CN111455053A

CN111455053A - Exosome RNA molecular marker combination for colorectal adenoma diagnosis and application thereof

Info

Publication number: CN111455053A
Application number: CN202010316557.1A
Authority: CN
Inventors: 张澍田; 闵力; 朱圣韬; 孔关义; 刘翔
Original assignee: Beijing Friendship Hospital
Current assignee: Beijing Friendship Hospital
Priority date: 2020-04-21
Filing date: 2020-04-21
Publication date: 2020-07-28
Anticipated expiration: 2040-04-21
Also published as: CN111455053B

Abstract

The invention relates to the field of biological detection, in particular to biological molecular detection, and more particularly relates to a group of exosome RNA markers related to colorectal adenoma and application thereof.

Description

Exosome RNA molecular marker combination for colorectal adenoma diagnosis and application thereof

Technical Field

Background

Colorectal adenomas are benign tumors originating from the epithelium of the colorectal mucosal gland, but are closely related to the development of colorectal cancer, and studies have shown that about 80% of colorectal cancers develop from colorectal adenomas, and thus colorectal adenomas are considered as precancerous lesions of colorectal cancer. With the increasing trend of colorectal cancer incidence and mortality of our country, research shows that the colorectal adenoma can be timely discovered and removed to reduce the colorectal cancer incidence by 76%, so that the research on the diagnosis and treatment of colorectal adenoma is crucial to the prevention of colorectal cancer.

The electronic colonoscopy is helpful for discovering and observing colorectal mucosal lesions, the accuracy and specificity of detection results can be improved by combining with biopsy, the method is an important means for detecting lesions such as colorectal adenoma and colorectal cancer, but the lesion detection rate is often influenced by various factors such as the experience of an examining physician, the capability level, electronic colonoscopy hardware equipment, the intestinal tract cleaning degree and the matching degree of an examined person, and complications such as bleeding and perforation are often caused by invasive operation. The current clinical noninvasive detection methods such as fecal occult blood detection and blood tumor marker detection are widely applied, but still have no good sensitivity and specificity, and can only be used as a reference index for colorectal adenoma diagnosis. Therefore, it is very important to develop a noninvasive colorectal adenoma detection method with high sensitivity and specificity.

As a research focus at home and abroad in recent years, the attention of Extracellular Vesicles (EVs) has been gradually paid, and the research shows that the Extracellular Vesicles are mainly vesicular bodies secreted by cells or formed by cell membrane shedding, have a double-layer membrane structure similar to a cell membrane, and have diameters of 30-1000 nm. Research shows that extracellular vesicles mainly comprise two major parts, namely MicroVesicles (MVs) and exosomes (exosomes), which participate in various biological functions in vivo after being secreted by cells, and particularly exosomes have important significance in the occurrence and development of diseases.

The exosome is also an extracellular vesicle in nature, is usually generated in an intracellular multivesicular body, is secreted and released to the outside of cells by the cells after being fused with cell membranes to form a bilayer membrane vesicle with the diameter usually between 30 and 150nm, and researches show that the exosome can be widely distributed in various body fluids such as blood, saliva, urine, milk, pleural effusion and ascites, is internally rich in various active substances such as DNA, RNA and protein, is used as an important medium for intercellular information transmission, and can participate in various pathophysiological processes such as antigen presentation, apoptosis, inflammatory reaction, tumorigenesis development and metastasis and the like in a human body.

Disclosure of Invention

Through years of research, the inventor obtains RNA molecular markers which are closely related to colorectal adenomas and can be used for clinical diagnosis, wherein the RNA molecular markers comprise a long-chain transcript (mRNA) which does not code two genes of RNAlnc-MKRN2-42: 1: TOP1 and C19ORF43, and one miRNA: miR-106b-3 p. The combined use of the RNA molecular markers can indicate colorectal adenoma with high sensitivity and specificity, and has excellent diagnostic performance.

The first aspect of the disclosure provides an RNA molecular marker combination for colorectal adenoma diagnosis, prognosis judgment, efficacy monitoring and/or recurrence monitoring, which comprises lnc-MKRN2-42:1, TOP1 gene transcript, C19ORF43 gene transcript and miR-106b-3 p.

In one embodiment, the lnc-MKRN2-42:1 has the sequence set forth in SEQ ID NO:14, specifically:

GCCGGGCACAGUGGCGCGUGCCUGUAGUCCCAGCUACUCGGGAGGCUGAGGUGGGAGGAUCGCUUGAGCCCAGGAGUUCUGGGCUGUAGUGCGCUAUGCCGAUCGGGUGUCCGCACUAAGUUCGGCAUCAAUAUGGUGACCUCCCGGGAGCGGGGGACCACCAGGUUGCCUAAGGAGGGGUGAACCGGCCCAGGUCGGAAACAGAGCAGGUCAAAACUCCCGUGCUGAUCAGGAGACAGAGUUUGUGAGCAGACAACUGGUCUGACCAAAAUUUAUGAGGUGGGAAUUUCCUCU(SEQ ID NO:14)；

the sequence of the TOP1 gene transcript is shown as SEQ ID NO:15, specifically:

CAAAUGCGAACUUAGGCUGUUACACAACUGCUGGGGUCUGUUCUCGCCGCCCGCCCGGCAGUCAGGCAGCGUCGCCGCCGUGGUAGCAGCCUCAGCCGUUUCUGGAGUCUCGGGCCCACAGUCACCGCCGCUUACCUGCGCCUCCUCGAGCCUCCGGAGUCCCCGUCCGCCCGCACAGGCCGGUUCGCCGUCUGCGUCUCCCCCACGCCGCCUCGCCUGCCGCCGCGCUCGUCCCUCCGGGCCGACAUGAGUGGGGACCACCUCCACAACGAUUCCCAGAUCGAAGCGGAUUUCCGAUUGAAUGAUUCUCAUAAACACAAAGAUAAACACAAAGAUCGAGAACACCGGCACAAAGAACACAAGAAGGAGAAGGACCGGGAAAAGUCCAAGCAUAGCAACAGUGAACAUAAAGAUUCUGAAAAGAAACACAAAGAGAAGGAGAAGACCAAACACAAAGAUGGAAGCUCAGAAAAGCAUAAAGACAAACAUAAAGACAGAGACAAGGAAAAACGAAAAGAGGAAAAGGUUCGAGCCUCUGGGGAUGCAAAAAUAAAGAAGGAGAAGGAAAAUGGCUUCUCUAGUCCACCACAAAUUAAAGAUGAACCUGAAGAUGAUGGCUAUUUUGUUCCUCCUAAAGAGGAUAUAAAGCCAUUAAAGAGACCUCGAGAUGAGGAUGAUGCUGAUUAUAAACCUAAGAAAAUUAAAACAGAAGAUACCAAGAAGGAGAAGAAAAGAAAACUAGAAGAAGAAGAGGAUGGUAAAUUGAAAAAACCCAAGAAUAAAGAUAAAGAUAAAAAAGUUCCUGAGCCAGAUAACAAGAAAAAGAAGCCGAAGAAAGAAGAGGAACAGAAGUGGAAAUGGUGGGAAGAAGAGCGCUAUCCUGAAGGCAUCAAGUGGAAAUUCCUAGAACAUAAAGGUCCAGUAUUUGCCCCACCAUAUGAGCCUCUUCCAGAGAAUGUCAAGUUUUAUUAUGAUGGUAAAGUCAUGAAGCUGAGCCCCAAAGCAGAGGAAGUAGCUACGUUCUUUGCAAAAAUGCUCGACCAUGAAUAUACUACCAAGGAAAUAUUUAGGAAAAAUUUCUUUAAAGACUGGAGAAAGGAAAUGACUAAUGAAGAGAAGAAUAUUAUCACCAACCUAAGCAAAUGUGAUUUUACCCAGAUGAGCCAGUAUUUCAAAGCCCAGACGGAAGCUCGGAAACAGAUGAGCAAGGAAGAGAAACUGAAAAUCAAAGAGGAGAAUGAAAAAUUACUGAAAGAAUAUGGAUUCUGUAUUAUGGAUAACCACAAAGAGAGGAUUGCUAACUUCAAGAUAGAGCCUCCUGGACUUUUCCGUGGCCGCGGCAACCACCCCAAGAUGGGCAUGCUGAAGAGACGAAUCAUGCCCGAGGAUAUAAUCAUCAACUGUAGCAAAGAUGCCAAGGUUCCUUCUCCUCCUCCAGGACAUAAGUGGAAAGAAGUCCGGCAUGAUAACAAGGUUACUUGGCUGGUUUCCUGGACAGAGAACAUCCAAGGUUCCAUUAAAUACAUCAUGCUUAACCCUAGUUCACGAAUCAAGGGUGAGAAGGACUGGCAGAAAUACGAGACUGCUCGGCGGCUGAAAAAAUGUGUGGACAAGAUCCGGAACCAGUAUCGAGAAGACUGGAAGUCCAAAGAGAUGAAAGUCCGGCAGAGAGCUGUAGCCCUGUACUUCAUCGACAAGCUUGCUCUGAGAGCAGGCAAUGAAAAGGAGGAAGGAGAAACAGCGGACACUGUGGGCUGCUGCUCACUUCGUGUGGAGCACAUCAAUCUACACCCAGAGUUGGAUGGUCAGGAAUAUGUGGUAGAGUUUGACUUCCUCGGGAAGGACUCCAUCAGAUACUAUAACAAGGUCCCUGUUGAGAAACGAGUUUUUAAGAACCUACAACUAUUUAUGGAGAACAAGCAGCCCGAGGAUGAUCUUUUUGAUAGACUCAAUACUGGUAUUCUGAAUAAGCAUCUUCAGGAUCUCAUGGAGGGCUUGACAGCCAAGGUAUUCCGUACAUACAAUGCCUCCAUCACGCUACAGCAGCAGCUAAAAGAACUGACAGCCCCGGAUGAGAACAUCCCAGCGAAGAUCCUUUCUUAUAACCGUGCCAAUCGAGCUGUUGCAAUUCUUUGUAACCAUCAGAGGGCACCACCAAAAACUUUUGAGAAGUCUAUGAUGAACUUGCAAACUAAGAUUGAUGCCAAGAAGGAACAGCUAGCAGAUGCCCGGAGAGACCUGAAAAGUGCUAAGGCUGAUGCCAAGGUCAUGAAGGAUGCAAAGACGAAGAAGGUAGUAGAGUCAAAGAAGAAGGCUGUUCAGAGACUGGAGGAACAGUUGAUGAAGCUGGAAGUUCAAGCCACAGACCGAGAGGAAAAUAAACAGAUUGCCCUGGGAACCUCCAAACUCAAUUAUCUGGACCCUAGGAUCACAGUGGCUUGGUGCAAGAAGUGGGGUGUCCCAAUUGAGAAGAUUUACAACAAAACCCAGCGGGAGAAGUUUGCCUGGGCCAUUGACAUGGCUGAUGAAGACUAUGAGUUUUAGCCAGUCUCAAGAGGCAGAGUUCUGUGAAGAGGAACAGUGUGGUUUGGGAAAGAUGGAUAAACUGAGCCUCACUUGCCCUCGUGCCUGGGGGAGAGAGGCAGCAAGUCUUAACAAACCAACAUCUUUGCGAAAAGAUAAACCUGGAGAUAUUAUAAGGGAGAGCUGAGCCAGUUGUCCUAUGGACAACUUAUUUAAAAAUAUUUCAGAUAUCAAAAUUCUAGCUGUAUGAUUUGUUUUGAAUUUUGUUUUUAUUUUCAAGAGGGCAAGUGGAUGGGAAUUUGUCAGCGUUCUACCAGGCAAAUUCACUGUUUCACUGAAAUGUUUGGAUUCUCUUAGCUACUGUAUGCAAAGUCCGAUUAUAUUGGUGCGUUUUUACAGUUAGGGUUUUGCAAUAACUUCUAUAUUUUAAUAGAAAUAAAUUCCUAAACUCCCUUCCCUCUCUCCCAUUUCAGGAAUUUAAAAUUAAGUAGAACAAAAAACCCAGCGCACCUGUUAGAGUCGUCACUCUCUAUUGUCAUGGGGAUCAAUUUUCAUUAAACUUGAAGCAGUCGUGGCUUUGGCAGUGUUUUGGUUCAGACACCUGUUCACAGAAAAAGCAUGAUGGGAAAAUAUUUCCUGACUUGAGUGUUCCUUUUUAAAUGUGAAUUUUUAUUUCUUUUUAAUUAUUUUAAAAUAUUUAAACCUUUUUCUUGAUCUUAAAGAUCGUGUAGAUUGGGGUUGGGGAGGGAUGAAGGGCGAGUGAAUCUAAGGAUAAUGAAAUAAUCAGUGACUGAAACCAUUUUCCCAUCAUCCUUUGUUCUGAGCAUUCGCUGUACCCUUUAAGAUAUCCAUCUUUUUCUUUUUAACCCUAAUCUUUCACUUGAAAGAUUUUAUUGUAUAAAAAGUUUCACAGGUCAAUAAACUUAGAGGAAAAUGAGUAUUUGGUCCAAAAAAAGGAAAAAUAAUCAAGAUUUUAGGGCUUUUAUUUUUUCUUUUGUAAUUGUGUAAAAAAUGGAAAAAAACAUAAAAAGCAGAAUUUUAAUGUGAAGACAUUUUUUGCUAUAAUCAUUAGUUUUAGAGGCAUUGUUAGUUUAGUGUGUGUGCAGAGUCCAUUUCCCACAUCUUUCCUCAAGUAUCUUCUAUUUUUAUCAUGAAUUCCCUUUUAAUCAACUGUAGGUUAUUUAAAAUAAAUUCCUACAACUUAAUGGAAA(SEQ ID NO:15)；

the sequence of the C19ORF43 gene transcript is shown as SEQ ID NO:16, specifically:

GUCCUUUGCGCGGCACCUGGCGACAAAAUGGCUGCCCGAGGGAGACGGGCGGAGCCUCAGGGCCGGGAGGCUCCGGGCCCCGCGGGCGGUGGCGGUGGCGGGAGCCGUUGGGCUGAGUCGGGAUCGGGGACGUCGCCCGAGAGCGGGGACGAGGAGGUGUCGGGCGCGGGUUCGAGCCCGGUGUCGGGCGGCGUGAACUUGUUCGCCAACGACGGCAGCUUCCUGGAGCUGUUCAAGCGGAAGAUGGAGGAGGAGCAGCGGCAGCGGCAGGAGGAGCCGCCCCCGGGUCCGCAGCGACCCGACCAGUCGGCCGCCGCCGCUGGCCCCGGGGAUCCGAAGAGGAAGGGCGGUCCGGGCUCCACACUUAGCUUCGUGGGCAAACGCAGAGGCGGGAACAAACUAGCCCUCAAGACGGGAAUAGUAGCCAAGAAGCAGAAGACGGAGGAUGAGGUAUUAACAAGUAAAGGUGACGCGUGGGCCAAGUACAUGGCAGAAGUGAAAAAGUACAAAGCUCACCAGUGCGGUGACGAUGAUAAAACUCGGCCCCUGGUGAAAUGACGCCCCUCCCCCACCUGCCCAUGGCCUGGGACUCUCUGCGAUGUACAUAACUAUUUAAUGCAGCGGCAGCGGCGACAGCCUUCCCUGAGAGGACUUAAAAGCAGAAGGAAACCGAGAUGCUUCCCGCAGCCGUGGACGAUUCUCCAGGACUCUUUUUUUACCUUGAGCACUUGCCUCGUGAGACUUCAUAGAACAGUGGUUUACUGUCCCCCCCUUCUCACCUCCUCAUUCUCUCUGGCUCUUUCUGUCUUCCUCUUCUCACCCUCCUCCCUCCCCUUAGCCAUCACUUCUGGGAAGUAAAGAACUUGACUUAGUGCCGGA (SEQ ID NO: 16); and

the sequence of the miR-106b-3p is shown as SEQ ID NO: 17, specifically:

CCGCACUGUGGGUACUUGCUGC(SEQ ID NO:17)。

in another embodiment, the RNA molecule marker is from an exosome, preferably the exosome is from a bodily fluid, such as blood, urine, saliva or sputum.

A second aspect of the present disclosure provides a reagent, e.g., a primer and/or a probe, for detecting a marker of an RNA molecule according to any of the embodiments of the first aspect described above.

In one embodiment, the primers for detecting lnc-MKRN2-42:1 are: an upstream primer sequence shown as SEQ ID NO.1 and a downstream primer sequence shown as SEQ ID NO. 2; the probe sequence for detecting lnc-MKRN2-42:1 is shown as SEQ ID NO. 3;

primers for detecting TOP1 gene transcripts were: an upstream primer sequence shown as SEQ ID NO. 4 and a downstream primer sequence shown as SEQ ID NO. 5; the probe sequence for detecting the TOP1 gene transcript is shown as SEQ ID NO. 6;

the primers for detecting the transcript of the C19ORF43 gene are as follows: an upstream primer sequence shown as SEQ ID NO. 7 and a downstream primer sequence shown as SEQ ID NO. 8; the probe sequence for detecting the transcript of the C19ORF43 gene is shown as SEQ ID NO. 9; and

the primers for detecting miR-106b-3p are as follows: an upstream primer sequence shown as SEQ ID NO. 11 and a downstream primer sequence shown as SEQ ID NO. 13; the sequence of the probe for detecting miR-106b-3p is shown in SEQ ID NO 12.

A third aspect of the present disclosure provides a use of the RNA molecular marker of the first aspect or the reagent of the second aspect in preparing a kit for colorectal adenoma diagnosis, prognosis, efficacy monitoring and/or recurrence monitoring.

In four aspects of the present disclosure, a kit for colorectal adenoma diagnosis, prognosis, efficacy monitoring and/or recurrence monitoring is provided, which comprises the RNA molecular marker of the first aspect or the reagent of the second aspect, optionally, the kit further comprises an RNA extraction reagent, a reverse transcription reagent, and/or a PCR amplification reagent.

A fifth aspect of the present disclosure provides a method of colorectal adenoma diagnosis, prognosis, efficacy monitoring and/or recurrence monitoring, comprising the steps of:

(1) collecting body fluid samples of the subject, e.g. blood, urine, sputum, saliva

(2) Isolating exosomes from the body fluid sample;

(3) extracting exosome RNA;

(4) detecting the expression level of the RNA molecule marker combination comprising the long single-stranded non-coding RNA lnc-MKRN2-42:1, TOP1 gene transcript, C19ORF43 gene transcript and miR-106b-3p, preferably further compared with the expression level of a healthy control, to determine whether the subject has colorectal adenoma or has the size of the risk of colorectal adenoma, the therapeutic effect and/or prognosis of colorectal adenoma, and/or whether the colorectal adenoma recurs or the size of the risk of recurrence.

In one embodiment, the detection in step (4) comprises reverse transcription and quantitative PCR, preferably, the reagent used in the quantitative PCR is the reagent described in the second aspect.

In another embodiment, step (4) further comprises the step of normalizing said expression levels using an external reference gene, preferably cel-miR-39; preferably, the normalized expression level value of the molecular marker is further subjected to a logic (logistic) regression process to obtain an output value, and the output value is compared with a judgment threshold, wherein when the output value is higher than the judgment threshold, the output value indicates that the subject to be detected has colorectal adenoma or has a high risk of colorectal adenoma, the treatment effect and/or prognosis condition of colorectal adenoma is poor, and/or the colorectal adenoma recurs after the treatment or has a high risk of recurrence.

The tumor marker, the kit and the detection method provided by the invention realize a noninvasive detection method based on the plasma of a patient, have the characteristics of rapidness and high efficiency, have good patient compliance, have extremely high sensitivity and specificity on early colorectal adenoma species, and have important value on diagnosis of colorectal adenoma.

Drawings

FIG. 1 shows the performance of lnc-MKRN2-42:1, TOP1, C19ORF43 and miR-106b-3p in combination to diagnose early colorectal adenoma.

Detailed Description

The invention may be further understood by the examples, however, it is to be understood that these examples are not limiting of the invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the invention as described herein and claimed below.

Definition of

The term "colorectal adenoma" refers to a neoplastic polyp located in mucosal glandular epithelial tissue originating from the colorectal region. In the present invention, "colorectal adenoma" may include a case where adenoma occurs only at the site of colon (may also be referred to as "colorectal adenoma"), adenoma occurs only at the site of rectum (may also be referred to as "rectal adenoma"), or adenoma occurs simultaneously with colorectal adenoma.

The term "RNA molecule marker" is an RNA molecule associated with a particular disease, indication or trait that can be used to indicate that particular disease, indication or trait. In the present invention, the RNA molecular marker is used to indicate colorectal adenoma, and thus can be used as a colorectal adenoma RNA molecular marker.

In the present invention, the RNA molecular marker comprises a long non-coding RNA, specifically lnc-MKRN2-42:1, an exemplary sequence is shown in SEQ ID NO: 14; and mRNA, specifically TOP1 and C19ORF 43; and miRNA, in particular miR-106b-3p, an exemplary sequence is set forth in SEQ ID NO: shown at 17. Wherein, TOP1 gene transcript used as RNA molecular marker in the invention represents mRNA molecule obtained by transcription of TOP1 gene, and exemplary sequence is shown as SEQ ID NO. 15; the C19ORF43 gene transcript is an mRNA molecule transcribed from the C19ORF43 gene, and an exemplary sequence is shown in SEQ ID NO: 16.

It is noted that the sequences of the transcripts of lncRNA, miRNA and genes in actual detection may be different from the exemplary sequences because of the wide occurrence of sequence polymorphisms in human genes, and thus the detected sequences may be variant sequences of the above exemplary sequences, including, for example, alterations, insertions or deletions of one or more nucleotides.

The term "exosome" refers to a small extracellular vesicle, which is secreted by a cell to be released to the outside of the cell after fusion of an intracellular vesicle with a cell membrane, and has a double-layer membrane structure. The RNA molecule used as the marker of the RNA molecule in the present invention is present in and isolated from the exosome.

Examples

Example 1 screening of exosome RNA molecular markers associated with colorectal adenomas based on high throughput sequencing

Blood from 15 patients with early colorectal adenoma and 15 healthy subjects in the control group was not less than 10m L, plasma was isolated and then the differential expression of the mRNA was determined by classical ultracentrifugation (Evaluation of circulating small extracellular derived miRNAs as biomarkers of early color cancer: a complex with plasma total miRNAs, &lltttt transfer = L "&l/t &ttttm Min, S Zhu, che L n, X L iu, R Wei, cane 5 Zhao, Y Zhang, Z Zhang, GKong, Journal of extracellular derived mirvesal 8(1), 64) in plasma, AGEN = RNA was extracted using early colorectal tumor mini-array, the resulting differential expression of the prepared miRNA in the sample library was analyzed, and the final transcript RNA was analyzed for the differential expression of two sets of specific mRNA, final mRNA was found in the sample library of early colorectal miRNA, (5 mRNA: RNA, 5 mRNA was found to have a differential expression in the sample library, and was analyzed in the final sample library of early colorectal miRNA expression (5 mRNA).

TABLE 1 RNA molecular marker List

Example 2 fluorescent quantitative PCR platform-based miRNA, mRNA and lncRNA detection system

Detection System for miRNA

1) miRNA reverse transcription reaction system

miRNA reverse transcription reagents, enzymes and oligdT were purchased from TAKARA, standards were synthesized from the Shanghai Yinyi Weiji base, and primers for specific reverse transcription were synthesized by Suzhou hong, using a reverse transcription system of 20 μ L, as shown in Table 2.

TABLE 2 reverse transcription System for miRNA

2) qPCR reaction system:

the PCR reaction mixture was purchased from TAKARA, the upstream primer, the probe, i.e., the universal downstream primer, was synthesized by Honghong, Suzhou, and the fluorescent quantitative PCR instrument was ABI 7500. The PCR reaction system is shown in Table 3:

TABLE 3 qPCR reaction System for miRNA

mRNA and lncRNA detection system

1) Reverse transcription system

PrimeScript by TAKARA^TMRT reagent Kit (Perfect Real Time) and PremixEx Taq^TM(Probe qPCR) kit for reverse transcription and qPCR detection.

Preparing a reverse transcription reaction system (reaction solution is prepared on ice) according to the components listed in Table 4, then putting the reaction system into a PCR instrument for reaction under the reaction conditions of 37 ℃ for 60min, 85 ℃ for 5s and 12 ℃ infinity, and adding 50 mu L DEPC-H after the reverse transcription is finished₂O dilution, 3u L as a template, PCRShould be used.

TABLE 4 reverse transcription System for Long-chain RNAs

2) qPCR system

The primer and the probe for detecting the exosome miRNA molecular marker comprise:

primers and probes for detection of lnc-MKRN2-42: 1: the upstream primer of the lnc-MKRN2-42:1 is a nucleotide sequence shown as SEQ ID NO.1, the downstream primer is a nucleotide sequence shown as SEQ ID NO. 2, and the probe is a nucleotide sequence shown as SEQ ID NO. 3;

primers and probes for detection of TOP1 gene transcripts: the upstream primer of TOP1 is a nucleotide sequence shown as SEQ ID NO. 4, the downstream primer is a nucleotide sequence shown as SEQ ID NO. 5, and the probe is a nucleotide sequence shown as SEQ ID NO. 6;

primers and probes for detecting C19ORF43 gene transcripts: the upstream primer of the C19ORF43 is a nucleotide sequence shown as SEQ ID NO. 7, the downstream primer is a nucleotide sequence shown as SEQ ID NO. 8, and the probe is a nucleotide sequence shown as SEQ ID NO. 9.

Primers and probes for detection of miR-106b-3 p: the reverse transcription primer of the miR-106b-3p is shown as SEQ ID NO. 10; the upstream primer is a nucleotide sequence shown as SEQ ID NO. 11, the downstream primer is a nucleotide sequence shown as SEQ ID NO. 13, and the probe is a nucleotide sequence shown as SEQ ID NO. 12.

All the primer and probe sequences described above are shown in Table 5.

TABLE 5 primer and Probe sequences used in the examples

A qPCR reaction system was prepared according to the components listed in Table 6 (reaction solution was prepared on ice), and a no-template control was set as a negative control. Then, the mixture was put into a real-time fluorescence PCR apparatus (ABI7500) to perform amplification detection under the following reaction conditions.

TABLE 6 qPCR reaction System for Long-chain RNAs

TABLE 7 qPCR reaction conditions for Long-chain RNAs

Example 3 evaluation of the Effect of the kit for early diagnosis and detection of colorectal adenoma

1) Sample collection

Blood 10m L of early stage (stage I and II) colorectal adenoma patients (24 cases), benign colorectal patients and healthy human control samples (53 cases in total) diagnosed in hospitals were collected and plasma was isolated.

2) Exosome RNA extraction

Plasma exosomes were isolated using a Qiagen commercial ExoEasy kit or exosur from engitai, and RNA in exosomes was extracted using a Qiagen miReasy mini kit, and RNA concentration and quality were measured using Agilent 2100, and RNA concentration was recorded.

3) RNA two-step detection system

Detecting the plasma exosome RNA extracted in the step 2) by adopting the mRNA and lncRNA detection system based on the fluorescent quantitative PCR platform in the embodiment 2), detecting the Ct value of the target RNA, and calculating the relative expression amount according to the Ct value and a relative quantitative formula.

4) As a result:

ct values of lnc-MKRN2-42:1, miR-106b-3p, TOP1 and C19ORF43 were measured on plasma exosomes from 24 and 53 control samples (healthy human and benign lesions) of early colorectal adenoma patients, and the results of all samples are shown in Table 8. Further, the Δ Ct of the target RNA relative to the reference gene was calculated based on the Ct value of the target gene and the Ct value of the reference RNAcel-39-3p (shown in Table 9).

TABLE 8 Ct values for all RNA markers detected in each sample

TABLE 9 Delta Ct values of target RNAs relative to reference genes

Joint detection of markers lnc-MKRN2-42:1, miR-106b-3p, TOP1 and C19ORF43

Using the relative quantitative formula value 2^-ΔCtCalculating the fold change of the relative expression amount of each RNA molecular marker, and then knowing the relative expression amount of each RNA molecular markerThe relative expression level of the reference gene. On the basis, logistic regression is further adopted for training (using an R language glm function), and the ROC characteristic curve and AUC (using an R language ROC function) are used for evaluating the accuracy of the RNA molecule marker combination applied to detecting the colorectal adenoma.

As shown in fig. 1, based on the detection results in tables 8 and 9, p < ═ 0.05 was obtained by t-test analysis using the R language, indicating that the combined marker was significantly associated with early colorectal adenoma. The results show that the AUC of the combination diagnosis of early colorectal adenoma by lnc-MKRN2-42:1, miR-106b-3p, TOP1 and C19ORF43 is 0.83, the negative predictive value is 94.87%, the sensitivity is 91.67% and the specificity is 69.81%. The sensitivity and specificity meet the requirements of clinical application, and can be used as an effective clinical diagnosis marker.

Sequence listing

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aaggagaaga aaagaaaacu agaagaagaa gaggauggua aauugaaaaa acccaagaau 780

aaagauaaag auaaaaaagu uccugagcca gauaacaaga aaaagaagcc gaagaaagaa 840

gaggaacaga aguggaaaug gugggaagaa gagcgcuauc cugaaggcau caaguggaaa900

uuccuagaac auaaaggucc aguauuugcc ccaccauaug agccucuucc agagaauguc 960

aaguuuuauu augaugguaa agucaugaag cugagcccca aagcagagga aguagcuacg 1020

uucuuugcaa aaaugcucga ccaugaauau acuaccaagg aaauauuuag gaaaaauuuc 1080

uuuaaagacu ggagaaagga aaugacuaau gaagagaaga auauuaucac caaccuaagc 1140

aaaugugauu uuacccagau gagccaguau uucaaagccc agacggaagc ucggaaacag 1200

augagcaagg aagagaaacu gaaaaucaaa gaggagaaug aaaaauuacu gaaagaauau 1260

ggauucugua uuauggauaa ccacaaagag aggauugcua acuucaagau agagccuccu 1320

ggacuuuucc guggccgcgg caaccacccc aagaugggca ugcugaagag acgaaucaug 1380

cccgaggaua uaaucaucaa cuguagcaaa gaugccaagg uuccuucucc uccuccagga 1440

cauaagugga aagaaguccg gcaugauaac aagguuacuu ggcugguuuc cuggacagag 1500

aacauccaag guuccauuaa auacaucaug cuuaacccua guucacgaau caagggugag 1560

aaggacuggc agaaauacga gacugcucgg cggcugaaaa aaugugugga caagauccgg 1620

aaccaguauc gagaagacug gaaguccaaa gagaugaaag uccggcagag agcuguagcc 1680

cuguacuuca ucgacaagcu ugcucugaga gcaggcaaug aaaaggagga aggagaaaca 1740

gcggacacug ugggcugcug cucacuucgu guggagcaca ucaaucuaca cccagaguug 1800

gauggucagg aauauguggu agaguuugac uuccucggga aggacuccau cagauacuau 1860

aacaaggucc cuguugagaa acgaguuuuu aagaaccuac aacuauuuau ggagaacaag 1920

cagcccgagg augaucuuuu ugauagacuc aauacugguauucugaauaa gcaucuucag 1980

gaucucaugg agggcuugac agccaaggua uuccguacau acaaugccuc caucacgcua 2040

cagcagcagc uaaaagaacu gacagccccg gaugagaaca ucccagcgaa gauccuuucu 2100

uauaaccgug ccaaucgagc uguugcaauu cuuuguaacc aucagagggc accaccaaaa 2160

acuuuugaga agucuaugau gaacuugcaa acuaagauug augccaagaa ggaacagcua 2220

gcagaugccc ggagagaccu gaaaagugcu aaggcugaug ccaaggucau gaaggaugca 2280

aagacgaaga agguaguaga gucaaagaag aaggcuguuc agagacugga ggaacaguug 2340

augaagcugg aaguucaagc cacagaccga gaggaaaaua aacagauugc ccugggaacc 2400

uccaaacuca auuaucugga cccuaggauc acaguggcuu ggugcaagaa gugggguguc 2460

ccaauugaga agauuuacaa caaaacccag cgggagaagu uugccugggc cauugacaug 2520

gcugaugaag acuaugaguu uuagccaguc ucaagaggca gaguucugug aagaggaaca 2580

gugugguuug ggaaagaugg auaaacugag ccucacuugc ccucgugccu gggggagaga 2640

ggcagcaagu cuuaacaaac caacaucuuu gcgaaaagau aaaccuggag auauuauaag 2700

ggagagcuga gccaguuguc cuauggacaa cuuauuuaaa aauauuucag auaucaaaau 2760

ucuagcugua ugauuuguuu ugaauuuugu uuuuauuuuc aagagggcaa guggauggga 2820

auuugucagc guucuaccag gcaaauucac uguuucacug aaauguuugg auucucuuag 2880

cuacuguaug caaaguccga uuauauuggu gcguuuuuac aguuaggguu uugcaauaac 2940

uucuauauuu uaauagaaau aaauuccuaa acucccuucc cucucuccca uuucaggaau 3000

uuaaaauuaa guagaacaaaaaacccagcg caccuguuag agucgucacu cucuauuguc 3060

auggggauca auuuucauua aacuugaagc agucguggcu uuggcagugu uuugguucag 3120

acaccuguuc acagaaaaag caugauggga aaauauuucc ugacuugagu guuccuuuuu 3180

aaaugugaau uuuuauuucu uuuuaauuau uuuaaaauau uuaaaccuuu uucuugaucu 3240

uaaagaucgu guagauuggg guuggggagg gaugaagggc gagugaaucu aaggauaaug 3300

aaauaaucag ugacugaaac cauuuuccca ucauccuuug uucugagcau ucgcuguacc 3360

cuuuaagaua uccaucuuuu ucuuuuuaac ccuaaucuuu cacuugaaag auuuuauugu 3420

auaaaaaguu ucacagguca auaaacuuag aggaaaauga guauuugguc caaaaaaagg 3480

aaaaauaauc aagauuuuag ggcuuuuauu uuuucuuuug uaauugugua aaaaauggaa 3540

aaaaacauaa aaagcagaau uuuaauguga agacauuuuu ugcuauaauc auuaguuuua 3600

gaggcauugu uaguuuagug ugugugcaga guccauuucc cacaucuuuc cucaaguauc 3660

uucuauuuuu aucaugaauu cccuuuuaau caacuguagg uuauuuaaaa uaaauuccua 3720

caacuuaaug gaaa 3734

<210>16

<211>879

<212>RNA

<213> human

<400>16

guccuuugcg cggcaccugg cgacaaaaug gcugcccgag ggagacgggc ggagccucag 60

ggccgggagg cuccgggccc cgcgggcggu ggcgguggcg ggagccguug ggcugagucg 120

ggaucgggga cgucgcccga gagcggggac gaggaggugu cgggcgcggg uucgagcccg 180

gugucgggcg gcgugaacuu guucgccaac gacggcagcu uccuggagcu guucaagcgg 240

aagauggagg aggagcagcg gcagcggcag gaggagccgc ccccgggucc gcagcgaccc 300

gaccagucgg ccgccgccgc uggccccggg gauccgaaga ggaagggcgg uccgggcucc 360

acacuuagcu ucgugggcaa acgcagaggc gggaacaaac uagcccucaa gacgggaaua 420

guagccaaga agcagaagac ggaggaugag guauuaacaa guaaagguga cgcgugggcc 480

aaguacaugg cagaagugaa aaaguacaaa gcucaccagu gcggugacga ugauaaaacu 540

cggccccugg ugaaaugacg ccccuccccc accugcccau ggccugggac ucucugcgau 600

guacauaacu auuuaaugca gcggcagcgg cgacagccuu cccugagagg acuuaaaagc 660

agaaggaaac cgagaugcuu cccgcagccg uggacgauuc uccaggacuc uuuuuuuacc 720

uugagcacuu gccucgugag acuucauaga acagugguuu acuguccccc ccuucucacc 780

uccucauucu cucuggcucu uucugucuuc cucuucucac ccuccucccu ccccuuagcc 840

aucacuucug ggaaguaaag aacuugacuu agugccgga 879

<210>17

<211>22

<212>RNA

<213> human

<400>17

ccgcacugug gguacuugcu gc 22

Claims

1. An RNA molecular marker combination for colorectal adenoma diagnosis, prognosis judgment, curative effect monitoring and/or relapse monitoring comprises lnc-MKRN2-42:1, TOP1 gene transcript, C19ORF43 gene transcript and miR-106b-3 p.

2. The RNA molecular marker of claim 1, wherein the lnc-MKRN2-42:1 has a sequence shown in SEQ ID NO:14 and the sequence of TOP1 is shown in SEQ ID NO:15 and the sequence of C19ORF43 is shown as SEQ ID NO:16 and the sequence of miR-106b-3p is shown in SEQ ID NO: shown at 17.

3. The RNA molecule marker according to claim 1 or 2, wherein the RNA molecule marker is present in an exosome derived from a bodily fluid.

4. The RNA molecular marker of claim 3, wherein the body fluid is blood, urine, saliva or sputum.

5. A reagent for detecting a marker for the RNA molecule of any one of claims 1 to 4, the reagent comprising a primer and/or a probe, wherein

The primers for detecting lnc-MKRN2-42:1 are as follows: an upstream primer sequence shown as SEQ ID NO.1 and a downstream primer sequence shown as SEQ ID NO. 2; the sequence of a probe for detecting lnc-MKRN2-42:1 is shown as SEQ ID NO. 3;

primers for detecting TOP1 gene transcripts were: an upstream primer sequence shown as SEQ ID NO. 4 and a downstream primer sequence shown as SEQ ID NO. 5; the sequence of a probe for detecting TOP1 is shown as SEQ ID NO. 6;

6. Use of the RNA molecule marker of any one of claims 1 to 4 or the reagent of claim 5 for the preparation of a kit for colorectal adenoma diagnosis, prognosis, efficacy monitoring and/or recurrence monitoring.

7. A kit for colorectal adenoma diagnosis, prognosis, efficacy monitoring and/or recurrence monitoring comprising the RNA molecule marker of any one of claims 1 to 4 or the reagent of claim 5.

8. The kit of claim 7, further comprising an RNA extraction reagent, a reverse transcription reagent, and/or a PCR amplification reagent.

9. A method of colorectal adenoma diagnosis, prognosis, efficacy monitoring and/or recurrence monitoring comprising the steps of:

(1) collecting a body fluid sample of a to-be-detected object;

(2) isolating exosomes from the body fluid sample;

(3) extracting exosome RNA;

(4) detecting the expression level of the RNA molecule marker combination comprising the long single-stranded non-coding RNA lnc-MKRN2-42:1, TOP1 gene transcript, C19ORF43 gene transcript and miR-106b-3p, and further comparing the expression level with the expression level of a healthy control to judge whether the subject has colorectal adenoma or has the risk size of colorectal adenoma, the treatment effect and/or prognosis condition of colorectal adenoma, and/or whether the colorectal adenoma recurs or the risk size of recurrence is high.

10. The method according to claim 9, wherein the detection of step (4) comprises the steps of reverse transcription and quantitative PCR using the reagent of claim 5.

11. The method of claim 9, wherein step (4) further comprises the step of normalizing the expression level with an exogenous gene.

12. The method of claim 11, wherein the exogenous gene of step (4) is cel-miR-39.

13. The method according to claim 11, wherein after the normalization in step (4), the normalized expression level values of the molecular markers are subjected to logistic regression to obtain output values, and the output values are compared with a determination threshold, wherein when the output values are higher than the determination threshold, the output values indicate that the subject to be tested has colorectal cancer or has a high risk of colorectal adenoma, the treatment effect and/or prognosis condition of colorectal adenoma is poor, and/or the colorectal adenoma has recurred after the treatment or has a high risk of recurrence.