CN111073981B - Application of circular RNA circEMB_003 as a marker of colorectal cancer - Google Patents

Abstract

The present invention relates to the application of a circular RNA circEMB_003 as a colorectal cancer marker. The expression level of this marker was negatively correlated with the size (p<0.05), TNM stage (p<0.05) and distant metastasis (p<0.05) of colorectal cancer tumors, and was correlated with the patient's age, gender, and tumor differentiation. There was no correlation (p>0.05). At the same time, the stability of circEMB_003 was verified by RNase R digestion and actinomycin D treatment, which can better reflect the occurrence and development of colorectal tumors, so that it can be used to monitor the response of colorectal tumors to therapeutic measures. The discovery of this marker provides a new experimental theoretical basis and a new direction for further research on the pathogenesis of colorectal cancer and exploration of therapeutic targets for colorectal cancer, and can be applied to the preparation of colorectal cancer diagnosis and/or prognosis evaluation The reagents or kits have enriched the diagnosis and detection methods of colorectal cancer.

Description

Application of circular RNA circEMB_003 as a marker for colorectal cancer

technical field

The invention relates to the field of colorectal cancer, in particular to the application of a circular RNA circEMB_003 as a colorectal cancer marker.

Background technique

Colorectal cancer is a common digestive tract malignant tumor that seriously endangers human health. Its incidence ranks third among various malignant tumors in the world, and its tumor-related mortality ranks second. With the development of early tumor screening and the application of new treatment methods, the mortality rate of colorectal cancer has decreased, but its incidence has increased significantly and is getting younger and younger. Therefore, in-depth research on the occurrence and development of colorectal cancer and the search for key molecular markers and drug targets have important scientific significance and clinical value for developing effective treatment measures and improving the survival rate of colorectal cancer patients.

Circular RNAs (circRNAs) are a class of non-coding RNAs that are widely found in mammals in recent years. They are abundant in expression and are formed by alternative splicing of special reverse-complementary precursor mRNAs. Different from traditional linear RNA (linear RNA) which contains 5' and 3' ends, the end-to-end connection of circRNA forms a closed loop structure. The closed circular structure of circRNA makes it more stable than linear RNA and resistant to degradation by exonuclease. Meanwhile, the expression of circRNAs is tissue- and cell-type specific, and abundant circRNAs can also be detected in blood, saliva, urine or exosomes. These characteristics of circRNA suggest its potential as a disease diagnostic marker. CircRNAs with diagnostic and prognostic value have been found in a variety of tumors. For example, Josh N.Vo et al. detected the expression of circRNAs in various tumors by exon capture RNA sequencing, and screened circRNAs that can be used as potential molecular markers of prostate cancer. Weng et al. found that the expression level of circRNA ciRS-7 was increased in colorectal cancer and correlated with the prognosis and survival rate of patients. These studies all show that the screening of circRNAs as early tumor markers is an effective detection method, and it is expected to become a new tumor molecular therapeutic target. At present, there are still many circRNAs related to colorectal cancer that have not been discovered. It is of great research and clinical value to find these circRNAs for the prevention and diagnosis of colorectal cancer.

SUMMARY OF THE INVENTION

Based on this, it is necessary to provide the application of a circular RNA circEMB_003 as a marker for colorectal cancer.

The application of a quantitative detection agent of circular RNA circEMB_003 in the preparation of a kit for colorectal cancer diagnosis and/or prognosis evaluation, characterized in that the circular RNA circEMB_003 has the SEQ ID NO. Nucleotide sequence.

In one embodiment, the quantitative detection reagent for circular RNA circEMB_003 includes a reagent suitable for at least one of the following methods:

Fluorescent dye method, digital PCR, resonance light scattering, real-time PCR, sequencing or biological mass spectrometry.

In one embodiment, the quantitative detection agent for circular RNA circEMB_003 is a probe or primer that can specifically bind to circular RNA circEMB_003 or a cDNA corresponding to circular RNA circEMB_003.

In one embodiment, the probe or primer is detectably labeled.

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

In one embodiment, the quantitative detection agent of circular RNA circEMB_003 is the qRT-PCR primer of circular RNA circEMB_003, and the upstream primer is shown in SEQ ID NO.2, and the downstream primer is shown in SEQ ID NO.3.

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

In one embodiment, the internal reference primer pair includes one or more of a 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 respectively shown in SEQ ID NO. 4 and SEQ ID NO. 5, and the nucleotide sequences of the GAPDH amplification primer pair are respectively shown in SEQ ID NO. ID NO.6 and SEQID NO.7.

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

Through statistical analysis of the relationship between the relative expression levels of the above colorectal cancer molecular markers (circEMB_003) in colorectal cancer tissues and the clinicopathological parameters of colorectal cancer patients, it was found that the expression level of circEMB_003 was associated with the size of colorectal cancer tumors (p< 0.05), TNM stage (p<0.05), and distant metastasis (p<0.05) were negatively correlated, but had no correlation with the patient's age, gender, and tumor differentiation (p>0.05). At the same time, the stability of circEMB_003 was verified by RNase R digestion and actinomycin D treatment, which can better reflect the occurrence and development of colorectal tumors, which can be used to monitor the response of colorectal tumors to therapeutic measures. In addition, by using the relative expression of circEMB_003 in colorectal cancer (CRC) tissues and adjacent tissues or the relative expression of circEMB_003 in peripheral blood plasma of CRC patients and normal people, a receiver operating characteristic curve (ROC) was prepared to evaluate its performance as a Potential value of diagnostic markers for CRC. Results The area under the ROC curve (AUC) was 0.768 (95% confidence interval, 0.669-0.876), and the specificity was 0.9275 (95% confidence interval, 0.8389-0.9761). The closer the area under the ROC curve is to 1, the more diagnostic value it is, which indicates that colorectal cancer can be efficiently differentiated from normal tissues by detecting the expression of circEMB_003, and circEMB_003 has potential value as a diagnostic marker for colorectal cancer. The discovery of this marker provides a new experimental theoretical basis and a new direction for further research on the pathogenesis of colorectal cancer and exploration of therapeutic targets for colorectal cancer, and can be applied to the preparation of colorectal cancer diagnosis and/or prognosis evaluation The reagents or kits have enriched the diagnosis and detection methods of colorectal cancer.

Description of drawings

Fig. 1 is the pattern diagram that CircEMB_003 forms;

Fig. 2 is the agarose gel electrophoresis figure of CircEMB_003 amplified fragment;

Fig. 3 is the Sanger sequencing result graph of CircEMB_003;

Fig. 4 is the qRT-PCR amplification curve of CircEMB_003;

Fig. 5 is the qRT-PCR dissolution curve of CircEMB_003;

Figure 6 is a graph showing the fold ratio of CircEMB_003 expression in 43 cases of matched colorectal cancer tissues and adjacent tissues;

Figure 7 is a graph showing the average expression level of CircEMB_003 in 43 matched colorectal cancer tissues and adjacent tissues;

Figure 8 is a graph showing the stability results of RNase R digestion to verify CircEMB_003, wherein A is an agarose gel electrophoresis graph, and B is a qRT-PCR result graph;

Figure 9 is a graph showing the stability results of Actinomycin D treatment to verify CircEMB_003;

Figure 10 is the receiver operating characteristic curve for evaluating the potential diagnostic value of circEMB_003;

Figure 11 shows the results of FISH probe detection of circEMB_003 expression in colorectal cancer tissue;

Figure 12 is a graph showing the expression results of CircEMB_003 in 7 strains of colorectal cancer cells and normal intestinal epithelial cells NCM460.

Detailed ways

In order to facilitate understanding of the present invention, the present invention will be described more fully below, and preferred embodiments of the present invention will be given. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure is provided.

Unless otherwise defined, 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 terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present 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 only be understood as a detection agent for circular RNA circEMB_003, but should include other detections known to those skilled in the art that can reflect the expression level of circular RNA circEMB_003 reagents. For example, the cDNA obtained by reverse transcription of circular RNA circEMB_003 can be quantitatively detected, or the expression level of circular RNA circEMB_003 can be indirectly detected.

In the present invention, unless otherwise stated, "circular RNA circEMB_003" can be used as a "marker", "marker gene", "biochemical marker", "colorectal cancer tumor marker" or "colorectal cancer cancer marker" thing" is replaced. It specifically refers to a molecule to be used as a target for the analysis of a patient's experimental sample.

The circular RNA circEMB_003 used as a marker in the present invention is expected to include its full-length ribonucleotide sequence, or a naturally-occurring variant, or a fragment of the full-length sequence and the variant, in particular, the specific sequence can be detected and determined , more preferably fragments that can be distinguished from other RNA sequences in colorectal tissue. Preferably at least 7, 8, 9, 10, 11, 12, 15 or 20 contiguous ribonucleotides of the full length ribonucleotide sequence are included.

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

A "naturally occurring variant" is to be understood as a gene of a higher animal usually accompanied by a high frequency of polymorphisms. There are also many molecules that, during splicing, produce isoforms containing mutually different amino acid sequences. Any gene associated with a cancer-related disease that has an activity similar to that of the marker gene is included in the marker gene, even if it has nucleotide sequence differences due to polymorphism or being an isotype.

The quantitative detection reagent of RNA can be selected from reagents known to those skilled in the art, such as nucleic acid that can hybridize with the RNA and is labeled with fluorescent label, etc.; Primers for amplifying the product of RT-PCR - cDNA.

The following describes the colorectal cancer-related circular RNA genes, colorectal cancer molecular markers and their applications in further detail mainly in conjunction with the specific embodiments and the accompanying drawings.

1. Extraction of tissue and cellular RNA

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

(1) Wrap the mortar and scissors with tin foil and bake in a 180°C oven for 6-8 hours to remove RNase;

(2) Take the mortar and other items out of the oven and cool to room temperature, and pre-cool the mortar with liquid nitrogen; cut tissue pieces about the size of soybeans, put them into the mortar and grind them carefully, and add liquid nitrogen while grinding, until the tissue crushing;

(3) After the tissue is ground into powder, add RNAiso ^TM Plus reagent (1mL/50mg～100mg) and perform homogenization treatment. After the tissue is fused with the RNAiso ^TM Plus mixture, transfer the ground material to 1.5 RNase-free 1.5 mL centrifuge tube;

(4) If the total RNA of the cells is extracted, collect the cells in good growth state, digest them with trypsin and collect them into a 1.5 mL centrifuge tube, wash 3 times with PBS, add 1 mL of RNAiso ^TM Plus per 9.6 cm ² area, and let stand for a while Pipette with a pipette tip until there is no cell precipitation, and let stand at room temperature for 5 min;

(5) Add 200 μL of chloroform per 1 mL of TRIzol reagent. After adding chloroform, cover the centrifuge tube tightly and shake vigorously up and down for 20 seconds to ensure that the aqueous phase and the organic phase can fully contact, and let stand for 10 minutes at room temperature. The centrifuge tube was centrifuged in a low temperature centrifuge at 4°C, 12000 rpm, for 15 minutes. After centrifugation, the mixed solution was stratified, the upper layer was a substantially colorless aqueous phase containing total RNA, and the lower layer was a light red phenol-chloroform phase;

(6) Transfer the upper water phase into a new centrifuge tube, add isopropanol with the same volume as the water phase liquid in the centrifuge tube, gently invert it up and down to make it fully mixed, let stand for 10 minutes at room temperature, 4°C, Centrifuge at 12000 rpm for 15 minutes.

(7) Carefully discard the supernatant, wash the precipitated RNA, wash twice with 1 ml of 75% ethanol (prepared with 0.1% DEPC water after autoclaving), and then centrifuge in a low temperature centrifuge at 4°C, 12000rpm Centrifuge for 5 minutes, discard the supernatant, dry at room temperature for 10 minutes, and add about 20 μL of DEPC water to dissolve.

(8) The integrity of total RNA was detected by 1% agarose gel electrophoresis, and the concentration and purity of RNA were detected by spectrophotometer, and labeled.

2. RNA reverse transcription

(1) RNA reverse transcription is carried out according to the instructions of PrimeScript ^TM RT reagent Kit with gDNA Eraser (TaKaRa), first remove the gDNA that may contaminate the RNA, and configure the reaction system according to the following table:

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

(2) Procedure for removing gDNA: 42°C, 2min;

(3) Add the following reagents on ice to the aforementioned reaction system from which gDNA has been removed according to the following table:

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

(4) Reverse transcription program: 37°C, 15min; 85°C, 5sec;

(5) Dilute the reverse transcription product 5-10 times with sterile ultrapure water, store it at -20°C and use it as soon as possible or directly perform PCR and qRT-PCR reactions.

3. Hsa_circEMB_003 sequence (the following is the cDNA sequence)

Location: chr5:49694940-49707217strand:-

Length: 954bp

RNA sequence (circBank):

aacattctagtatgccagtagaaaaaaatatcactttagaaaggccttctaatgtaaatctcacatgccagttcacaacatctggggatttgaatgcagtaaatgtgacttggaaaaaagatggtgaacaacttgagaataattatcttgtcagtgcaacaggaagcaccttgtatacccaatacaggttcaccatcattaatagcaaacaaatgggaagttattcttgtttctttcgagaggaaaaggaacaaaggggaacatttaatttcaaagtccctgaacttcatgggaaaaacaagccattgatctcttacgtaggggattctactgtcttgacatgtaaatgtcaaaattgttttcctttaaattggacctggtacagtagtaatgggagtgtaaaggttcctgttggtgttcaaatgaataaatatgtgatcaatggaacatatgctaacgaaacaaagctgaagataacacaacttttggaggaagatggggaatcttactggtgccgtgcactattccaattaggcgagagtgaagaacacattgagcttgtggtgctgagctatttggtgcccctcaaaccatttcttgtaatagtggctgaggtgattcttttagtggccaccattctgctttgtgaaaagtacacacaaaagaaaaagaagcactcagatgaggggaaagaatttgagcagattgaacagctgaaatcagatgatagcaatggtatagaaaataatgtccccaggcatagaaaaaatgagtctctgggccagtgaatacaaaacatcatgtcgagaatcattggaagatatacagagttcgtatttcagctttgtttatccttcctgttaagagcctctgagtttttagttttaaaaggatgaaaagcttatgcaacatgctcagcaggagcttcatcaacgatatatgtcagatctaaag

4. Primer Design

Both EMB and circEMB_003 primers were designed with Oligo7. NCBI queried the EMB mRNA sequence, and selected its conserved region to design primers. The circEMB_003 sequence is the sequence of exons 3 to 9 of EMB, and a part of its 5'-end sequence (about 150 bp) is connected to the 3'-end, and the pattern formed is shown in Figure 1. Input the sequence into Oligo7, set the primer length to be 18bp to 23bp, the amplified fragment length to be 100bp to 250bp, and select the primers with high scores, close Tm values of upstream and downstream primers, and try to have no hairpin structure, no dimers, and no mismatches. The upstream and downstream primers of circEMB_003 need to span the cleavage site (ie, the junction between the 5' and 3' ends). The sequences of GAPDH and 18S rRNA primers are the valid sequences verified by the laboratory. All primer sequences were blasted on NCBI to preliminarily rule out non-specific binding to other genes. The primers were synthesized by Sangon Bioengineering Co., Ltd. The powdered primers were first centrifuged at high speed and then diluted with nuclease-free water to 100 μM stock solution, and then diluted with sterile water to 10 μM working solution, and the dissolution curve was observed by qRT-PCR. Specific and dimer primers were used for subsequent experiments. The specific primer sequences are shown in the table below:

Five, polymerase chain reaction (polymer chain reaction, PCR)

(1) According to the instructions of the PrimeSTAR GXL DNAPolymerase (Takara, R050Q) kit, first configure the following reaction system on ice:

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

(2) PCR reaction program: 98°C, 10sec denaturation; 55°C or 60°C, 15sec annealing (primer Tm value less than 55°C set to 55°C, greater than 55°C set to 60°C); 68°C, 18sec extension (according to the product 1kb is set as 1min calculation); 35 cycles;

(3) Store the PCR product at 4°C and perform agarose gel electrophoresis as soon as possible.

6. Agarose gel electrophoresis and Sanger sequencing

According to the length of the product fragment, choose and configure 2% agarose gel, that is, add 2g agar powder to 100mL 1×TAE, microwave repeatedly to dissolve completely, the solution is clear, pay attention to avoid the liquid evaporation caused by excessive boiling and affect the final concentration, cool down When the temperature reaches about 60°C, add 10 μL of GelStain, shake well, pour it into the glue dispensing tank with the comb inserted, and solidify for about 40 minutes at room temperature. Pull out the comb and put the gel into the electrophoresis tank. Note that the sample hole is on the negative side. 1×TAE was poured into the tank for about 1mm below the glue surface, 10μL of PCR products added with loading buffer were loaded into each well, 5μL of DNA marker was added to the leftmost well, and the electrophoresis was performed at 100V for about 25min-40min. Images acquired within the gel imaging system, as shown in Figure 2, confirmed the reverse shearing sequence of CircEMB_003. Then, the gel was cut with a clean blade and sent for sequencing. Sanger sequencing was completed by Beijing Ruibo Xingke Biotechnology Co., Ltd. The results are shown in Figure 3, which further confirmed the reverse shearing sequence of circEMB_003.

7. Fluorescence quantitative RT-PCR (qRT-PCR)

(1) According to the instructions of SybrGreen qPCR Master mix (DBI), configure the qRT-PCR reaction system on ice under dark conditions as follows:

reagent Volume (μL) SybrGreen qPCR master mix 10 Forward primer (10μM) 0.5 Reverse primer (10μM) 0.5 cDNA 2 ddH₂O (sterile ultrapure water) 7 Total 20

(2) Add 3 duplicate wells to each sample, mix well and centrifuge briefly, and put the eight tubes into the ABI7500 fluorescence quantitative PCR instrument;

(3) qRT-PCR reaction program: 95°C, pre-denaturation for 1 min; 95°C, denaturation for 15sec; 60°C, annealing and extension for 34sec; 40 cycles;

(4) Data analysis: use the 2- ^△△Ct method to organize and analyze the data, and subtract the Ct value of each sample (Ct value refers to the total number of cycles experienced when the fluorescence signal in the measured reaction tube reaches the set threshold) The Ct value of the internal reference (△Ct=Ct target gene-Ct internal reference) is the △Ct value, 18S rRNA was used as the internal reference in the actinomycin D treatment experiment, and GAPDH was used as the internal reference for the rest, △△Ct=△Ct experimental group-△Ct In the control group, the relative expression was calculated by formula 2 ^-ΔΔCt . The experiments were repeated three times, and the mean and standard deviation of the three times were calculated.

The amplification curve is shown in Figure 4, showing that the amplification efficiency is high and specific. The melting curve is shown in Figure 5, showing a single peak, indicating the absence of non-specific amplification and primer dimers. The fold ratio of CircEMB_003 expression in 43 paired colorectal cancer tissues and adjacent tissues is shown in Figure 6 (T/N, N represents normal colorectal tissue, T represents colorectal cancer tissue). Down-regulated expression in rectal cancer tissues. The average expression levels of CircEMB_003 in 43 paired colorectal cancer tissues and adjacent tissues are shown in Figure 7. Normal represents normal colorectal tissue, and Tumor represents colorectal cancer tissue. The higher the △Ct value, the lower the expression level of circEMB_003. , the results showed that the expression level of circEMB_003 in colorectal cancer tissues was significantly lower than that in adjacent tissues.

8. RNase R digestion to verify the stability of circEMB_003

RNA from adjacent tissues of colorectal cancer was extracted and divided into 1 μg×4 portions, one without RNase R, the other 3 with 0.05 μL RNase R and 0.7 μL RNase R buffer, and nuclease-free sterile water. Make up to 7 μL, the digestion reaction is carried out in a PCR machine, and digest at 37°C for 15min and 30min respectively. After the digestion is completed, add 2μL of gDNAbuffer and 1μL of gDNA Eraser to each tube together with the undigested RNA, and 42°C for 2min to remove possible RNA Contaminated gDNA was reverse transcribed according to the instructions of PrimeScript ^TM RT reagent Kit with gDNAEraser (TaKaRa), the reverse transcribed cDNA was diluted 4 times with sterilized water, and 2 μL was taken for PCR and qRT-PCR, circEMB_003, EMB and GAPDH mRNA levels Both were compared with the undigested RNA.

The results are shown in Figure 8. The RNA of (+) colorectal cell SW480 was digested with RNase R for 15 min and 30 min, respectively, and 0 was the RNA without RNase R (-), and PCR amplification was performed by agarose electrophoresis (Figure 8A). And (Fig. 8B) qRT-PCR detection of circEMB_003, EMB and GAPDH mRNA levels (*, p<0.05; **, p<0.01). The results showed that, compared with linear RNA, circEMB_003 was indeed able to tolerate RNase R digestion.

9. Actinomycin D treatment of cells to verify the stability of circEMB_003

(1) After centrifugation, actinomycin D powder (1 mg) was fully dissolved in 100 μL DMSO (concentration 10 μg/μL) in a clean bench, stored at 4°C, and used as soon as possible;

(2) SW480 cells were plated in 6-well plates, and after 24 hours, the experimental group was added with actinomycin D (to make the final concentration 100ng/mL), and the control group was added with the same dose of DMSO, with 3 duplicate wells in each group;

(3) At 0h, 12h, and 24h, the cells were digested with trypsin to collect the RNA extracted from the cells, and 500 ng was taken for reverse transcription and subsequent qRT-PCR reaction after quantification.

The results are shown in Figure 9. The application of actinomycin D inhibited the nascent RNA synthesis of colorectal cancer cells SW480 to verify the stability of circEMB_003. The qRT-PCR results showed that 12h after the application of actinomycin D, EMB had been degraded by more than 60% (p <0.0001), while circEMB_003 was not significantly degraded (p>0.05), indicating that circEMB_003 is more stable than linear RNA EMB.

10. Receiver operating characteristic curve (ROC) to evaluate the potential diagnostic value of circEMB_003

The relative expression of circEMB_003 in CRC tissues and adjacent tissues was used to generate receiver operating characteristic (ROC) curves to evaluate its potential value as a diagnostic marker for CRC. The results are shown in Figure 10. The area under the ROC curve (AUC) was 0.768 (95% confidence interval, 0.669-0.876), and the specificity was 0.9275 (95% confidence interval, 0.8389-0.9761). The closer the area under the ROC curve is to 1, the more diagnostic value it is, which indicates that colorectal cancer can be efficiently identified from normal tissues by detecting the expression of circEMB_003, and circEMB_003 has potential value as a diagnostic marker for colorectal cancer.

11. The relationship between the expression of CircEMB_003 in colorectal cancer tissues and the clinicopathological characteristics of patients

We statistically analyzed the relationship between the relative expression of circEMB_003 in colorectal cancer tissues and the clinicopathological parameters of colorectal cancer patients. The results are shown in Table 1. The expression level of circEMB_003 was negatively correlated with the tumor size (p<0.05), TNM stage (p<0.05) and distant metastasis (p<0.05) of colorectal cancer, and was negatively correlated with the patient's age, There was no correlation between gender and tumor differentiation (p>0.05).

Table 1 The relationship between the expression level of CircEMB_003 and the clinicopathological characteristics of colorectal cancer patients

aGrouped by mean age.

bTumor diameters are grouped by mean.

*p<0.5.

12. Fluorescence In situ hybridization (FISH)

After deparaffinization and gradient dehydration of paraffin sections of 30 colorectal cancer specimens, the slides were immersed in 0.2N HCl for 5 min, washed with 40 μg/mL proteinase K at 37 °C for 10 min, and washed with PBS with Ethanol gradient dehydration: 70%, 80%, 90%, absolute ethanol soaking. The tissue taken out of ethanol was quickly added dropwise with 1× hybridization buffer, placed at room temperature for 5 min, the buffer was gently poured, and the green fluorescent labeled circEMB_003 probe (50 nM) diluted with 1× hybridization buffer was added, and covered with a coverslip 56 ℃ for 1 hour. SSC gradient washing, 5×SSC at 56°C for 5min, 1×SSC at 56°C for 5min×2 times, 0.2×SSC at 56°C for 5min×2 times, 0.2×SSC at room temperature for 5min, and PBS for 5min×3 times. DAPI working solution (1:1000) was added dropwise, incubated at room temperature for 10 min, and washed with PBS for 5 min × 3 times. An appropriate amount of anti-fluorescence decay mounting medium was added dropwise to mount the slides, and then observed and photographed using a fluorescence microscope. The results are shown in Figure 11, indicating that the expression of circEMB_003 is down-regulated in colorectal cancer tissues.

13. Expression of CircEMB_003 in colorectal cancer cell lines

The expression of circEMB_003 in seven colorectal cancer cell lines SW620, HCT116, RKO, SW480, LoVo, Caco-2, HCT8 and normal colon epithelial cell line NCM460 was detected by qRT-PCR. The results are shown in Figure 12. showed that the expression level of circEMB_003 was significantly down-regulated in colorectal cancer cells compared with NCM460.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

sequence listing

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ggctgttgtc atacttctca tgg 23

Claims (10)

1. The application of a quantitative detection agent of circular RNA circEMB_003 in the preparation of a kit for colorectal cancer diagnosis, wherein the nucleotide sequence of the circular RNA circEMB_003 is shown in SEQ ID NO.1. 2. application according to claim 1, is characterized in that, the quantitative detection reagent of described circular RNA circEMB_003 comprises the reagent that is applicable to following at least one method: Digital PCR, resonance light scattering, real-time PCR, sequencing or biological mass spectrometry. 3 . The application according to claim 1 , wherein the quantitative detection agent for circular RNA circEMB_003 is a probe or primer that can specifically bind to circular RNA circEMB_003 or cDNA corresponding to circular RNA circEMB_003. 4 . 4. The use according to claim 3, wherein the probe or primer has a detectable label. 5. The application according to claim 4, wherein the label is a fluorescent label or an isotope label. 6. The application according to claim 1, wherein the quantitative detection agent of the circular RNA circEMB_003 is the qRT-PCR primer of the circular RNA circEMB_003, and the upstream primer is shown in SEQ ID NO. As shown in SEQID NO.3. 7. The application according to any one of claims 1 to 6, wherein the test kit also includes an internal reference primer pair. 8. The application according to 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 application according to claim 8, wherein the nucleotide sequences of the 18S rRNA amplification primer pair are respectively shown in SEQ ID NO.4 and SEQ ID NO.5, and the GAPDH amplification primer The nucleotide sequences of the pairs are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively. 10 . The application according to claim 7 , wherein the kit further comprises at least one of RNA extraction reagents, PCR reaction buffer, dNTPs and DNA polymerase. 11 .

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