AU2021103717A4 - An Exosomal CircRNA PVT1 As A Diagnostic Marker For Gastric Cancer - Google Patents
An Exosomal CircRNA PVT1 As A Diagnostic Marker For Gastric Cancer Download PDFInfo
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Abstract
of Descriptions
The invention belongs to the field of biomedical technology, and specifically
relates to the application of exosomal circRNA PVT1 as a diagnostic marker for
gastric cancer. Successfully separated and identified plasma exosomes. The exosomal
circRNA PVT1 in the gastric cancer group was significantly increased compared with
the control group (P<0.05). The diagnostic value of exosomal circRNA PVT1 is
better than traditional serum markers. The expression level of exosomal circRNA
PVT1 in the plasma of patients with gastric cancer is related to a number of
clinicopathological features. Exosomal circRNA PVT1 can be used to distinguish
gastric cancer patients with different clinicopathological characteristics. By detecting
the expression level of this marker, it can not only judge whether the patient has
gastric cancer, but also make a preliminary judgment on the condition of gastric
cancer patients, which has significant clinical application prospects.
Description
Descriptions
An exosomal circRNA PVT1 as a diagnostic marker for gastric
cancer
Technical field:
The invention belongs to the technical field of biomedicine, and specifically
relates to the application of exosomal circRNA PVT1 as a diagnostic marker for
gastric cancer.
Background technique:
According to the latest GLOBOCAN database, gastric cancer (GC) has become
the world's fifth most common cancer and the third most common cause of death from
cancer, threatening human life and health. Gastric cancer has the characteristics of
insidious onset, easy metastasis, early missed diagnosis and high recurrence rate.
Many patients are already in the advanced stage of gastric cancer when they are
diagnosed, and the prognosis is poor. However, the clinically commonly used tumor
markers such as CEA, sugar chain antigen CA19-9, CA72-4, etc., are not specific and
sensitive for screening gastric cancer. Therefore, it is very urgent to introduce new and
more reliable non-invasive diagnostic markers for gastric cancer.
In the early 1980s, Trams et al. discovered exosomes during the maturation of
reticulocytes. It is a vesicle with a double membrane structure formed by most cells
(including platelets, T cells and cancer cells) through a series of regulatory processes
such as "endocytosis-fusion-efflux", with a diameter of about 50-150nm. In recent
years, a large number of studies have found that almost all types of cells can secrete
such vesicles, which play an important role in the exchange of information between
Descriptions
cells, and have confirmed that they play an important role in the occurrence and
development of tumors. Because exosomes have good secretion characteristics and
stable vesicle-like structure, and contain a large number of disease-specific proteins
and nucleic acids, they can be detected in a variety of body fluids. Therefore, the
internal components of exosomes are expected to become new tumor diagnostic
molecular markers.
Circular RNA (circRNA) is a type of non-coding RNA molecule that is different
from traditional linear RNA. It was first discovered in RNA viruses in 1976. Due to
the limitations of technology at that time, circRNA was only considered to be a type
of low-abundance RNA formed by wrong splicing of exon transcripts, so the research
on them was not thorough enough. In recent years, with the wide application of
high-throughput sequencing technology and the rapid development of bioinformatics,
circRNA has been found to be stable, abundant and ubiquitous in mammals. And the
circRNA present in the body will be differentially expressed in different tissues or the
same tissue but the body is at different stages of disease development, and has cell
phenotype specificity and developmental stage specificity. CircRNA does not have a
'end cap and 3'end poly A tail structure, but they are connected to form a covalently
closed loop structure, which makes the expression in the cell more stable and is not
easily degraded by exonuclease. Some researchers detected the existence of circRNA
in exosomes in 2015, and circRNA plays an important regulatory role in a variety of
diseases, especially tumors, with its high conservation and strong stability, which
makes exosomal circRNA have great potential in the diagnosis of tumor diseases.
Descriptions
Although in previous studies, the regulatory role of exosomal circRNA in gastric
cancer has been extensively studied, the significance of circulating exosomal circRNA
in peripheral blood for molecular diagnosis of gastric cancer is still unclear. Finding
new and suitable exosomal circRNAs and exploring them as biomarkers for the
diagnosis and judgment of gastric cancer is crucial for assisting clinical screening and
diagnosis of gastric cancer.
Summary of the invention:
In view of the problems in the prior art, the purpose of the present invention is to
provide a circRNA marker for diagnosis and treatment of gastric cancer and its
application. The present invention uses experiments to prove that the relative
expression of exosomal circRNA PVT1 in the gastric cancer group is significantly
higher than that in the control group (P<0.05). The diagnostic performance of
exosomal circRNA PVT1 for gastric cancer is better than traditional serum
biomarkers, and it also has a certain diagnostic value for gastric cancer patients with
different clinical pathological characteristics. Therefore, exosomal circRNA PVT1
can be used as a molecular marker for the diagnosis and treatment of gastric cancer.
In order to achieve the above objective, the present invention adopts the
following technical solutions.
The circular RNA involved in the present invention is named circRNA PVT1 or
hsacirc_0001821 in NCBI. CircRNA PVT1 nucleotide sequence fragments (SEQ ID
NO. 1) of the present invention is:
Descriptions
The upstream primer sequence circRNA PVT1-F (SEQ ID NO.2) is:
CGACTCTTCCTGGTGAAGCATCTGAT, the downstream primer sequence
circRNA PVT1-R (SEQ ID NO. 3) is: TACTTGAACGAAGCTCCATGCAGC.
The invention relates to a plasma/serum exosome extraction kit, an RNA
extraction kit, a reverse transcription kit and a fluorescence quantitative kit. The
identification of exosomes involves transmission electron microscopy, NTA
(Nanoparticle Tracking Analysis) particle size analyzer and Western blot.
The invention adopts a special plasma/serum exosome extraction kit to separate
and extract exosomes. Real-time fluorescent quantitative polymerase chain reaction
(qRT-PCR) technology was used to analyze the expression and difference of gastric
cancer-specific circRNA PVT1 in the plasma exosomes of individual gastric cancer
patients and benign gastric disease and healthy individuals. Exosomal circRNA PVT1
shows a high diagnostic value for gastric cancer screening and disease judgment.
The positive effects of the present invention are: The plasma exosomal circRNA
PVT1 is disclosed as a diagnostic marker for gastric cancer. Successfully separated
Descriptions
and identified plasma exosomes. The level of individual plasma exosomal circRNA
PVT1 in gastric cancer patients was significantly higher than that in controls (P<
0.05). The diagnostic performance of exosomal circRNA PVT1 for gastric cancer is
better than traditional serum biomarkers, and it also has a certain diagnostic value for
gastric cancer patients with different clinical pathological characteristics.
Description of the figures:
Figure 1 is the characterization of exosomes.
Figure 2 is the show of the relative expression results of exosomal circRNA
PVT1 preliminary experiments. Note: The upper limit, lower limit of the box and the
line in the box indicate the 75th percentile, the 25th percentile and the median value
*P< 0.05, **P < 0.01.
Figure 3 is the show of the relative expression levels of exosomal circRNA PVT1,
serum CA72-4, CEA and CA19-9 in 50 patients with gastric cancer and 40 controls.
Note: The upper limit, lower limit of the box and the line in the box indicate the 75th
percentile, the 25th percentile and the median value *P < 0.05, **P < 0.01.
Detailed Description of the Presently Preferred Embodiments
The present invention will be further described in detail below with reference to
the drawings and embodiments. The experimental methods that do not indicate
specific conditions in the examples usually follow conventional conditions, such as
the conditions described in textbooks and experimental guides, or the conditions
Descriptions
suggested by the manufacturer, which are well known or easily known to those of
ordinary skill in the art. The following embodiments are only preferred embodiments
of the present invention, and do not limit the present invention. For those skilled in
the art, the present invention can have various modifications and changes. Any
modification, equivalent replacement, improvement, etc., made within the spirit and
principle of the present invention shall be included in the protection scope of the
present invention.
Example research experiment of circRNA PVT1 in molecular diagnosis of gastric
cancer.
1 Materials
1.1 Research Objects
From May 2019 to May 2020, 50 patients were diagnosed with gastric cancer in
Qingdao University Affiliated Hospital, Qingdao, aged 42-79 years old, with an
average age of (61.609.66) years old, including 27 males and 23 females.
Gastroscopy was performed in our hospital during the same period, 20 patients with
benign pathology and 20 healthy subjects (no obvious abnormalities in blood routine,
biochemical, tumor markers, etc.) were selected as the control group. They were 38 to
74 years old, with an average of (58.43+8.24) years old, including 18 males and 22
females. Among them, 12 cases were superficial gastritis and 8 cases were gastric
polyps. All the included cases were diagnosed to exclude gastric cancer. There was no
statistical difference in age and gender between the gastric cancer group and the
control group, and peripheral blood plasma was collected as experimental samples.
Descriptions
According to the American Joint Committee on Cancer (AJCC) and the Union for
International Cancer Control (UICC) in 2009, the TNM staging standards for
malignant tumors were staged for gastric cancer patients, including 22 cases of stage I
to II , 28 cases of stage III to IV.
1.2 Data Collection
Adopting retrospective analysis method, using Hospital Information System (HIS)
and Laboratory Information System (Laboratory Information System, LIS) to collect
gastric cancer patient information including: specimen number, name, age, gender,
hospitalization registration number, specimen collection date, specimen period
(preoperative or postoperative specimens), operation date, diagnosis, tumor size,
degree of differentiation, TNM staging, lymph node metastasis, liver metastasis,
peritoneal metastasis, traditional tumor markers (CA72-4, CEA, CA19-9) results.
Inclusion criteria: All cases were diagnosed as primary gastric cancer by
histopathological examination, and no other malignant tumors or organ failure were
combined. None of the patients experienced acute infection, acute cardiovascular and
cerebrovascular diseases, and severe trauma within 3 months, and none of them
received any tumor-related surgery, radiotherapy, chemotherapy, or immunotherapy
history.
Exclusion criteria: patients with unstable vital signs; patients during pregnancy
and lactation; patients with RNA virus infection, such as influenza virus, hepatitis A,
hepatitis C, hepatitis E virus, etc.; suffering from other diseases that may affect the
determination of circRNA expression level (such as neuro system diseases, ischemic
Descriptions
stroke, etc.); some common diseases (chronic hepatitis, pancreatitis, etc.) that can
cause changes in CA72-4, CEA and CA19-9 indicators.
1.3 Reagents and Instruments
Plasma exosome extraction kit: HieffTM Quick exosome isolation kit (for
Serum/Plasma) (Yeasen, China). RNA extraction kit: Trizol LS Reagent (Invitrogen
life technologies, USA). Reverse transcription PCR: Goldenstar RT6 cDNA
Synthesis Kit Ver.2 (TSINGKE, China). Real-Time PCR: 2xTSINGKE Master qPCR
Mix-SYBR(+UDG) (TSINGKE China). Chloroform, absolute ethanol.
-80°C refrigerator (Panasonic, Japan). High-speed refrigerated centrifuge
(Thermo, USA). BioDrop UV spectrophotometer (Baidian, UK). TOOTM Thermal
Cycler PCR machine (Bio-Rad, USA). CFX ConnectTM Real-Time System
fluorescence quantitative PCR instrument (Bio-Rad, USA).
2 Methods
2.1 Plasma Sample Collection and Processing
Before using any treatment, collect 5 mL of EDTA peripheral blood from all
subjects, centrifuge at 3,000xg for 5 min, separate the plasma, aliquot it into
RNAse-free centrifuge tubes, and store it at -80°C. Before extraction of exosomes,
thawed or fresh plasma was centrifuged at 3,000xg for 10 min and 10,000xg for 20
min at 4°C, then the precipitate was discarded and the supernatant was retained.
2.2 Selection of Target circRNA
The circRNA PVT1 that has been functionally confirmed in gastric cancer related
research is determined by consulting the literature and related data database.
Descriptions
2.3 Determination of primer sequence
Design the primer sequence of the target gene and internal reference gene by
consulting the literature or database, as shown in Table 1.
Table 1 Primer Sequence of circRNA PVT1 and GAPDH
Primer name Direction Primer sequence (5' - 3')
GAPDH Forward GTCGTGGAGTCTACTGGCGTCTTCA
Reverse TCGTGGTTCACACCCATCACAAACA
circPVT1 Forward CGACTCTTCCTGGTGAAGCATCTGAT
Reverse TACTTGAACGAAGCTCCATGCAGC
2.4 Extraction and Identification of Exosomes from Plasma Samples
(1) Preparation before experiment: Aseptic preparation. Take out the samples and
reagents and place them at room temperature (15°C-25°C), and thaw them for later
use.
(2) Add 4 times the volume of 1xPBS to the pretreated sample and mix well.
(3) In the sample diluted with PBS, continue to add the corresponding amount of
41202-A reagent; cover the centrifuge tube cap tightly, vortex for 1 min, and place in
a refrigerator at 4°C for 2 h.
(4) Take out the centrifuge tube containing the mixed solution, centrifuge at
10,000xg for 60 min at 4°C, discard the supernatant (absorb the supernatant as much
as possible), and collect the exosomes-rich precipitate.
(5) Take a certain volume of 1xPBS solution and blow the centrifuged sediment
evenly, and after it is fully suspended in PBS, transfer the suspension to a new 1.5ml
Descriptions
centrifuge tube.
(6) Centrifuge the 1.5ml centrifuge tube containing the resuspension solution at
4°C at 12,000xg for 2 minutes, discard the precipitate, and retain the supernatant,
which is a solution rich in exosomal particles.
(7) Use transmission electron microscope, NTA particle size analyzer and
Western blot to identify the characteristics of the extracted serum exosomes. As
shown in Figure 1, under the transmission electron microscope, the serum exosomes
were found to be a hemispherical concave on one side. CD81 and Alix proteins are
protein markers located on and in the membrane of exosomes, respectively, which can
be successfully displayed by Western blot verification. NTA shows that the size of
exosomes is about 60-150nm.
2.5 Total RNA Extraction of Exosomes from Plasma Samples
(1) Preparation before experiment: aseptic preparation. Take out the samples and
reagents and place them at room temperature (15°C-25°C), and thaw them for later
use.
(2) Homogenization: Take out the exosomes solution sample from the -80°C
refrigerator. After thawing, centrifuge at 12,000xg for 10 min at 4°C to remove
possible impurities. Take 250ul sample, transfer to a 1.5ml centrifuge tube, 750pl
Trizol reagent and 20pl glacial acetic acid, manually shake the tube vigorously to mix.
(3) Two-phase separation: After homogenization, the sample is incubated at 15 to
°C for 5 minutes to allow the nucleic acid protein complex to dissociate completely.
Add 0.2ml of chloroform to every 750d1of Trizol reagent homogenized sample and
Descriptions
close the tube cap tightly. After shaking the tube vigorously by hand for 15 seconds,
incubate at 15 to 30°C for 2 to 3 minutes. Centrifuge at 12,000xg for 15 min at 4°C.
After centrifugation, the mixed liquid will be divided into the lower red
phenol-chloroform phase, the middle layer and the upper colorless water phase. The
RNA is all distributed in the water phase. The volume of the water phase is
approximately 60% of the Trizol added during homogenization.
(4) RNA precipitation: transfer the aqueous phase to a new centrifuge tube, and
add 500 1 isopropanol, and mix well to precipitate the RNA. After mixing, incubate
at 15 to 30°C for 10 minutes, and centrifuge at 12,000xg for 10 minutes at 4°C. At
this time, the RNA precipitate that is not visible before centrifugation will form a
gel-like precipitate on the bottom and side walls of the tube.
(5) RNA washing: remove the supernatant, add at least 1 ml of 75% ethanol to the
sample homogenized with 750 1 Trizol reagent to wash the RNA precipitate. After
shaking, centrifuge at 7,500xg for 5 min at 4°C.
(6) Re-dissolve the RNA precipitate: remove the ethanol solution, dry the RNA
precipitate in the air for 5-10 minutes, do not dry it by vacuum centrifugation. Note
that the RNA precipitation should not be completely dried, otherwise the solubility of
RNA will be greatly reduced. The A260/280 ratio of partially dissolved RNA samples
will be less than 1.6. When dissolving RNA, first add RNase-free water and pipe
repeatedly with a gun, and then incubate at 55 to 60°C for 10 minutes. The obtained
RNA solution was stored at -80°C.
2.6 Reverse Transcription Reaction
Descriptions
(1) Preparation before experiment: aseptic preparation; take out samples and
reagents and thaw on ice for later use.
(2) System configuration: The reaction solution is configured on ice. In order to
ensure the accuracy of the reaction solution prepared by subassembly, the reaction
solution is prepared in a volume slightly larger than the amount of reagents (the
number of samples n+2). The system preparation ratio is carried out according to the
instructions.
(3) Use T100TM Thermal Cycler PCR instrument for RT-PCR reaction. The
reaction conditions are set as follows: 25°C, 10min; 55°C, 15min; 85°C, 5min.
(4) Temporarily store the obtained cDNA sample at 4°C.
2.7 Real-Time PCR Reaction
(1) Preparation before experiment: aseptic preparation. The primer melts. Warm
up the fluorescent quantitative PCR machine.
(2) System configuration: The reaction solution is configured on ice. In order to
ensure the accuracy of the reaction solution prepared by subassembly, the reaction
solution is prepared in a volume slightly larger than the amount of reagents (the
number of samples n+2). The system preparation ratio is carried out according to the
instructions. When adding the cDNA template, pipetting with a sampler repeatedly to
make the components in the system fully mixed.
(3) Test the prepared samples simultaneously. The reaction conditions were set as
follows: 95°C, 2min (pre-denaturation). 95°C, 15sec (denaturation). 60°C, 30sec
(annealing). A total of 40 cycles. Fluorescence signals were collected at 60°C.
Descriptions
(4) Interpretation of amplification curve and melting curve: the reaction
specificity and amplification efficiency are measured by the amplification curve and
melting curve. Adjust the primer concentration and PCR reaction conditions
according to the results.
2.8 Statistical Analysis
We used SPSS 23.0 software for statistical analysis of data. The ACT method was
used to calculate the expression level of circRNA. The higher the ACt value, the lower
the expression level of circRNA. Measurement data are expressed by ( x s), and
comparison between groups is by t test. Enumeration data were expressed by the
number of cases (n) or percentage (%), and the comparison between groups was
performed by the /test. GraphPad Prism 5.0 software and SPSS 23.0 software were
used to draw Receiver Operating Characteristic (ROC) curve and graph. The
difference was statistically significant when P<0.05.
3 Results
3.1 Pre-test Results
In the preliminary experiment, the plasma samples of 5 cases in the gastric cancer
group and 10 cases in the control group (5 cases of benign gastric disease and 5 cases
of healthy physical examination) were selected for the experiment. The target gene
was circRNA PVT1 and the internal reference gene was GADPH. The material
method and PCR reaction conditions are as above.
As shown in Figure 2, the relative expression of the target gene circRNA PVT1 in
plasma exosomes of patients with gastric cancer was significantly higher than that of
Descriptions
patients with benign gastric disease and healthy subjects (all P<0.05). There was no
significant difference in the expression level of plasma exosomes in patients with
benign gastric disease and healthy subjects. Therefore, patients with benign gastric
disease and healthy physical examination were selected as the control group, and
circRNA PVT1 was determined as the target gene for follow-up experiments.
3.2 Relative Expression Results of Exosomal circRNA PVT1 in Gastric Cancer
Group and Control Group
As shown in Figure 3, the relative expression level of plasma exosomal circPVT1
in the gastric cancer group was significantly higher than that in the control group, and
the difference was statistically significant (P<0.05). Similarly, the levels of serum
CA72-4, CEA and CA19-9 in the gastric cancer group were also significantly higher
than those in the control group, and the differences were statistically significant (all
P<0.05). The results show that exosomal circRNA PVT1 can achieve the effect of
screening for gastric cancer.
3.3 ROC Curve Analysis of Plasma Exosomal circRNA PVT1 for Screening Gastric
Cancer
3.4 Correlation between the Relative Expression of Plasma Exosomal circRNA PVT1
in Gastric Cancer Group and Clinicopathological Parameters in Patients with Gastric
Cancer
The expression levels and clinicopathological characteristics of plasma exosomal
circRNA PVT1 are shown in Table 2. The expression level of plasma exosomal
Descriptions
circRNA PVT1 was significantly correlated with gender, tumor diameter, TNM stage,
lymph node metastasis and peritoneal metastasis (all P < 0.05). The results show that
the expression level of plasma exosomal circRNA PVT1 is related to a number of
clinicopathological parameters in patients with gastric cancer.
Table 3 The relationship of exosomal circRNA PVT1 expression levels (ACt) in
plasma from GC patients and clinicopathological factors of GC patients.
circPVT1 Factors No. of patients(%) Ma+DPvau Mean±SD P value Age(years) <60 17(34.0) 2.86+0.47 0.21 >60 33(66.0) 3.07+0.62 Gender Male 27(54.0) 2.83+0.51 0.028 Female 23(46.0) 3.19+0.61 Diamater(cm) >5 28(56.0) 2.73+0.36 <0.01 <5 22(44.0) 3.34+0.63 TNM stage I-II 22(44.0) 3.28+0.54 <0.01 III-IV 28(56.0) 2.78+0.52 Lymphatic metastasis Positive 23(46.0) 2.78+0.41 0.010 Negative 27(54.0) 3.19+0.64 Liver metastasis Positive 12 (24.0) 3.01+0.59 0.916 Negative 38 (76.0) 2.99+0.58 Peritoneal metastasis Positive 19 (38.0) 2.61+0.48 <0.01 Negative 31 (62.0) 3.23+0.51 CA72-4 Positive 22(44.0) 2.98+0.56 0.847 Negative 28(56.0) 3.01+0.60 CEA Positive 29(58.0) 3.07+0.63 0.334 Negative 21(42.0) 2.91+0.51 CA19-9 Positive 23(46.0) 3.02+0.61 0.814 Negative 27(54.0) 2.98+0.57
Descriptions
Note: Statistical significance (P < 0.05) is shown in bold.
3.5 ROC curves of plasma exosomes circRNA PVT1 for distinguishing patients with
gastric cancer in different groups
The ROC curve was used to analyze the diagnostic value of plasma exosomal
circRNA PVT1 in distinguishing different groups of patients with gastric cancer.
Claims (5)
1. A gastric cancer diagnostic marker is characterized in that the
diagnostic marker is exosomal circRNA PVT1, and its nucleotide
sequence is shown in SEQ ID NO.1.
2. The application according to claim 1, wherein the detection
reagents include: a plasma/serum exosome extraction kit, an RNA
extraction kit, a reverse transcription kit, and a fluorescence
quantification kit.
3. The application according to claim 1, characterized in that the
identification of exosomes includes: transmission electron microscopy,
NTA (Nanoparticle Tracking Analysis) particle size analyzer and Western
blot.
4. The application according to any one of claims 1 and 2, wherein
the upstream primer sequence circRNA PVT1-F of circRNA PVT1 is
shown in SEQ ID NO. 2, and the downstream primer sequence circRNA
PVT1-R is shown in SEQ ID NO. 3.
5. The application according to claims 1-4, wherein the detection
equipment for exosomal circRNA PVT1 includes: a high-speed
refrigerated centrifuge, an ordinary PCR machine, and a real-time
fluorescent quantitative PCR machine.
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