CN113493828A - Application of circular RNA in molecular marker of intestinal polyp - Google Patents

Application of circular RNA in molecular marker of intestinal polyp Download PDF

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CN113493828A
CN113493828A CN202110783778.4A CN202110783778A CN113493828A CN 113493828 A CN113493828 A CN 113493828A CN 202110783778 A CN202110783778 A CN 202110783778A CN 113493828 A CN113493828 A CN 113493828A
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circular rna
circhadha
primer
polyps
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施颖
李金莹
黄卫
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Jinan University
University of Jinan
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Abstract

The invention discloses application of circular RNA in molecular markers of intestinal polyps. The invention discovers that circHADHA, namely hsa _ circ _0053063 has expression difference in intestinal polyp patients and normal human plasma by utilizing real-time fluorescent quantitative PCR (polymerase chain reaction), stably expresses the circHADHA in intestinal epithelial cells, can promote the expression of autophagy related genes in the intestinal epithelial cells which are induced to generate inflammatory injury by LPS (lipo lipid), has the effect of promoting the repair of the intestinal epithelial injury, and can realize the aim of preventing the recurrence of the intestinal polyp. Therefore, the circular RNA can be used as a molecular marker for early screening and/or auxiliary diagnosis of intestinal polyps and a drug target for preventing and treating relapse and/or malignant change, and provides a theoretical basis for clinicians to formulate personalized treatment schemes.

Description

Application of circular RNA in molecular marker of intestinal polyp
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to application of circular RNA in a molecular marker of intestinal polyps.
Background
Intestinal polyps are a widespread and common disease, usually occuring in hidden and easily recurring. Therefore, a novel marker is searched, and the method has positive significance for auxiliary screening and prevention and treatment of intestinal polyp. Circular RNA (circular RNA ) is extensiveExists in eukaryotic cells, is generated by variable shearing, mostly has highly conserved sequences, does not have a 5 'end cap and a 3' end ploy A tail structure, is in a closed ring structure, is not easy to be degraded by RNA exonuclease, and is more stable than linear RNA expression[1]. Most circular RNAs are non-coding RNAs that exert a regulatory effect at the transcriptional or post-transcriptional level, but a few are also translatable into polypeptides.
Research shows that the circular RNA has differential expression in various human diseases and plays an important role. The circular RNA can regulate transcription, splicing and chromatin interaction, and participate in the regulation and control of transcription, alternative splicing and chromatin cyclization; can play the role of competitive endogenous RNA, and the spongiform absorbs the micro RNA to further regulate the expression of target genes. The circRNA may also bind to a different protein, act as a protein scaffold, or competitively bind to proteins[2]. In addition, the expression of the circular RNA in human peripheral blood has higher abundance, and the circular RNA can become a novel marker related to early diagnosis and disease course of diseases[3]。
Disclosure of Invention
The first purpose of the present invention is to overcome the shortcomings and drawbacks of the prior art, and to provide an application of circular RNA in preparing a drug for preventing and treating intestinal polyps, and a tool for early screening and/or auxiliary diagnosis of intestinal polyps.
The second purpose of the invention is to provide the application of the reagent for detecting the expression level of the circular RNA in the plasma in the preparation of the medicine for preventing and treating intestinal polyps and the tool for early screening and/or auxiliary diagnosis of the intestinal polyps.
The third purpose of the invention is to provide the application of the cyclic RNA as a molecular marker (drug target) in screening drugs for preventing and treating intestinal polyps and drugs for preventing and treating recurrence and/or malignant transformation of the intestinal polyps.
The fourth purpose of the invention is to provide the application of the circular RNA in the preparation of tools for diagnosing the intestinal mucosa injury.
The fifth purpose of the invention is to provide the application of the circular RNA in the preparation of the medicine for promoting the repair of the intestinal mucosa injury.
The purpose of the invention is realized by the following technical scheme:
the application of the circular RNA in preparing medicines for preventing and treating intestinal polyps and tools for early screening and/or auxiliary diagnosis of the intestinal polyps; wherein, the circular RNA is circHADHA, namely hsa _ circ _ 0053063.
The medicine for preventing and treating intestinal polyps comprises a medicine for preventing and treating intestinal polyps and a medicine for preventing (preventing) and treating recurrence and malignant transformation of the intestinal polyps.
The circHADHA (circRNA ID of circbase database: hsa _ circ _0053063) is in a closed ring structure and stably exists in peripheral blood, and the ring RNA molecule contains a micro RNA response element and can be used as competitive endogenous RNA to be combined with micro RNA-361 so as to regulate the expression level of a target gene.
The circHADHA improves the expression level of autophagy-related gene ATG13 in the intestinal epithelial cells by regulating miR-361, inhibits the expression level of cell inflammatory factors in the intestinal epithelial cells, and achieves the purpose of preventing and treating intestinal polyps or recurrence and malignant change of the intestinal polyps.
The cell inflammatory factors include but are not limited to IL-1 beta, IL-17A, IL-1 alpha, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-12, IL-13, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, TNF-beta and TGF-beta.
The circhacdha can be natural or artificially synthesized, or obtained by transfecting cells with a vector capable of expressing a DNA fragment of the circhacdha; such as circHADHA in blood plasma, is more suitable for clinical detection, and makes the operation more feasible.
The vector comprises a viral vector and a eukaryotic vector.
The viral vector may be any suitable vector, including but not limited to a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes viral vector, an alphaviral vector.
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 pLCDH-ciR expression vector, pCMT-Myc expression vector, pcDNA3.0 expression vector, pcDNA6.0 expression vector, pEGFP expression vector, Pef Bos expression vector, pTet expression vector, pTRE expression vector, or a vector modified based on known expression vectors, such as pBin438, pCAMBIA1301, etc.
The tools include, but are not limited to, kits, test strips, chips, high throughput sequencing platforms.
The tool comprises a reagent for detecting the expression level of circhacdha.
The kit comprises a primer and/or a probe for detecting circHADHA.
Further, the primer and/or probe for detecting circhacdha in the kit may further comprise a primer and/or probe for detecting the expression level of the circular RNA reported in the prior art.
The chip comprises a solid phase carrier and a circular RNA probe fixed on the solid phase carrier.
The circular RNA probe is a FISH probe, and the sequence of the circular RNA probe is shown as follows:
5’-CAAGCGGCTAACATGCCAGGGGT-3’。
further, the circular RNA probe immobilized on the chip can also comprise a circular RNA probe which is reported in the prior art and can be used for detecting the expression level of circHADHA.
Further, the circular RNA probe comprises a specific partial or whole sequence corresponding to circHADHA.
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 paper comprises a primer and/or a probe for detecting circHADHA.
The high-throughput sequencing platform comprises primers and/or probes for detecting circhacdha.
The primer sequence for detecting circHADHA comprises at least one of a PCR detection primer, a divergent primer and a PCR product sequencing primer; wherein the content of the first and second substances,
(1) the PCR detection primers are shown below:
an upstream primer: 5'-AAAATGGGACTGGTTGACCAACT-3', respectively;
a downstream primer: 5'-CTGTGCTTCTTGTGATAGCTGTG-3', respectively;
(2) divergent primers (Divergent primers) are shown below:
an upstream primer: 5'-TGGTGGAACCCCTGGCATGT-3', respectively;
a downstream primer: 5'-CAGGCAGGATCCATTGATGGC-3', respectively;
(3) PCR product sequencing primers are shown below:
an upstream primer: 5'-GGACATGATGCTGACTGGTAGA-3', respectively;
a downstream primer: 5'-ATGGCAACCTCAAGTCCTCCTC-3' are provided.
The probe for detecting circHADHA is a FISH probe, and the sequence of the probe is as follows:
5’-CAAGCGGCTAACATGCCAGGGGT-3’。
the application of the circular RNA in preparing a medicament for improving the expression of the autophagy-related gene ATG13 is provided.
The circular RNA (circHADHA) can promote the expression level of the autophagy-related gene ATG13 in intestinal epithelial cells by regulating miR-361.
The application of the reagent for detecting the expression level of the circular RNA in the plasma in preparing the medicine for preventing and treating intestinal polyps and the tool for early screening and/or auxiliary diagnosis of the intestinal polyps.
The application of the circular RNA as a molecular marker (drug target) in screening drugs for preventing and treating intestinal polyps and drugs for preventing and treating recurrence and/or malignant change of the intestinal polyps.
After the close correlation between the circHADHA and the intestinal polyps is known, substances for regulating the expression of the circHADHA can be screened based on the characteristics, and then drugs really useful for preventing and treating the recurrence and the malignant transformation of the intestinal polyps can be found from the substances, so the invention also provides a method for promoting the potential substances for repairing the intestinal mucosa injury, and the method comprises the following steps: treating the intestinal epithelium-related cell system in a damaged state with a candidate substance, wherein if the candidate substance can promote the expression or increase the activity of circhacdha as described above, the candidate substance is a potential substance for promoting repair of intestinal epithelium damage.
The medicine for preventing and treating intestinal polyps comprises medicines for preventing and treating intestinal polyps, such as medicines for inhibiting the growth of the intestinal polyps and the like.
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, and the like); preferably, the obtained potential substance is subjected to further cell experiments and/or animal experiments to further select and identify a substance that is truly useful for promoting repair of damage to the intestinal mucosa.
The application of the circular RNA in preparing a tool for diagnosing intestinal mucosa injury.
The tool for diagnosing the intestinal mucosa injury comprises but is not limited to a kit, test paper, a chip and a high-throughput sequencing platform.
The tool for diagnosing the intestinal mucosa injury comprises a reagent for detecting the expression level of circHADHA.
The application of the circular RNA in preparing the medicine for promoting the repair of the intestinal mucosa injury.
Further, the medicament comprises an effective dose of a promoter of circhacdha; the promoter can promote the expression of the circhacdha, or can improve the activity of the circhacdha, or can promote the effective action time of the circhacdha, or can improve the stability of the circhacdha.
Further, the target of said enhancer is not limited to circhacdha itself, but also includes upstream and downstream of circhacdha, such as: a genomic sequence encoding circhacdha, a regulated gene or protein of circhacdha, a gene or protein regulating circhacdha.
Further, the promoter includes proteins, oligonucleotides, small molecule compounds, expression plasmids (including oligonucleotide expression vectors), slow virus fluid, and/or small interfering RNA.
Further, the medicament can also comprise an effective dose of an inhibitor of circhacdha; the inhibitor can inhibit the degradation of circhacdha.
Further, the target of the inhibitor is not limited to circhacdha itself, but includes upstream and downstream of circhacdha, for example: a genomic sequence encoding circhacdha, a regulated gene or protein of circhacdha, a gene or protein regulating circhacdha.
Further, the inhibitor comprises protein, oligonucleotide, small molecule compound and oligonucleotide expression vector.
The vector for expressing the oligonucleotide comprises a viral vector and a eukaryotic vector.
Further, 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.
Further, the herpes virus includes herpes simplex virus, vaccinia virus, EB virus, etc.
Further, the eukaryotic expression vector may be any suitable vector, including but not limited to pLCDH-ciR expression vector, pCMT-Myc expression vector, pcDNA3.0 expression vector, pcDNA6.0 expression vector, pEGFP expression vector, Pef Bos expression vector, pTet expression vector, pTRE expression vector, or a vector modified based on known expression vectors, such as pBin438, pCAMBIA1301, etc.
The medicament also comprises pharmaceutically acceptable carriers, including but 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 medicament can be prepared into various dosage forms according to the conventional method in the field of pharmacology, including but not limited to microinjection technology, dosage forms suitable for transfection, injection, tablets, powder, granules and capsules.
The medicine can be independently applied, or can be applied by being combined with other medicines capable of preventing and treating intestinal polyp growth, or medicines for preventing and treating intestinal polyp recurrence and malignant transformation, or medicines for promoting intestinal mucosa injury repair; the subject may be a human or other mammal; more specifically, the subject is an organ, tissue, cell.
The medicament may be administered ex vivo; the ex vivo administration is realized by the following modes: the circHADHA expression vector is introduced or transfected into human body self or heterogenous cell (or heterogenous cell) in vitro, expanded in vitro and returned to human body.
The drug may also be administered in vivo; the in vivo administration is realized by the following modes: directly introducing an expression vector of circHADHA into a human body; the expression vector may be viral or non-viral, even naked DNA or RNA.
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 circhacdha in accordance with the invention may vary depending on the mode of administration and the severity of the disease to be treated, among other things. 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 circhacdha promoter include bioavailability, metabolism, half-life, and the like; 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 fluorescence quantitative polymerase chain reaction (Real-time PCR), In situ hybridization (In hybridization), RNA-seq sequencing (RNA sequencing), and biochip. In a specific embodiment of the present invention, a reverse transcription polymerase chain reaction method (RT-PCT) and a Real-time fluorescent quantitative polymerase chain reaction method (Real-time PCR) are used.
The "circular RNA" used in the present invention is commonly used as "circular RNA" and "circular RNA".
As used herein, "early diagnosis and/or assisted screening of intestinal polyps" includes prognosis of intestinal polyp lesions, i.e., determining whether a subject is at risk for intestinal polyps; also comprises the prejudgment of intestinal polyp lesion, namely the time judgment of judging whether the testee needs to receive definite diagnosis of colonoscopy; also included is the diagnosis of a recurrence of intestinal polyps, i.e., determining whether the subject is likely to have a recurrence or predicting that the subject has had a recurrence.
As used herein, "preventing and treating intestinal polyp recurrence and/or malignant transformation" includes amelioration, delay, remission, and cure of the disease.
The invention uses intestinal epithelial cells cultured in vitro to research the effects of circHADHA on promoting the repair of intestinal mucosa injury and preventing and treating intestinal polyp recurrence.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention utilizes real-time fluorescence quantitative PCR experiment to prove that the expression difference of circHADHA, namely, the circRNA ID, hsa _ circ _0053063 exists between intestinal polyp patients and normal human plasma, and the stable expression of the circHADHA in intestinal epithelial cells can promote the expression of autophagy related genes in the intestinal epithelial cells induced by LPS to generate inflammatory injury, so that the circHADHA is considered as a molecular marker for early screening and/or auxiliary diagnosis of intestinal polyp and a drug target for preventing and treating relapse and/or malignant change, can provide theoretical basis for clinical doctors to formulate personalized treatment schemes, and has clinical application prospect.
(2) The invention discovers that circHADHA is related to intestinal polyps for the first time, and by detecting the expression of the circHADHA of a subject, the invention can assist in judging whether the subject has the intestinal polyps, receiving the time of colonoscopy or judging whether the subject has the recurrence risk of having the intestinal polyps, thereby guiding a clinician to provide a screening or treatment scheme for the subject.
(3) Compared with the traditional detection means, the method has the characteristics of no wound, economy, convenience and the like in peripheral blood circular RNA diagnosis, can realize early screening and/or auxiliary diagnosis of intestinal polyps, has the effect of promoting repair of intestinal epithelial injury, and can realize prevention of recurrence of the intestinal polyps, so that the method can be used for preparing an intestinal polyp early screening and/or auxiliary diagnosis tool, an intestinal polyp recurrence prevention preparation or a medicine for promoting repair of intestinal mucosa injury.
Drawings
FIG. 1 is a graph showing that circHADHA promotes the expression of autophagy-related genes in intestinal epithelial cells by regulating miR-361.
FIG. 2 is a graph showing the inhibition of the expression level of cellular inflammatory factors in intestinal epithelial cells with LPS-induced inflammatory injury by circHADHA.
Detailed Description
The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure. 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: CircHADHA plasma screening and detection of regulation of cell autophagy-related gene expression in intestinal epithelial cells
1. Sample collection and preparation
Colonoscopy was performed in asymptomatic populations, 10 patients with intestinal polyps and 10 healthy volunteers were collected, 4ml of whole blood was drawn each, centrifuged at 12,000 Xg for 10 minutes at 4 ℃ to remove possible impurities, 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 the mixture was incubated at 60 ℃ for 10 minutes. The RNA solution obtained was stored at-80 ℃.
cDNA Synthesis
The annealing mixture was formulated as follows: 600ng of RNA template, 1. mu.l of gene-specific primer Mix (10uM) (available from Invitrogen), 1.6. mu.l of dNTPs Mix (2.5mM each) and RNase-free water were added to make the total volume 13.5. mu.l, and the mixture was incubated in a water bath at 65 ℃ for 5 minutes and then left on ice for 2 minutes. Subsequently, RT reaction solution was added as follows: 5 XFirst-Strand Buffer 4. mu.l, 0.1M Dithiothreitol (DTT) 1. mu.l, RNase Inhibitor 0.5. mu.l, SuperScript III RT 1. mu.l. After mixing, the mixture was incubated at 37 ℃ for 1 minute, 50 ℃ for 60 minutes and 70 ℃ for 15 minutes. The cDNA obtained is placed in an ice bath for later use or stored at-20 ℃.
4.Real-time PCR
A Real-time PCR reaction system is prepared according to the following system configuration: 2 × Arraystar PCR Master Mix 5 μ l, 10uM PCR specific forward primer 0.5 μ l, 10uM PCR specific reverse primer 0.5 μ l (forward primer: 5'-AAAATGGGACTGGTTGACCAACT-3'; reverse primer: 5'-CTGTGCTTCTTGTGATAGCTGTG-3'), RNase-free water to a total volume of 8 μ l, Mix the solutions at the bottom of the flick tube and centrifuge briefly. Add 8ul of the mixture to each corresponding well of 384-well PCR plate and add 2. mu.l of the corresponding cDNA, respectively. Sealing and mixing centrifugally for a short time, placing on a Realtime PCR instrument, and carrying out PCR reaction according to the following conditions: at 95 ℃ for 10 min; 95 ℃ for 10 seconds; 60 ℃ for 60 seconds (fluorescence collected); 95 ℃ for 10 seconds; 60 ℃ for 60 seconds; at 95 ℃, 15 seconds, 40 cycles; the heating was slowly brought to 99 deg.C (instrument auto-Ramp Rate 0.05 deg.C/sec).
Construction of circHADHA expression vector and lentiviral packaging
A full-length 362bp sequence of circHADHA/hsa _ circ _0053063 (circRNA ID: hsa _ circ _0053063, circbase database: http:// www.circbase.org /) was amplified by PCR and ligated into a modified lentiviral over-expression vector pLCDH-ciR containing the loop-forming elements (pLCDH-ciR from Genesed). Transfecting 293T cells with the constructed vector, extracting total RNA of a cell sample, carrying out qPCR detection after reverse transcription (an upstream primer: 5'-TGGTGGAACCCCTGGCATGT-3'; a downstream primer: 5'-CAGGCAGGATCCATTGATGGC-3'); meanwhile, sequencing primers are applied, PCR is carried out, then bands are recovered, and sequencing is carried out to verify the circularization site adaptor (upstream primer: 5'-GGACATGATGCTGACTGGTAGA-3'; downstream primer: 5'-ATGGCAACCTCAAGTCCTCCTC-3'). The sequencing peak map is normal, no miscellaneous peak or overlapping band exists, the sequence comparison is identical, and the results show that the circHADHA is successfully inserted into pLCDH-ciR and the over-expression vector is successfully constructed. 293T cells were used for simultaneous transfection (lipo2000, Invitrogen) of an overexpression plasmid and a helper packaging plasmid (pspAx.2, PMD2.G) (packaging plasmids were purchased from Geneseed). After 6 hours the medium was changed, cells were observed one day after transfection, and all supernatants were collected after 1 day of further culture in the incubator. Centrifuging for 10min to remove exfoliated cells and large cell debris, collecting circhacdha lentiviral fluid, concentrating, and storing at-80 deg.c.
6. Construction of intestinal epithelial cells stably expressing circHADHA
Culturing the intestinal epithelial cell line HCoEpic (purchased from ScienCell) in a culture medium containing 10% (v/v) fetal calf serum under the conditions of 37 ℃ and 5% (v/v) CO2And 95% humidity. The original medium was replaced with fresh medium containing 6. mu.g/ml polybrene (polybrene), and the appropriate amount of circHADHA lentivirus suspension obtained above was added. After 24 hours of incubation at 37 ℃, the virus-containing medium was replaced with fresh medium for 48 hours. Screening by puromycin (puromycin) with the final concentration of 2mg/L, and establishing an intestinal epithelial cell line for stably expressing circHADHA for about 2-3 weeks.
7. Autophagy-related gene expression detection
LPS (1ug/ml) was used to induce inflammatory injury of HCoEpic (purchased from ScienCell) in intestinal epithelial cells, and the expression of the autophagy-related gene ATG13 in intestinal epithelial cells was detected under the conditions of circHADHA and miR-361, respectively. The specific grouping is as follows:
1) after the intestinal epithelial cells were treated with induction by LPS (1ug/ml) to HCoEpic, miR-361(Gene ID: MIMAT0004682, having the sequence: 5'-UCCCCCAGGUGUGAUUCUGAUUU-3', respectively; GenePharma corporation) or miR-ctrl (miR-ctrl is a negative control; GenePharma corporation) was assigned, and 24 hours later, real-time fluorescent quantitative PCR was performed to detect the expression of the autophagy-associated gene ATG 13.
2) LPS (1ug/ml) induced intestinal epithelial cell HCoEpic treated with circHADHA overexpression (obtained in step (6)) or pLCDH-ciR (purchased from Geneseed Inc.) (circRNA control), and real-time fluorescent quantitative PCR was performed to detect the expression of autophagy-related gene ATG 13.
3) After the intestinal epithelial cells HCoEpic of circHADHA overexpression or pLCH-ciR (circRNA control) are treated by LPS (1ug/ml) induction, miR-ctrl (miR control) is transfected respectively, and after 24 hours, real-time fluorescence quantitative PCR is carried out to detect the expression of the autophagy-related gene ATG 13.
4) After intestinal epithelial cells HCoEpic of circHADHA overexpression or pLCH-ciR (circRNA control) are treated by LPS (1ug/ml) induction, miR-361 is transfected respectively, and real-time fluorescent quantitative PCR is carried out after 24 hours to detect the expression of the autophagy-related gene ATG 13.
8. Results
As shown in FIG. 1, circHADHA can promote the expression level of the autophagy-related gene ATG13 in intestinal epithelial cells with inflammatory lesions induced by LPS.
Example 2: detection of cytokine mediated by circhacdha in intestinal epithelial cells
1. Cellular inflammatory injury inducing treatment
After inflammatory injury of intestinal epithelial cells HCoEpic of circHADHA over-expressed or pLCH-ciR (circRNA control) obtained in the step (6) of example 1 was induced by LPS (1ug/ml), miR-361 was transfected respectively, RNA was extracted 24 hours later, and real-time fluorescence quantitative PCR was performed to detect a cell inflammatory factor.
RNA extraction
After adding Trizol to the cells, standing the cells at room temperature for 5min to fully crack the cells; chloroform was added thereto, followed by vigorous shaking for 15 seconds and standing at room temperature for 5 min. Centrifuging at 4 deg.C and 12,000g for 15min, sucking upper water phase, transferring to another centrifuge tube, adding isopropanol, mixing, and standing at room temperature for 10 min. Centrifuge at 12,000g for 10min at 4 ℃ and discard the supernatant. Add 75% (v/v) ethanol, gently shake the centrifuge tube, suspend the pellet. Centrifuging at 4 deg.C and 7,500g for 5min, and discarding supernatant as much as possible. Drying at room temperature for 5-10 min, and adding 20 μ l of RNase-free water to dissolve the precipitate.
3. Reverse transcription
Preparing a first strand cDNA synthesis reaction solution according to the following system: 2 × RT Mix 10 μ l, Genesed Enzyme Mix 1 μ l, primers 2 μ l, total RNA 1 μ g, make up to 20 μ l with RNase-free water.
The first strand cDNA synthesis reaction was performed as follows: 10min at 25 ℃, 15min at 42 ℃ and 5min at 85 ℃.
qPCR experiment
The qPCR reaction system is prepared according to the following system: geneseed qPCR SYBR Green Master Mix 10. mu.l, forward primer (10. mu.M) 0.5. mu.l, reverse primer (10. mu.M) 0.5. mu.l, 50 ROX Reference Dye 20.4. mu.l, template DNA 2. mu.l, the total system was made up to 20. mu.l with RNase-free water.
qPCR reaction program set-up was performed as follows: 95 ℃ for 5 min; 95 ℃, 10 seconds, 60 ℃, 34 seconds (signal acquisition), 40 cycles; 95 deg.C, 15 seconds, 60 deg.C, 60 seconds, 95 deg.C, 15 seconds.
The qPCR primer sequences were as follows:
①GAPDH：
an upstream primer: 5'-AGAAGGCTGGGGCTCATTTG-3', respectively;
a downstream primer: 5'-GCAGGAGGCATTGCTGATGAT-3', respectively;
②IL-1β：
5'-AGGAAGATGCTGGTTCCCTG-3' as upstream primer;
5'-GCATCGTGCACATAAGCCTC-3' as downstream primer;
③IL-17A：
5'-CAAGAACTTCCCCCGGACTG-3' as upstream primer;
the downstream primer is 5'-CTCTCAGGGTCCTCATTGCG-3'.
5. Results
As shown in FIG. 2, circHADHA inhibited the expression level of cellular inflammatory factors in LPS-induced inflammatory damaged intestinal epithelial cells.
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.
Reference to the literature
[1]Chen LL.The expanding regulatory mechanisms and cellular functions of circular RNAs.Nat Rev Mol Cell Biol 2020,21(8):475-490.
[2]Gao X,Xia X,Li F,Zhang M,Zhou H,Wu X,et al.Circular RNA-encoded oncogenic E-cadherin variant promotes glioblastoma tumorigenicity through activation of EGFR-STAT3signalling.Nat Cell Biol 2021.
[3]Wang S,Zhang K,Tan S,Xin J,Yuan Q,Xu H,et al.Circular RNAs in body fluids as cancer biomarkers:the new frontier of liquid biopsies.Mol Cancer 2021,20(1):13.
Sequence listing
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Claims (10)

1. The application of the circular RNA in preparing the medicine for preventing and treating intestinal polyps and the tool for early screening and/or auxiliary diagnosis of the intestinal polyps is characterized in that: the circular RNA is circHADHA, namely hsa _ circ _ 0053063.
2. Use according to claim 1, characterized in that:
the medicine for preventing and treating intestinal polyps comprises a medicine for preventing and treating intestinal polyps and a medicine for preventing and treating recurrence and malignant transformation of the intestinal polyps.
3. Use according to claim 2, characterized in that:
the circHADHA improves the expression level of autophagy-related gene ATG13 in the intestinal epithelial cells by regulating miR-361, inhibits the expression level of cell inflammatory factors in the intestinal epithelial cells, and achieves the purpose of preventing and treating intestinal polyps or recurrence and malignant change of the intestinal polyps.
4. Use according to claim 1, characterized in that:
the tool comprises a kit, test paper, a chip or a high-throughput sequencing platform;
the kit, the test paper and the high-throughput sequencing platform comprise primers and/or probes for detecting circHADHA;
the primer sequence for detecting circHADHA comprises at least one of a PCR detection primer, a divergent primer and a PCR product sequencing primer; wherein the content of the first and second substances,
(1) the PCR detection primers are shown below:
an upstream primer: 5'-AAAATGGGACTGGTTGACCAACT-3', respectively;
a downstream primer: 5'-CTGTGCTTCTTGTGATAGCTGTG-3', respectively;
(2) divergent primers are shown below:
an upstream primer: 5'-TGGTGGAACCCCTGGCATGT-3', respectively;
a downstream primer: 5'-CAGGCAGGATCCATTGATGGC-3', respectively;
(3) PCR product sequencing primers are shown below:
an upstream primer: 5'-GGACATGATGCTGACTGGTAGA-3', respectively;
a downstream primer: 5'-ATGGCAACCTCAAGTCCTCCTC-3', respectively;
the probe for detecting circHADHA is a FISH probe, and the sequence of the probe is as follows:
5’-CAAGCGGCTAACATGCCAGGGGT-3’；
the chip comprises a solid phase carrier and a circular RNA probe fixed on the solid phase carrier;
the circular RNA probe is a FISH probe, and the sequence of the circular RNA probe is shown as follows:
5’-CAAGCGGCTAACATGCCAGGGGT-3’。
5. use of the circular RNA of claim 1 for the preparation of a medicament for increasing the expression of the autophagy-related gene ATG 13.
6. Use of an agent for detecting the expression level of a circular RNA as described in claim 1 in plasma for the preparation of a medicament for the prevention of intestinal polyps, an early screening and/or a tool for the auxiliary diagnosis of intestinal polyps.
7. Use of the cyclic RNA as claimed in claim 1 as a molecular marker for screening drugs for the prevention and treatment of intestinal polyps, and drugs for the prevention and treatment of recurrence and/or malignant transformation of intestinal polyps.
8. Use of the circular RNA as claimed in claim 1 for the preparation of a tool for diagnosing damage to the intestinal mucosa.
9. Use according to claim 8, characterized in that: the tool for diagnosing the intestinal mucosa injury comprises a kit, test paper, a chip or a high-throughput sequencing platform.
10. Use of the circular RNA of claim 1 for the preparation of a medicament for promoting repair of damage to the intestinal mucosa.
CN202110783778.4A 2021-07-12 2021-07-12 Application of circular RNA in molecular marker of intestinal polyp Pending CN113493828A (en)

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