CN117448320A - PCR internal reference of blood small extracellular vesicle miRNA, preparation method and application - Google Patents

PCR internal reference of blood small extracellular vesicle miRNA, preparation method and application Download PDF

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CN117448320A
CN117448320A CN202210884557.0A CN202210884557A CN117448320A CN 117448320 A CN117448320 A CN 117448320A CN 202210884557 A CN202210884557 A CN 202210884557A CN 117448320 A CN117448320 A CN 117448320A
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mirna
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年宝宁
张大东
李志宽
许晓雅
陈升
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Shanghai 3D Medicines Co Ltd
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Abstract

The invention provides a PCR internal reference of blood small extracellular vesicle miRNA, a preparation method and application thereof, belonging to the technical field of genetic engineering. The internal reference is at least one of miR-532-5p, miR-30d-5p, let-7i-5p, miR-148b-3p, miR-128-3p, miR-30e-5p, miR-7-5p, miR-101-3p, miR-181a-2-3p and miR-106b-3p. The invention adopts two blood samples from independent queues to find and verify miR-532-5p as an internal reference molecule for quantitative detection of the liver cancer patient blood small extracellular vesicle miRNA, which is beneficial to improving the accuracy and reliability of the liver cancer blood small extracellular vesicle miRNA as an early diagnosis or treatment biomarker.

Description

PCR internal reference of blood small extracellular vesicle miRNA, preparation method and application
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a PCR internal reference of blood small extracellular vesicle miRNA, a preparation method and application thereof.
Background
Liver cancer is one of the most common cancer types, the fourth leading cause of cancer death worldwide, and is expected to rise to the third in 2030, with the disease incidence currently being the sixth worldwide. Among them, hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for about 90% of cases. Because of hidden onset and difficult early diagnosis of hepatocellular carcinoma, most patients have middle and late stage of disease, and cannot perform radical operation. The prognosis of hepatocellular carcinoma is poor, the survival rate of patients in five years is less than 10%, the patients are easy to relapse, and the survival rate of early liver cancer patients in five years is 70% higher than that of patients in late liver cancer treated systematically, wherein the survival period of early liver cancer patients is only 1-1.5 years after effective treatment. At present, the diagnosis method of the hepatocellular carcinoma comprises abdominal imaging, fine needle aspiration biopsy (EUS-FNA) under the guidance of endoscopic ultrasound, detection of tumor marker Alpha Fetoprotein (AFP) and the like, but the methods have certain limitations and have unsatisfactory detection effect. For example, lesions with a diameter of less than 1cm for imaging methods such as abdominal ultrasound are not HCC or are difficult to identify, with only 45% sensitivity; EUS-FNA is an invasive detection method, which is easy to cause intraperitoneal infection, hemorrhage and tumor diffusion; the sensitivity of AFP is only improved to 63% when combined with ultrasonic examination, which is easy to cause missed diagnosis of liver cancer patients in early stages of tumor, and can not be an effective diagnostic marker. Therefore, finding early diagnostic biomarkers with high sensitivity and specificity is an important point and hot spot of the current clinical diagnosis research of hepatocellular carcinoma.
Small extracellular vesicles (SmallExtracellular Vesicles, sEVs), also known as exosomes, are membranous vesicles with diameters of about 30-150nm actively secreted by a variety of cell types, can be found in all biological fluids, and the contents contain bioactive molecules such as proteins, lipids, nucleic acids (e.g., microRNA, mRNA, lncRNA and circRNA), and can reflect cell origin and physiological status. Exosomes are a promising biomarker of disease circulation, and their role in the fields of early diagnosis, prognostic monitoring, immunotherapy, etc. of cancer has been widely focused and studied in depth. Sohn et al found that miR-18a, miR-221, miR-222 and miR-224 expression levels were significantly increased in serum exosomes of hepatocellular carcinoma patients (P < 0.05), compared to Chronic Hepatitis B (CHB) and cirrhosis (LC) patients, and miR-101, miR-106b, miR-122 and miR-195 levels were lower than in CHB patients, indicating that these blood exosomes miRNAs could be biomarkers for liver cancer diagnosis (PMID: 26380927). Wang et al found that serum levels of exosome miR-21 were significantly higher in hepatocellular carcinoma patients than in CHB patients or healthy volunteers (P < 0.0001), and in addition, this study showed that serum levels of miR-21 were also higher in hepatocellular carcinoma patients than in CHB patients and healthy volunteers, but that the sensitivity of detection was much lower than in exosome miR-21 (PMID: 24963487). Therefore, detection of miRNAs in blood exosomes by non-invasive methods has great potential to provide indicative significance for early diagnosis of liver cancer.
The real-time fluorescent quantitative PCR technology is the most common and effective method for detecting the expression level of the small extracellular vesicle miRNA, and has the advantages of rapidness, economy, easiness in use and the like. However, since there are differences in RNA extraction quality, reverse transcription efficiency, PCR amplification efficiency, experimental operation errors, and the like between different samples, the reliability of the target gene expression results is affected. One or more endogenous molecules are used as reference molecules for detecting the expression of the target genes, so that errors caused by operation can be effectively avoided, and the influence caused by the biological difference of individuals can be reduced. However, there is no known reference molecule in the detection process of exosome miRNA, and some snRNA or miRNA is selected for reference by researchers in the past mainly according to references or self experience, such as the most commonly used reference snRNA, such as U6, which is expressed only in cell nuclei, and the detection of U6 expression is likely to be caused by insufficient purity of the extracted exosome; mirnas such as miR-16 and miR-451, which are often used as internal references, are extremely susceptible to hemolysis interference; there are also some potential molecules that can be used as references to exosome mirnas that are not stable in expression under different physio-pathological conditions, so blind use of unverified reference molecules may lead to erroneous and even opposite conclusions. The exosome method is used as another alternative strategy for quantitative analysis of exosome miRNA, and is mainly used for correcting quality control RNA extraction, reverse transcription and qPCR processes, but cannot control the most complex exosome separation process in the earlier stage, so that the exosome method cannot completely meet the requirements of exosome quantitative research.
At present, the quantitative detection of the blood small extracellular vesicle miRNA has the problem that the quantitative detection is difficult to correct and standardize, so that the consistency of sequencing data and qPCR detection results is poor, the possibility of biomarker discovery is reduced, and the clinical transformation of related research results of the extracellular vesicle miRNA biomarker is limited.
Studies have shown (PMID: 33429910) that miR-26a-5p can be used for the standardization of the pediatric hematological malignancy patient plasma exosome miRNA qRT-PCR by analyzing public plasma exosome RNA-seq data to determine and select a set of miRNAs with stable abundance as candidate standardized molecules, and then detecting the abundance changes of the candidate miRNAs in the plasma exosomes of healthy controls and four representative pediatric lymphoma patients by qRT-PCR. In addition, researchers in Chinese patent application CN109536502A find reference molecules which are stably expressed in the plasma exosome miRNA of the pregnant trophoblastic tumor patient based on a small sample size (comprising 10 MTX sensitive and 10 MTX drug resistant patients), and the reference molecules are used as candidate reference molecules to be verified in another group of independent queues, so that a novel reference molecule miR-129-5p suitable for qRT-PCR quantitative detection of the plasma exosome miRNA of the pregnant trophoblastic tumor patient is disclosed. However, the study involved a too small number of samples in the discovery cohort, and the analysis results hardly represented the entire gestational trophoblastic tumor patient population.
However, whether miR-26a-5p and miR-129-5p can be used for quantitative detection of plasma small extracellular vesicle miRNA qRT-PCR of liver cancer patients in the above study is to be verified by other studies.
Disclosure of Invention
The invention aims to discover and disclose a PCR internal reference of blood small extracellular vesicle miRNA, a preparation method and application thereof, which are used for correcting errors in experimental processes between different samples, correcting and standardizing quantitative data better and improving the accuracy and reliability of the blood small extracellular vesicle miRNA as a biomarker for early diagnosis or treatment of liver cancer.
The technical scheme of the invention is realized as follows:
the invention provides a PCR internal reference of blood extracellular vesicle miRNA, which is characterized in that the internal reference is at least one of miR-532-5p, miR-30d-5p, let-7i-5p, miR-148b-3p, miR-128-3p, miR-30e-5p, miR-7-5p, miR-101-3p, miR-181a-2-3p and miR-106b-3 p; the nucleotide sequence of miR-532-5p is shown as SEQ ID NO. 1; the nucleotide sequence of the miR-30d-5p is shown as SEQ ID NO. 2; the nucleotide sequence of the let-7i-5p is shown as SEQ ID NO. 3; the nucleotide sequence of miR-148b-3p is shown as SEQ ID NO. 4; the nucleotide sequence of miR-128-3p is shown as SEQ ID NO. 5; the nucleotide sequence of the miR-30e-5p is shown as SEQ ID NO. 6; the nucleotide sequence of miR-7-5p is shown as SEQ ID NO. 7; the nucleotide sequence of miR-101-3p is shown as SEQ ID NO. 8; the nucleotide sequence of miR-181a-2-3p is shown in SEQ ID NO. 9; the nucleotide sequence of miR-106b-3p is shown as SEQ ID NO. 10.
As a further improvement of the invention, the internal reference is at least one selected from the group consisting of miR-532-5p, let-7i-5p and miR-148b-3p.
As a further improvement of the invention, the internal reference is miR-532-5p.
The invention further protects the application of the internal reference in preparing biomarkers or kits for diagnosing liver cancer.
The invention further provides a preparation method of the PCR internal reference of the blood small extracellular vesicle miRNA, which comprises the following steps:
s1: extracting small extracellular vesicles from blood;
s2: extracting miRNA from the extracted blood small extracellular vesicles and performing high-throughput sequencing;
s3: sequentially carrying out sequencing comparison, annotation, filtration and expression quantity standardization treatment on the miRNA obtained by extraction to obtain the standardized expression quantity of the miRNA;
s4: screening miRNA according to screening criteria, screening stable miRNA as a candidate internal reference to be verified;
s5: and adopting second generation sequencing, screening out internal references through a training queue, and then verifying by using a verification queue.
As a further improvement of the present invention, the method for extracting blood small extracellular vesicles in step S1 comprises: at least one of reagent extraction, ultracentrifugation extraction, gradient density centrifugation extraction, ultrafiltration centrifugation extraction, and magnetic bead immunity extraction.
As a further improvement of the present invention, the specific method for extracting the blood small extracellular vesicle miRNA in step S1 and step S2 is as follows: the mirnas of the extracellular vesicles of the blood were extracted using the miRNeasy Serum/Plasma Kit.
As a further improvement of the present invention, the screening criteria in step S4 are:
1) The expression level is higher in benign and malignant liver tumor patients, and the expression level after standardization is more than 8;
2) The coefficient of variation is small and the expression is stable.
As a further improvement of the present invention, the step S4 specifically includes:
s41: eliminating miRNA with the standardized expression quantity less than 8;
s42: calculating the variation coefficient of each rest miRNA, and sequencing the variation coefficients from small to large;
s43: selecting the expression quantity of miRNAs with variation coefficients in the first N sequences, and carrying out stability analysis on the selected expression quantity of the miRNAs;
s44: selecting the first M miRNAs with the front comprehensive stability as candidate internal references to be verified;
wherein, N and M are natural numbers, and N is larger than M.
As a further improvement of the invention, the value of N is 40-60, and the value of M is 6-10.
As a further improvement of the invention, the candidate internal references to be verified comprise miR-532-5p, let-7i-5p and miR-148b-3p.
As a further improvement of the present invention, PCR internal references comprising the blood small extracellular vesicle miRNA of claim 1 or 2 are included.
The invention further provides a detection kit comprising the detection reagent.
The invention has the following beneficial effects:
(1) The invention discovers and discloses 10 potential internal reference molecules miR-532-5p, miR-30d-5p, let-7i-5p, miR-148b-3p, miR-128-3p, miR-30e-5p, miR-7-5p, miR-101-3p, miR-181a-2-3p and miR-106b-3p for the detection of blood small extracellular vesicles of liver cancer patients through miRNAqRT-PCR for the first time.
(2) Based on the potential internal reference molecules, three stable internal references of miR-532-5p, let-7i-5p and miR-148b-3p are screened out and used as preferred internal references for detecting blood small extracellular vesicle miRNA qRT-PCR of liver cancer patients. And miR-532-5p is further preferred as a preferred internal reference.
(3) According to the invention, blood of healthy people, liver cirrhosis and hepatocellular carcinoma patients is taken as a research sample, a small extracellular vesicle (exosome) separating reagent L3525 (REX 015S,3DMed, shangghai, china) independently developed by Shanghai Di biomedical science and technology is adopted to extract blood exosomes, a small RNA sequencing is further utilized to obtain a miRNA expression profile of the subject blood small extracellular vesicle, firstly miRNAs with the 10 th position before comprehensive stability ranking are screened out from sequencing data of 61 training queue samples by using RefFinder software to serve as candidate internal reference molecules, then another group of sequencing data of 40 independent groups is used to verify candidate internal references, and finally, the expression stability of miR-532-5p in the candidate internal reference molecules is determined to be optimal, so that an internal reference molecule suitable for miqRT-PCR quantitative analysis of liver cancer blood small extracellular vesicles is disclosed.
(4) According to the invention, two independent queues (n=61 and n=40) are adopted for blood samples, and miR-532-5p is used as an internal reference molecule for quantitative detection of the liver cancer patient blood small extracellular vesicle miRNA, so that the accuracy and reliability of the liver cancer blood small extracellular vesicle miRNA as an early diagnosis or treatment biomarker can be improved.
(5) The invention selects the blood of healthy people, liver cirrhosis and hepatocellular carcinoma patients as a research sample, and can reflect the disease condition of the real world liver cancer patients, so that the internal reference molecule for liver cancer blood small extracellular vesicle miRNA qRT-PCR detection provided by the invention has higher reliability.
(6) The invention discovers total relatively stable blood extracellular vesicle miRNAs in healthy people, liver cirrhosis patients and hepatocellular carcinoma patients according to a linear model fitting (limma-voom) method in an R language limma analysis package, and gives stability ranking based on detection and analysis results.
(7) In the invention, extraction reagent L3525 is independently researched and developed by Shanghai Di biomedical science and technology limited company to precipitate blood extracellular vesicles, and miRNA of the blood extracellular vesicles is extracted for subsequent qRT-PCR detection experiments.
(8) According to the invention, by using another batch of independent research queue data, the fact that the internal reference molecule miR-532-5p suitable for the detection of the blood small extracellular vesicle miRNA qRT-PCR of a liver cancer patient has high stability is verified, so that the internal reference molecule miR-532-5p is used for correcting errors in experimental processes between different samples, quantitative data are better corrected and standardized, and the accuracy and reliability of research of using the liver cancer blood small extracellular vesicle miRNA as a diagnosis or treatment biomarker are improved.
Of course, the above advantages are not required to be present in one embodiment of the present invention.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 shows the identification result of small extracellular vesicles by transmission electron microscopy;
FIG. 2 shows the results of identification of extracellular vesicles NTA;
FIG. 3 is a graph showing comparison of integrated gene stability;
FIG. 4 is a graph comparing the stability of genes analyzed by the DeltaCt method;
FIG. 5 is a graph comparing the stability of genes analyzed using BestKeeper software;
FIG. 6 is a graph comparing the stability of genes analyzed using the norm Finder software;
FIG. 7 is a graph comparing the stability of genes analyzed using Genorm software.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
(1) Blood sample collection and small extracellular vesicle separation
The study included a total of 101 samples from training and validation cohorts, blood samples were collected at K 2 EDTA anticoagulated vacuum blood collection tubes (6 mL, REF367863, BD, USA), gently inverted several times for mixing, and the tubes were stored upright and then transported on ice within 1h after collection. The received samples were centrifuged at 1600g for 10min at 4℃and the supernatant carefully transferred to a new 1.5mL centrifuge tube while the level of hemolysis was determined, recorded (samples with a level of hemolysis no more than 4 were used for subsequent experiments), followed by centrifugation of 16000g for 15min at 4℃and the supernatant was dispensed into new 1.5mL centrifuge tubes, 1mL each, and stored at-80℃for further use.
Small extracellular vesicles were isolated from plasma using L-type exosome precipitants developed by ideas (L3525, 3d, shanghai, china). After 5min in a 37℃water bath, the plasma samples were centrifuged at 12000g for 10min at 4 ℃. The supernatant was transferred to a 0.45 μm filter (CLS 8163-100EA, costar, USA), centrifuged at 12000g for 5min at 4℃and then transferred to a 0.22 μm filter (CLS 8161-100EA, costar, USA), centrifuged at 12000g for 5min at 4℃and finally transferred to a fresh 1.5mL centrifuge tube. To the filtrate, 1/4 volume of the separation reagent L3525 was added, mixed upside down and incubated at 4℃for 30min, followed by centrifugation at 4700g at 4℃for 30min. Finally, the supernatant was discarded and the particles of small extracellular vesicles were resuspended using 0.2mL Phosphate Buffer (PBS). The collected small extracellular vesicles are subjected to characterization and identification and RNA extraction.
(2) Characterization of blood small extracellular vesicles
1) TEM (Transmission Electron Microscopy, transmission electron microscope)
The morphology of small extracellular vesicles was detected using transmission electron microscopy scanning techniques. Blood small extracellular vesicles suspended in PBS were fixed with 4% paraformaldehyde and transferred into carbon-coated electron microscopy grids. The cells were washed twice with PBS and the third with PBS containing 50mM glycine for 3min each. Then, it was incubated with PBS containing 0.5% BSA for 10min. Finally, the grids were stained with 2% uranyl acetate and the morphology of small extracellular vesicles was analyzed using TEM (H-7650,Hitachi High-Technologies, japan).
2) NTA (Nanoparticle Tracking Analysis )
The NTA technology is adopted to detect the particle size characteristics of the small extracellular cyst. Small extracellular vesicles were first diluted to 1X 10 with PBS 7 -1×10 9 /ml, blow and mix well. Subsequently, the NTA (NanoSight NS300, malvern, UK) instrument was turned on to inject the diluted sample into the sample cell, and the camera lens parameters, shutter value selection 890, gain value selection 146, detection threshold set to 7 were set using the 488nm excitation module. Each video analyzes and obtains at least 200 complete tracks. Finally, nanoparticle tracking data of small extracellular vesicles were analyzed using NTA analysis software (version 2.3).
(3) Extraction of blood small extracellular vesicle miRNA and high throughput sequencing
1) Extraction of blood small extracellular vesicle miRNA
Total RNA from the extracellular vesicles was extracted using a MIRNeasy Serum/Plasma Kit (21789, qiagen, USA) and the concentration and fragment distribution of total RNA from the blood extracellular vesicles were measured using an Agilent 2100Bioanalyzer and associated chip and reagents Agilent Small RNA Kit (5067-1548, agilent, USA).
2) High throughput sequencing of blood small extracellular vesicle miRNAs
First, a library of miRNAs for high throughput sequencing was prepared using NEBNext Multiplex SmallRNA Library Prep Set for Illumina (E7300L, NEB, USA), the library was purified using NucleoSpin Gel and PCR Clean-up (740609.50, MN, germany), and 30. Mu.L of enzyme-free water was added to elute the library DNA. Library concentrations were then measured using Qubit4.0 and associated reagents QubitdsDNA HS Assay Kit (Q32854, life, USA), and fragment sizes of the library were measured using an Agilent 2100Bioanalyzer and associated chips and reagents Agilent High Sensitivity DNA Kit (5067-4626, agilent, USA). Finally, the qualified library is sequenced by using an IlluminaHiSeqX platform, the sequencing strategy is 2 multiplied by 150bp, and the data requirement of each sample is 5G.
(4) Bioinformatics analysis of miRNA sequencing data
1) Sequencing data alignment. After removal of small RNA sequencing data sequencing adaptors, sequencing data was aligned to human reference genome hg19 (genome download link: http:// hgdownload. Soe. Ucsc. Edu/goldenPath/hg 19/bigmaps /) using BWA software (version: 0.7.12-r 1039) and the number of reads aligned to miRNAs was counted.
2) miRNA annotation. Mirnas were annotated with the gencodeev 25 and miRBasev21 databases, retaining mature mirnas annotated as known for subsequent analysis.
3) miRNA filtration. The length of the reserved is less than or equal to 30nt and each sample is covered with at least 2 ready mature mirnas for subsequent analysis.
4) miRNA expression level is standardized. And (3) respectively carrying out miRNA expression quantity standardization treatment on the found queue samples by using an M value weighted tail-biting average value method (TMM, trimmed mean of M-values) in a limma analysis packet in R language.
(5) Selection of miRNA candidate reference molecules
Based on small RNA sequencing detection technology, expression profiles of blood small extracellular vesicle mirnas of 61 training cohort samples (including 8 healthy people, 21 liver cirrhosis and 32 hepatocellular carcinoma patients) were obtained, and candidate reference molecules were selected according to the following principles:
1) In healthy peopleThe patients with liver cirrhosis and hepatocellular carcinoma all have higher expression level, and the standardized expression log is required 2 RPM value greater than 8;
2) Statistical analysis was performed according to the miRNA expression variation coefficient (CV% = standard deviation/mean), and miRNAs with top 60 rank from small to large CV values were selected for stability analysis. The stability analysis of the expression of each miRNA gene uses an on-line based tool RefFinder (http:// fulxie.0fees. Us /), which integrates four different commonly used standardized tools: bestkeepers (Pfaffl et al, 2004), delta-Ct (Silver et al, 2006), normFinder (Andersen et al, 2004), and gemum (vanderompale et al, 2002).
(6) Candidate reference molecule stability validation
Another batch of 40 independent samples (including 4 healthy, 18 cirrhosis and 18 hepatocellular carcinoma patients) was selected for blood small extracellular vesicle miRNA expression profile data as a validation queue, and the stability of the top 10 candidate reference molecules was validated using the evaluation software reffilter.
Example 2
(1) Study cohort and clinical information
The study included a total of 101 samples of training and validation cohorts, including 12 healthy people, 39 patients with cirrhosis, and 50 patients with imaging detection results suspected to be liver cancer (blood samples of the patients were collected before surgery), and the patients in the group were accurately diagnosed after surgery according to pathological examination results. Clinical information for study cohort samples is shown in table 1.
Table 1 study cohort sample clinical information
(2) Characterization of blood small extracellular vesicles
The invention extracts the small extracellular vesicles (exosomes) of the blood of a patient by adopting a small extracellular vesicles (exosomes) separation reagent L3525 independently researched and developed by Shanghai thought Di biomedical technology limited company, then adopts TEM to detect the morphological characteristics of the small extracellular vesicles, and adopts NTA to detect the particle size distribution of the small extracellular vesicles. TEM detection results show that small extracellular vesicles show a typical horseshoe-shaped form (see figure 1), and NTA detection results show that the main particle size peak of the small extracellular vesicles in a representative sample extracted by the patent is 146nm, and the small extracellular vesicles accord with the particle size distribution of the small extracellular vesicles (see figure 2).
(3) Candidate reference molecule screening
From the second generation sequencing data of blood small extracellular vesicle miRNAs of healthy people, liver cirrhosis and hepatocellular carcinoma patients, miRNAs with high expression level and small variation coefficient and stable expression are selected as candidate internal reference molecules. First, log is removed 2 RPM value<8, calculating the coefficient of variation (CV% = standard deviation/mean) of each miRNA, and sorting the coefficient of variation from small to large. Then, the expression level of the first 60 miRNAs was introduced into an on-line stability analysis software RefFinder, and the evaluation results are shown in Table 2. The 10-position miRNAs with the highest comprehensive stability ranking are selected as candidate reference molecules to be verified, namely hsa-miR-532-5p, hsa-miR-30d-5p, hsa-let-7i-5p, hsa-miR-148b-3p, hsa-miR-128-3p, hsa-miR-30e-5p, hsa-miR-7-5p, hsa-miR-101-3p, hsa-miR-181a-2-3p and hsa-miR-106b-3p, and the sequence information of the 10 miRNAs is shown in Table 3.
TABLE 2 expression stability score results for four algorithms
TABLE 3 candidate internal control molecule sequences for miRNAs
miRNAs Accession number Sequence (5 '-3') Description of the invention
hsa-miR-532-5p MIMAT0002888 CAUGCCUUGAGUGUAGGACCGU SEQ ID NO.1
hsa-miR-30d-5p MIMAT0000245 UGUAAACAUCCCCGACUGGAAG SEQ ID NO.2
hsa-let-7i-5p MIMAT0000415 UGAGGUAGUAGUUUGUGCUGUU SEQ ID NO.3
hsa-miR-148b-3p MIMAT0000759 UCAGUGCAUCACAGAACUUUGU SEQ ID NO.4
hsa-miR-128-3p MIMAT0000424 UCACAGUGAACCGGUCUCUUU SEQ ID NO.5
hsa-miR-30e-5p MIMAT0000692 UGUAAACAUCCUUGACUGGAAG SEQ ID NO.6
hsa-miR-7-5p MIMAT0000252 UGGAAGACUAGUGAUUUUGUUGUU SEQ ID NO.7
hsa-miR-101-3p MIMAT0000099 UACAGUACUGUGAUAACUGAA SEQ ID NO.8
hsa-miR-181a-2-3p MIMAT0004558 ACCACUGACCGUUGACUGUACC SEQ ID NO.9
hsa-miR-106b-3p MIMAT0004672 CCGCACUGUGGGUACUUGCUGC SEQ ID NO.10
(4) Candidate reference molecule stability validation
Another batch of 40 independent samples (including 4 healthy, 18 cirrhosis and 18 hepatocellular carcinoma patients) was selected for blood small extracellular vesicle miRNA expression profile data as a validation queue, and the stability of the top 10 candidate reference molecules was validated using the evaluation software reffilter. The results of the software analysis (see fig. 3-7) show that: the expression stability of miR-532-5p, let-7i-5p and miR-148b-3p which are arranged at the front in 10 miRNAs candidate reference molecules is very high in consistency with the results verified by two analysis methods of NormFinder and Delta-Ct respectively, so that the 3 miRNAs can be used as potential reference molecules, especially the miR-532-5p can be used for correcting the deviation in the experimental process between different samples, and the accuracy and reliability of the liver cancer blood extracellular vesicle miRNA as diagnosis and treatment biomarker can be improved.
Compared with the prior art, the invention aims to solve the technical problems described in the background art and is specifically divided into the following two parts:
(1) Screening miRNAs with stable expression among different individuals as candidate internal reference molecules through high-throughput sequencing data analysis of blood small extracellular vesicle miRNA expression profiles of healthy people, liver cirrhosis and hepatocellular carcinoma patients;
(2) The stability of the candidate reference molecule selected in (1) above was validated using sequencing data from an additional independent set of healthy, cirrhosis and hepatocellular carcinoma patient populations.
By combining the research results of (1) and (2), the invention adopts two blood samples from independent queues to find and verify miR-532-5p as an internal reference molecule for quantitative detection of the liver cancer patient blood small extracellular vesicle miRNA, thereby being beneficial to improving the accuracy and reliability of the liver cancer blood small extracellular vesicle miRNA as an early diagnosis or treatment biomarker.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (12)

1. A PCR internal reference of blood extracellular vesicle miRNA, which is characterized in that the internal reference is at least one of miR-532-5p, miR-30d-5p, let-7i-5p, miR-148b-3p, miR-128-3p, miR-30e-5p, miR-7-5p, miR-101-3p, miR-181a-2-3p and miR-106b-3 p; the nucleotide sequence of miR-532-5p is shown as SEQ ID NO. 1; the nucleotide sequence of the miR-30d-5p is shown as SEQ ID NO. 2; the nucleotide sequence of the let-7i-5p is shown as SEQ ID NO. 3; the nucleotide sequence of miR-148b-3p is shown as SEQ ID NO. 4; the nucleotide sequence of miR-128-3p is shown as SEQ ID NO. 5; the nucleotide sequence of the miR-30e-5p is shown as SEQ ID NO. 6; the nucleotide sequence of miR-7-5p is shown as SEQ ID NO. 7; the nucleotide sequence of miR-101-3p is shown as SEQ ID NO. 8; the nucleotide sequence of miR-181a-2-3p is shown in SEQ ID NO. 9; the nucleotide sequence of miR-106b-3p is shown as SEQ ID NO. 10.
2. The PCR internal reference to blood small extracellular vesicle miRNA of claim 1, wherein the internal reference is selected from at least one of miR-532-5p, let-7i-5p, and miR-148b-3p.
3. The PCR internal reference to claim 1, wherein said internal reference is miR-532-5p.
4. A method for preparing a PCR internal reference for a blood small extracellular vesicle miRNA according to any one of claims 1-3, comprising the steps of:
s1: extracting small extracellular vesicles from blood;
s2: extracting miRNA from the extracted blood small extracellular vesicles and performing high-throughput sequencing;
s3: sequentially carrying out sequencing comparison, annotation, filtration and expression quantity standardization treatment on the miRNA obtained by extraction to obtain the standardized expression quantity of the miRNA;
s4: screening miRNA according to screening criteria, screening stable miRNA as a candidate internal reference to be verified;
s5: and adopting second generation sequencing, screening out internal references through a training queue, and then verifying by using a verification queue.
5. The method according to claim 4, wherein the method for extracting the small extracellular vesicles in step S1 comprises: at least one of reagent extraction, ultracentrifugation extraction, gradient density centrifugation extraction, ultrafiltration centrifugation extraction, and magnetic bead immunity extraction.
6. The preparation method according to claim 4, wherein the specific method for extracting the blood small extracellular vesicle miRNA in the step S2 in the step S1 is as follows: the mirnas of the extracellular vesicles of the blood were extracted using the miRNeasy Serum/Plasma Kit.
7. The method according to claim 4, wherein the screening criteria in step S4 are:
1) The expression level is higher in benign and malignant liver tumor patients, and the expression level after standardization is more than 8;
2) The coefficient of variation is small and the expression is stable.
8. The preparation method according to claim 4, wherein the step S4 specifically comprises:
s41: eliminating miRNA with the standardized expression quantity less than 8;
s42: calculating the variation coefficient of each rest miRNA, and sequencing the variation coefficients from small to large;
s43: selecting the expression quantity of miRNAs with variation coefficients in the first N sequences, and carrying out stability analysis on the selected expression quantity of the miRNAs;
s44: selecting the first M miRNAs with the front comprehensive stability as candidate internal references to be verified;
wherein, N and M are natural numbers, and N is larger than M.
9. The method of claim 8, wherein N is 40-60 and M is 6-10.
10. The method of preparation of claim 8, wherein the candidate internal references to be validated comprise miR-532-5p, let-7i-5p, and miR-148b-3p.
11. A detection reagent comprising a PCR internal reference to a blood small extracellular vesicle miRNA according to any one of claims 1-3.
12. A test kit comprising the test reagent of claim 11.
CN202210884557.0A 2022-07-25 2022-07-25 PCR internal reference of blood small extracellular vesicle miRNA, preparation method and application Pending CN117448320A (en)

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