CN111808956A - Primer and kit of serum exosome RP11-470P21-2 and application of primer and kit in diagnosis and treatment of gastric cancer liver metastasis - Google Patents
Primer and kit of serum exosome RP11-470P21-2 and application of primer and kit in diagnosis and treatment of gastric cancer liver metastasis Download PDFInfo
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Abstract
The invention relates to a primer, a kit and a detection method for detecting serum exosome RP11-470P21-2 and application thereof in gastric cancer liver metastasis diagnosis and treatment, wherein the kit comprises an RNA extraction reagent of a serum lncRNA sample, a primer, a genome DNA removal reaction system, a reverse transcription reaction system and a qPCR operation reaction system, the invention discloses the expression conditions of lncRNA in serum exosomes of gastric cancer patients and normal persons, and the fluorescent quantitative PCR is used for detecting RP11-470P 21-2. The serum exosome RP11-470P21-2 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 a serum exosome RP11-470P21-2 and application thereof in diagnosis and treatment of 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 serum exosome RP11-470P21-2 and application thereof, wherein RP11-470P21-2 in the serum exosome can be used for early diagnosis of gastric cancer liver metastasis, judging disease progression and prognosis conditions and evaluating the treatment effect of gastric cancer liver metastasis.
In order to solve the technical problems, the invention provides a primer for detecting serum exosome RP11-470P21-2, wherein the nucleotide sequence of the primer is shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
The invention also provides a kit for detecting the serum exosome RP11-470P21-2, which comprises the primer.
Further comprises an RNA extraction reagent of a serum exosome, a genome DNA removing reaction system, a reverse transcription reaction system and a qPCR reaction system.
The invention also provides a method for detecting the serum exosome RP11-470P21-2, which adopts the kit and comprises the following steps:
extracting exosomes in serum 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;
further, the conditions for the reaction for removing 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; 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.
Further, the method for extracting exosomes in the serum 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 ℃.
Furthermore, the serum 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 diagnosis and treatment reagent for liver metastasis of gastric cancer.
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 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 lncRNAs from the serum exosomes of the gastric cancer patients, carries out gene re-annotation analysis, merges data results, and finds that RP11-470P21-2 is obviously up-regulated in the serum 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 RP11-470P21-2 in the invention exists in serum exosomes, and the exosomes are mainly used as transport vectors. In the invention, the high expression of the molecule is closely related to the liver metastasis of the gastric cancer, but the molecule is not related to helicobacter pylori infection and CEA level, which indicates that the molecule is an independent prognostic molecule in predicting the liver metastasis of the gastric cancer.
(3) The invention researches the correlation between RP11-470P21-2 and clinical parameters of gastric cancer patients, and the result shows that: the expression level of the serum exosome RP11-470P21-2 is obviously related to the cancer tissue infiltration depth, lymph node metastasis, liver metastasis and clinical stages of a patient (the P values are 0.006, 0.023, 0.009 and 0.001 respectively), so that the molecular marker RP11-470P21-2 highly related to the liver metastasis of the gastric cancer is creatively obtained, 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 RP11-470P21-2 by reverse transcription and RT-PCR, and the detection sensitivity and specificity are 82.50 percent and 95.00 percent respectively.
Drawings
FIG. 1 is an amplification curve of detection of RP11-470P21-2 in serum exosomes of gastric cancer patient group and healthy person group;
FIG. 2 shows the expression of RP11-470P21-2 in serum exosomes of gastric cancer patient group and healthy person group;
FIG. 3 is the area under the curve of RP11-470P21-2 in serum exosomes of gastric cancer patient group and healthy person group;
FIG. 4 is a graph of the relationship between the expression of the serum exosome RP11-470P21-2 and OS in gastric cancer patients.
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 RP11-470P21-2 in serum exosomes
1. Obtaining a serum sample to be detected:
serum samples from 120 gastric cancers and 120 normal persons.
The samples were coagulant serum samples from the gastric cancer patients and from normal persons.
2. Extracting exosome from serum sample to be detected and verifying exosome
The exosome in the gastric serum 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 serum sample to be tested
0.25mL of serum sample or frozen serum sample was transferred to a centrifuge tube, 0.75mL of RNAasso Blood was added, and pipetting up and down was repeated with a pipette until the cells were completely lysed. Chloroform (1/5 volumes based on the volume of the sample solution + RNAlsoblood) 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.
Setting circulation parameters:
qPCR data processing according to 2-△△C tCalculating the expression condition of the target gene by a formula:
setting CtA1Ct value of target gene of sample No. 1B1Is 1Ct value of reference gene in sample No; ctA2Ct value of target gene of No. 2 sampleB2The reference gene Ct value of the No. 2 sample, the ratio of the target gene expression levels of the No. 1 sample and the No. 2 sample can be roughly calculated as (2-delta Ct method): Δ Ct ═ Ct (Ct)A2-CtB2)-(CtA1-CtB1) X represents the expression level of the target gene in sample No. 2 as 2 in sample No. 1-XAnd (4) doubling.
The experimental results are as follows:
the fluorescent real-time quantitative PCR method is adopted to detect the expression of RP11-470P21-2 in the serum exosomes of the gastric cancer patient group and the healthy patient group. 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.
Fluorescent real-time quantitative PCR detection finds that the expressions of RP11-470P21-2 in the serum of patients in a gastric cancer group and healthy groups are 5.731806 +/-0.2676 and 2.298398 +/-0.2618 respectively, and compared with the expression levels of RP11-470P21-2 in the serum of the gastric cancer group, the expression levels are obviously up-regulated and the difference is obvious (P is less than 0.0001). See fig. 2.
Further, the working characteristic curve of the subject was plotted, and the results showed that the area under the curve (AUC) of RP11-470P21-2 for diagnosing gastric cancer in serum was 0.92, the optimal cutoff value of RP11-470P21-2 for diagnosing gastric cancer was 4.478, and the results showed that the sensitivity and specificity were 82.50% and 95.00%, respectively. See fig. 3.
Example 2 clinical significance of serum exosomes RP11-470P21-2
Further analyzing the correlation between the expression of the serum exosome RP11-470P21-2 and clinical parameters such as gastric cancer gender, age, tumor diameter, clinical stage, tissue differentiation degree, lymph node metastasis condition, liver metastasis and the like. As a result, as shown in tables 4 and 5, the expression level of serum exosome RP11-470P21-2 was found to be significantly related to the infiltration depth, lymph node metastasis, liver metastasis, and clinical stage of the patients (P values of 0.006, 0.023, 0.009, and 0.001, respectively), but not significantly related to the sex, age, tumor onset site, tumor diameter, tissue differentiation degree, helicobacter pylori infection, and CEA level of the patients. The result of multi-factor analysis indicates that the high expression of the serum exosome RP11-470P21-2 is one of independent prognostic factors of the gastric cancer liver metastasis.
TABLE 4 clinical significance of serum exosomes RP11-470P21-2
TABLE 5 Multi-factor analysis results
120 serum exosomes RP11-470P21-2 of gastric cancer patients are divided into two groups according to expression levels, namely a high expression group and a low expression group. As shown in FIG. 4, the analysis shows that the high expression group OS time is less than that of the low expression group OS time, which indicates that the high expression of the serum exosome RP11-470P21-2 is closely related to the shortening of the Overall Survival (OS) of the patients.
The results show that the abnormal expression of the serum exosome RP11-470P21-2 in the serum exosome of a gastric cancer patient is up-regulated, so that the serum exosome 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 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> first-person hospital in Zhenjiang city
Primer and kit for <120> serum exosome RP11-470P21-2 and application of primer and kit in diagnosis and treatment of gastric cancer liver metastasis
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
aggcaaagcc ctcctgttc 19
<210>2
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
Claims (8)
1. The primer for detecting the serum exosome RP11-470P21-2 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 serum exosome RP11-470P21-2, characterized in that it comprises the primer of claim 1.
3. The kit for detecting the serum exosome RP11-470P21-2 according to claim 2, which is characterized by further comprising an RNA extraction reagent of the serum exosome, a genome DNA removal reaction system, a reverse transcription reaction system and a qPCR reaction system.
4. A method for detecting serum exosome RP11-470P21-2, characterized in that the kit of claim 2 or 3 is used, comprising:
extracting exosomes in serum 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.
5. The method for detecting serum exosome RP11-470P21-2 according to claim 4, wherein the conditions for removing genomic DNA 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.
6. The method for detecting the serum exosome RP11-470P21-2 as claimed in claim 4, wherein the method for extracting exosomes in the serum 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 ℃.
7. The method for detecting serum exosome RP11-470P21-2 as claimed in claim 4, wherein the serum exosome samples to be detected are from gastric cancer patients and normal persons.
8. The primer of claim 1 or the kit of any one of claims 2 to 3, which is used as a reagent for diagnosing and treating liver metastasis of gastric cancer.
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|---|---|---|---|---|
| CN106701964A (en) * | 2017-01-17 | 2017-05-24 | 中国人民解放军第三军医大学第附属医院 | Serum exosome miRNA biological marker and kit for early diagnosis of gastric cancer |
| CN107488740A (en) * | 2017-10-24 | 2017-12-19 | 南阳师范学院 | Detect the LncRNA combinations of stomach cancer prognosis situation and the kit containing the combination |
| CN108277282A (en) * | 2018-03-07 | 2018-07-13 | 镇江市第人民医院 | Detect primer, kit and detection method and its application of circular rna hsa_circ_0006148 |
| 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 (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701964A (en) * | 2017-01-17 | 2017-05-24 | 中国人民解放军第三军医大学第附属医院 | Serum exosome miRNA biological marker and kit for early diagnosis of gastric cancer |
| CN107488740A (en) * | 2017-10-24 | 2017-12-19 | 南阳师范学院 | Detect the LncRNA combinations of stomach cancer prognosis situation and the kit containing the combination |
| CN108277282A (en) * | 2018-03-07 | 2018-07-13 | 镇江市第人民医院 | Detect primer, kit and detection method and its application of circular rna hsa_circ_0006148 |
| 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: 20201023 |