CN111073981B

CN111073981B - Application of circular RNA circEMB-003 as colorectal cancer marker

Info

Publication number: CN111073981B
Application number: CN202010053100.6A
Authority: CN
Inventors: 胡育菡; 张哲莹; 马帅; 李健; 钟加滕
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2022-07-05
Anticipated expiration: 2040-01-17
Also published as: CN111073981A

Abstract

The invention relates to application of circular RNA circEMB _003 as a colorectal cancer marker. The expression level of this marker is negatively correlated with colorectal cancer tumor size (p <0.05), TNM staging (p <0.05) and distant metastasis (p <0.05), while there is no correlation with patient age, sex, tumor differentiation (p > 0.05). Meanwhile, the stability of circEMB _003 is verified through RNA enzyme R digestion and actinomycin D treatment, and the occurrence and development of colorectal tumors can be well reflected, so that the method can be used for monitoring the reaction of the colorectal tumors to treatment measures and the like. The marker provides a new experimental theoretical basis and a new direction for further researching the pathogenesis of the colorectal cancer and exploring the treatment target of the colorectal cancer, can be applied to the preparation of a reagent or a kit for colorectal cancer diagnosis and/or prognosis evaluation, and enriches the means for diagnosis and detection of the colorectal cancer.

Description

Application of circular RNA circEMB-003 as colorectal cancer marker

Technical Field

The invention relates to the field of colorectal cancer, in particular to application of circular RNA circEMB _003 as a colorectal cancer marker.

Background

Colorectal cancer is a common malignancy of the digestive tract that seriously harms human health. The incidence rate of the cancer is higher in the third place among various malignant tumors in the world, and the tumor-related mortality rate is higher in the second place. With the development of early tumor screening work and the application of new therapeutic methods, the mortality rate of colorectal cancer is reduced, but the morbidity rate is obviously increased and the colorectal cancer is increasingly younger. Therefore, the occurrence and development process of the colorectal cancer is deeply researched, key molecular markers and drug targets are searched, and the method has important scientific significance and clinical value for developing effective treatment measures and improving the survival rate of colorectal cancer patients.

Circular RNA (circRNA) is a non-coding RNA which is widely found in mammals in recent years, is rich in expression and is formed by performing variable shearing on special precursor mRNA with reverse complementary ends. Unlike conventional linear RNA (linear RNA) which has 5 'and 3' ends, circRNA is joined end to form a closed loop structure. The closed loop structure of circRNA makes it more stable than linear RNA and is resistant to degradation by exonucleases. Meanwhile, the expression of circRNA has tissue and cell type specificity, and abundant circRNA can be detected in blood, saliva, urine or exosomes. These features of circRNA suggest its potential as a diagnostic marker for disease. circRNA with diagnostic and prognostic value has now been found in a variety of tumors. For example, Josh N.vo et al detected the expression of circRNA in various tumors by exon capture RNA sequencing, and screened circRNA that could be used as a potential molecular marker for prostate cancer. Weng et al found that circRNA cirS-7 was expressed at increased levels in colorectal cancer and correlated with prognostic survival in patients. The research shows that the screening of the circRNAs as early tumor markers is an effective detection means and is expected to become a novel tumor molecular treatment target. At present, many circular RNAs related to colorectal cancer are not discovered, and research and search of the circular RNAs have important research value and clinical value for prevention and treatment diagnosis of colorectal cancer.

Disclosure of Invention

Based on this, it is necessary to provide an application of circular RNA circEMB _003 as a marker of colorectal cancer.

The application of a quantitative detection agent of circular RNA circEMB _003 in preparing a kit for colorectal cancer diagnosis and/or prognosis evaluation is characterized in that the circular RNA circEMB _003 has a nucleotide sequence shown in SEQ ID NO. 1.

In one embodiment, the quantitative detection agent for circular RNA circEMB _003 comprises a reagent suitable for use in at least one of the following methods:

fluorescent dye method, digital PCR, resonance light scattering method, real-time fluorescent quantitative PCR, sequencing or biomass spectrometry.

In one embodiment, the quantitative detection agent for circular RNA circEMB _003 is a probe or primer capable of specifically binding circular RNA circEMB _003 or a cDNA corresponding to circular RNA circEMB _ 003.

In one embodiment, the probe or primer carries a detectable label.

In one embodiment, the label is a fluorescent label, a chemiluminescent probe, or an isotopic label.

In one embodiment, the quantitative detection agent for circular RNA circEMB _003 is qRT-PCR primer of circular RNA circEMB _003, wherein the upstream primer is shown as SEQ ID NO.2, and the downstream primer is shown as SEQ ID NO. 3.

In one embodiment, the kit further comprises an internal reference primer pair.

In one embodiment, the reference primer pair comprises one or more of an 18S rRNA amplification primer pair and a GAPDH amplification primer pair.

In one embodiment, the nucleotide sequences of the 18S rRNA amplification primer pair are shown as SEQ ID NO.4 and SEQ ID NO.5, respectively, and the nucleotide sequences of the GAPDH amplification primer pair are shown as SEQ ID NO.6 and SEQ ID NO.7, respectively.

In one embodiment, the kit further comprises at least one of RNA extraction reagents, PCR reaction buffers, dNTPs, and DNA polymerase.

Through statistical analysis of the relation between the relative expression quantity of the colorectal cancer molecular marker (circEMB _003) in colorectal cancer tissues and clinical pathological parameters of colorectal cancer patients, the expression level of the circEMB _003 is negatively correlated with the size (p <0.05), the TNM stage (p <0.05) and the distant metastasis (p <0.05) of colorectal cancer tumors, but has no correlation with the age, the sex and the tumor differentiation of patients (p > 0.05). Meanwhile, the stability of circEMB _003 is verified through RNA enzyme R digestion and actinomycin D treatment, and the occurrence and development of colorectal tumors can be well reflected, so that the method can be used for monitoring the reaction of the colorectal tumors to treatment measures and the like. In addition, the potential value as a diagnostic marker for CRC is evaluated by preparing a receiver operating characteristic curve (ROC) by using the relative expression level of circEMB _003 in colorectal cancer (CRC) tissues and tissues around the cancer or the relative expression level of circEMB _003 in the plasma of the peripheral blood of a CRC patient and a normal human. The result was that the area under the ROC curve (AUC) was 0.768 (95% confidence interval of 0.669-0.876) and the specificity was 0.9275 (95% confidence interval of 0.8389-0.9761). The area under the ROC curve is closer to 1, and the diagnostic value is higher, which shows that the colorectal cancer can be efficiently identified from normal tissues by detecting the expression of circEMB _003, and the circEMB _003 has potential value as a colorectal cancer diagnostic marker. The marker provides a new experimental theoretical basis and a new direction for further researching the pathogenesis of the colorectal cancer and exploring the treatment target of the colorectal cancer, can be applied to the preparation of a reagent or a kit for colorectal cancer diagnosis and/or prognosis evaluation, and enriches the means for diagnosis and detection of the colorectal cancer.

Drawings

FIG. 1 is a schematic diagram of the formation of CircEMB _ 003;

FIG. 2 is a diagram of agarose gel electrophoresis of an amplified fragment of CircEMB _ 003;

FIG. 3 is a graph of Sanger sequencing results for CircEMB _ 003;

FIG. 4 is a qRT-PCR amplification curve for CircEMB _ 003;

FIG. 5 is a qRT-PCR dissolution curve for CircEMB _ 003;

FIG. 6 is a graph of the fold ratio of CircEMB _003 expression in 43 paired colorectal cancer tissues versus paracancerous tissues;

FIG. 7 is a graph of mean expression levels of CircEMB _003 in 43 paired colorectal cancer tissues and para-carcinoma tissues;

FIG. 8 is a graph showing the results of the stability verification of CircEMB _003 by digestion with RNase R, in which A is an agarose gel electrophoresis pattern and B is a graph showing the results of qRT-PCR;

FIG. 9 is a graph of stability results for actinomycin D treatment verification CircEMB _ 003;

figure 10 is a plot of the working characteristics of subjects evaluating the potential diagnostic value of circEMB _ 003;

FIG. 11 is a chart showing the results of FISH probe detection of circEMB _003 expression in colorectal cancer tissues;

FIG. 12 is a graph showing the results of the expression of CircEMB _003 in 7 lines of colorectal cancer cells and normal intestinal epithelial cells NCM 460.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The term "quantitative detection agent for circular RNA circEMB _ 003" in the present invention should not be understood as merely a detection agent for circular RNA circEMB _003, but should include the remaining detection agents known to those skilled in the art that reflect the expression level of circular RNA circEMB _ 003. For example, the reverse transcription of circular RNA circEMB _003 can be performed to obtain cDNA, or the expression level of circular RNA circEMB _003 can be indirectly measured.

In the present invention, "circular RNA circEMB _ 003" may be replaced by "marker", "marker gene", "biochemical marker", "colorectal cancer tumor marker" or "colorectal cancer marker", unless otherwise specified. It refers in particular to molecules to be used as targets for the analysis of patient test samples.

Circular RNA circEMB _003 used as a marker in the present invention is intended to include full-length ribonucleotide sequences thereof, or naturally occurring variants, or fragments of both full-length and variants, particularly fragments that can be detected and determined for a particular sequence, more preferably fragments that are distinguishable from other RNA sequences in colorectal tissue. Preferably at least 7, 8, 9, 10, 11, 12, 15 or 20 consecutive ribonucleotides of said full-length ribonucleotide sequence.

One skilled in the art will recognize that ribonucleotides released by cells or present in the extracellular matrix may be damaged (e.g., during inflammation) and may be degraded or cleaved into such fragments. As will be appreciated by the skilled artisan, mRNA or fragments thereof may also be present as part of the complex. Such complexes may also be used as markers in the sense of the present invention.

By "naturally occurring variant" it is understood that the genes of higher animals are often associated with a high frequency of polymorphisms. There are also many molecules that produce isoforms containing amino acid sequences that differ from each other during splicing. Any gene associated with a cancer-related disease having an activity similar to that of the marker gene is included in the marker gene even if it has a nucleotide sequence difference due to polymorphism or isotype.

As the quantitative detection agent for RNA, there can be used a known agent known to those skilled in the art, for example, a nucleic acid capable of hybridizing with the RNA and labeled with a fluorescent label; the detection agent for RNA can be selected from primers for RT-PCR and primers for amplification of cDNA, the product of RT-PCR in common.

The colorectal cancer related circular RNA gene, the colorectal cancer molecular marker and the application thereof are further explained in detail mainly by combining the detailed description and the attached drawings.

Extraction of tissue and cellular RNA

Total RNA was extracted from colorectal cancer tissues and cells using TRIzol reagent.

(1) Wrapping the mortar and scissors with tinfoil paper, and baking in an oven at 180 deg.C for 6-8 hr to remove RNA enzyme;

(2) taking out the mortar and other articles from the oven, cooling to room temperature, and precooling the mortar by using liquid nitrogen; shearing tissue blocks with the size of soybeans, placing the tissue blocks into a mortar for careful grinding, and adding liquid nitrogen while grinding until the tissue is ground;

(3) after the tissue is ground into powderAdding RNAiso^TMPlus reagent (1mL/50 mg-100 mg) and homogenizing until tissue and RNAioso are obtained^TMAfter fusing the Plus mixture, transferring the ground material into a 1.5mL centrifuge tube without RNase;

(4) if total RNA of cells is extracted, cells with good growth state are collected, trypsinized and collected into a 1.5mL centrifuge tube, washed 3 times with PBS, and the concentration of each 9.6cm²Area add 1mL of RNAiso^TMPlus, after standing for a moment, blowing and beating the mixture by using a gun head until no cell precipitates, and standing the mixture for 5min at room temperature;

(5) add 200. mu.L chloroform to 1mL TRIzol reagent, cover the tube after adding chloroform, shake it up and down vigorously for 20 seconds to ensure adequate contact between the aqueous and organic phases, and allow to stand at room temperature for 10 minutes. The tube was centrifuged in a low temperature centrifuge at 12000rpm for 15 minutes at 4 ℃. Centrifuging to separate the mixture, wherein the upper layer is a colorless aqueous phase containing total RNA and the lower layer is a light red phenol-chloroform phase;

(6) transferring the upper aqueous phase into a new centrifuge tube, adding isopropanol with the same volume as the aqueous phase liquid into the centrifuge tube, gently turning upside down to mix the mixture fully, standing for 10 minutes at room temperature, and centrifuging for 15 minutes at 12000 rpm.

(7) The supernatant was carefully discarded, the precipitated RNA washed, washed 2 times with 1ml of 75% ethanol (made up of autoclaved 0.1% DEPC water), centrifuged again in a low temperature centrifuge at 12000rpm for 5 minutes at 4 ℃ and the supernatant discarded, dried for 10 minutes at room temperature and dissolved in about 20. mu.L of DEPC water.

(8) The integrity of the total RNA is detected by 1% agarose gel electrophoresis, and the concentration and purity of the RNA are detected by a spectrophotometer and marked.

II, reverse transcription of RNA

(1) Reverse transcription of RNA according to PrimeScript^TMThe RT reagent Kit with gDNA Eraser (TaKaRa) instructions were performed by first removing gDNA that may be contaminating in RNA, and configuring the reaction system according to the following table:

reagent	Volume (μ L)
		5×gDNA Eraser Buffer	2
gDNA Eraser	1
		RNA	2(500ng/μL)
RNase Free dH₂O	up to 10

(2) gDNA removal program: at 42 ℃ for 2 min;

(3) the following reagents were added to the reaction system on ice as described below with the gDNA removed:

reagent	Volume (μ L)
		5×PrimeScript Buffer	4
PrimeScript RT Enzyme Mix I	1
		Random 6mers(100μM)	1
RNase Free dH₂O	4

(4) Reverse transcription procedure: 15min at 37 ℃; 85 ℃ for 5 sec;

(5) the reverse transcription product is diluted by 5-10 times by using sterile ultrapure water, is stored at the temperature of-20 ℃ and is used as soon as possible or is directly used for PCR and qRT-PCR reaction.

Third, Hsa _ circEMB _003 sequence (hereinafter, cDNA sequence)

Position: chr5:49694940-

Length: 954bp

RNA sequence(circBank):

aacattctagtatgccagtagaaaaaaatatcactttagaaaggccttctaatgtaaatctcacatgccagttcacaacatctggggatttgaatgcagtaaatgtgacttggaaaaaagatggtgaacaacttgagaataattatcttgtcagtgcaacaggaagcaccttgtatacccaatacaggttcaccatcattaatagcaaacaaatgggaagttattcttgtttctttcgagaggaaaaggaacaaaggggaacatttaatttcaaagtccctgaacttcatgggaaaaacaagccattgatctcttacgtaggggattctactgtcttgacatgtaaatgtcaaaattgttttcctttaaattggacctggtacagtagtaatgggagtgtaaaggttcctgttggtgttcaaatgaataaatatgtgatcaatggaacatatgctaacgaaacaaagctgaagataacacaacttttggaggaagatggggaatcttactggtgccgtgcactattccaattaggcgagagtgaagaacacattgagcttgtggtgctgagctatttggtgcccctcaaaccatttcttgtaatagtggctgaggtgattcttttagtggccaccattctgctttgtgaaaagtacacacaaaagaaaaagaagcactcagatgaggggaaagaatttgagcagattgaacagctgaaatcagatgatagcaatggtatagaaaataatgtccccaggcatagaaaaaatgagtctctgggccagtgaatacaaaacatcatgtcgagaatcattggaagatatacagagttcgtatttcagctttgtttatccttcctgttaagagcctctgagtttttagttttaaaaggatgaaaagcttatgcaacatgctcagcaggagcttcatcaacgatatatgtcagatctaaag

Design of primers

The EMB and circEMB _003 primers were designed using Oligo 7. The NCBI inquires an EMB mRNA sequence and selects a conserved region thereof to design a primer. The circEMB _003 sequence is an EMB exon 3-9 sequence, and a pattern diagram formed by connecting a part of the sequence (about 150bp) at the 5 'end to the 3' end is shown in FIG. 1. Inputting the sequence into Oligo7, setting the length of the primer to be 18 bp-23 bp, setting the length of the amplified fragment to be 100 bp-250 bp, selecting the primer with high score, approximate Tm values of the upstream primer and the downstream primer, no hairpin structure, no dimer and no mismatch as far as possible, and the upstream primer and the downstream primer of circEMB-003 need to cross the shearing site (namely the junction of the 5 'end and the 3' end). The GAPDH and 18S rRNA primer sequences are sequences which are verified to be effective in the laboratory. All primer sequences were blastd at NCBI to initially exclude the presence of non-specific binding amplification to other genes. The primers are synthesized by the company of bioengineering GmbH, the powdery primers are firstly centrifuged at high speed and then diluted into 100 mu M storage solution by non-ribozyme water, then diluted into 10 mu M working solution by sterile water, and the primers without non-specificity and dimer are selected by observing the dissolution curve through qRT-PCR for subsequent experiments. The specific primer sequences are shown in the following table:

polymerase Chain Reaction (PCR)

(1) Following the protocol of the PrimeSTAR GXL DNApolymerase (Takara, R050Q) kit, the following reaction system was first prepared on ice:

reagent	Volume (μ L)
		5×PrimeSTAR GXL Buffer	10
dNTP Mixture(2.5mM each)	4
		Forward Primer	1.5
Reverse Primer	1.5
		cDNAor DNA	1
PrimeSTAR GXL DNA Polymerase	1
		Sterilized distilled water	up to 50

(2) PCR reaction procedure: denaturation at 98 ℃ for 10 sec; annealing at 55 ℃ or 60 ℃ for 15sec (the Tm value of the primer is 55 ℃ or less and 60 ℃ or more) and the Tm value of the primer is 60 ℃ or more); extension at 68 ℃ for 18sec (calculated on the basis of the 1kb product set to 1 min); 35 cycles;

(3) after the PCR product was finished, it was stored at 4 ℃ and subjected to agarose gel electrophoresis as soon as possible.

Sixthly, agarose gel electrophoresis and Sanger sequencing

Selecting agarose gel with the preparation of 2 percent according to the length of a product fragment, namely adding 2g of agar powder into 100mL of 1 xTAE, repeatedly heating by microwave until the agarose gel is completely dissolved, clarifying the solution, paying attention to avoid the influence of liquid evaporation caused by excessive boiling on final concentration, cooling to about 60 ℃, adding 10 uL of GelStain, shaking uniformly, pouring into a glue preparation tank in which a comb is inserted, solidifying at room temperature for about 40min, pulling out the comb, putting glue into an electrophoresis tank, paying attention to the fact that a sample loading hole is arranged on the negative side, pouring 1 xTAE into the tank until the glue surface does not reach about 1mm, loading 10 uL of PCR product of loading buffer into each hole, adding 5 uL of DNAker into the hole on the extreme left side, carrying out electrophoresis for about 25 min-40 min under the voltage of 100V, collecting images in a gel imaging system after the electrophoresis is finished, and as shown in figure 2, verifying the reverse shear sequence of the CircEMB 003. Sequencing was then performed by clean-blade gel cutting and Sanger sequencing was performed by Beijing Rui Boxing Biotechnology Inc., and the results are shown in FIG. 3, which further confirmed the reverse cleavage sequence of circEMB _ 003.

Seventhly, fluorescent quantitative RT-PCR (qRT-PCR)

(1) The qRT-PCR reaction system was configured on ice under dark conditions according to the SybrGreen qPCR Master mix (DBI) instructions as follows:

reagent	Volume (μ L)
		SybrGreen qPCR master mix	10
Forward primer(10μM)	0.5
		Reverse primer(10μM)	0.5
cDNA	2
		ddH₂O (ultra pure water sterilization)	7
Total	20

(2) Adding 3 compound holes into each sample, fully and uniformly mixing, then performing instantaneous centrifugation, and putting the eight connecting tubes into an ABI7500 fluorescent quantitative PCR instrument;

(3) qRT-PCR reaction procedure: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15 sec; annealing and extending for 34sec at 60 ℃; 40 cycles;

(4) and (3) data analysis: by 2^-△△CtThe method is used for data sorting and analysis, the Ct value (the Ct value refers to the total number of cycles that the fluorescence signal in the reaction tube reaches a set threshold value) of each sample is subtracted by the Ct value of the internal reference (the delta Ct (Ct target gene) -Ct internal reference), namely the delta Ct value, 18S rRNA is used as the internal reference for actinomycin D treatment experiments, GAPDH is used as the internal reference for the rest, and the delta Ct (delta Ct) experimental group-delta Ct control group are used as the formula 2^-△△CtAnd calculating the relative expression amount. The experiments were repeated 3 times, and the mean and standard deviation were calculated 3 times.

The amplification curve is shown in FIG. 4, which shows high amplification efficiency and specificity. The melting curve is shown in FIG. 5 and is shown as a single peak, indicating no non-specific amplification and primer dimer. The graph of the expression of CircEMB _003 in 43 paired colorectal cancer tissues and paracarcinoma tissues is shown in fig. 6 (T/N, N for normal colorectal tissue and T for colorectal cancer tissue), which shows that CircEMB _003 is down-regulated in colorectal cancer tissue. The average expression level of CircEMB _003 in 43 paired colorectal and paracancerous tissues is shown in fig. 7, Normal represents Normal colorectal tissue, Tumor represents colorectal tissue, and the higher the Δ Ct value, the lower the expression level of CircEMB _003, indicating that the expression level of CircEMB _003 in colorectal tissue is significantly lower than that in paracancerous tissue.

Eighthly, RNA enzyme R digestion verification of circEMB _003 stability

Extracting RNA of para-carcinoma tissue of colorectal cancer, dividing the RNA into 1 microgram multiplied by 4 parts, adding no RNase R in 1 part, adding 0.05 microliter of RNase R and 0.7 microliter of RNase R buffer in the other 3 parts, adding sterile water without nuclease to supplement the sterile water to 7 microliter, carrying out digestion reaction in a PCR instrument, digesting for 15min and 30min at 37 ℃, adding 2 microliter of gDNAbuffer and 1 microliter of gDNA Eraser in each tube together with the undigested RNA after the digestion is finished, removing gDNA possibly polluted in the RNA at 42 ℃ for 2min, and removing the gDNA possibly polluted in the RNA according to PrimeScript^TMRT reagent Kit with gDNAeraser (TaKaRa) instruction for reverse transcription, reverse transcriptionThe transcribed cDNA was diluted 4-fold with sterile water, and 2. mu.L of the cDNA was subjected to PCR and qRT-PCR, and the mRNA levels of circEMB-003, EMB and GAPDH were compared with the RNA before the digestion.

As shown in FIG. 8, RNA from (+) colorectal SW480 was digested with RNase R for 15min and 30min, 0 was RNA without RNase R (-), and mRNA levels of circEMB _003, EMB and GAPDH were determined by PCR amplification agarose electrophoresis (FIG. 8A) and qRT-PCR (FIG. 8B) (. p < 0.05;. p, p < 0.01). The results show that circEMB _003 is indeed able to tolerate the digestion by rnase R compared to linear RNA.

Ninth, verification of circEMB _003 stability by Actinomycin D-treated cells

(1) Actinomycin D powder (1mg) was centrifuged and dissolved well in 100. mu.L DMSO (concentration 10. mu.g/. mu.L) in a super clean bench, stored at 4 ℃ and used as soon as possible;

(2) SW480 cells are paved in a 6-well plate, after 24h, actinomycin D (enabling the final concentration to be 100ng/mL) is added into an experimental group, DMSO with the same dosage is added into a control group, and 3 multiple wells are arranged in each group;

(3) collecting cells for extracting RNA after 0h, 12h and 24h of pancreatin digestion respectively, and taking 500ng after quantification for reverse transcription and subsequent qRT-PCR reaction.

The results are shown in figure 9, inhibition of the synthesis of SW480 nascent RNA in colorectal cancer cells by actinomycin D confirmed the stability of circEMB _003, and qRT-PCR results showed that EMB had degraded by more than 60% (p <0.0001) 12h after actinomycin D was applied, while circEMB _003 had not seen significant degradation (p >0.05), indicating that circEMB _003 was more stable than linear RNA EMB.

Ten, receiver operating characteristic curve (ROC) evaluation of the potential diagnostic value of circEMB _003

Relative expression of circEMB _003 in CRC tissues and tissues beside cancer is used for preparing a receiver operating characteristic curve (ROC) and evaluating the potential value of the ROC as a CRC diagnostic marker. As shown in FIG. 10, the area under the ROC curve (AUC) was 0.768 (95% confidence interval of 0.669 to 0.876), and the specificity was 0.9275 (95% confidence interval of 0.8389 to 0.9761). The area under the ROC curve is closer to 1, and the diagnostic value is higher, which shows that the colorectal cancer can be efficiently identified from normal tissues by detecting the expression of circEMB _003, and the circEMB _003 has potential value as a colorectal cancer diagnostic marker.

Eleven, relation between expression of CircEMB _003 in colorectal cancer tissues and clinical pathological characteristics of patients

We statistically analyzed the relationship between the relative expression of circEMB _003 in colorectal cancer tissues and the clinical pathological parameters of colorectal cancer patients. As shown in Table 1, the expression level of circEMB-003 was negatively correlated with the size of colorectal cancer tumor (p <0.05), TNM staging (p <0.05) and distant metastasis (p <0.05), but not with the age, sex, and differentiation of tumor of the patient (p > 0.05).

TABLE 1 relationship between expression levels of CircEMB _003 and clinical pathology of colorectal cancer patients

a are grouped by mean age.

b tumor diameters were grouped by mean.

*p<0.5。

Twelve, Fluorescence In Situ Hybridization (FISH)

Paraffin sections of 30 colorectal cancer specimens are subjected to paraffin removal and gradient dehydration, the glass slide is immersed in 0.2N HCl for 5min, after washing, the glass slide is digested with 40 mu g/mL proteinase K at 37 ℃ for 10min, and after washing, the glass slide is subjected to gradient dehydration with ethanol: 70 percent, 80 percent, 90 percent and absolute ethyl alcohol. The tissue taken out of the ethanol was quickly dropped with 1 × hybrid buffer, left at room temperature for 5min, gently decanted, and treated with green fluorescently labeled circEMB _003 probe (50nM) diluted with 1 × hybrid buffer for 1 hour at 56 ℃ with a cover slip. SSC gradient washing, 5min at 5 XSSC 56 deg.C, 5min X2 times at 1 XSSC 56 deg.C, 5min X2 times at 0.2 XSSC 56 deg.C, 5min at 0.2 XSSC normal temperature, 5min X3 times with PBS wash. DAPI working solution (1:1000) was added dropwise, incubated at room temperature for 10min, and washed with PBS for 5min × 3 times. A proper amount of the anti-fluorescence attenuation mounting agent mounting was added dropwise, and then observation and photography were performed using a fluorescence microscope, and the result is shown in FIG. 11, which indicates that circEMB-003 expression is down-regulated in colorectal cancer tissues.

Thirteen, the expression of CircEMB _003 in colorectal cancer cell lines

The qRT-PCR technology is used for respectively detecting the expression of circEMB _003 in 7 colorectal cancer cell strains SW620, HCT116, RKO, SW480, LoVo, Caco-2, HCT8 and normal colon epithelial cell strain NCM460, and the result is shown in figure 12, which shows that the expression level of the circEMB _003 in the colorectal cancer cells is obviously reduced compared with the NCM 460.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Xinxiang medical college

Application of <120> circular RNA circEMB _003 as colorectal cancer marker

<160> 7

<170> SIPOSequenceListing 1.0

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auggugaaca acuugagaau aauuaucuug ucagugcaac aggaagcacc uuguauaccc 180

aauacagguu caccaucauu aauagcaaac aaaugggaag uuauucuugu uucuuucgag 240

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cagaugauag caaugguaua gaaaauaaug uccccaggca uagaaaaaau gagucucugg 780

gccagugaau acaaaacauc augucgagaa ucauuggaag auauacagag uucguauuuc 840

agcuuuguuu auccuuccug uuaagagccu cugaguuuuu aguuuuaaaa ggaugaaaag 900

cuuaugcaac augcucagca ggagcuucau caacgauaua ugucagaucu aaag 954

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<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggacatctaa gggcatcaca 20

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggagcgagat ccctccaaaa t 21

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggctgttgtc atacttctca tgg 23

Claims

1. The application of the quantitative detection agent of the circular RNA circEMB _003 in preparing the kit for diagnosing the colorectal cancer is characterized in that the nucleotide sequence of the circular RNA circEMB _003 is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein the quantitative detection agent for circular RNA circEMB _003 comprises a reagent suitable for use in at least one of the following methods:

digital PCR, resonance light scattering, real-time fluorescent quantitative PCR, sequencing or biomass spectrometry.

3. The use according to claim 1, wherein the quantitative detector for circular RNA circEMB _003 is a probe or primer capable of specifically binding circular RNA circEMB _003 or a cDNA corresponding to circular RNA circEMB _ 003.

4. The use of claim 3, wherein the probe or primer is detectably labeled.

5. Use according to claim 4, wherein the label is a fluorescent label or an isotopic label.

6. The use according to claim 1, wherein the quantitative detector for circular RNA circEMB _003 is qRT-PCR primer for circular RNA circEMB _003, the upstream primer is shown as SEQ ID No.2, and the downstream primer is shown as SEQ ID No. 3.

7. The use of any one of claims 1 to 6, wherein the kit further comprises an internal reference primer pair.

8. The use of claim 7, wherein the pair of internal reference primers comprises one or more of a pair of 18S rRNA amplification primers and a pair of GAPDH amplification primers.

9. The use of claim 8, wherein the 18S rRNA amplification primer pair has nucleotide sequences shown as SEQ ID No.4 and SEQ ID No.5, respectively, and the GAPDH amplification primer pair has nucleotide sequences shown as SEQ ID No.6 and SEQ ID No.7, respectively.

10. The use of claim 7, wherein the kit further comprises at least one of an RNA extraction reagent, PCR reaction buffer, dNTPs, and DNA polymerase.