CN109234401B - Molecular marker for diagnosing gastric adenocarcinoma - Google Patents

Molecular marker for diagnosing gastric adenocarcinoma Download PDF

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CN109234401B
CN109234401B CN201811431328.3A CN201811431328A CN109234401B CN 109234401 B CN109234401 B CN 109234401B CN 201811431328 A CN201811431328 A CN 201811431328A CN 109234401 B CN109234401 B CN 109234401B
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荣大伟
曹红勇
唐薇薇
付凯
卢琛
郑吴彬
江伟
王路明
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Abstract

The invention relates to a molecular marker circ _ PSMC3 for diagnosing gastric adenocarcinoma, and the nucleotide sequence of the molecular marker circ _ PSMC3 is shown in SEQ ID NO. 1. The expression level of circ _ PSMC3 in a patient blood sample is detected through qRT-PCR, early diagnosis and prognosis evaluation of gastric adenocarcinoma can be realized, and specific primers for detecting the molecular markers comprise an upstream primer shown in SEQ ID NO.2 and a downstream primer shown in SEQ ID NO. 3. The expression level of the molecular marker in a gastric adenocarcinoma patient is remarkably lower than that of a normal person by combining early-stage chip screening with clinical large-sample verification. The clinical detection only needs to collect trace peripheral blood, has small wound, is easy to be accepted by an examinee, can become an effective means for early diagnosis, disease progress judgment and prognosis evaluation of gastric adenocarcinoma patients, and the index has strong specificity, high sensitivity and stable result when being used for gastric adenocarcinoma diagnosis.

Description

Molecular marker for diagnosing gastric adenocarcinoma
Technical Field
The invention relates to a molecular marker for diagnosing gastric adenocarcinoma, belonging to the technical field of biology.
Background
Gastric cancer is one of the most common malignant tumors that seriously threaten human health, and there are two main pathological types: adenocarcinoma and other types. Most stomach cancers are adenocarcinomas with a distinct geographic difference in their distribution. The incidence rate of gastric cancer in China is at a high level all over the world, the prognosis is poor, and the overall survival rate in 5 years is low. In 2016, the clinician journal of cancer reports that gastric cancer has become one of the five most prevalent tumors in China, with the prevalence ranking second in men and third in women.
At present, the diagnosis rate of gastric adenocarcinoma is very low, clinical common diagnosis technologies such as gastroscope, upper gastrointestinal angiography, CT and the like are difficult to popularize due to higher examination cost and certain pain and risk to be born, and tumor markers such as CEA and the like in conventional serological examination are not satisfactory in terms of sensitivity and specificity, so that the search for novel tumor markers is an important subject in the field of tumor research.
Circular RNA (circRNAs) is a new endogenous non-coding RNA, which was identified as transcripts of out-of-order exons in the early 90 s of the 20 th century, and its structure, function and mechanism were reported in succession, and became a research hotspot in the next 20 years. Unlike traditional linear RNAs that terminate in 5 'and 3' ends, circRNAs are special closed loop structures lacking a 5 'end cap and a 3' poly (a) tail, and have higher stability and sequence conservation compared to micro RNAs (mirnas) and long non-coding RNAs (lncrnas) in mammalian cells because of their nuclease resistance. A number of recent studies have reported the expression of circRNAs in different species. Based on the rapid development of bioinformatics analysis and high throughput sequencing technology, researchers find tens of thousands of circRNAs, and find that they are involved in the development of diseases such as vasculopathy, nervous system diseases, tumors, and the like. The CircRNAs chip analysis technique has been applied to many tumors including digestive system tumors, nervous system tumors, urinary system tumors, head and neck tumors, etc., such as pharyngeal squamous cell carcinoma, laryngeal squamous cell carcinoma, basal cell carcinoma, pancreatic ductal adenocarcinoma, esophageal carcinoma, hepatocellular carcinoma, papillary thyroid carcinoma, etc. However, in gastric cancer, plasma circRNAs are rarely reported.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide a molecular marker for diagnosing gastric adenocarcinoma, which judges whether a patient has gastric adenocarcinoma or the risk of the patient with the gastric adenocarcinoma by detecting the expression level of the molecular marker, so as to guide a clinician to perform early intervention and treatment and improve the survival rate and the quality of the patient.
Technical scheme
A molecular marker for diagnosing gastric adenocarcinoma is circ _ PSMC3, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1.
The circular RNA has the characteristics of mechanism stability, abundance degree, sample specific expression and the like, and the inventor finds that the expression of circ _ PSMC3 in blood samples of gastric adenocarcinoma patients and normal people is different and is related to staging by detecting the expression level of circ _ PSMC3 in the blood samples of gastric adenocarcinoma patients and normal people.
The specific primers for detecting the molecular markers comprise an upstream primer shown by SEQ ID NO.2 and a downstream primer shown by SEQ ID NO. 3.
The sequence of the upstream primer is as follows: 5'-GTTTAGGGTCCCTGCCCTTTG-3', respectively;
the sequence of the downstream primer is as follows: 5'-GTGTTGGGCTGGAAGCCATC-3' are provided. The specificity of primer amplification was verified by sequencing the PCR product of the primer.
The application of the specific primer in preparing or screening gastric adenocarcinoma diagnostic drugs.
A diagnostic kit for gastric adenocarcinoma comprises the specific primer.
Has the advantages that: the invention provides a molecular marker circ _ PSMC3 for gastric adenocarcinoma diagnosis, wherein the expression level of the circ _ PSMC3 in a blood sample of a patient is detected through qRT-PCR (quantitative reverse transcription-polymerase chain reaction), so that early diagnosis and prognosis evaluation of gastric adenocarcinoma can be realized, early-stage chip screening is combined with large clinical samples to verify and find, the expression level of the circ _ PSMC3 molecular marker in a gastric adenocarcinoma patient is obviously lower than that of a normal person, and the expression level is highly related to clinical stages and the survival period of the patient. The clinical detection only needs to collect trace peripheral blood (5ml), has small wound, is more easily accepted by the examinee, can become an effective means for early diagnosis, disease progress judgment and prognosis evaluation of gastric adenocarcinoma patients, and reflects that the specificity and the sensitivity of the index for gastric adenocarcinoma diagnosis are high by ROC curve analysis, wherein the AUC value is 0.9326, and P is less than 0.0001. Stable result and wide clinical application prospect.
Drawings
FIG. 1 is a schematic diagram of the structure of circ _ PSMC 3;
FIG. 2 is a ROC curve of circ _ PSMC3 for gastric adenocarcinoma patients versus healthy population;
FIG. 3 shows the expression level of circ _ PSMC3 in the plasma of gastric adenocarcinoma patients and healthy people detected by qRT-PCR;
FIG. 4 shows the expression level of circ _ PSMC3 in cancer tissues and paracancerous tissues of patients with gastric adenocarcinoma;
FIG. 5 is a survival curve analysis of the correlation of circ _ PSMC3 expression levels with patient survival.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
Example 1
1. Case selection
Study patient tissues and blood were derived from patients with gastric adenocarcinoma who received radical or palliative surgical resection at the Nanjing Hospital affiliated to Nanjing medical university during the period from 2013 to 2016 8 months. The normal control blood samples are all from the health examination population in the first hospital in Nanjing, and the health control population excludes the history of malignant tumors and the history of gastrointestinal related diseases. All specimens in the experiment are signed by patients or clients thereof to give informed consent, and all researches related to human body specimens are approved by the ethical committee of the first hospital in Nanjing City.
Inclusion criteria for study patients were as follows:
a. a post-operative pathology confirmed as gastric adenocarcinoma (clinical staging according to the 7 th edition (2009) stomach adenocarcinoma TNM staging criteria);
b. patients who received radical or palliative resection during hospitalization were excluded from preoperative chemoradiotherapy;
c. the current medical history, family history, personal history and various inspection data are complete.
In this study, there were 106 blood samples from patients with GC cancer and 21 blood samples from normal persons.
The clinical data collection is mainly divided into gastric adenocarcinoma patients and normal persons, and the general data are required to be included: including the patient's name, gender, age, occupation, past medical history, family history, female menstrual history, and marriage and childbirth history. The gastric adenocarcinoma patients are mainly obtained by the hospitalization data of the gastric adenocarcinoma patients. The clinical characteristics of the tumor include histological type, location, size, infiltration degree, lymph node metastasis, tumor cell differentiation degree, tumor TNM stage, operation time, radiotherapy and chemotherapy after operation, and conventional auxiliary examination. Meanwhile, follow-up visit agreement of all people is obtained, and the inspection result data is perfectly stored.
2. Specimen collection
Is the collection of blood, and each gastric adenocarcinoma patient takes blood once (5 ml/tube) before operation for subsequent experimental study. All blood samples should be protected from hemolysis. Wherein the blood collecting tube for collecting the plasma sample is an Ethylene Diamine Tetraacetic Acid (EDTA) anticoagulation tube, and the whole blood sample is centrifugally collected and frozen within 0.5 h; the blood collecting tube for collecting the serum sample is a conventional biochemical procoagulant tube, and the sample can be separated out, centrifuged and frozen in 2 h. The whole blood sample was centrifuged in two steps to remove the influence of cellular components, first at 2000g, 4 ℃ for 10min, then the supernatant was carefully transferred to a new EP tube, then 12000g, 4 ℃ for 10min, and finally plasma and serum samples were dispensed (400. mu.l/tube) and placed in a-80 ℃ freezer for long-term storage.
3. Total RNA extraction
The mirVana PARIS Kit (Ambion1566, USA) is used for extracting total RNA from plasma and serum, and the specific steps are as follows:
1) all reagents need to be recovered to be used at room temperature, a plasma/serum sample is taken out and melted on an ice surface for standby, and eluent is preheated at 95 ℃ for standby;
2) adding 400 mul of plasma/serum sample into an EP tube added with 2X Denaturing Solution with the same volume, and fully shaking and uniformly mixing;
3) adding Acid-Phenol of the same volume as Chloroform, shaking and mixing for 1min, and centrifuging at room temperature at 10000g for 5 min;
4) carefully transfer the upper aqueous phase to a new EP tube and record the volume of transferred aqueous phase, then add 1.25 volumes of 100% ethanol to the EP tube, transfer the lysate/ethanol mixture through a filter after mixing well (10000g centrifuge for 30 s);
5) add 700. mu.l mi RNA Wash Solution 1 filter 1 times (10000g centrifugation 15s), then add 500. mu.l Wash Solution 2/3 filter 2 times (10000g centrifugation 15 s);
6)50 μ l RNA on 95 deg.C preheated non-ribonuclease water elution filter (10000g centrifugation 30s), -80 deg.C long term storage;
7) and measuring the concentration and purity of the sample by using an ultraviolet spectrophotometer.
RNA reverse transcription and qPCR amplification
(1) Configuration of reverse transcription System
Taking out the reverse transcription kit from the refrigerator at-20 deg.c, inserting 2 kinds of enzyme into ice box, thawing other reagent ice, and RNase Free H2Melting the O at normal temperature for later use.
Secondly, calculating the volume of RNA required by reverse transcription of each tube according to the concentration of the RNA to be measured, and simultaneously calculating the volume of DEPC water. The total volume of RNA and DEPC water cannot exceed 7 ul. Note that: RNA is extremely easy to degrade by repeated freezing and thawing, and once the quality of the RNA extracted from a sample is determined to be better, the RNA is completely reverse transcribed into cDNA at one time as far as possible according to the cancer and cancer side pairing standard. If the RNA amount of the matched sample is large and exceeds 10 parts of reverse transcription amount, the reverse transcription can be performed only in 10 parts in consideration of the cost of the reverse transcription.
Thirdly, placing the PCR tube frame on ice, placing the PCR tube frame into a sterile 200ul PCR tube according to the requirement and marking the PCR tube frame according to the number. After a pipette sucks a proper amount of RNA, the pipette tip is vertically inserted into the bottom of the PCR tube, and liquid is gently beaten out at a constant speed, so that bubbles can be reduced or even not generated, and no obvious residue of the pipette tip is confirmed after the liquid is beaten out.
Fourthly, a pipette sucks a proper amount of DEPC water, and the gun head is stuck to the rear wall and is driven into the PCR tube. Because the previous sample RNA is vertically added into the bottom of the tube, the peripheral side walls of the tube can be regarded as uncontaminated areas, liquid can be slowly ejected at a constant speed by adhering to the front/rear/left/right side walls of the eight-connected tube at any angle by the habit of the user, and bubbles can be reduced or even not generated.
Reverse transcription Step 1: add gDNAeraser 1ul and 5x gDNA Eraser Buffer 2ul per tube. And (4) respectively sticking the side walls in different directions for adding according to the operation of the step 4.
Placing on an eight-tube centrifuge, slightly bouncing off bubbles at the bottom or on the wall of the tube, and centrifuging for a short time.
Seventhly, placing the reaction product on ice for later use after the reaction is carried out for 2 minutes at 42 ℃ on an RT-PCR instrument.
B, reverse transcription Step 2: 1.5ml of EP tube was used, and each tube was prepared according to the following formulation
5x PrimerScript Buffer2 4ul
Rnase Free dH2O 4ul
PrimerScript RT Enzyme Mix I 1ul
RTPrimer Mix 1ul
Setting reaction conditions: at 37 deg.C for 15min, at 85 deg.C for 5sec, and at 4 deg.C.
Ninthly, fastening the pipe cover, taking the pipe cover off the pipe support, placing the pipe support in a centrifuge, flicking the bottom of the pipe by a person with bubbles, centrifuging for a short time, confirming that the liquid level of each pipe is consistent and the pipe has no bubbles, and placing the pipe cover on ice again.
Operating on the R, setting reaction condition, storing cDNA at-20 deg.C after 15-20 min.
(2) QPCR amplification
The specific primer for detecting the molecular marker circ _ PSMC3 comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is as follows: 5'-GTTTAGGGTCCCTGCCCTTTG-3' (SEQ ID NO.2), and the sequence of the downstream primer is: 5'-GTGTTGGGCTGGAAGCCATC-3' (SEQ ID NO. 3).
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control, GAPDH _ F: 5'-TGCACCACCAACTGCTTAGC-3', GAPDH _ R: 5'-GGCATGGACTGTGGTCATGAG-3' are provided. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the results.
Preparing an ice box, taking the cDNA, the primers and the PCR reagent (SYBR Premix Ex TaqTM II and ROC Reference Dye II) of a sample to be detected out of a refrigerator at the temperature of-20 ℃, and unfreezing on ice.
Preparing eight-linked pipes, pipe covers, eight-linked pipe racks, tweezers, sterile 1.5ml EP pipes, EP pipe racks, pipettors with appropriate measuring ranges (2ul and 20ul are necessary measuring ranges) and sterile film gloves.
Thirdly, placing the unfrozen cDNA, the primers and the PCR reagent on an oscillator in sequence for short-time oscillation, then placing the oscillator on a centrifuge for short-time centrifugation, and standing on ice for later use.
Taking 1.5ml of EP tube, preparing the eight-tube PCR Mix (according to the total volume of 19 ul) according to the following formula of each hole:
SYBR Premix Ex TaqTM II 10ul
Forward Primer
1ul 3 for simplified operation can be formulated into mixed primers
Reverse Primer
ROC Reference Dye/II 0.4ul*4
cDNA 1ul
dH2O 7.6ul
Reaction conditions are as follows:
ABI 7300/7500 Real-Time PCR System，StepOnePlusTM
Stage1:95℃30sec Reps 1
Stage2:95℃5sec,60℃30-34sec,Reps 40
Stage3:95℃15sec,60℃1min,95℃5sec,Reps 1
note that: stage2 time setting at 60 ℃: 7500 Fast Real-Time PCR System/StepOnePlusTM 30 sec, 7300 31 sec, 7500:34 sec.
Fifthly, adjusting the measuring range of the 2ul pipette to 1ul according to the preset position of each sample and the position of the multiple holes in the eight-connected pipe, and sequentially adding the cDNA of the samples.
Sixthly, the daylight lamp is turned off, all bright light sources are shielded, the ROC Reference Dye/II is added into the EP tube which is placed still before and is used for containing the PCR Mix, the mixture is shaken and evenly mixed, and then the mixture is centrifuged for a short time to ensure that the split mixed Mix is evenly mixed.
Seventhly, adjusting the measuring range of the 20ul pipette to 19ul, sucking the Mix, sequentially attaching the Mix to the rear wall of the eight-link pipe to beat out liquid, and confirming that no obvious residue exists in the gun head after beating each time.
And eighthly, clamping one side edge of the eight-joint pipe cover by using tweezers, and lightly placing the eight-joint pipe cover at the pipe opening. Wearing new sterile film gloves, lightly taking the eight-tube linkage and the eight-tube linkage frame out of the ice box and placing the eight-tube linkage and the eight-tube linkage frame on a table, lightly pressing the tube cover on one side for fixing, and then rapidly and forcefully fastening the tube cover. Marking marks on two ends of the tube cover by the marker pens, lightly buckling bottoms of two ends of the eight-connected tube from bottom to top, taking the eight-connected tube off the tube frame, placing the eight-connected tube on an eight-connected centrifugal frame, slightly bouncing the bottom of the tube by a person with bubbles, centrifuging for a short time, and placing the eight-connected tube on ice after confirming that the liquid level of each tube is consistent and no bubbles exist in the tube.
Ninthly, operating on the computer, setting reaction conditions, adding and running a dissolution curve, determining a target band through dissolution curve analysis and electrophoresis, and performing relative quantification by a delta CT method.
5. Data analysis and results
Correlation of circ _ PSMC3 with GC patients clinical staging data and prognosis using the t-test and chi-square test and survival analysis. Receiver Operating Characteristic (ROC) curves were performed to evaluate their diagnostic value. All statistical analyses were performed using SPSS for Windows v.17.0. For all results, P <0.05 was considered statistically significant.
FIG. 1 is a schematic diagram of the structure of circ _ PSMC3, and it can be seen that circ _ PSMC3 is from PSMC gene and has a length of 502 bp.
Fig. 2 is a ROC curve of circ _ PSMC3 for patients with gastric adenocarcinoma and healthy people, and it can be seen that AUC is 0.9326 (95% CI 0.8862-0.9791, specificity is 95.24%, sensitivity is 85.85%, and P is less than 0.0001), which indicates that circ _ PSMC3 has higher accuracy in diagnosis of gastric adenocarcinoma, and can be used as a molecular marker for diagnosis of patients with gastric adenocarcinoma.
FIG. 3 shows the expression level of Circ _ PSMC3 in the plasma of gastric adenocarcinoma patients and healthy population plasma (106 plasma of gastric adenocarcinoma patients and 21 plasma of healthy population) detected by qRT-PCR, and it can be seen that the expression level of Circ _ PSMC3 in the plasma of gastric cancer patients is lower than that of healthy population, and the expression level of lymph metastasis gastric cancer patients is lower than that of non-metastasis gastric cancer patients.
Fig. 4 shows the expression level of circ _ PSMC3 in cancer tissues and paracarcinoma tissues of gastric adenocarcinoma patients (106 cases of cancer tissues and paracarcinoma tissues of gastric adenocarcinoma patients), and it can be seen that circ _ PSMC3 is significantly lower in cancer tissues of gastric carcinoma patients than in the corresponding paracarcinoma tissues.
The prognosis survival curves of 106 patients with gastric adenocarcinoma are analyzed, and fig. 5 is a graph of survival curves for analyzing the correlation between the expression level of Circ _ PSMC3 and the survival rate of the patients, so that the expression level of Circ _ PSMC3 and the survival rate of the patients with gastric adenocarcinoma are in negative correlation, which indicates that the Circ _ PSMC3 can be used as a marker for diagnosis and prognosis evaluation of the patients with gastric adenocarcinoma.
Sequence listing
<110> Cao hong Yao
<120> molecular marker for gastric adenocarcinoma diagnosis
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 502
<212> DNA
<213> Cyclic RNA (circ _ PSMC3)
<400> 1
ctttgacagt gagaaggctg gggaccggga ggtgcagagg acaatgctgg agcttctgaa 60
ccagctggat ggcttccagc ccaacaccca agttaaggta attgcagcca caaacagggt 120
ggacatcctg gaccccgccc tcctccgctc gggccgcctt gaccgcaaga tagagttccc 180
gatgcccaat gaggaggccc gggccagaat catgcagatc cactcccgaa agatgaatgt 240
cagtcctgac gtgaactacg aggagctggc ccgctgcaca gatgacttca atggggccca 300
gtgcaaggct gtgtgtgtgg aggcgggcat gatcgcactg cgcaggggtg ccacggagct 360
cacccacgag gactacatgg aaggcatcct ggaggtgcag gccaagaaga aagccaacct 420
acaatactac gcctagggca cacaggccag ccccagtctc acggctgaag tgcgcaataa 480
aagatggttt agggtccctg cc 502
<210> 2
<211> 21
<212> DNA
<213> Cyclic RNA (circ _ PSMC3)
<400> 2
gtttagggtc cctgcccttt g 21
<210> 3
<211> 20
<212> DNA
<213> Cyclic RNA (circ _ PSMC3)
<400> 3
gtgttgggct ggaagccatc 20

Claims (2)

1. The application of a molecular marker circ _ PSMc3 in preparing a gastric adenocarcinoma diagnostic kit is disclosed, wherein the nucleotide sequence of the circ _ PSMc3 is shown as SEQ ID No. 1.
2. The use according to claim 1, wherein the gastric adenocarcinoma diagnostic kit comprises specific primers for detecting the molecular marker circ _ PSMc3, wherein the specific primers are an upstream primer shown as SEQ ID No.2 and a downstream primer shown as SEQ ID No. 3.
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CN110894528A (en) * 2019-09-22 2020-03-20 潘文胜 CircRNA marker for diagnosing gastric poorly differentiated adenocarcinoma and application thereof

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