communication by carrying proteins, lipids, nucleic acids, and metabolic products, and are involved in immune response, viral infection, metabolism, cardiovascular disease, neurodegenerative disease, and cancer progression, and other physiological and pathological processes. Studies have shown that circRNAs are abundant in exosomes, circulate through exosomes and reach target cells, thereby exerting their various biological functions. As a stable biomarker, circRNA can be enriched in exosomes, so studying exosomal circRNA markers in circulating body fluids is of great significance for disease diagnosis.

[0005] Currently, the research on using exosomal circRNA as a biomarker for the diagnosis and prognosis of intrahepatic cholangiocarcinoma is still limited, and its clinical transformation application has not been fully established. SUMMARY

[0006] In order to overcome the above problems, the present application provides an intrahepatic cholangiocarcinoma diagnosis, grading or prognosis marker, detection reagent or kit, system and application thereof.

[0007] To achieve the above technical purposes, the present application adopts the following technical solutions: In a first aspect, the present application provides an intrahepatic cholangiocarcinoma diagnosis, grading and prognosis marker, which comprises an exosome-derived circUBR5, and the nucleic acid sequence of the circUBR5 is shown as SEQ ID NO. 1.

[0008] In one or more embodiments, the exosome is a blood, plasma or serum exosome, preferably a serum exosome.

[0009] In a second aspect, the present application provides the use of a reagent or kit for detecting the marker of the first aspect in the preparation of an intrahepatic cholangiocarcinoma diagnosis, grading and prognosis marker.

[0010] In one or more embodiments, the detection reagent or kit comprises a primer pair for detecting circUBR5, and the nucleotide sequences of the upstream primer and the downstream primer are shown as SEQ ID NO. 2 and SEQ ID NO. 3.

[0011] In one or more embodiments, the detection reagent or kit further comprises an exosome extraction reagent and an RT-qPCR detection reagent.

[0012] In one or more embodiments, the detection reagent or kit further comprises: the internal reference primer for homogenization is a primer pair with beta-actin as the internal reference, and the nucleotide sequences of the upstream primer and the downstream primer are shown as SEQ ID NO. 4 and SEQ ID NO. 5.

[0013] In a third aspect, the present application provides an intrahepatic cholangiocarcinoma diagnosis, grading and prognosis marker detection reagent or kit, which comprises a primer for detecting circUBR5, and the nucleotide sequences are shown as SEQ ID NO. 2 and SEQ ID NO. 3.

[0014] In one or more embodiments, the detection reagent or kit comprises a primer pair for detecting circUBR5, and the nucleotide sequences of the upstream primer and the downstream primer are shown as SEQ ID NO. 2 and SEQ ID NO. 3.

[0015] In one or more embodiments, the detection reagent or kit further comprises an exosome extraction reagent and an RT-qPCR detection reagent.

[0016] In one or more embodiments, the detection reagent or kit further comprises: the internal reference primers for homogenization are a primer pair with β-actin as the internal reference, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 4 and SEQ ID NO. 5.

[0017] In a fourth aspect of the present application, a system for diagnosing, grading and prognosing intrahepatic cholangiocarcinoma is provided, comprising a detection unit and an evaluation unit; The detection unit is used to detect the expression amount of the marker of the first aspect in the subject; The evaluation unit diagnoses, grades and / or prognostically evaluates the subject according to the expression amount of the marker of the subject obtained in the detection unit.

[0018] In one or more embodiments, the diagnosis comprises diagnosing intrahepatic cholangiocarcinoma in the subject according to the expression amount of the marker of the subject: When the expression amount of the marker of the subject is higher than the threshold value, the subject is positive for intrahepatic cholangiocarcinoma; When the expression amount of the marker of the subject is lower than the threshold value, the subject is negative for intrahepatic cholangiocarcinoma.

[0019] The threshold value is determined by ROC curve analysis and based on the maximum Youden index; the threshold value is 2.7.

[0020] In one or more embodiments, the grading comprises grading intrahepatic cholangiocarcinoma in the subject according to the expression amount of the marker of the subject: The threshold value is 3.42, which is the median of the relative expression amount of circUBR5 in patients with intrahepatic cholangiocarcinoma, and the patients are divided into a high expression group and a low expression group.

[0021] In one or more embodiments, the prognosis comprises prognostically judging intrahepatic cholangiocarcinoma in the subject according to the expression amount of the marker of the subject.

[0022] The threshold value is 3.42, which is the median of the relative expression amount of circUBR5 in patients with intrahepatic cholangiocarcinoma, and the patients are divided into a high expression group and a low expression group.

[0023] The present application has the following beneficial effects: (1) The present application relates to the technical field of medical diagnosis, in particular to an intrahepatic cholangiocarcinoma diagnosis, grading or prognosis marker, detection reagent or kit, system and application thereof. In the present application, based on a clinical large sample cohort, it is first found that the expression of serum exosome-derived circUBR5 in the serum exosomes of intrahepatic cholangiocarcinoma patients is significantly up-regulated, the receiver operating characteristic curve (ROC) analysis result shows that the area under the curve (AUC) of circUBR5 in the diagnosis of intrahepatic cholangiocarcinoma reaches 0.833, which is significantly better than that of traditional tumor markers CA19-9 (AUC 0.662) and CEA (AUC 0.580), and the sensitivity and specificity also have significant advantages; and when circUBR5 is used in combination with CA19-9, the diagnostic AUC is further improved to 0.858. In addition, it is found that the expression level of circUBR5 is closely related to the TNM stage, vascular invasion, lymph node metastasis and distant metastasis of patients, and the high expression of circUBR5 is positively correlated with the significant shortening of the overall survival of patients; at the same time, a stable and repeatable serum exosome circUBR5 detection system is constructed, and the technical reliability of the detection system is verified.

[0024] (2) The present application provides a new serum exosome biomarker for early diagnosis, prognosis evaluation and metastasis risk prediction of intrahepatic cholangiocarcinoma, provides a theoretical basis and technical support for future precise diagnosis and treatment and targeted intervention of intrahepatic cholangiocarcinoma, and has broad clinical transformation prospects and industrialization application potential. BRIEF DESCRIPTION OF DRAWINGS

[0025] The drawings accompanying the specification of the present application serve to provide a further understanding of the present application, and the illustrative embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application.

[0026] Figure 1 The genomic location of the chromosome where circUBR5 is located and the specific splicing site of circUBR5; Figure 2 A serum exosome identification chart for intrahepatic cholangiocarcinoma patients, benign biliary disease patients and healthy people; wherein, A is electron microscope identification; B is nanoparticle size analysis, from left to right, in order, healthy people, benign biliary disease patients and intrahepatic cholangiocarcinoma patients; C is Western blot detection of the expression of exosome surface marker protein; Figure 3 RT-qPCR detection results of circUBR5 expression in serum exosomes of 48 healthy controls, 46 benign biliary disease patients and 57 intrahepatic cholangiocarcinoma patients in the examples; Figure 4The standard curve graphs of GAPDH, UBC and circUBR5, wherein A is the standard curve graph of GAPDH, B is the standard curve graph of UBC, and C is the standard curve graph of circUBR5; Figure 5 The ROC curve of serum exosome circUBR5, CA19-9 and CEA for distinguishing patients with intrahepatic cholangiocarcinoma and healthy people; Figure 6 The ROC curve of serum exosome circUBR5 for distinguishing patients with early intrahepatic cholangiocarcinoma and healthy people; Figure 7 The independent predictive value of circUBR5 in the diagnosis of iCCA was verified by single factor and multiple factor Logistic regression analysis, wherein A is the single factor Logistic regression analysis result table; B is the forest plot of the result of A; C is the multiple factor Logistic regression analysis table; and D is the forest plot of the result of C; Figure 8 The ROC curve analysis of circUBR5 combined with CA19-9 for diagnosing intrahepatic cholangiocarcinoma; Figure 9 The difference comparison between the expression level of circUBR5 and the TNM pathological stage (I-II period and III-IV period); Figure 10 The comparison of the expression level of circUBR5 in patients with and without vascular invasion; Figure 11 The comparison of the expression level of circUBR5 in patients with and without lymph node metastasis; Figure 12 The comparison of the expression level of circUBR5 in patients with and without distant metastasis; Figure 13 The survival curve graph of the overall survival (OS) of patients with intrahepatic cholangiocarcinoma after grouping according to the different expression amounts of serum exosome circUBR5; Figure 14 The influence analysis of circUBR5 on the OS of patients in single factor and multiple factor Cox regression analysis; wherein A is the single factor Cox regression analysis result table; B is the forest plot of the result of A; C is the multiple factor Cox regression analysis table; and D is the forest plot of the result of C. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0028] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0029] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

[0030] Example 1 Isolation and identification of serum exosomes (1) Sample collection: Peripheral blood of 57 patients with intrahepatic cholangiocarcinoma (iCCA), 46 patients with benign biliary disease (BBD) and 48 healthy (HC) controls were collected in vacuum blood collection tubes without anticoagulant, and after standing at room temperature for 30 min, centrifuged at 3000 g for 10 min, serum was separated and stored at -80 ℃ for use.

[0031] (2) Extraction of exosomes: Ultracentrifugation method was used to isolate serum exosomes. The specific steps are as follows: (1) The serum was centrifuged at 300 g for 10 min at 4 °C to remove cells; (2) After transferring the supernatant, centrifuge at 2000 g for 10 min to remove cell debris and nuclear debris; (3) Transfer the supernatant again and centrifuge at 10000 g for 30 min to further remove large particle impurities; (4) The final supernatant was centrifuged at 110000 g for 70 min (Beckman Coulter Optima L-100K ultracentrifuge, SW32Ti rotor) to obtain exosome precipitate; (5) Resuspend the exosomes with PBS and centrifuge again at 110000 g for 70 min to wash, discard the supernatant and collect the precipitate as exosomes.

[0032] (3) Identification of exosome characteristics: The extracted exosomes were used for subsequent morphological and molecular characteristic verification, and the main methods included: (1) Transmission electron microscope: observe the morphology and membrane structure of exosomes; (2) Nanoparticle tracking analysis: detect particle size distribution and concentration; (3) Western blot: detection of typical exosome markers (CD63, TSG101, Alix), Calnexin as negative control.

[0033] Example 2 Extraction of serum exosome RNA and quantification of circUBR5 (1) RNA extraction: ExoRNeasy Serum / Plasma Midi Kit (Qiagen, Germany) was used to extract serum exosome RNA. The steps are as follows: Add lysis buffer XBP (equal volume) to the exosome sample and incubate at room temperature for 10 min to fully lyse; Add the above lysis solution to the filter column and use gDNA Eliminator Column to remove impurities; Gradually add buffer XWP for column washing to remove protein and lipid impurities; Use RNase-free water to elute RNA, centrifuge at 12000 g for 1 min, repeat twice; Determine the RNA concentration, OD260 / 280=1.8~2.1 is a qualified sample.

[0034] (2) RNA reverse transcription and qPCR: Use HiScript III RT SuperMix (Vazyme) to synthesize cDNA, the reaction system is shown in Table 1 below; Table 1 cDNA synthesis reaction system

[0035] Reaction program: 50°C for 15 min; 85°C for 5 min inactivation; cool standby.

[0036] Use ChamQ Universal SYBR qPCR Master Mix (Vazyme) for qPCR, the reaction system is shown in Table 2 below; Table 2 qPCR reaction system

[0037] The primer sequences are shown in Table 3 below; Table 3 Primer sequences

[0038] PCR reaction conditions: Pre-denaturation: 95°C for 30 s; Denaturation: 95 °C 10 s, Annealing / extension: 60 °C 30 s, 40 cycles in total.

[0039] (3) Standard curve and system verification The synthetic circUBR5 circular RNA template was diluted according to a 10-fold gradient to establish a standard curve; The Ct values of each concentration gradient sample were detected by real-time fluorescent quantitative PCR, and a standard curve was drawn with each sample concentration and the corresponding Ct value, to obtain a linear equation Ct = -klogX0 + b. According to the slope (k), the amplification efficiency (E) was calculated, and the formula was: E = (10 (-1 / k) - 1) × 100%. The internal reference genes GAPDH and UBC were used for synchronous detection, and a standard curve was established in parallel with the circUBR5 standard curve to evaluate the stability and repeatability of the system.

[0040] Example 3 Statistical analysis process (1) Diagnostic efficiency evaluation: The R language pROC package was used to evaluate the diagnostic ability of circUBR5 in distinguishing intrahepatic cholangiocarcinoma (iCCA) from non-cancer (benign biliary tract disease + healthy control). The maximum Youden index (Youden Index = sensitivity + specificity - 1) was used as the optimal cutoff value for diagnosis, and the sensitivity and specificity at this point were recorded. In subsequent analysis, according to the median expression of circUBR5 in iCCA patients, the patients were divided into "circUBR5 high expression group" and "circUBR5 low expression group", which were used to evaluate their correlation with clinical characteristics and prognosis.

[0041] (2) Statistical analysis of clinical correlation: The samples were stratified according to clinical grouping standards, including TNM staging (I, II, III, IV), presence or absence of vascular invasion, presence or absence of lymph node metastasis, distant metastasis, and nerve invasion. For categorical variables, the Chi-square test or Fisher's exact probability method was used; for continuous variables, first perform Shapiro-Wilk test to determine normality, if normally distributed, use independent sample t-test; if not normally distributed, use Mann-Whitney U test.

[0042] (3) Logistic regression modeling: diagnostic model construction: Logistic regression model was constructed using R language glm() function to predict the risk of intrahepatic cholangiocarcinoma. The variables such as circUBR5, CA19-9, CEA, etc. were respectively put into the model for single factor analysis. All P<0.05 variables were included in the multivariate model for multivariate analysis. The OR value, 95% CI and P value were output.

[0043] (4) Survival analysis: The survival curves of circUBR5 high expression group and low expression group were drawn using Kaplan-Meier method, and the statistical significance of survival difference between the two groups was evaluated by Log-rank test.

[0044] (5) Cox proportional hazards model: prognostic factor analysis Cox regression model was constructed using R language survival package to analyze the factors affecting prognosis. The variables included age, gender, TNM stage, tumor size, circUBR5 expression, etc. for single factor Cox analysis. The variables with P<0.05 in single factor were included for multivariate Cox analysis. The hazard ratio (HR), 95% CI and P value were output.

[0045] All experiments were repeated at least three times independently. The significance criterion was set as P<0.05.

[0046] Experimental results: Based on circBase and circBank databases, circUBR5 was formed by reverse splicing of exons 2-5 of UBR5 gene on human chromosome 8 (chr8: 102360071-102361626), with a length of 324 bp. By Sanger sequencing of PCR products after qRT-PCR reaction, the specific splicing site of circUBR5 was successfully identified (as shown in Figure 1 ).

[0047] Transmission electron microscopy, nanoparticle tracking analysis and Western blot results showed that the extracted serum exosomes had a typical double-membrane structure (Fig. Figure 2 A), the particle size was mainly concentrated at about 100 nm (Fig. Figure 2 B), and positive expression of CD63, TSG101 and Alix was detected, while endoplasmic reticulum marker Calnexin was not detected (Fig. Figure 2 C).

[0048] The expression level of serum exosome circUBR5 in each group of serum exosomes was detected by qRT-PCR. The standard curve of GAPDH, UBC and circUBR5 showed that the Ct value had a good linear relationship with the logarithm of sample concentration, and the amplification efficiency met the requirements.Figure 3 ). The detection results showed that the expression of circUBR5 in patients with intrahepatic cholangiocarcinoma was significantly higher than that in the benign biliary disease group and the healthy control group ( Figure 4 ). ROC curve analysis showed that the AUC of circUBR5 for the diagnosis of intrahepatic cholangiocarcinoma was 0.833, the sensitivity was 0.737, and the specificity was 0.875, which was significantly better than the traditional tumor markers CA19-9 (AUC = 0.662) and CEA (AUC = 0.580) ( Figure 5 ). In patients with early cholangiocarcinoma, circUBR5 still showed high diagnostic ability ( Figure 6 , AUC = 0.738), and single factor and multiple factor Logistic regression analysis further showed that circUBR5 and CA19-9 were independent predictors for the diagnosis of iCCA ( Figure 7 ), and the AUC of combined diagnosis was increased to 0.858 ( Figure 8 ).

[0049] Further analysis of the relationship between circUBR5 and clinical pathological characteristics. The relative expression of circUBR5 in patients with intrahepatic cholangiocarcinoma was used as the threshold to divide the patients into high expression group and low expression group; here the threshold is 3.42. The results showed that the high expression of circUBR5 was significantly related to the late TNM stage (III, IV stage), vascular invasion, lymph node metastasis and distant metastasis (Table 4). Compared with patients with I, II stage, no metastasis and invasion, the serum exosome circUBR5 level was significantly increased in patients with III, IV pathological stage ( Figure 9 , P < 0.0001), with vascular invasion ( Figure 10 , P < 0.0001), lymph node metastasis ( Figure 11 , P < 0.0001) and distant metastasis ( Figure 12 , P < 0.0001).

[0050] Kaplan-Meier survival analysis showed that the overall survival (OS) of patients in the high expression group of circUBR5 was significantly shorter than that in the low expression group ( Figure 13 , P = 0.001). Single factor and multiple factor Cox regression analysis showed that the expression level of circUBR5, patient age and nerve invasion were independent adverse prognostic factors for the OS of patients with intrahepatic cholangiocarcinoma ( Figure 14 ).

[0051] In summary, serum exosome circUBR5 has good diagnostic and prognostic value for intrahepatic cholangiocarcinoma, especially in early detection, metastasis prediction and survival evaluation, which shows potential clinical application prospects.

[0052] Table 4. Significance statistics of serum exosomal circUBR5 expression levels and clinical characteristics of subjects

[0053] The above merely provides the preferred embodiments of the present application, but not for limiting the present application. For the person skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A diagnostic, grading and prognostic marker for intrahepatic bile duct carcinoma, characterized in that: The marker includes exosome-derived circUBR5, and the nucleic acid sequence of the circUBR5 is shown in SEQ ID NO.

1.

2. The diagnostic, grading and prognostic marker for intrahepatic cholangiocarcinoma according to claim 1, wherein: The exosomes are blood, plasma or serum exosomes, preferably serum exosomes.

3. Use of the diagnostic, grading and prognostic marker for intrahepatic cholangiocarcinoma according to claim 1 or 2 in the preparation of a detection reagent or kit for the diagnostic, grading and prognostic marker for intrahepatic cholangiocarcinoma.

4. The use according to claim 3, characterized in that The detection reagent or kit includes a primer pair for detecting circUBR5, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and SEQ ID NO.

3.

5. The use according to claim 3, characterized in that The detection reagent or kit also includes an exosome extraction reagent and an RT-qPCR detection reagent.

6. The use according to claim 3, characterized in that The detection reagent or kit further includes: an internal reference primer for normalization, which is a primer pair with β-actin as an internal reference, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.4 and SEQ ID NO.

5.

7. A detection reagent or kit for intrahepatic bile duct carcinoma diagnosis, grading and prognosis markers, characterized in that: It includes primers for detecting circUBR5, and the nucleotide sequences are shown in SEQ ID NO. 2 and SEQ ID NO.

3.

8. The detection reagent or kit according to claim 7, characterized in that The detection reagent or kit also includes an exosome extraction reagent and an RT-qPCR detection reagent.

9. The detection reagent or kit according to claim 8, characterized in that The detection reagent or kit further includes: an internal reference primer for normalization, which is a primer pair with β-actin as an internal reference, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 4 and SEQ ID NO.

5.

10. A system for diagnosis, grading and prognosis of intrahepatic bile duct cancer, characterized in that: It includes a detection unit and an evaluation unit; The detection unit is used to detect the expression level of the intrahepatic cholangiocarcinoma diagnosis, grading and prognosis marker according to claim 1 or 2 in the subject; The evaluation unit performs diagnosis, grading and / or prognostic evaluation on the subject according to the expression level of the subject marker obtained in the detection unit.

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