CN111560439B - Primer and kit for detecting peripheral blood exosome circ-LRP8 and application of primer and kit in gastric cancer liver metastasis - Google Patents
Primer and kit for detecting peripheral blood exosome circ-LRP8 and application of primer and kit in gastric cancer liver metastasis Download PDFInfo
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Abstract
The invention relates to a primer, a kit, a detection method and application of a peripheral blood exosome circ-LRP8, wherein the kit comprises an RNA extraction reagent, a primer, a genome DNA removal reaction system, a reverse transcription reaction system and a qPCR operation reaction system of the peripheral blood exosome, and the invention discloses the expression conditions of the circ-LRP8 in peripheral blood exosomes of gastric cancer patients and normal people, and uses fluorescent quantitative PCR to detect the peripheral blood exosome circ-LRP8. The circ-LRP8 can be used for early diagnosis of gastric cancer liver metastasis, judging disease progression and prognosis, and evaluating 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-LRP8 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. The national tumor registration center data shows that 67.9 new cases of gastric cancer and 49.8 new cases of gastric cancer die in 2015 in China, which are high in the second place of malignant tumors in China. Early gastric cancer is difficult to diagnose, most patients are in middle and late stages during diagnosis, invasion and metastasis of the middle and late gastric cancer often occur, the treatment of gastric cancer patients is seriously influenced, and the five-year survival rate is lower than 30 percent. 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 supply 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, the 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 possible 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 peripheral blood exosome circ-LRP8 and application thereof, wherein the peripheral blood exosome circ-LRP8 can be used for early diagnosis of gastric cancer liver metastasis, judgment of disease progression and prognosis conditions and evaluation of the treatment effect of gastric cancer liver metastasis.
In order to solve the technical problem, the invention provides a peripheral blood exosome circ-LRP8, wherein the nucleotide sequence of the circ-LRP8 is shown as SEQ ID NO:1 is shown.
The invention also provides a primer for detecting the peripheral blood exosome circ-LRP8, wherein the nucleotide sequence of the primer is shown as SEQ ID NO:2 and SEQ ID NO:3, respectively.
The invention also provides a kit for detecting the peripheral blood exosome circ-LRP8, which comprises the primer.
Further comprises an RNA extraction reagent of peripheral blood exosomes, a genome DNA removal reaction system, a reverse transcription reaction system and a qPCR reaction system.
Further, the conditions of the reaction for removing the genomic DNA were 42 ℃ for 2min and 4 ℃ infinity; the reverse transcription reaction conditions are 37 ℃ for 15min, 85 ℃ for 5s and 4 ℃ infinity; qPCR reaction conditions were stage 1: circulating at 95 ℃ for 1 time and 30 seconds; and (2) stage: circulating at 95 ℃ for 40 times in 5S; circulating for 40 times at 60 ℃ for 31S; and (3) stage: circulating at 95 ℃ for 1 time and 15 seconds; 60 ℃ and 1 cycle of 60 seconds; 95 ℃ and 15S, 1 cycle.
The invention also provides a method for detecting the peripheral blood exosome circ-LRP8, which adopts the kit and comprises the following steps:
obtaining 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 method.
Furthermore, the peripheral blood exosome samples to be detected are from gastric cancer patients and normal persons.
The invention also provides application of the primer or the kit as a reagent for diagnosing the liver metastasis of the gastric cancer, evaluating the treatment effect or evaluating the prognosis condition.
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 has 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-LRP8 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 the treatment of 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-LRP8 in the invention exists in blood exosomes, mainly takes the exosomes as transport vectors, has cell phenotype specificity and development stage specificity, and the research finds that the low expression of the molecule is closely related to gastric cancer liver transfer, which indicates that the molecule is an independent prognostic factor of the gastric cancer liver transfer.
(3) The invention researches the correlation between circ-LRP8 and clinical parameters of gastric cancer patients, and the result shows that: the expression level of peripheral blood exosome circ-LRP8 is obviously related to the cancer tissue infiltration depth, lymph node metastasis, liver metastasis and clinical stages of patients (the P values are 0.002,0.001 and 0.001 respectively), so that the molecular marker RNA hsa _ circ-LRP8 highly related to the liver metastasis of the gastric cancer is creatively obtained, and the molecular marker 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 the circ-LRP8 by reverse transcription and RT-PCR, and the detection sensitivity and specificity are respectively 98.33 percent and 98.33 percent.
Drawings
FIG. 1 is the amplification curve of the circ-LRP8 assay in peripheral blood exosome samples from the gastric cancer patient group and the healthy group;
FIG. 2 is the expression of circ-LRP8 in peripheral blood exosome samples from the gastric cancer patient group and the healthy group;
FIG. 3 is the area under the curve of circ-LRP8 in peripheral blood exosomes in the gastric cancer patient group and healthy person group.
Detailed Description
The invention is further described below with reference to the following examples and the accompanying drawings. 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-LRP8 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 10 cm) and cells or cell debris were removed. (2) Taking the upper liquid, putting the upper liquid into a centrifuge tube, adding 63 ul of ExoQuick reagent into 250 ul of serum, and standing at 4 ℃ for 30min; (3) Centrifuging the mixture at 1500 Xg (centrifugation radius 10 cm) at 4 deg.C for 30min; (4) Sucking out all the supernatant, centrifuging at 1500 Xg (centrifugation radius 10 cm) 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 exosome 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 pipetted into a centrifuge tube, 0.75mL of RNAasso Blood was added, and pipetting was repeated up and down with a pipette gun until the cells were completely lysed. Chloroform (1/5 volume of the sample solution plus RNAioso Blood) was added to the homogenate and the centrifuge tube was closed, and the mixture was mixed until the emulsion became milky white. 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. RNA precipitation occurred at the bottom of the tube after centrifugation at 12,000 Xg for 10 minutes at 4 ℃. 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 was subjected to reverse transcription into cDNA
The reaction system for reverse transcription was as shown in Table 2.
TABLE 2 reverse transcription reaction System
The reaction parameters of the reverse transcription operation are
37℃15min；
85℃5s；
4℃∞。
6. The obtained cDNA was subjected to reaction detection by the qPCR method
20 μ L of the reaction system are shown in Table 3.
TABLE 3qPCR reaction System
Circulation parameters:
the experimental results are as follows:
the expression of circ-LRP8 in peripheral blood exosomes of a gastric cancer patient group and a healthy patient group is detected by 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.
As shown in FIG. 2, the expression levels of circ-LRP8 in peripheral blood of patients in gastric cancer group and healthy group were 0.5830 + -0.0753 and 0.9425 + -0.0935, respectively, and compared with the healthy group, the expression levels of circ-LRP8 in peripheral blood of gastric cancer group showed significant down-regulation and difference (P < 0.0001).
Further working characteristic curves of the subjects were made, as shown in FIG. 3, and the results showed that the area under the curve (AUC) for diagnosing gastric cancer by cyclic RNA circ-LRP8 in plasma was 0.99, the optimum cutoff value for cyclic RNA circ-LRP8 for diagnosing gastric cancer was 0.7263, and the results showed that the sensitivity and specificity were 98.33% and 98.33%, respectively,
example 2 clinical significance of peripheral blood exosomes circ-LRP8
Further analyzing the correlation between the expression of peripheral blood exosome RNA hsa _ circ-LRP8 and clinical parameters of gastric cancer gender, age, tumor diameter, TNM stage, tissue differentiation degree, lymph node metastasis condition, liver metastasis and the like.
TABLE 4 analysis of clinical significance of peripheral blood exosomes circ-LRP8
TABLE 5 Multi-factor analysis results
From tables 4 and 5, it can be seen that: peripheral blood exosome RNA hsa _ circ-LRP8 expression levels were significantly associated with patient infiltration depth, lymph node metastasis, liver metastasis, TNM staging (P values 0.002,0.001, and 0.001, respectively), but not with their patient sex, age, tumor location, tumor diameter, and degree of tissue differentiation (P values 0.852,0.709,0.966,0.100, and 0.258, respectively). The result of multifactorial analysis indicates that the low expression of peripheral blood exosome RNA hsa _ circ-LRP8 is one of independent prognostic factors of gastric cancer liver metastasis.
The results show that the peripheral blood exosome RNA hsa _ circ-LRP8 is expressed and down-regulated in the peripheral blood exosomes of the gastric cancer patients, and can be used for early diagnosis, disease progression and prognosis of gastric cancer liver metastasis and 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.
Sequence listing
<110> Jiangsu Huameijian sanden Biotechnology Limited
<120> primers and kit for detecting peripheral blood exosome circ-LRP8 and application of primers and kit in gastric cancer liver metastasis
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<170> SIPOSequenceListing 1.0
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aaatgcatgc atgctctccc agcaccaggt tagtggtcta ctcccaggcc catgctgtcc 120
cagggggcca gcactggggt ctgtgggagg gtgtggggtt gtgtccttag g 171
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gtctactccc aggcccatg 19
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Claims (5)
1. The primer for detecting the peripheral blood exosome circ-LRP8 is characterized in that the nucleotide sequence of the primer is shown as SEQ ID NO:2 and SEQ ID NO:3, respectively.
2. A kit for detecting peripheral blood exosome circ-LRP8, comprising the primer according to claim 1.
3. The kit for detecting the peripheral blood exosome circ-LRP8 according to claim 2, which is characterized by further comprising an RNA extraction reagent of the peripheral blood exosome, a degenomic DNA reaction system, a reverse transcription reaction system and a qPCR reaction system.
4. The kit for detecting peripheral blood exosome circ-LRP8 according to claim 3, wherein the conditions for the genome DNA elimination reaction are 42 ℃ for 2min and 4 ℃ for infinity; the reverse transcription reaction conditions are 37 ℃ for 15min, 85 ℃ for 5s and 4 ℃ infinity; qPCR reaction conditions were stage 1: circulating at 95 ℃ for 1 time and 30 seconds; and (2) stage: circulating at 95 ℃ for 40 times in 5S; cycling at 60 deg.C for 31S and 40 times; and (3) stage: circulating at 95 ℃ for 1 time and 15 seconds; 60 ℃ and 60S, and 1 circulation; 95 ℃, 15S and 1 cycle.
5. Use of the primer according to claim 1 or the kit according to any one of claims 2 to 4 as a reagent for the preparation of a gastric cancer diagnosis.
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