CN109295221B

CN109295221B - Application of circular RNA as molecular marker of colorectal cancer

Info

Publication number: CN109295221B
Application number: CN201811168419.2A
Authority: CN
Inventors: 施颖
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-10-08
Filing date: 2018-10-08
Publication date: 2021-07-27
Anticipated expiration: 2038-10-08
Also published as: CN109295221A

Abstract

The invention discloses the application of circular RNA as a colorectal cancer molecular marker. The present invention finds for the first time that hsa_circ_0005758 is related to colorectal cancer, and the expression of hsa_circ_0005758 is decreased in the plasma of colorectal cancer patients. Therefore, by detecting the expression of hsa_circ_0005758 in the subject, it can be judged whether the subject has colorectal cancer or whether there is a patient suffering from colorectal cancer or not. Risk of colorectal cancer. The hsa_circ_0005758 of the present invention can be used as a molecular marker for the diagnosis and treatment of colorectal cancer, which can realize the early diagnosis of colorectal cancer; it can also be used as an effective target for screening the diagnosis and treatment of colorectal cancer, and can be made into diagnostic kits, test strips and chips and other diagnostic tools. Meanwhile, the present invention also proves that hsa_circ_0005758 can inhibit the proliferation of intestinal cancer cells, so it can be used in the field of anti-tumor drugs.

Description

Application of circular RNA as colorectal cancer molecular marker

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of circular RNA as a colorectal cancer molecular marker.

Background

Because early symptoms of gastrointestinal tumors are not obvious and the specificity of screening indexes is not high, early discovery and early diagnosis become a great problem, the accuracy and sensitivity of postoperative recurrence detection indexes are not ideal, and the research of circular RNA brings eosin for early diagnosis of gastrointestinal tumors.

The circular RNA belongs to the non-coding RNA, closed RNA formed by non-covalent linkage of exons and/or introns (Guarnerio, J., et al., oncogene Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal transitions. cell,2016.166(4): p.1055-1056.). Circular RNA is characterized mainly by the following: firstly, the circular RNA is widely existed in the eukaryotic organism, has high abundance and has the characteristic of space-time specificity; secondly, the structure of a 5 'end cap and a 3' end tail (ploy A) is not provided, so that the structure is not sheared by RNA ribonuclease and has the characteristic of structural stability in a eukaryotic organism; thirdly, most of the genes have highly conserved sequences, and most of the genes do not have variation in the biological evolution process; and fourthly, the polypeptide can be widely existed in human serum exosomes and has specific expression in tumor tissues.

Research shows that the Circular RNA plays an important role in malignant process of tumor, and the regulation pathway of the Circular RNA mainly plays a role of a micro RNA sponge body by adsorbing the micro RNA, removes the inhibition effect of the micro RNA on a target gene and further regulates and controls the expression of the target gene (Holdt, L.M., et al, Circular non-coding RNA ANRIL models RNA activation and apoptosis in human NatCommun,2016.7: p.12429.). Therefore, in future clinical diagnosis, monitoring the expression level of plasma circular RNA can provide reference for clinical diagnosis of diseases, and become a marker related to early diagnosis and disease course; by regulating the expression of the circular RNA, a new strategy can be provided for diagnosis and treatment of tumors. Although the research on circular RNA is increasing nowadays and becomes a potential biomarker for early diagnosis of tumors of a new generation, no report on plasma circular RNA markers for early diagnosis and screening of colorectal cancer exists so far.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and providing the application of the circular RNA as the colorectal cancer molecular marker.

The invention also aims to provide the application of the circular RNA in preparing the anti-tumor drugs.

The purpose of the invention is realized by the following technical scheme: use of circular RNA as a molecular marker for colorectal cancer.

The circular RNA is hsa _ circ _0005758, and the nucleotide sequence is shown as follows (SEQ ID NO. 1):

TCACCCCAAACCAGAAGGCCTGGCTGCTGCCAAGAAGCTTTGTGAGAATCTTTTGCAAACAGTTCATGCTGAATACTCTAGATTTGTGAATCAGATTAATACTGCTGTACCTTTACCAGGCTATACACAACCCTCTGCTATAAGTAGTGTCCCTCCTCAACCACCATATTATCCATCCAATGGCTATCAGTCTGGTTACCCTGTTGTTCCCCCTCCTCAGCAGCCAGTTCAACCTCCCTACGGAGTACCAAGCATAGTGCCACCAGCTGTTTCATTAGCACCTGGAGTCTTGCCGGCATTACCTACTGGAGTCCCACCTGTGCCAACACAATACCCGATAACACAAGTGCAGCCTCCAGCTAGCACTGGACAG；

the sequence of the cyclization sites of the hsa _ circ _0005758 in circbase (circbase database) is as follows (SEQ ID NO. 2): agctagcactggacagtcaccccaaaccagaagg, respectively; the RNA is formed by variable shearing, is in a closed ring structure, is not easily degraded by exonuclease and is more stable than linear RNA; part of the circular RNA molecules contain a micro RNA response element, can be used as competitive endogenous RNA, is combined with micro RNA, and plays the role of micro RNA sponge in cells, thereby relieving the inhibition effect of the micro RNA on target genes thereof and up-regulating the expression level of the target genes.

The hsa _ circ _0005758 can be natural or artificial hsa _ circ _0005758 or obtained by transfecting cells with a vector expressing the DNA fragment of hsa _ circ _ 0005758.

The vector comprises a viral vector and a eukaryotic vector.

The viral vector may be any suitable vector, including but not limited to retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, alphaviral vectors and the like.

The herpesvirus includes herpes simplex virus, vaccinia virus, EB virus and the like.

The eukaryotic expression vector may be any suitable vector, including but not limited to a pCMT-Myc expression vector, a pcDNA3.0 expression vector, a pcDNA6.0 expression vector, a pEGFP expression vector, a Pef Bos expression vector, a pTet expression vector, a pTRE expression vector, or a vector modified based on a known expression vector, such as pBin438, pCAMBIA1301, and the like.

The application of the circular RNA in preparing a kit, test paper, a chip, a high-throughput sequencing platform, an exosome or a liposome for diagnosing colorectal cancer is disclosed, wherein the circular RNA is hsa _ circ _ 0005758.

The kit includes primers or probes for diagnosis of hsa _ circ _ 0005758.

The nucleotide sequence of the primer for diagnosing hsa _ circ _0005758 is as follows:

an upstream primer: 5'-gcatagtgccaccagctgtt-3', respectively;

a downstream primer: 5'-tctggtttggggtgactgtc-3' are provided.

Further, the primers or probes for diagnosing hsa _ circ _0005758 in the kit may further comprise primers or probes that have been reported in the prior art and can be used for detecting the expression level of the circular RNA.

The chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier.

Further, the solid phase carrier can be made of various conventional materials based on the chip field, including but not limited to nylon membrane, glass or silicon slice modified by active groups (such as aldehyde group, amino group, etc.), unmodified glass slice, plastic slice, etc.

The test strip includes primers or probes for diagnosis of hsa _ circ _ 0005758.

an upstream primer: 5'-gcatagtgccaccagctgtt-3', respectively;

a downstream primer: 5'-tctggtttggggtgactgtc-3' are provided.

The high throughput sequencing platform includes primers or probes for diagnosis of hsa _ circ _ 0005758.

an upstream primer: 5'-gcatagtgccaccagctgtt-3', respectively;

a downstream primer: 5'-gcctggtaaaggtacagcagta-3' are provided.

Use of a circular RNA for the preparation of a reagent for detecting the expression level of hsa _ circ _0005758, said circular RNA being hsa _ circ _ 0005758.

The application of the circular RNA as a colorectal cancer molecular marker in screening drugs for treating colorectal cancer can screen substances for promoting the expression of hsa _ circ _0005758 based on the characteristics after knowing the complete close correlation between hsa _ circ _0005758 and colorectal cancer; and a drug which is really useful for treating colorectal cancer can be found from the substances. Accordingly, the present invention also provides a method of screening for potential substances for the treatment of colorectal cancer, the method comprising: treating a colorectal cancer-associated cell line with a candidate substance, if the candidate substance promotes the expression or activity of hsa _ circ _0005758 as described above, indicating that the candidate substance is a potential substance for treating colorectal cancer.

The cell system can be a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably a non-human mammal animal model, such as a mouse, a rabbit, a sheep, a monkey, etc.), etc.; preferably, the obtained potential substances are subjected to further cell experiments and/or animal experiments to further select and identify substances that are truly useful for the treatment of colorectal cancer.

The application of circular RNA in preparing antitumor drugs, wherein the circular RNA is hsa _ circ _ 0005758.

The tumor is a digestive tract tumor, including colorectal cancer and the like.

The anti-tumor drug is a drug for inhibiting the proliferation of colorectal cancer cells.

The anti-tumor medicine comprises an effective dose of an accelerant; the promoter is capable of promoting expression of hsa _ circ _0005758, or is capable of promoting activity of hsa _ circ _0005758, or is capable of promoting the effective duration of hsa _ circ _0005758, or is capable of promoting stability of hsa _ circ _ 0005758.

The targets of the enhancer are not limited to hsa _ circ _0005758 itself, but also include upstream and downstream of hsa _ circ _0005758, for example: a genomic sequence encoding hsa _ circ _0005758, a target gene of hsa _ circ _0005758, a protein or gene that regulates hsa _ circ _ 0005758.

Further, the promoter comprises proteins, oligonucleotides, small molecule compounds, oligonucleotide expression vectors and the like; promoters may also be slow viral fluid, expression plasmids and/or small interfering RNA.

The anti-tumor drug also comprises a pharmaceutically acceptable carrier, and the carrier comprises but is not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulating agents, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavors, adsorptive carriers, and the like.

The antitumor drug can be prepared into various dosage forms according to the conventional method in the pharmaceutical field, including but not limited to dosage forms suitable for microinjection technology and transfection, such as injection, tablets, powder, granules, capsules and the like.

The anti-tumor drug can be used alone or combined with other drugs for treating colorectal cancer; the subject may be a human or other mammal; more specifically, the subject is an organ, tissue, cell.

The anti-tumor drug can be applied in vitro, and specifically comprises the following components: the expression vector of the circular RNA is introduced or transfected into human body self or heterogenous cells (or heterogenous cells) in vitro, and the circular RNA is transferred back to the human body after the in vitro cell amplification.

The anti-tumor drug can be applied in vivo, and specifically comprises the following components: directly introducing an expression vector of the circular RNA into a body; the expression vector may be viral or non-viral, even naked DNA or RNA.

The circular RNA is hsa _ circ _ 0005758.

As used herein, an "effective amount" refers to an amount that is functional or active in humans and/or animals and is acceptable to humans and/or animals. The effective dose of hsa _ circ _0005758 may vary depending on the mode of administration and the severity of the disease to be treated. Selection of a preferred effective dose can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the hsa _ circ _0005758 promoter are exemplified by bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc.

The means for analyzing the expression profile of circular RNA in the present invention include, but are not limited to, the following: reverse transcription polymerase chain reaction (RT-PCT), Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR), In situ hybridization (In situ hybridization), Northern blot hybridization (Northern blotting), RNase protection assay (RNase protection assay), Solexa sequencing technology (Solexa sequencing technology), and biochips. In a specific embodiment of the present invention, a real-time fluorescent quantitative polymerase chain reaction method and an in situ hybridization method are used.

The "circular RNA" used in the present invention is commonly used as "circular RNA" and "circular RNA".

"diagnosing colorectal cancer" as used in the present invention includes prognosis of a lesion of colorectal cancer, i.e., determining whether a subject is at risk of having colorectal cancer, and also includes diagnosis of colorectal cancer, i.e., determining whether a subject has a possibility of recurrence or determining that a subject has relapsed.

The term "treating colorectal cancer" as used in the present invention includes improvement, alleviation and cure of the disease.

The present invention uses colorectal cancer cells cultured in vitro to study the therapeutic effect of hsa _ circ _0005758 on colorectal cancer.

Compared with the prior art, the invention has the following advantages and effects:

1. the invention discovers for the first time that hsa _ circ _0005758 is associated with colorectal cancer, and by detecting the expression of hsa _ circ _0005758 in a subject, whether the subject has colorectal cancer or not can be judged, or whether the subject is at risk of having colorectal cancer or not can be judged, so that a clinician can know to provide a prevention scheme or a treatment scheme for the subject.

2. The hsa _ circ _0005758 can also be used as an effective target for screening colorectal cancer diagnosis and treatment, and can be made into diagnostic tools such as diagnostic kits, test paper, chips and the like, and can be used for early screening and diagnosis of colorectal cancer.

3. Experiments prove that hsa _ circ _0005758 can inhibit the biological process of intestinal cancer cell proliferation, so that hsa _ circ _0005758 is considered as a drug target for treating intestinal cancer. As a new molecular marker for colorectal cancer-related diagnosis and treatment, compared with the traditional detection means, the circular diagnosis has more timeliness, specificity and sensitivity, and can realize early diagnosis of colorectal cancer, thereby reducing the mortality rate of colorectal cancer. The molecular marker provides a theoretical basis for a clinician to formulate a personalized treatment scheme, can be used for early screening, diagnosis and treatment of clinical digestive tract tumors, and has wide application prospects in clinic.

Drawings

FIG. 1 is a graph showing the results of detecting the expression of hsa _ circ _0005758 in the plasma of patients with intestinal cancer and non-intestinal cancer by Real-time PCR.

FIG. 2 is a graph showing the effect of hsa _ circ _0005758 expression on intestinal cancer cell proliferation.

FIG. 3 is a graph showing the effect of interfering with hsa _ circ _0005758 expression on intestinal cancer cell proliferation.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. In the following examples, the reagents used were all analytical grade and were commercially available unless otherwise indicated. Experimental procedures not specifically identified herein are generally carried out under conventional conditions such as those described in the molecular cloning guidelines, published by scientific Press 2002, edited by J. SammBruk et al, or under conditions recommended by the manufacturer. 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. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention.

Example 1: real-time PCR detection of expression of hsa _ circ _0005758 in colorectal cancer tissue

1. Sample collection and preparation

10 colorectal cancer patients, 10 colonic polyps and 10 healthy volunteers were collected, and 4ml of whole blood was collected, centrifuged at 12,000 Xg at 4 ℃ for 10 minutes, to remove impurities that may be present, and plasma specimens were collected. The collected plasma specimens were stored in a refrigerator at-80 ℃ for later use.

RNA extraction

The plasma samples were dissolved on ice, 750. mu.l of TRIzol LS Reagent and 20. mu.l of glacial acetic acid were added, and the tubes were shaken vigorously by hand until mixed. And after homogenizing, incubating the sample at room temperature for 5 minutes to completely dissociate the nucleic acid-protein complex, adding 0.2ml of chloroform, manually and violently shaking the tube body for 15 seconds, and then incubating at room temperature for 2-3 minutes. Centrifugation was carried out at 12,000 Xg for 15 minutes at 4 ℃. After centrifugation, the upper colorless aqueous phase was removed, 500. mu.l of isopropanol was added, and the mixture was mixed well to precipitate RNA therein. After incubation at room temperature for 10 minutes, and centrifugation at 12,000 Xg for 10 minutes at 4 ℃, RNA precipitated on the bottom and side walls of the tube to form a gelatinous pellet. The supernatant was removed, 1ml of 75% (v/v) ethanol was added, and the RNA pellet was washed. After shaking, the mixture was centrifuged at 7,500 Xg for 5 minutes at 4 ℃. Removing the ethanol solution, and drying the RNA precipitate in the air for 5-10 minutes. RNase-free water was added to dissolve the RNA and incubated at 60 ℃ for 10 minutes. The RNA solution obtained was stored at-80 ℃.

cDNA Synthesis

First, an annealing mixture was prepared, including RNA template, primer Mix (Random N9primers, 10uM), dNTPs Mix (2.5mM), RNase-free H₂O, the mixture was placed in a water bath at 65 ℃ for 5 minutes and on ice for 2 minutes. The system is configured as follows:

reaction components	System proportion
		RNA template	600ng
Primer mix (10uM)	1μl
		dNTPs Mix(2.5mM)	1.6μl
Addition of RNase-free H₂To total volume of O	13.5μl

And secondly, gently and uniformly mixing the solution, centrifuging for a short time, and then sequentially adding RT reaction liquid comprising 5X First-Strand Buffer, 0.1M DTT, RNase Ihibitor and SuperScript III RT into a centrifuge tube. After mixing uniformly, the mixture was kept at 37 ℃ for 1 minute. Incubate at 50 ℃ for 60 minutes. The enzyme was inactivated by incubation at 70 ℃ for 15 minutes. The cDNA was placed in an ice bath for future use or stored at-20 ℃. The system is configured as follows:

reaction components	System proportion
		5X First-Strand Buffer	4μl
0.1M DTT	1μl
		RNase Ihibitor	0.5μl
SuperScript III RT	1μl
		Annealing the mixture	13.5μl
Total volume	20μl

4.Real-time PCR

Firstly, respectively configuring a Realtime PCR reaction system for the cDNA samples, mixing the solution at the bottom of a flick tube, and centrifuging at 5000rpm for a short time. Wherein:

PCR specific primer F (upstream primer): 5'-gcatagtgccaccagctgtt-3', respectively;

PCR specific primer R (downstream primer): 5'-tctggtttggggtgactgtc-3' are provided.

The system is configured as follows:

reaction components	System proportion
		2×Master Mix	5μl
PCR specific primer F of 10uM	0.5μl
		PCR specific primer R of 10uM	0.5μl
Adding water to the total volume	8μl

② Add 8. mu.l of the mixture to each well of the 384-well-PCR plate. The corresponding 2. mu.l of cDNA was added. A Sealing Film of Sealing Film is carefully stuck on the reaction kettle, and after the mixture is subjected to short-time centrifugal mixing, the reaction kettle is placed on a Realtime PCR instrument for PCR reaction. All the indexes were carried out according to the following procedures: at 95 ℃ for 10 min; 40 PCR cycles (95 ℃, 10 seconds; 60 ℃, 60 seconds (fluorescence collection), after the amplification reaction is finished, heating to 99 ℃ from 60 ℃ slowly (instrument automation-Ramp Rate is 0.05 ℃/second) according to the conditions of 95 ℃, 10 seconds, 60 ℃, 60 seconds, 95 ℃, 15 seconds).

5. Results

As shown in FIG. 1, hsa _ circ _0005758 expression was reduced in plasma of colorectal cancer patients (intestinal cancer group) compared to colorectal polyps patients (non-intestinal cancer group).

Example 2: colorectal cancer cell proliferation assay to detect the effects of hsa _ circ _0005758 expression on colorectal cancer cells

1. Intestinal cancer cell culture

The intestinal cancer cell lines HCT116, HT29 (purchased from ATCC) were adherent cells, and were cultured in culture Medium (McCoy's 5A (modified) Medium, purchased from Thermo Fisher scientific) containing 10% (v/v) fetal bovine serum. Adjusting the cell concentration to 3-5 × 10⁶L, inoculated in a culture dish at 37 ℃ with 5% (v/v) CO₂And 95% humidity. The growth condition of the cells is observed every day, the culture solution is changed, and the cells grow in a monolayer adherent manner.

Hsa _ circ _0005758 transfection

Intestinal cancer cells at a ratio of 1 × 10⁵Hole is spreadCulturing in 24-well plate for 18-24 hr to obtain cell number of 2 × 10⁵Perwell, transfection with hsa _ circ _0005758 lentivirus (purchased from Gisey Biotech, Guangzhou). 24 hours after transfection, the original medium was replaced with 2ml of fresh medium containing 6. mu.g/ml polybrene (polybrene), and the appropriate amount of hsa _ circ _0005758 lentiviral suspension was added. Incubation was performed at 37 ℃. After 4 hours 2ml fresh medium was added to dilute the coagulum. The culture was continued for 24 hours and the virus-containing medium was replaced with fresh medium for 72 hours.

3. Cell proliferation assay

Inoculating cell suspension of the intestinal cancer cells hsa _ circ _0005758 and control cells thereof in 96-well plate, and culturing at 37 deg.C with 5% (v/v) CO₂The incubator of (1) was pre-incubated for 24 hours. After 10ml of CCK-8 solution was added to each well and incubated for 1 to 4 hours, absorbance at 450nm was measured with a microplate reader.

4. Results

As shown in FIG. 2, hsa _ circ _0005758 inhibited intestinal cancer cell proliferation.

Example 3: colorectal cancer cell proliferation assay to detect effects of interference with hsa _ circ _0005758 expression on colorectal cancer cells

1. Intestinal cancer cell culture

The intestinal cancer cell lines HCT116, HT29 (purchased from ATCC) were adherent cells, and were cultured in culture Medium (McCoy's 5A (modified) Medium, purchased from Thermo Fisher Scientific) containing 10% (v/v) fetal bovine serum. Adjusting the cell concentration to 3-5 × 10⁶L, inoculated in a culture dish at 37 ℃ with 5% (v/v) CO₂And 95% humidity. The growth condition of the cells is observed every day, and the culture solution is replaced, so that the cells grow in a monolayer adherent manner.

2. Interference with hsa _ circ _0005758 transfection

The target sequence for interference hsa _ circ _0005758 is: GCTAGCACTGGACAGTCAC are provided. RNA interfering with hsa _ circ _0005758 was synthesized (si-hsa _ circ _0005758, siRNA). Using a six well plate as an example, 5X 10 of the day before transfection⁵The intestinal cancer cells are inoculated on a 6-well plate and 2ml of complete culture medium, and the cell confluence before transfection reaches 70-90%. Mu.l of siRNA (final concentration 50nM) was added to 100. mu.l of serum-free medium and mixed gently.Mix the lipofectamine reagent, dilute 4 μ l lipofectamine reagent with 100 μ l serum-free medium, mix gently, and stand at room temperature for 5 min. And mixing the diluted siRNA and lipofectamine reagent, gently mixing the mixture evenly, and standing the mixture for 20min at room temperature so as to form the siRNA-lipofectamine compound. Add 200 u lsiRNA-lipofectamine complexes to the cell wells that had been changed to 800 u l serum free medium, gently shake the cell culture plate back and forth. Cells were incubated at 37 ℃ and 5% (v/v) CO₂And (5) in the incubator, after 5-6 h of culture, absorbing the transfection medium, replacing the complete culture medium, and continuing to culture for 24 h.

3. Cell proliferation assay

The intestinal cancer cells interfering with hsa _ circ _0005758 and the cell suspension of the control group (no siRNA added in the control group) are inoculated in a 96-well plate, and 5% (v/v) CO is added at 37 DEG C₂The incubator of (1) was pre-incubated for 24 hours. After 10ml of CCK-8 solution was added to each well and incubated for 1 to 4 hours, absorbance at 450nm was measured with a microplate reader.

4. Results

As shown in FIG. 3, the interference hsa _ circ _0005758 promotes intestinal cancer cell proliferation.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

Application of <120> circular RNA as colorectal cancer molecular marker

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 373

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hsa_circ_0005758

<400> 1

tcaccccaaa ccagaaggcc tggctgctgc caagaagctt tgtgagaatc ttttgcaaac 60

agttcatgct gaatactcta gatttgtgaa tcagattaat actgctgtac ctttaccagg 120

ctatacacaa ccctctgcta taagtagtgt ccctcctcaa ccaccatatt atccatccaa 180

tggctatcag tctggttacc ctgttgttcc ccctcctcag cagccagttc aacctcccta 240

cggagtacca agcatagtgc caccagctgt ttcattagca cctggagtct tgccggcatt 300

acctactgga gtcccacctg tgccaacaca atacccgata acacaagtgc agcctccagc 360

tagcactgga cag 373

<210> 2

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> cyclization site sequence

<400> 2

agctagcact ggacagtcac cccaaaccag aagg 34

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer

<400> 3

gcatagtgcc accagctgtt 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer

<400> 4

tctggtttgg ggtgactgtc 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 2

<400> 5

gcatagtgcc accagctgtt 20

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 2

<400> 6

gcctggtaaa ggtacagcag ta 22

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> interference with the target sequence of hsa _ circ _0005758

<400> 7

gctagcactg gacagtcac 19

Claims

1. The application of the circular RNA in the preparation of a kit, test paper, a chip or a high-throughput sequencing platform for diagnosing colorectal cancer is characterized in that: the circular RNA is hsa _ circ _ 0005758.

2. Use according to claim 1, characterized in that: the kit and the test paper both comprise a primer or a probe for detecting hsa _ circ _0005758, and the nucleotide sequence of the primer for detecting hsa _ circ _0005758 is as follows:

an upstream primer: 5'-gcatagtgccaccagctgtt-3', respectively;

a downstream primer: 5'-tctggtttggggtgactgtc-3', respectively;

the high-throughput sequencing platform comprises a primer or a probe for detecting hsa _ circ _0005758, and the nucleotide sequence of the primer for detecting hsa _ circ _0005758 is as follows:

an upstream primer: 5'-gcatagtgccaccagctgtt-3', respectively;

a downstream primer: 5'-gcctggtaaaggtacagcagta-3' are provided.

3. The application of the circular RNA as a colorectal cancer molecular marker in screening medicaments for treating colorectal cancer is characterized in that: the circular RNA is hsa _ circ _ 0005758.

4. The application of the circular RNA in preparing the antitumor drug is characterized in that: the circular RNA is hsa _ circ _ 0005758;

the tumor is colorectal cancer.

5. The use of the circular RNA as claimed in claim 4 for the preparation of an antitumor drug, wherein: the anti-tumor drug is a drug for inhibiting the proliferation of colorectal cancer cells.

6. The use of the circular RNA according to any one of claims 4 to 5 for the preparation of an antitumor drug, wherein: the anti-tumor medicine comprises an effective dose of accelerant of hsa _ circ _ 0005758; the promoter is protein, oligonucleotide, small molecular compound, slow virus liquid and/or expression plasmid.

7. The use of the circular RNA of claim 6 for the preparation of an antitumor drug, wherein: the promoter is an oligonucleotide expression vector.

8. The use of the circular RNA of claim 6 for the preparation of an antitumor drug, wherein: the promoter is small interfering RNA.

9. The use of the circular RNA according to any one of claims 4 to 5 for the preparation of an antitumor drug, wherein: the anti-tumor medicine comprises a pharmaceutically acceptable carrier; the carrier is diluent, buffer, suspension, emulsion, granule, encapsulating agent, excipient, filler, adhesive, spray, transdermal absorbent, wetting agent, disintegrating agent, absorption enhancer, surfactant, colorant, correctant, or adsorption carrier.