CN111549141A - Primer and kit for detecting peripheral blood exosome circ-PPCDC (circulating-positive-negative-positive - Google Patents
Primer and kit for detecting peripheral blood exosome circ-PPCDC (circulating-positive-negative-positive Download PDFInfo
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Abstract
The invention relates to a primer and a kit for detecting peripheral blood exosome circ-PPCDC and application thereof, wherein the kit comprises an RNA extraction reagent of a peripheral blood exosome sample, the primer, a genome DNA removal reaction system, a reverse transcription reaction system and a qPCR operation reaction system. The peripheral blood exosome circ-PPCDC can be used for early diagnosis of gastric cancer liver metastasis, judgment of disease progression and prognosis, and evaluation of the treatment effect of gastric cancer liver metastasis.
Description
Technical Field
The invention relates to a primer and a kit for detecting peripheral blood exosome circ-PPCDC and application thereof in gastric cancer liver metastasis, belonging to the technical field of molecular diagnosis.
Background
Stomach cancer seriously threatens human health, is one of five cancers around the world, and the statistical report of the world health organization shows that about 95.1 ten thousand new stomach cancer cases and about 72.3 ten thousand death cases are found around the world in 2012. The incidence of gastric cancer is high in east asia (particularly china, korea, mongolia and japan), central europe, eastern europe, south america and most parts of africa. China is the world with the highest incidence and mortality of gastric cancer. Data of national tumor registration centers show that about 67.9 ten thousand cases of new gastric cancer and about 49.8 ten thousand cases of new gastric cancer in 2015 are high in China and second-place malignant tumors. Early gastric cancer is difficult to diagnose, most patients are in middle and late stages during treatment, invasion and metastasis of the gastric cancer in the middle and late stages often occur, the treatment of the gastric cancer patients is seriously influenced, and the five-year survival rate is lower than 30%. The liver is the main target organ of gastric cancer metastasis, the incidence rate of the liver metastasis is up to 44%, the resection operation of patients with gastric cancer liver metastasis is very limited, the treatment difficulty is high, and the five-year survival rate of the patients with gastric cancer liver metastasis is only about 10%.
Gastric cancer is prone to liver metastasis and closely related to the biological characteristics of the liver. The liver is the largest glandular organ of a human body and is supplied by dual blood of a hepatic portal vein and a hepatic artery, wherein the hepatic portal vein accounts for 80 percent, the hepatic artery accounts for 20 percent, blood of digestive tract organs flows back through the hepatic portal vein, and the liver has rich blood flow. Liver capillaries are porous blood sinus-like structures with strong permeability, and liver sinus duct lacks subcutaneous basement membrane, and compared with other organs such as brain, lung and bone, tumor cells are more easily exosmosed in liver, so liver is the most important organ for digestive tract tumor metastasis. Gastric cancer, colorectal cancer, pancreatic cancer, etc. are first transferred to the liver through the pathways of vascular metastasis, lymphatic return, etc. The abundant vascular system of the liver provides sufficient nutrients for the growth of tumor cells. In addition to the function of the blood circulation system, gastric cancer cells are also easily adhered to the endothelial cells of the liver sinuses. The liver capillary vessel can block a large amount of tumor cells, and after the tumor cells interact with vascular endothelial cells, cytokines are secreted to shrink the vascular endothelial cells and promote the tumor cells to invade blood vessels, so that the tumor cells can be planted in liver parenchyma tissues.
At present, early diagnosis of gastric cancer liver metastasis is still a great problem. Some tumor molecular markers have been used for clinical early detection and auxiliary diagnosis, such as carcinoembryonic antigens CEA, CA199, CA125, AFP, etc., but the specificity is still low. Molecules such as CD44v6, OPN, YB-1, MAGE-A10 and the like are reported to be potential markers of liver metastasis of gastric cancer, but are still to be further verified. Therefore, the discovery of the new tumor-related gene for regulating and controlling the gastric cancer cell liver metastasis not only helps to deeply understand the molecular mechanism of the occurrence and development of the gastric cancer liver metastasis, supplements and perfects the existing cell canceration theoretical system, but also helps to identify the new specific biomarker and drug target of the gastric cancer liver metastasis, and provides an important theoretical basis for the prevention, diagnosis and treatment of the gastric cancer liver metastasis.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a primer and a kit for detecting the circ-PPCDC in the peripheral blood exosomes and application thereof.
In order to solve the technical problems, the invention provides a primer for detecting peripheral blood exosome circ-PPCDC, wherein the nucleotide sequence of the primer is shown as SEQ ID NO: 1 and SEQ ID NO: 2, wherein the nucleotide sequence of the circ-PPCDC is shown as SEQ ID NO: 3, respectively.
The invention also provides a kit for detecting the peripheral blood exosome circ-PPCDC, which comprises the primer.
Further comprises an RNA extraction reagent of peripheral blood exosomes, a reverse transcription reaction system and a qPCR reaction system.
Further, a genome DNA removing reaction system is also included.
The invention also provides application of the primer or the kit in serving as a gastric cancer and liver metastasis monitoring reagent.
Further, circ-PPCDC in peripheral blood exosomes was detected using the above-described primers or kit.
Further, a method of detecting circ-PPCDC in peripheral blood exosomes, comprising:
extracting exosomes in peripheral blood to be detected;
extracting RNA from the exosome sample;
removing genomic DNA from the extracted RNA;
performing reverse transcription on the obtained RNA to cDNA;
the obtained cDNA was detected by qPCR.
Furthermore, the peripheral blood exosome samples to be detected are from gastric cancer patients and normal persons.
Further, the method for extracting exosomes in the peripheral blood to be detected comprises the following steps:
whole blood was centrifuged at 3000 Xg for 15min to remove cells or cell debris;
taking the upper layer liquid, putting the upper layer liquid into a centrifuge tube, adding 63 mul of ExoQuick reagent into 250 mul of serum, and standing at 4 ℃ for 30 min;
centrifuging the mixture at 1500 Xg for 30min at 4 deg.C;
sucking out all the supernatant, centrifuging at 1500 Xg for 5min at 4 deg.C, and sucking out all the supernatant;
the pellet was dissolved in 200. mu.l of 1 XPBS and stored at-20 ℃.
The exosome is an efflux vesicle with the diameter of about 30-100nm, which is released into an extracellular matrix after an intracellular multivesicular body is fused with a cell membrane. The exosome can appear outside cells in body fluid such as blood, urine or hydrothorax and ascites, and move or transfer in vivo along with blood flow or body fluid, or stay in tissues or organs to form a new tumor marker, and can be used for screening, early diagnosis, prognosis prediction, monitoring individualized treatment and the like. Exosomes include the corresponding mRNA, microRNA, and other non-coding RNAs, all of which are functionally transduced into recipient cells. The circular RNA is a reverse RNA splicing product, has a covalent closed circular structure, lacks a 5 'end cap structure and a 3' poly-A tail structure, can resist hydrolysis of RNA exonuclease, has a half-life of 48h, is obviously higher than a linear RNA with a half-life of 10h, and has higher stability.
The invention achieves the following beneficial effects:
(1) the invention comprehensively adopts the second generation sequencing technology and the high-throughput chip technology, screens a series of abnormal circRNAs from the peripheral blood exosomes of the gastric cancer patients, carries out gene re-annotation analysis, merges data results, and finds that the circ-PPCDC is obviously reduced in the peripheral blood exosomes of the gastric cancer, thereby being used as a diagnosis marker of the gastric cancer.
(2) The bottleneck of treating malignant tumors such as gastric cancer and the like is to a great extent that the tumor is difficult to sample in real time, the curative effect evaluation is delayed, the drug-resistant site is difficult to discover and the like, and the difficulty of tissue sampling is overcome by a liquid biopsy technology mainly comprising exosomes. The circ-PPCDC exists in blood exosomes, mainly takes the exosomes as transport vectors, has cell phenotype specificity and development stage specificity, and researches show that the low expression of the molecule is closely related to gastric cancer liver metastasis, which indicates that the molecule is an independent postrain factor of the gastric cancer liver metastasis.
(3) The invention researches the correlation between circ-PPCDC and clinical parameters of gastric cancer patients, and the result shows that: the expression level of the peripheral blood exosome circ-PPCDC is obviously related to the cancer tissue infiltration depth, lymph node metastasis, liver metastasis and TNM stage of a patient (the P values are 0.018, 0.023, 0.016 and 0.015 respectively), so that the molecular marker RNA hsa _ circ-PPCDC highly related to the liver metastasis of the gastric cancer is creatively obtained, and the expression level can be used for early diagnosis of the liver metastasis of the gastric cancer, judging the disease progression and prognosis conditions and evaluating the treatment effect of the liver metastasis of the gastric cancer.
(4) The invention designs a corresponding primer sequence, realizes the detection of circ-PPCDC by reverse transcription and RT-PCR, and the sensitivity and specificity of the detection are respectively 72.50 percent and 82.50 percent.
Drawings
FIG. 1 is an amplification curve of the circ-PPCDC assay in peripheral blood exosomes in gastric cancer patient group and healthy person group;
FIG. 2 shows the expression of circ-PPCDC in peripheral blood exosomes in the gastric cancer patient group and healthy person group;
FIG. 3 is the area under the circ-PPCDC curve in peripheral blood exosomes of gastric cancer patient group and healthy person group.
Detailed Description
The present invention will be further described with reference to the following examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example 1 detection of circ-PPCDC in peripheral blood exosomes
1. Obtaining a peripheral blood sample to be detected:
peripheral blood samples from 120 gastric cancers and 120 normal persons.
Samples were coagulant-added peripheral blood samples from the gastric cancer patients and from normal persons.
2. Extracting exosome from peripheral blood sample to be detected and verifying exosome
The exosome in peripheral blood of the stomach is extracted according to the exosome extraction kit specification by operation, and the steps are as follows: (1) whole blood was centrifuged at 3000 Xg for 15min (centrifugation radius 10cm) and cells or cell debris were removed. (2) Taking the upper layer liquid, putting the upper layer liquid into a centrifuge tube, adding 63 mul of ExoQuick reagent into 250 mul of serum, and standing at 4 ℃ for 30 min; (3) centrifuging the mixture at 1500 Xg (centrifugation radius 10cm) at 4 deg.C for 30 min; (4) sucking out all the supernatant, centrifuging at 1500 Xg (centrifugation radius 10cm) at 4 deg.C for 5min, and sucking out all the supernatant; (5) the pellet was dissolved in 200. mu.l of 1 XPBS and stored at-20 ℃. And identifying the exosomes by adopting a projection electron microscope.
3. Extraction of RNA from peripheral blood sample to be examined
0.25mL of peripheral blood sample or frozen peripheral blood sample was transferred to a centrifuge tube, 0.75mL of RNAiosobood was added, and pipetting was repeated up and down with a pipette until the cells were completely lysed. Chloroform (1/5 volumes based on the volume of the sample solution + RNAlso Blood) was added to the homogenate and the mixture was mixed until the solution was emulsified in a milky white state. The mixture was allowed to stand at room temperature for 5 minutes. Centrifuge at 12,000 Xg for 15 minutes at 4 ℃. The centrifuge tube was carefully removed from the centrifuge, and the homogenate was divided into three layers at this time, i.e.: a colorless supernatant (containing RNA), an intermediate white protein layer (mostly DNA), and a colored lower organic layer. The supernatant was aspirated and transferred to a new centrifuge tube. Adding equal volume of isopropanol into the supernatant, turning the centrifuge tube upside down, mixing well, and standing for 10 minutes at room temperature. When centrifuged at 12,000 Xg for 10 minutes at 4 ℃ RNA precipitates appear at the bottom of the tube. The supernatant was discarded, an equal amount of 75% ethanol was added, and after washing the precipitate with Vortex, the precipitate was centrifuged at 7,500 Xg at 4 ℃ for 5 minutes, and the supernatant was discarded. The precipitate was dried at room temperature. After the precipitate is dried, an appropriate amount of RNase-free water (20-30. mu.L) is added to dissolve the precipitate.
4. Removal of genomic DNA from extracted RNA
The genome DNA removal reaction system is shown in Table 1.
TABLE 1 Degenomic DNA reaction System
The parameters of the genome-free DNA reaction are as follows:
42℃ 2min;
4℃ ∞。
5. the obtained RNA is subjected to reverse transcription to cDNA
The reaction system for reverse transcription was as shown in Table 2.
TABLE 2 reverse transcription reaction System
The reverse transcription operation has reaction parameters of
37℃ 15min;
85℃ 5s;
4℃ ∞。
6. The obtained cDNA was subjected to reaction detection by the qPCR method
The 20. mu.L reaction system is shown in Table 3.
TABLE 3qPCR reaction System
Circulation parameters:
the experimental results are as follows:
the expression of circ-PPCDC in peripheral blood exosomes of the gastric cancer patient group and the healthy patient group is detected by adopting a fluorescent real-time quantitative PCR method. The amplification curve index has an amplification stage and a plateau stage, and the curve is smooth. Indicating that the amplification efficiency is higher. See fig. 1.
The fluorescent real-time quantitative PCR results are shown in FIG. 2, the expression levels of circ-PPCDC in peripheral blood of patients in the gastric cancer group and healthy group are 0.1801 + -0.2314 and 0.5995 + -0.4060, respectively, and compared with the healthy group, the expression levels of circ-PPCDC in peripheral blood of patients in the gastric cancer group show that the expression levels are obviously reduced and the difference is obvious (P < 0.0001).
Further working characteristic curves of the subjects were made, and as shown in FIG. 3, the results showed that the area under the curve (AUC) for diagnosing gastric cancer by circular RNA circ-PPCDC in plasma was 0.86, the optimum cutoff value for diagnosing gastric cancer by circular RNA circ-PPCDC was 0.1737, and the results showed that the sensitivity and specificity were 72.50% and 82.50%, respectively.
Example 2 clinical significance of the peripheral blood exosomes circ-PPCDC
Further analyzing the correlation between the peripheral blood circ-PPCDC expression and clinical parameters such as gastric cancer gender, age, tumor diameter, TNM stage, tissue differentiation degree, lymph node metastasis condition, liver metastasis and the like.
TABLE 4 clinical significance of the peripheral blood exosomes circ-PPCDC
TABLE 5 Multi-factor analysis results
From tables 4 and 5, it can be seen that: the peripheral blood exosome circ-PPCDC expression level was significantly correlated with the depth of infiltration, lymph node metastasis, liver metastasis, TNM staging in patients (P values of 0.018, 0.023, 0.016 and 0.015, respectively), but not with their patients' sex, age, tumor location, tumor diameter and degree of tissue differentiation (P values of 0.707,0.584,0.801,0.100 and 0.215, respectively). The multi-factor analysis result indicates that the low expression of the peripheral blood exosome circ-PPCDC is one of the independent prognostic factors of the gastric cancer liver metastasis.
The results show that the abnormal expression of the peripheral blood exosome circ-PPCDC in the peripheral blood exosomes of the gastric cancer patients is reduced, and the method can be used for early diagnosis of gastric cancer liver metastasis, judgment of disease progression and prognosis and evaluation of the treatment effect of the gastric cancer liver metastasis.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
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<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
ggtacataca tatatgtgtg t 21
<210>3
<211>38
<212>DNA
<213> Intelligent (Homo sapiens)
<400>3
gttgtccagt ctggcaagag aatacggagc ccttcagg 38
Claims (9)
1. The primer for detecting the peripheral blood exosome circ-PPCDC is characterized in that the nucleotide sequence of the primer is shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
2. A kit for detecting the peripheral blood exosome circ-PPCDC, comprising the primer according to claim 1.
3. The kit for detecting peripheral blood exosome circ-PPCDC according to claim 2, which further comprises a peripheral blood exosome RNA extraction reagent, a reverse transcription reaction system and a qPCR reaction system.
4. The kit for detecting peripheral blood exosome circ-PPCDC according to claim 3, characterized by further comprising a decogenom DNA reaction system.
5. The primer of claim 1 or the kit of any one of claims 2 to 4, for use as a reagent for monitoring liver metastasis of gastric cancer.
6. The use according to claim 5, wherein the primers of claim 1 or the kit of any one of claims 2 to 4 are used to detect circ-PPCDC in peripheral blood exosomes.
7. The use according to claim 6, wherein the method for detecting circ-PPCDC in peripheral blood exosomes comprises:
extracting exosomes in peripheral blood to be detected;
extracting RNA from the exosome sample;
removing genomic DNA from the extracted RNA;
performing reverse transcription on the obtained RNA to cDNA;
the obtained cDNA was detected by qPCR.
8. The use according to claim 7, wherein the sample of peripheral blood exosomes to be detected is from gastric cancer patients and normal persons.
9. The use according to claim 7, wherein the method for extracting exosomes from peripheral blood to be detected comprises:
whole blood was centrifuged at 3000 Xg for 15min to remove cells or cell debris;
taking the upper layer liquid, putting the upper layer liquid into a centrifuge tube, adding 63 mul of ExoQuick reagent into 250 mul of serum, and standing at 4 ℃ for 30 min;
centrifuging the mixture at 1500 Xg for 30min at 4 deg.C;
sucking out all the supernatant, centrifuging at 1500 Xg for 5min at 4 deg.C, and sucking out all the supernatant;
the pellet was dissolved in 200. mu.l of 1 XPBS and stored at-20 ℃.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107858435A (en) * | 2017-12-25 | 2018-03-30 | 镇江市第人民医院 | Detect circular rna circRNA_101835 primer, kit and detection method and application |
CN110129324A (en) * | 2019-05-14 | 2019-08-16 | 江苏大学 | The extraction of gastric cancer serum excretion body marker hsa_circ_001477 a kind of and detection method and application |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN107858435A (en) * | 2017-12-25 | 2018-03-30 | 镇江市第人民医院 | Detect circular rna circRNA_101835 primer, kit and detection method and application |
CN110129324A (en) * | 2019-05-14 | 2019-08-16 | 江苏大学 | The extraction of gastric cancer serum excretion body marker hsa_circ_001477 a kind of and detection method and application |
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Application publication date: 20200818 |