CN117051103A - Plasma exosome circular RNA biomarker for diagnosing colorectal cancer and application thereof - Google Patents

Abstract

The utility model discloses a plasma exosome annular RNA biomarker for diagnosing colorectal cancer and application thereof. The biomarker is derived from the circRNA ETLET that is specifically stable in the plasma exosomes. The utility model discloses that the exosome circRNA can be used as a novel biological diagnosis index in early clinical diagnosis of colorectal cancer for the first time, lays a theoretical foundation for deep research of the exosome circRNA of colorectal cancer in the future, further provides a novel research direction for early diagnosis of colorectal cancer, and has important reference significance and application value.

Description

Plasma exosome circular RNA biomarker for diagnosing colorectal cancer and application thereof

Technical Field

The utility model belongs to the field of biological detection, and relates to a plasma exosome annular RNA biomarker for diagnosing colorectal cancer and application thereof.

Background

Colorectal cancer has become one of the most common malignant tumors worldwide, with the number average of new and death cases being the third most common tumor. In China, along with the improvement of living standard of residents and the popularity of western diet and life style, the incidence rate of colorectal cancer is in an annual rising trend, and the health of the residents in China is seriously threatened. Patients with early colorectal cancer are usually in middle and late stages when they are admitted to the hospital due to lack of specific clinical symptoms, and about 25% of patients have already metastasized at the time of first diagnosis, which seriously affects the therapeutic effect and prognosis. The high incidence and younger colorectal cancer not only brings serious health damage to patients, but also brings heavy mental and economic burden to families and society.

A large number of crowd data confirm that early diagnosis of colorectal cancer is a key way to effectively reduce its mortality and increase survival rate. Colorectal cancer is generally diagnosed by colonoscopy, double contrast barium enema, CT imaging and other technologies. Although endoscopic detection is the gold standard for colorectal cancer screening, early colorectal cancer discovery and treatment is severely impacted due to high cost and poor compliance. Currently, some tumor marker detection (such as carcinoembryonic antigen (CEA) and the like) and noninvasive screening methods such as fecal occult blood and the like are widely used for early screening of colorectal cancer. However, each detection method has the problems of insufficient sensitivity, difficult sampling, high detection technical difficulty and the like, and cannot be completely applied to screening of large-scale people. Thus, the search for sensitive and specific biomarkers that can identify early colorectal cancer is critical to achieving early prevention, diagnosis and treatment of colorectal cancer.

In recent years, exosomes (exosomes) have been of interest as potential biomarkers of tumorigenic development. Exosomes are vesicles formed by endocytosis of normal or pathological intracellular multivesicular bodies, are fused with the envelope and secreted outside the cell to form a group of vesicle with a double-layer membrane structure, and have diameters of about 30-120 nm. Research shows that exosomes released by stress of the exosome cells enter the receptor cells (receptor cells) through modes of near secretion, membrane fusion, ligand-receptor combination, phagocytosis and the like, release carried bioactive substances derived from abnormal expression of the exosome cells, such as mRNA, non-coding RNA (cricRNA, microRNA, lncRNA) and lipid and the like, influence the biological functions of the receptor cells, and further participate in the occurrence and development of diseases. In view of the fact that the exosomes contain bioactive substances which reflect physiological or pathological changes of host cells or origin tissues, the exosome double-layer membrane structure can enable the carried contents to stably exist in various body fluids such as blood, saliva, urine and the like for a long time; therefore, exosomes carry biologically active substances with great potential as disease diagnostic biomarkers.

Among the numerous biologically active substances encapsulated by exosomes, circular RNAs (circrnas) have been widely studied in recent years as important members of the family of human largest transcripts. The circRNA is a non-coding RNA molecule which has a closed loop structure and does not have a 5 '-cap end and a 3' -end, the source and the type of the non-coding RNA molecule are complex and various, the molecular structure is considered to be mainly formed through two model approaches of transcript exon nonlinear reverse splicing cyclization and/or intron pairing/self cyclization, and the non-coding RNA molecule can play an important role in various malignant tumors of human beings by playing an oncogene or cancer suppressor gene-like role. Studies have shown that circRNA has mainly the following 5 major biological properties in physiological and pathological (including tumor) activities: the positioning is clear: the circRNA is enriched in the cytoplasm, and a small number exists in the nucleus; (2) high abundance: the circRNA is widely expressed in normal and tumor tissues/cells, and the abundance can be up to more than 10 times higher than that of the corresponding linear isomer; (3) high stability: the closed loop structure enables the circRNA to resist nuclease degradation and repeated freeze thawing, has longer half-life period, and has potential of being used as a circulating biomarker; (4) high conservation: most circrnas have highly conserved sequences; (5) high specificity: the circRNA expression has tissue and tumor specificity. The data indicate that the circRNA can play an epigenetic regulation role in transcription and post-transcription level, or can combine with transcription factor cis-regulation gene expression, or can play a competitive endogenous RNA (ceRNA) role as a 'cavernous' adsorbing miRNA, and participate in tumorigenesis and development.

There is currently little research on the circRNA in colorectal cancer plasma exosomes, none of the prior art relates to systematically identifying colorectal cancer specific plasma exosomes circRNA and assessing its potential as a biomarker.

Disclosure of Invention

The technical problems to be solved are as follows: the utility model aims to provide a plasma exosome circRNA biomarker for diagnosing colorectal cancer and application thereof, comprehensively evaluate the application value of the biomarker for early diagnosis of colorectal cancer, provide theoretical basis for developing early diagnosis colorectal cancer noninvasive biomarkers in the future, and have certain clinical practical value.

The aim of the utility model can be achieved by the following technical scheme:

the application of a reagent for detecting the plasma exosome hsa_circ_0087960 in preparing a colorectal cancer auxiliary diagnosis kit.

As a preferred embodiment of the present utility model, the reagent for detecting hsa_circ_0087960 is a specific primer pair and/or probe for detecting hsa_circ_ 0087960.

As a further preferred aspect of the utility model, the specific primer pair for detecting hsa_circ_0087960 is shown in SEQ ID NO.2 and SEQ ID NO. 3.

A colorectal cancer auxiliary diagnosis kit, which contains a specific primer pair and/or a probe for detecting hsa_circ_ 0087960.

As a preferred embodiment of the present utility model, the colorectal cancer auxiliary diagnosis kit comprises a specific primer pair for detecting hsa_circ_0087960 as shown in SEQ ID NO.2 and SEQ ID NO. 3.

The beneficial effects are that:

the present utility model found that the expression level of exosome circETLET (i.e., hsa_circ_0087960, supra) in the plasma of colorectal cancer patients was significantly lower than that of colorectal polyp patients (p=0.030) and healthy control population (P < 0.001), and that the expression level of exosome circETLET in the plasma of colorectal polyp patients was also significantly down-regulated (p=0.039) compared to healthy control population; meanwhile, the expression level of exosome circetelet in the plasma of colorectal cancer patients is significantly different from the expression level of exosome circetelet in the plasma of gastric cancer, breast cancer, bladder cancer, cervical cancer, kidney cancer and lung cancer patients (P < 0.001). After the blood plasma is repeatedly frozen and thawed for 0 times, 2 times, 4 times or 8 times at the room temperature and the temperature of minus 80 ℃, the expression level of the exosome circETLET in the blood plasma of the patients, colorectal polyps and the control group population is not changed significantly; at the same time, there was no significant difference in the exosome circetetest in plasma at room temperature for 0h, 4h, 8h or 24h (fig. 7), suggesting that the exosome circetetest can still exist stably when the plasma is in an extreme environment. ROC curve analysis shows that plasma exosome circetelet is effective in screening colorectal cancer patient population, with auc=0.858 (95% ci=0.786-0.930), and sensitivity and specificity for diagnosing colorectal cancer of 76.2% and 92.1%, respectively. The utility model discloses that the exosome circETLET can be used as a novel biological diagnosis index in early clinical diagnosis of colorectal cancer for the first time, lays a theoretical foundation for deep research of the exosome circRNA of colorectal cancer in the future, further provides a novel research direction for early diagnosis of colorectal cancer, and has important reference significance and application value.

Description of the drawings:

the following is a further detailed description of embodiments of the utility model with reference to the accompanying drawings:

FIG. 1 is an identification of colorectal cancer and normal cell supernatant exosomes;

panel A shows identification of the exosome forms of supernatant of colorectal normal cells and colorectal cancer cells, and panel B shows detection of exosome marker proteins in supernatant of colorectal normal cells and colorectal cancer cells

FIG. 2 shows the expression of circETLET in cell supernatant exosomes;

FIG. 3 is an exosome circETLET primer specific assay;

FIG. 4 is an exosome circETLET loop identification;

panel A shows the result of agarose gel electrophoresis of the amplification product of the circETLET, panel B shows the relative expression level of the amplification product of the circETLET, panel C shows the schematic diagram of the circETLET, and panel D shows the result of Sanger sequencing of the amplification product of the circETLET

FIG. 5 is an identification of plasma exosomes in colorectal cancer, colorectal polyps, healthy control populations;

panel A shows the identification of the plasma exosome profile of healthy controls, colorectal polyps, colorectal cancer patients, and panel B shows the detection of exosome marker proteins in healthy controls, colorectal polyps, colorectal cancer patients

FIG. 6 shows the expression levels of exosome circETLET in plasma of various tumor and healthy control populations;

FIG. 7 shows the stability of exosome circETLET in plasma of colorectal cancer and healthy control population;

figure 8 is a graph of subject performance characteristics evaluating the ability of plasma exosomes circetetest to discriminate colorectal cancer patients from healthy populations.

The specific embodiment is as follows:

the utility model is illustrated in more detail by the following examples: the present examples are given as detailed embodiments and procedures on the premise of the technical solution of the present utility model, but the scope of the present utility model is not limited to the following examples. Conditions, methods, etc., not specified in the following examples were carried out in accordance with conventional or manufacturer-suggested conditions.

1. Recognition of colorectal cancer exosome circRNA

The colorectal cancer tissue and its corresponding paracancerous normal tissue are collected 52. Tissue sources from 2017 month 1 to 2018 month 1, and all subjects signed informed consent after pathological diagnosis by doctors. Total RNA in colorectal cancer tissues and corresponding paracancerous normal tissues was extracted by Trizol method and subjected to high-throughput sequencing, and 14,221 circRNA were identified by the find_circ software. According to Fold Change>|3|,P<1×10 ^-3 Screening criteria, a total of 3 circrnas expressed significantly differentially in colorectal cancer tissues. The supernatant exosomes of colorectal normal epithelial cells (FHC) and two colorectal cancer cell lines (HCT 116 and DLD 1) were further extracted and identified (see fig. 1), and circular or quasi-circular cystic vesicles with a diameter of about 100nm having a membrane structure were found by transmission electron microscopy, which fit the morphology and size of the exosomes, while significantly expressing the exosome-specific marker proteins TSG101 and Alix. Based on the cell supernatant exosome model, hsa_circ_0087960 (SEQ ID No. 1) was found compared to colorectal normal epithelial cells FHC; in colorectal cancerCell lines and their secreted exosomes were significantly under-expressed, see fig. 2 and table 1. To show origins and colorectal cancer specificity, it is designated herein as circumcetlet (exosome-transmitted low expression transcript).

TABLE 1 colorectal cancer specific exosome circRNA

2. Primer design and loop forming identification of exosome circRNA

(1) Design of specific primers for circETLET

Specific circular and linear primers for circETLET were designed, circETLET primers: 5'-TGTTCACCACCTACAACCAC-3' (SEQ ID No. 2), 5'-GAGAAGCTGTGTACCTGATGC-3' (SEQ ID No. 3); ETLET linear primer 5'-GCCACCTTTAGGCAGATCCT-3' (SEQ ID No. 4), 5'-AGCCAAGATGGTGTGGTTGA-3' (SEQ ID No. 5); internal reference GAPDH:5'-GGAGCGAGATCC CTCCAAAAT-3' (SEQ ID No. 6), 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID No. 7).

(2) Reverse transcription of exosome circRNA

Genomic DNA was removed using PrimeScript RT reagent Kit With gDNA Eraser reverse transcription kit (Takara) and the reaction system comprised 2. Mu.l of 5X gDNA Eraser Buffer, 1. Mu.l of gDNA Eraser, 1,000ng RNA and DEPC water, with a total reaction system of 10. Mu.l. The reaction procedure was 42℃for 2min; preserving at 4 ℃. RNA reverse transcription was then performed, and the reaction system consisted of 10. Mu.l of the genomic DNA-depleted reaction solution, 1. Mu. l PrimeScript RT Enzyme Mix I, 1. Mu.l of RT Primer Mix, 4. Mu.l of 5X PrimerScript Buffer and 4. Mu.l of DEPC water, with a total reaction system of 20. Mu.l. The reaction conditions of the method are as follows: 15min at 37 ℃; 5s at 85 ℃; preserving at 4 ℃.

(3) Specific detection of circETLET primer

Extracting normal colorectal epithelial cells, colorectal cancer cells, cell culture fluid exosomes thereof and total RNA of human plasma exosomes, and analyzing melting curves by using a Roche RT-PCR LC480 II type system after enzyme digestion and reverse transcription reaction. As a result, the curve was found to have a single peak, indicating that the circular primer was specific and was available for subsequent quantitative analysis (see FIG. 3).

(4) circETLET loop identification

The products of the real-time fluorescent quantitative PCR reaction were subjected to agarose gel electrophoresis (see FIG. 4), the amplified products of the circular primers of the circETLET were not significantly changed after digestion with RNase R enzyme, while the amplified products of the linear primers were significantly reduced after digestion with RNase R enzyme, the bands were lighter, indicating that the circETLET was resistant to digestion with RNase R enzyme, and that the circETLET was stably present in a circular form.

3. Quantitative detection of plasma exosome circRNA, evaluation of tissue specificity and stability

(1) Basic information of study crowd

200 plasma samples were collected, including 112 colorectal cancer patients, 28 colorectal polyp patients, and 60 healthy control populations, with basic information on the population being shown in Table 2. In addition, patients with gastric cancer (74 cases), lung cancer (42 cases), cervical cancer (32 cases), bladder cancer (24 cases), renal cancer (19 cases) and breast cancer (18 cases) were also recruited, and plasma samples were collected (see table 3).

TABLE 2 verification of crowd basic information

TABLE 3 essential information of the population suffering from cancer

(2) Collecting a plasma sample

Collecting peripheral venous blood 3ml by adopting an EDTA vacuum anticoagulation blood collection tube, and centrifugally separating into plasma and blood cells at the temperature of 4 ℃ at 3,000rpm for 10 min; the supernatant, plasma, was transferred to a fresh 1.5ml EP tube, with no contact with the middle layer white blood cells; the collected plasma and blood cell samples were kept in a-80℃refrigerator for further use. All colorectal cancer patients were subjected to pathological diagnosis by a senior pathologist, and had no other tumors prior to inclusion in the study, and had not received surgery, radiation or chemotherapy. The subjects were subjected to on-site epidemiological investigation in a face-to-face manner using a unified health questionnaire. The investigation content comprises clinical relevant information such as general demographic characteristics, tumor parts of patient tissues, tumor differentiation conditions, stage and the like. All investigators were uniformly trained.

(3) Extraction and identification of plasma exosomes

Firstly, centrifuging the plasma at 3,000g and 4 ℃ for 15min to remove cell debris or impurities; then, the subject plasma exosomes were extracted with reference to SBI company ExoQuick Plasma Prep with Thrombin kit, then 63 μl ExoQuick reagent was added, the EP tube was turned upside down or flicked to mix them uniformly, and incubated in a refrigerator at 4 ℃ for 30min without turning the EP tube during incubation; finally, centrifuging for 30min at 10,000rpm and 4 ℃, wherein the bottom of the tube can be seen as white or light yellow sediment; centrifuging at-80deg.C for 5min 1,500g after absorbing supernatant, carefully absorbing supernatant, and collecting precipitate as exosome. Exosomes with small vesicle structures with diameters of about 50-100 nm and uneven distribution are found by a transmission electron microscope, and exosome specific marker proteins TSG101 and Alix are detected by a Western blot experiment, as shown in figure 5.

(4) Extraction of plasma exosome circRNA

The plasma was aspirated 500. Mu.l and filtered through a 0.8 μm filter for pretreatment. Total Plasma RNA was extracted with reference to the Qiangen company exoRNeasy Serum/Plasma Maxi Kit, and then the RNA was purified and eluted from the spin column filter by centrifugation at 16,000g for 1min at room temperature, and the mass of total RNA was analyzed by agarose gel electrophoresis according to the ratio of 28S rRNA to 18S rRNA.

(5) Real-time fluorescent quantitative PCR (polymerase chain reaction) of exosome circRNA (circRNA)

Real-time quantitative PCR was performed using SYBR GREEN (Vazyme Biotech co., ltd) method, the reaction system comprising: mu.l SYBR Green Mix, 2.4. Mu.l double distilled water, 0.3. Mu.l primer F, 0.3. Mu.l primer R, 2.0. Mu.l cDNA template, and the total reaction system was 10. Mu.l. Amplification was performed using the Rogowski RT-PCR LC480 type II system, the procedure being: pre-denaturation: 300s at 95 ℃; annealing and extending: 95 ℃ for 15s;60 ℃ for 60s (40 cycles, each cycle does not collect fluorescence signal); adding a melting curve: 95 ℃ for 10s;60 s at 60 ℃;97℃for 1s (observing the specificity of the amplified product); and (3) cooling: 30s at 37 ℃. Three replicates were designed for each sample and normalized with GAPDH as an internal reference. Detection of the expression level of circETLET (SEQ ID No.2 and SEQ ID No. 3) and of the internal reference GAPDH (SEQ ID No.6 and SEQ ID No. 7)

(6) Analysis of results

Plasma exosome circRNA expression levels are expressed as Ct values in the examples. The Ct value refers to the number of cycles each reaction tube undergoes for the fluorescent signal to reach a set threshold. The more the initial copy number of the target circRNA is, the smaller the Ct value is, and the larger the reverse is. Under the condition that the amplification efficiency of the target exosome circRNA is the same as that of the internal reference, the quantitative delta Ct=Ct of the target exosome circRNA relative to the internal reference can be directly obtained _Purpose(s) -Ct _{Internal reference} . By 2 ^-ΔCt The fold change in target plasma exosome circRNA expression relative to the internal reference GAPDH expression is shown. All statistical tests were double-sided probability tests, P<A difference of 0.05 is statistically significant. The next analysis was performed using R software (version 3.6.0).

The results show that the expression level of exosome circetetlet in the plasma of colorectal cancer patients is significantly lower than that of colorectal polyp patients (p=0.030) and healthy control population (P < 0.001), and that the expression level of exosome circetelet in the plasma of colorectal polyp patients is also significantly down-regulated compared to healthy control population (p=0.039); meanwhile, the expression level of exosome circetelet in the plasma of colorectal cancer patients is significantly different from the expression level (P < 0.001) of exosome circetelet in the plasma of gastric cancer, breast cancer, bladder cancer, cervical cancer, kidney cancer and lung cancer patients, as shown in fig. 6. After the blood plasma is repeatedly frozen and thawed for 0 times, 2 times, 4 times or 8 times at the room temperature and the temperature of minus 80 ℃, the expression level of the exosome circETLET in the blood plasma of the patients, colorectal polyps and the control group population is not changed significantly; at the same time, there was no significant difference in the exosome circetetest in plasma at room temperature for 0h, 4h, 8h or 24h (fig. 7), suggesting that the exosome circetetest can still exist stably when the plasma is in an extreme environment. ROC curve analysis shows that plasma exosome circetelet can effectively screen colorectal cancer patient population, auc=0.858 (95% ci=0.786-0.930), and sensitivity and specificity of colorectal cancer diagnosis are 76.2% and 92.1%, respectively, as shown in fig. 8.

Sequence listing

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Claims (5)

1. The application of a reagent for detecting the plasma exosome hsa_circ_0087960 in preparing a colorectal cancer auxiliary diagnosis kit.

2. The use according to claim 1, characterized in that the reagent for detecting hsa_circ_0087960 of the plasma exosome is a specific primer pair and/or probe for detecting hsa_circ_ 0087960.

3. The use according to claim 2, characterized in that the specific primer pair for the detection hsa_circ_0087960 is shown in SEQ ID NO.2 and SEQ ID NO. 3.

4. A colorectal cancer auxiliary diagnosis kit, which is characterized by comprising a specific primer pair and/or a probe for detecting hsa_circ_ 0087960.

5. The auxiliary diagnostic kit for colorectal cancer according to claim 4, which comprises a specific primer pair for detecting hsa_circ_0087960 as shown in SEQ ID NO.2 and SEQ ID NO. 3.