CN117959447A - New use of cyclic RNA CIRCHP BP3 expression promoter in colorectal cancer diagnosis and treatment - Google Patents
New use of cyclic RNA CIRCHP BP3 expression promoter in colorectal cancer diagnosis and treatment Download PDFInfo
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- CN117959447A CN117959447A CN202410363136.2A CN202410363136A CN117959447A CN 117959447 A CN117959447 A CN 117959447A CN 202410363136 A CN202410363136 A CN 202410363136A CN 117959447 A CN117959447 A CN 117959447A
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Abstract
The invention discloses a new application of a circular RNACIRCHP BP3 expression promoter in colorectal cancer diagnosis and treatment, and discovers that circular RNACIRCHP BP3 is a brand-new colorectal cancer diagnosis and treatment target for the first time, and the circular RNACIRCHP BP3 can exert an in-vivo treatment effect of effectively killing colorectal cancer cells and can be used in preparation of tumor immunotherapy medicaments. The invention provides a new target for diagnosis and treatment of colorectal cancer, and has wide application prospect in clinical treatment of colorectal cancer.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a novel application of a cyclic RNA CIRCHP BP3 expression promoter in colorectal cancer diagnosis and treatment.
Background
Colorectal cancer, also known as carcinoma of large intestine, includes malignant tumors of the colon and rectum. It is one of the most common malignant tumors worldwide and the incidence is increasing. In China, the incidence and mortality rate of colorectal cancer also have an increasing trend. The hazard of colorectal cancer is mainly manifested in four aspects, firstly, early symptoms are not obvious, colorectal cancer often has no obvious symptoms in early stages, is easy to ignore or misdiagnose, and causes the worsening of the disease, when the obvious symptoms appear in patients, the patients often enter middle and late stages, and the treatment difficulty and risk are greatly increased; secondly, the disease progress is rapid, the colorectal cancer cells grow fast, and surrounding tissues and organs are easy to invade, so that serious complications such as intestinal obstruction, perforation, bleeding and the like are caused; again, the treatment means are limited, and although the treatment methods of colorectal cancer comprise surgery, chemotherapy, radiotherapy, targeted therapy and the like, the curative effect is different according to individual differences, and part of patients do not respond well to the existing treatment means; finally, the recurrence rate is high: even after treatment, the recurrence rate of colorectal cancer is still high, which has a serious impact on the quality of life and prognosis of the patient. Therefore, developing accurate detection methods and effective therapeutic means is an important scientific research direction.
Circular RNAs (circrnas) are a unique class of non-coding RNA molecules that exhibit a closed circular structure and are resistant to exonucleases, thus exhibiting high stability in cells. With advances in molecular biology, the role of circRNA in the development and progression of tumors, particularly colorectal cancer, is increasingly recognized. Research shows that the circRNA plays a key regulatory function in colorectal cancer, and the circRNA can be used as a oncogene or an anticancer gene to participate in the processes of tumor growth, invasion, metastasis and the like. Thus, circRNA is expected to be an important biomarker for colorectal cancer diagnosis, treatment and prognosis evaluation.
To date, no related studies or reports have been found of the use of circular RNA CIRCHP BP3 in the diagnosis and/or treatment of colorectal cancer.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a novel application of the annular RNA CIRCHP BP3 in colorectal cancer diagnosis and treatment.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides the use of a cyclic RNA CIRCHP BP3 expression promoter in the manufacture of a medicament for the treatment and/or prophylaxis of colorectal cancer;
further, circBase ID of the loop RNA CIRCHP BP3 is hsa_circle_ 0005782.
Further, the cDNA sequence corresponding to the circular RNA CIRCHP BP3 is shown as SEQ ID NO. 1;
The RNA sequence corresponding to the annular RNA CIRCHP BP3 is shown as SEQ ID NO. 2;
the annular RNA CIRCHP BP3 is an end-to-end annular structure formed by splicing after transcription of the nucleotide sequence shown in SEQ ID NO. 1;
The annular RNA CIRCHP BP3 is of an annular structure formed by connecting the nucleotide sequences shown in SEQ ID NO. 2 end to end.
Further, the medicaments comprise molecular targeted medicaments, biological agents and pharmaceutical compositions for the treatment and/or prevention of colorectal cancer.
Further, the cyclic RNA CIRCHP BP3 expression-promoting agent comprises a natural purified substance, a modified natural purified substance, a semisynthetic substance, a chemically synthesized substance, and/or any combinations thereof capable of promoting expression of cyclic RNA CIRCHP BP 3.
Further, the circular RNA CIRCHP BP3 expression promoter comprises circular RNA CIRCHP BP3, a recombinant vector comprising circular RNA CIRCHP BP3, a nanoparticle comprising circular RNA CIRCHP BP3, a protein microsphere comprising circular RNA CIRCHP BP3, a liposome comprising circular RNA CIRCHP BP3, a PEG-modified protein comprising circular RNA CIRCHP BP3, an extracellular vesicle comprising circular RNA CIRCHP BP3, and/or any combination thereof;
Preferably, the vector comprises a DNA plasmid vector, a lentiviral vector, a retroviral vector, a poxviral vector, a herpes simplex viral vector, an adenoviral vector, an adeno-associated viral vector, a liposome that binds a DNA plasmid, a molecular conjugate that binds a DNA plasmid, and/or a multimer that binds a DNA plasmid.
In some embodiments, the cyclic RNA CIRCHP BP3 expression promoter refers to a substance capable of promoting expression of cyclic RNA CIRCHP BP3, including, but not limited to: any natural purified substance, modified natural purified substance, semisynthetic substance, chemically synthesized substance, and/or any combinations thereof capable of promoting expression of cyclic RNA CIRCHP1BP3 are within the scope of the invention.
In some embodiments, the vector is not particularly limited as long as a vector capable of delivering the circular RNA CIRCHP BP3 of the present invention to overexpress circHP1BP3 is within the scope of the present invention, and in particular embodiments of the present invention, the vector is a pcdna3.1 vector.
In a second aspect the present invention provides a pharmaceutical composition for the treatment and/or prophylaxis of colorectal cancer.
Further, the pharmaceutical composition comprises the cyclic RNA CIRCHP BP3 expression-promoting agent of the first aspect of the invention;
Preferably, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and/or adjuvant;
Preferably, the pharmaceutical composition may further comprise other drugs for the treatment and/or prevention of colorectal cancer;
More preferably, the additional drug comprises an antiviral drug, a chemotherapeutic drug, a targeted therapeutic drug, an immunotherapeutic drug, a mesogenic drug, and/or any combination thereof;
most preferably, the antiviral drug comprises entecavir, lamivudine, sofosbu Weida norprevir, tenofovir disoproxil, adefovir dipivoxil, oseltamivir, telbivudine, ritonavir;
most preferably, the chemotherapeutic agent comprises fluorouracil, cyclophosphamide, doxorubicin, cisplatin, carboplatin, mitomycin, daunorubicin, epirubicin, gemcitabine, irinotecan, oxaliplatin, mitoxantrone;
Most preferably, the targeted therapeutic comprises sorafenib, regorafenib, lenvatinib, dorafinib, regorafenib, apatinib, cabotinib;
Most preferably, the immunotherapeutic agent comprises atilizumab, melittin Li Shan, garelizumab, tirelizumab, bevacizumab, na Wu Liyou mab, palbociclizumab;
Most preferably, the Chinese patent medicine comprises compound plaque chelating capsules, anticancer Ping Wan, huabufonin capsules, zhenxiang capsules and Zhenqi Fuzheng granules.
In some embodiments, specific illustrative examples of the pharmaceutically acceptable carrier and/or adjuvant include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as wetting agents, e.g., sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc.
In some embodiments, suitable pharmaceutically acceptable carriers and/or excipients are described in detail in Remington's Pharmaceutical Sciences (19 th ed., 1995) which are used as needed to aid stability of the formulation or to aid in enhancing the bioavailability of the active or active substance or to impart an acceptable mouthfeel or odor in the case of oral administration, and formulations which may be used in such pharmaceutical compositions may be in the form of the original compound itself, or optionally in the form of a pharmaceutically acceptable salt thereof. The pharmaceutical composition so formulated may be administered by any suitable means known to those skilled in the art, as desired, and when used, a safe and effective amount of the pharmaceutical composition of the present invention is administered to a human.
In some embodiments, the pharmaceutical compositions of the present invention are suitable for administration in a variety of formulations depending on factors such as the method of formulation, the mode of administration, the age, weight, sex, condition, diet, time of administration, route of administration, rate of excretion and sensitivity of the reaction of the patient, and the like, and the skilled practitioner will typically be able to readily determine the formulation and the dosage of the formulation effective for the desired treatment and/or prophylaxis.
In a third aspect the invention provides the use of cyclic RNACIRCHP BP3 as a diagnostic marker in the manufacture of a reagent for diagnosing and/or assessing colorectal cancer.
Further, the loop RNACIRCHP BP3 is a loop RNACIRCHP BP3 according to the first aspect of the present invention.
Further, the reagent comprises a primer for specifically amplifying the circular RNACIRCHP BP3 and/or a probe for specifically recognizing the circular RNACIRCHP BP 3;
Preferably, the sequence of the primer for specifically amplifying the circular RNACIRCHP BP3 is shown as SEQ ID NO:3-SEQ ID NO: 4.
In a fourth aspect the invention provides a product for diagnosing colorectal cancer.
Further, the product comprises an agent according to the third aspect of the invention;
preferably, the product further comprises reagents for detecting the expression level of circular RNA CIRCHP BP3 by sequencing techniques, nucleic acid hybridization techniques and/or nucleic acid amplification techniques;
preferably, the product comprises a kit, a chip and/or a test strip;
Preferably, the product diagnoses colorectal cancer by detecting the expression level of circular RNACIRCHP BP3 in the test sample.
In some embodiments, the kit is an RT-PCR kit, which may further comprise the elements necessary for reverse transcription polymerase chain reaction. The RT-PCR kit comprises a pair of primers specific for circular RNA CIRCHP BP 3. The primer is a nucleotide having a nucleic acid sequence specific for the circular RNA, which may be about 7 to 50 bp, more particularly about 10-39 bp, in length.
In some embodiments, the RT-PCR kit may further comprise a test tube or suitable vessel, reaction buffers (different pH values and magnesium concentrations), deoxynucleotides (dntps), enzymes (e.g., taq polymerase and reverse transcriptase), deoxyribonuclease inhibitors, ribonuclease inhibitors, DEPC-water, and sterile water.
In some embodiments, the kit is a DNA chip kit, which may further comprise elements necessary for manipulating a DNA chip. The DNA chip kit may comprise a substrate to which cDNA corresponding to the circular RNA CIRCHP BP3 or an oligonucleotide corresponding to a fragment thereof is bound, and reagents, agents and enzymes for constructing a fluorescent-labeled probe. In addition, the substrate may comprise a control cDNA or an oligonucleotide corresponding to a fragment thereof.
In a fifth aspect the present invention provides a method for inhibiting colorectal cancer cell proliferation, inhibiting colorectal cancer cell migration, inhibiting colorectal cancer cell invasion and/or inhibiting colorectal cancer tissue formation, for an in vitro non-therapeutic destination, the method comprising the steps of: an effective amount of a cyclic RNACIRCHP BP3 expression-promoting agent according to the first aspect of the invention is added to a system in need thereof.
The present invention also provides a method of treating and/or preventing colorectal cancer, the method comprising the steps of: administering to a subject in need thereof an effective amount of a cyclic RNA CIRCHP BP3 and/or cyclic RNA CIRCHP BP3 expression-promoting agent as described in the first aspect of the invention, and/or a pharmaceutical composition as described in the second aspect of the invention.
In some embodiments, when the circular RNA CIRCHP BP3 expression-promoting agent described in the first aspect of the invention, and/or the pharmaceutical composition described in the second aspect of the invention, are administered, the agent may be administered systemically, or the agent may be administered directly to a specific site where cancerous or precancerous cells are present. Thus, administration can be accomplished in any manner effective to deliver the agent to the cancerous or precancerous cells.
In some embodiments, the mode of administration includes, but is not limited to: the substance is administered topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, orally, intranasally instilled, intracavity or intravesically instilled, intraocularly, intraarterially, intralesionally or by application to mucous membranes such as the nose, throat and bronchi.
The present invention also provides a method of diagnosing and/or aiding in the diagnosis of colorectal cancer, the method comprising the steps of: detecting the expression level of circular RNA CIRCHP BP3 according to the first aspect of the invention in a subject-derived sample, wherein the subject is diagnosed as having colorectal cancer or as having a suspected patient at higher risk of having colorectal cancer if the expression level of circular RNA CIRCHP BP3 is significantly reduced in the subject-derived sample compared to a normal human.
In some embodiments, the subject refers to any animal, and also refers to human and non-human animals. The non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any domestic animals or pets; and non-mammals, such as chickens, amphibians, reptiles, etc., in particular embodiments of the invention, the subject is preferably a human.
In some embodiments, the sample refers to a composition obtained or derived from a subject of interest comprising cellular entities and/or other molecular entities to be characterized and/or identified, e.g., based on physical, biochemical, chemical, and/or physiological characteristics. The sample may be obtained from blood and other fluid samples of biological origin and tissue samples of the subject, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample may be solid tissue, such as tissue from fresh, frozen and/or preserved organs or tissue samples, biopsy tissue or aspirates; blood or any blood component; body fluid; cells from any time of gestation or development of an individual; or plasma. The sample includes biological samples that have been treated in any way after they have been obtained, such as by treatment with reagents, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning purposes. Samples described in the present invention include, but are not limited to: blood, tissue, blood-derived cells, serum, plasma, lymph, synovial fluid, cell extracts, and combinations thereof, in preferred embodiments, the sample is selected from a tissue sample or a blood sample of the subject.
Compared with the prior art, the invention has the advantages and beneficial effects that:
The invention discovers that the annular RNA CIRCHP BP3 is a brand new colorectal cancer diagnosis and treatment target point for the first time, and results of in vitro cell experiments and in vivo function experiments show that the expression of the annular RNA CIRCHP BP3 in colorectal cancer is obviously reduced, the over-expression circHP BP3 can obviously inhibit the cell activity, the cell proliferation capacity, the cell migration and invasion capacity and the tumor formation capacity of colorectal cancer cells, and the annular RNA CIRCHP1BP3 can play an in vivo treatment effect of effectively killing the colorectal cancer cells and can be used for preparing tumor immunotherapy medicaments. The invention provides a new target for diagnosis and treatment of colorectal cancer, and has wide application prospect in clinical treatment of colorectal cancer.
Drawings
FIG. 1 is a diagram of the characteristic structure of circHP BP 3;
FIG. 2 is a first generation sequencing view of circHP BP3 amplification product splice sites;
FIG. 3 is a graph showing the relative expression levels of RNA after digestion of circHP BP3 and mHP1BP3 by RNase R;
FIG. 4 is a graph showing comparison of the results of QPCR detection of circHP BP3 expression in colorectal and paracancestral tissues;
FIG. 5 is a graph showing the comparison of the results of QPCR detection of the expression of circHP BP3 in colorectal cancer cells CACO2, DLD-1 and normal colorectal cancer cells NCM 460;
FIG. 6 is a graph comparing cell viability of colorectal cancer cells overexpressing circHP BP3 with control cell lines, wherein colorectal cancer cell CACO2 is on the left and colorectal cancer cell DLD-1 is on the right; the abscissa is the group, the ordinate is the OD450 value (representing relative cell viability), vector is a control cell line transfected with empty vector pCDNA3.1-vector, circHP BP3 is a cell line transfected with pCDNA3.1-circHP1BP3 plasmid (overexpressing circHP1BP 3);
Fig. 7 is a graph of EDU cell proliferation assay results, wherein, graph a: results, panel B: statistical plots, pcdna3.1-vector was the control cell line transfected with empty vector, circHP BP3 was the cell line transfected with pcdna3.1-circHP BP3 plasmid (overexpressing circHP1BP 3);
Fig. 8 is a graph of cell scratch assay results, wherein, graph a: results, panel B: a statistical chart;
FIG. 9 is a graph showing the results of a clone formation experiment, wherein, panel A: results, panel B: a statistical chart;
FIG. 10 is the effect of overexpression circHP of 1BP3 on colorectal tumor proliferation in NCG mice, wherein, panel A: NCG mice on day 28 after intratumoral injection circHP BP3 overexpressing nanoparticles were grown subcutaneously as shown in panel B: NCG mice subcutaneous tumor volume increase profile.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In order to facilitate an understanding of the present invention, the following terms referred to in the present invention are explained herein:
As used herein, the term "colorectal cancer" refers to colon or rectal cancer. "colon cancer" refers to cancers and/or tumors that form in the colon tissue (the longest portion of the large intestine). Most colon cancers are adenocarcinomas (cancers that originate in cells that produce and release mucus and other fluids). "rectal cancer" refers to cancers and/or tumors that form in the rectal tissue (the last few inches of the large intestine before the anus).
As used herein, the term "primer" refers to 7-50 nucleic acid sequences that are capable of forming base pairs (basepair) complementary to the template strand and serve as starting points for replication of the template strand. Primers are usually synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer need not be exactly the same as the sequence of the template, but may be sufficiently complementary to hybridize with the template. Additional features may be incorporated that do not alter the basic properties of the primer. Examples of additional features that can be incorporated include methylation, capping, substitution of one or more nucleic acids with homologs, and modification between nucleic acids, but are not limited thereto.
As used herein, the term "probe" refers to a nucleic acid fragment, e.g., RNA or DNA, as short as a few to as long as hundreds of bases, which can establish specific binding with mRNA and can determine the presence of a particular mRNA due to a Labeling effect. Probes can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, and the like. Probes and hybridization conditions can be appropriately selected based on what is known in the art.
As used herein, the term "expression level" and "level" refer to the absolute or relative amount of expression of the cyclic RNA CIRCHP1BP3 of the invention, the expression level of which cyclic RNA CIRCHP1BP3 can be determined by a variety of techniques, in particular, the absolute or relative amount of cyclic RNA CIRCHP BP3 of the invention can be detected by using methods well known to those skilled in the art.
As used herein, the term "treatment" generally relates to the treatment of a human or animal (e.g., as applied by a veterinarian) in which certain desired therapeutic effects can be achieved, for example, inhibiting the development of a disorder (including reducing the rate of development of a disorder, halting the development of a disorder), ameliorating a disorder, and curing a disorder. Also included are treatments as a prophylactic measure (e.g., prophylaxis). The use of a patient who has not yet developed, but is at risk of developing, a disorder is also included in the term "treatment".
As used herein, the term "preventing" refers to complete or partial inhibition of the development, recurrence, onset or spread of a colorectal cancer disease disorder or condition caused by administration of the cyclic RNA CIRCHP BP3 and/or cyclic RNA CIRCHP BP3 expression-promoting agent described in the first aspect of the invention, and/or the pharmaceutical composition described in the second aspect of the invention.
As used herein, the term "diagnosis" refers to the discovery, judgment, or cognition of an individual's state of health or condition based on one or more symptoms, data, or other information associated with the individual. The health status of an individual may be diagnosed as healthy/normal (i.e., no disease or condition present) or may be diagnosed as unhealthy/abnormal (i.e., disease or condition present), the terms diagnosis, early diagnosis, making a diagnosis and variations of these terms include early detection of a disease/condition associated with a particular disease or condition (in the present invention, colorectal cancer); characteristics or classification of disease; discovery of progression, cure, or recurrence of disease; discovery of the response to disease following treatment or therapy in an individual, in the present invention, the diagnosis and/or assisted diagnosis of colorectal cancer includes distinguishing between individuals not suffering from colorectal cancer and individuals suffering from colorectal cancer.
As used herein, the term "pharmaceutical composition" may have any one of the formulations selected from the group consisting of: solutions, granules, suspensions, tablets, pills, powders, capsules, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, lyophilized formulations and suppositories. Furthermore, the pharmaceutical composition may be administered one or more times. In this case, the pharmaceutical composition may be administered in the form of a liquid formulation, powder, aerosol, capsule or suppository. In particular embodiments, the pharmaceutical compositions provided herein can be formulated into various dosage forms according to actual needs, and the dosage beneficial to the patient can be determined by the clinician based on the type, age, weight and general disease condition of the subject, mode of administration, and the like. The mode of administration may be, for example, injection or any other suitable mode of administration known to those skilled in the art.
As used herein, the term "effective amount" refers to an amount that has a therapeutic effect or is required to produce a therapeutic effect in a subject. For example, a pharmaceutically or pharmaceutically effective amount refers to the amount of drug required to produce a desired therapeutic effect, which can be reflected by the results of a clinical trial, a model animal study, and/or an in vitro study. The pharmaceutically effective amount depends on several factors, including but not limited to: the characteristic factors of the subject (such as height, weight, sex, age and history of administration), the severity of the disease, etc.
As used herein, the term "administering" refers to the act of injecting or physically delivering a substance present outside the body (e.g., the cyclic RNA CIRCHP BP3, cyclic RNA CIRCHP BP3 expression-promoting agent, and/or pharmaceutical composition described herein) into a subject, e.g., by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other physical delivery method known in the art. When a disease, disorder or condition, or symptom thereof is treated, administration of the substance is typically performed after the onset of the disease, disorder or condition, or symptom thereof. When a disease, disorder or condition, or symptom thereof is prevented, administration of the substance is typically performed prior to the onset of the disease, disorder or condition, or symptom thereof.
The invention is further illustrated below in conjunction with specific examples, which are intended to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The experimental procedure, in which no specific conditions are noted in the examples below, is generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.
The principal materials and reagent information used in the present invention are shown in Table 1 below.
TABLE 1 Main materials and reagents
| < B > sequence number > | < B > reagent name > | < B > production Co Ltd | < B > goods number > |
| 1 | EasyScript® First-Strand cDNA Synthesis SuperMix | Beijing full-type gold organism | AE301-02 |
| 2 | RNase R enzyme | Shanghai Biyun biotechnology Co., ltd | R7092S |
| 3 | SuperReal fluorescent quantitative premix reagent enhanced version (SYBR Green) | Tiangen Biochemical technology (Beijing) Co., ltd | FP205 |
| 4 | Cell Counting Kit-8 (CCK-8 kit) | Shanghai Biyun biotechnology Co.Ltd | C0038 |
| 5 | BeyoClick ™ EdU-555 cell proliferation detection kit | Shanghai Biyun biotechnology Co.Ltd | C0075S |
| 6 | Human normal colon epithelial cell NCM460 | Shang En Biotech Co., ltd | SNL-519 |
| 7 | CACO2 of human colorectal adenocarcinoma cells | North Biotech Co.Ltd | BNCC350769 |
| 8 | Human colorectal adenocarcinoma epithelial cell DLD-1 | North Biotech Co.Ltd | BNCC100190 |
The circular RNA CIRCHP BP3 (CircBase ID: hsa_circ_ 0005782) is derived from the No. 5 and No. 62 exons of 13 exons of the HP1BP3 gene on chromosome 1, the cyclized nucleotide sequence has 304 bases, and no report on the function of circHP1BP3 and colorectal cancer cells exists at present.
CircHP1 the cDNA sequence corresponding to BP3 of the nucleic acid sequence is shown as SEQ ID NO:1 is a circular structure formed by splicing after transcription of the nucleotide sequence shown in SEQ ID NO. 1. The RNA sequence corresponding to circHP BP3 is shown as SEQ ID NO. 2, and the structure of circHP BP3 is shown as SEQ ID NO:2, and the nucleotide sequence is connected end to form a circular structure.
CircHP 1A cDNA sequence corresponding to BP3 (SEQ ID NO: 1):
TGAGAAAGATCAGTCTAAAGAAAAGGAGAAGAAAGTGAAAAAAACAATTCCTTCCTGGGCTACCCTTTCTGCCAGCCAGCTAGCCAGGGCCCAGAAACAAACACCGATGGCTTCTTCCCCACGTCCCAAGATGGATGCAATCTTAACTGAGGCCATTAAGGCATGCTTCCAGAAGAGTGGTGCATCAGTGGTTGCTATTCGAAAATACATCATCCATAAGTATCCTTCTCTGGAGCTGGAGAGAAGGGGTTATCTCCTTAAACAAGCACTGAAAAGAGAATTAAATAGAGGAGTCATCAAACAG
circHP1BP3 corresponding RNA sequence (SEQ ID NO: 2):
UGAGAAAGAUCAGUCUAAAGAAAAGGAGAAGAAAGUGAAAAAAACAAUUCCUUCCUGGGCUACCCUUUCUGCCAGCCAGCUAGCCAGGGCCCAGAAACAAACACCGAUGGCUUCUUCCCCACGUCCCAAGAUGGAUGCAAUCUUAACUGAGGCCAUUAAGGCAUGCUUCCAGAAGAGUGGUGCAUCAGUGGUUGCUAUUCGAAAAUACAUCAUCCAUAAGUAUCCUUCUCUGGAGCUGGAGAGAAGGGGUUAUCUCCUUAAACAAGCACUGAAAAGAGAAUUAAAUAGAGGAGUCAUCAAACAG
EXAMPLE 1 construction of circular RNA
In this example, the inventors designed a specific primer pair capable of amplifying circHP1BP3 (CircBase ID: hsa_circ_ 0005782), and amplified the circular RNA of the HP1BP3 gene using the primer pair, confirmed the splice site (backsplicing junction site) of the circular RNA by a one-generation sequencing method, and then determined circHP1BP3 as a circular RNA molecule expressing a closed circular structure by RNase R digestion experiments. The specific experimental method is as follows:
1. Cell total RNA extraction and concentration determination
(1) When the cells of the 6-well plate exceed 90%, the old culture solution is discarded, after the cells are washed once by PBS, 1mLTrizol is added to each well, and then the cells are transferred to a 1.5 mL EP tube;
(2) The EP tube was left at room temperature for 10 minutes in order for the nucleic acid protein complex to be sufficiently separated;
(3) 0.2mL of chloroform was added to the EP tube, shaken for about 10s, and then allowed to stand at room temperature for 5min; chloroform is a nonpolar molecule, and can effectively inhibit the activity of RNase, when the cell solution added with Trizol is mixed with chloroform, water molecules of protein are removed by chloroform, so that the protein is denatured due to water loss and state, and the separation of aqueous phase and organic phase is accelerated;
(4) 12000g, centrifuging at 4deg.C for 15min, wherein the EP tube solution is divided into 3 layers, the bottom layer is red organic matter, the upper layer is colorless water phase, and RNA exists in the water phase;
(5) The supernatant (about 450 mL) was transferred to a fresh EP tube, then the same volume of isopropanol was added and left at room temperature for 10min; isopropanol absorbs water around RNA to precipitate it;
(6) 12000g, centrifuging at 4deg.C for 10min, observing white RNA precipitate at the bottom and side of the tube, and discarding supernatant;
(7) Washing the RNA precipitate with 75% DEPC-ethanol solution, centrifuging at 4deg.C for 5min, and discarding supernatant;
(8) Placing the RNA precipitate in a biosafety cabinet for 5min, and adding 20 microliters of DEPC water to dissolve RNA after airing, wherein the steps are operated on ice;
(9) The total RNA purity and concentration of the cells were measured using ScanDrop100,100 ultra-micro nucleic acid assay.
2. CDNA reverse transcription
Preparation of a reverse transcription reaction system (for example, 20. Mu.L system) was performed on ice, and after completion of the preparation, prepared total RNA of cells was added for reverse transcription, and the reverse transcription reaction system was as shown in Table 2.
TABLE 2 reaction system
The reverse transcription reaction conditions were as follows: the reaction was carried out at 37℃for 15 minutes, followed by 85℃for 5 seconds. Cooling to 4 deg.c, diluting the cDNA to 5 times, and storing in-20 deg.c refrigerator.
3. Primer design
Primers were designed using Primer3.0 on-line tool and verified with NCBI Blast.
The design principle of the primer is as follows: (1) the GC content of the primer is 50-60%; (2) the primer length is 17-25bp; (3) the Tm of the primer is 57-63 ℃; (4) the position of the primer avoids the tertiary structure of the target sequence; (5) avoiding repeating G or C bases a number of times; (6) avoiding the primer terminal base to be A; (7) the primer and the product avoid forming a secondary structure; (8) the product length is between 100 and 150 bp; the product of (9) avoids 4 single base repeats.
By the design principle, the primer 5 pair is designed together, and the primer pair adopted in the embodiment is as follows, wherein the primer pair is synthesized by the company Boxing of the Beijing Rui:
F：5 '-GGAGCTGGAGAGAAGGGGTTATC-3 '（SEQ ID NO:3）
R：5 '-AGAAAGGGTAGCCCAGGAAGG-3 '（SEQ ID NO:4）
In one or more embodiments, other primer pairs than those described above may be used for amplification.
4. First generation sequencing
The cDNA obtained by reverse transcription is amplified by using the primer set described above, and then the amplified product is subjected to first-generation sequencing.
5. RNase R digestion experiment
RNase R enzyme is an RNase capable of digesting linear RNA but has little effect on circular RNA.
Mu.g of total RNA from cells was incubated with 3U RNase R enzyme at 37℃for 30 minutes in a volume of 10. Mu.L, after which the RNase R enzyme was inactivated by heating to 75℃for 10 minutes and finally analyzed by RT-qPCR for the effect of RNase R addition on circHP BP3 and mHP1BP 3.
6. Experimental results
The characteristic structure diagram of circHP BP3 and the first generation sequencing result diagram of the splice site of the circHP BP3 amplified product are shown in FIG. 1 and FIG. 2 respectively, circHP BP3 is derived from an exon of the HP1BP3 gene, and the first generation sequencing result proves that circHP BP3 has reverse shearing connection and accurately shows the splice site of the amplified product, which indicates that circHP1BP3 is formed not due to recombination mismatch of genome.
The comparison of RNase R digestion with circHP BP3 and mHP1BP3 is shown in fig. 3, where linear mHP1BP3 is significantly reduced in expression after RNase R enzyme digestion, while circHP BP3 is resistant to RNase R enzyme digestion, confirming its cyclic structure.
Example 2 detection of expression of circHP1BP3 in clinical sample tissues and cell lines
The expression level of circHP BP3 in colorectal cancer and paracancestral tissues of 12 clinical patients and the expression difference between colorectal cancer cell lines and corresponding Normal liver cell lines were detected by fluorescence Quantitative PCR (QPCR), and it was found that circHP BP3 was significantly lower in colorectal cancer tissues (Tumor) than paracancestral tissues (Normal), and that circHP BP3 was able to be used for colorectal cancer diagnosis. The specific experimental method is as follows:
1. total RNA extraction and concentration determination in cells
(1) 1ML Trizol was added to the 6-well cell culture plate and lysed for 10min, followed by 1.5 mL EP tube;
(2) The EP tube is left at room temperature for about 10 minutes so that the nucleic acid protein complex can be sufficiently separated;
(3) 0.2 mL of chloroform was added to the EP tube, shaken for about 10s, and then allowed to stand at room temperature for 5min;
(4) 12000g, centrifuging at 4deg.C for 15min, wherein the EP tube solution is divided into 3 layers, the bottom layer is red organic matter, the upper layer is colorless water phase, and RNA exists in the water phase;
(5) The supernatant (about 450 mL) was transferred to a fresh EP tube, then the same volume of isopropanol was added and left at room temperature for 10min;
(6) 12000g, centrifuging at 4deg.C for 10min, observing white RNA precipitate at the bottom and side of the tube, and discarding supernatant;
(7) Washing the RNA precipitate with 75% DEPC-ethanol solution, centrifuging at 4deg.C for 5min, and discarding supernatant;
(8) Placing the RNA precipitate in a biosafety cabinet for 5min, airing, and adding 20 mu L of DEPC water to dissolve the RNA, wherein the steps are operated on ice;
(9) RNA purity and concentration were measured using ScanDrop100,100 ultra-micro nucleic acid assay.
2. QPCR amplification assay
Tissue RNA reverse transcription was performed using the cDNA reverse transcription method of example 1.
Preparation of PCR reaction system (for example, 20. Mu.L system) was performed on ice, and after completion of the preparation, cDNA template obtained by reverse transcription was added. Wherein, the PCR reaction system is shown in Table 3:
TABLE 3 PCR reaction system
The primer set used for amplifying circular RNA CIRCHP BP3 was the one shown in example 1 as SEQ ID NO:3-SEQ ID NO: 4:
F：5 '-GGAGCTGGAGAGAAGGGGTTATC-3 '（SEQ ID NO:3）
R：5 '-AGAAAGGGTAGCCCAGGAAGG-3 '（SEQ ID NO:4）
The reaction conditions include:
The first step, pre-denaturation, 95 ℃ for 5 minutes;
second, PCR (40 cycles), 95℃for 20 seconds; 60 ℃ for 20 seconds; 72℃for 20 seconds.
Thirdly, analyzing a melting curve, namely, at 65 ℃ for 5 seconds; 95℃for 5 seconds.
Quantitative analysis was performed after PCR amplification. The calculation formula of the relative expression quantity of the target gene is as follows: 2- ΔΔct=2- [ Δct ] Test- (. DELTA.ct) Control ]. Wherein, Δct=ct target-Ct housekeeping, ct target is target gene Ct value, ct housekeeping is housekeeping gene Ct value, Δct represents phase Ct value of each sample target gene relative to housekeeping gene, ΔΔct= (Δct) Test- (Δct) Control, represents normalization of the treatment group relative to the Control group, and 2- Δct represents relative expression amount of the treatment group relative to the Control group, and represents relative expression multiple of the target gene.
3. Experimental results
As shown in the above QPCR experiment results in FIGS. 4 and 5, the expression level of circHP BP3 in colorectal cancer tissue (Tumor) is obviously lower than that of paracancestral tissue (Normal), and the expression levels in colorectal cancer cells CACO2 and DLD-1 are obviously lower than that of Normal colorectal cancer cell NCM460, which shows that circHP BP3 has great significance in colorectal cancer occurrence and development, can be used as an ideal prognosis marker of colorectal cancer patients, and can play a positive role in colorectal cancer diagnosis.
Example 3 Effect of overexpression circHP BP3 on colorectal cancer cell proliferation, invasion and metastasis
CircHP1BP3 and a random sequence vector are constructed on a pCDNA3.1 vector special for over-expressing circular RNA, pCDNA3.1-circHP1BP3 and pCDNA3.1-vector are transfected on CACO2 cells as a treatment group and a control group, the effect of circHP BP3 over-expression on the activity of colorectal cancer cells is detected through CCK8 cell activity, the effect of circHP BP3 over-expression on the proliferation capacity of colorectal cancer cells is detected through EDU cell proliferation experiments, the effect of circHP BP3 over-expression on the migration and invasion capacity of colorectal cancer cells is examined through cell scratch experiments, and the effect of circHP BP3 over-expression on the oncolytic capacity of colorectal cancer cells is examined through clone formation experiments.
1. CCK-8 cell Activity assay
The dehydrogenase in the mitochondria of living cells can react with the WST-8 compound, and finally the dehydrogenase is reduced into hydrophilic formazan dye, the dye is yellow after being dissolved, the number of generated yellow formazan is positively correlated with the number of living cells, namely, the more the number of living cells is, the higher the degree of yellow of the solution is, so that the characteristic is utilized for detecting the proliferation of cells.
(1) 10000 CACO2 cells are inoculated in each well of a 96-well plate, and pCDNA3.1-circHP1BP3 and pCDNA3.1-vector are respectively transfected as a treatment group and a control group after 12h of cell attachment is cultivated;
(2) Add 10. Mu.L of CCK-8 reagent to the corresponding wells at 48 h;
(3) Placing the mixture in a constant temperature incubator with the temperature of 37 ℃ and the concentration of 5% CO 2 for culturing for about 2 hours;
(4) Finally, the absorbance was measured at 450nm using an microplate reader.
2. EDU cell proliferation assay
A cell proliferation assay kit (BeyoClick ™ EdU Cell Proliferation Kit with Alexa Fluor, 555) (available from Shanghai Biyun biotechnology Co., ltd.) is a kit for simply, rapidly and highly sensitively detecting cell proliferation based on the incorporation of thymidine (thymidine) analogue EdU (5-ethynyl-2' -deoxyuridine) during DNA synthesis, and the subsequent Click reaction (Click reaction) to label the EdU with Alexa Fluor 555.
(1) Inoculating 30 ten thousand cancer cells into a 6-hole plate, and respectively transfecting pCDNA3.1-circHP1BP3 and pCDNA3.1-vector as a treatment group and a control group after 12h of cell adherence is cultured;
(2) After 48h of culture, the culture medium is discarded, PBS is added for three times, 1ml of 4% paraformaldehyde is added for fixation at room temperature for 10min;
(3) Discarding 4% paraformaldehyde, adding PBS, washing for three times, adding 1ml of 0.1% triton X-100, and incubating for 10min at room temperature;
(4) The solution of triton X-100 at 0.1% was discarded, washed three times with PBS, and then detected using the BeyoClick ™ EdU-555 cell proliferation assay kit described above.
3. Cell scratch assay
(1) Scribing on a 24-hole cell culture plate, firstly scribing 3-5 lines on the back of the 24-hole plate by using a Mark pen, then inoculating 6 ten thousand CACO2 cells, and respectively transfecting pCDNA3.1-circHP1BP3 and pCDNA3.1-vector as a treatment group and a control group after 12h of cell adherence is cultivated;
(2) After six hours of transfection, a vertical trace is marked in the middle of the culture hole by using a yellow gun head of 200ul, then the culture hole is washed once by using PBS, and a fresh culture medium is added to take a picture under a lens and record for 0 hour;
(3) After 48h, washing with PBS for one time, taking a picture, and recording as 48h;
(4) Scratch healing rate was calculated using image J-processed pictures.
4. Cloning formation experiments
(1) 1000 Cells/well were inoculated in each experimental group in a 6-well plate, and pcdna3.1-circHP BP3 and pcdna3.1-vector were transfected as a treatment group and a control group, respectively, after 12h of cell attachment had been cultured;
(2) Culturing continuously until the number of cells in 14 days or most single clones is greater than 50, changing liquid every 3 days in the middle, and observing the cell state;
(3) After cloning is completed, photographing the cells under a microscope, washing the cells for 1 time by using PBS, adding 1mL of 4% paraformaldehyde into each hole for fixation for 30-60 min, and washing the cells for 1 time by using PBS;
(4) 1ml of crystal violet dye solution is added into each hole, and cells are dyed by 10-20 min;
(5) Washing the cells with PBS for several times, airing, and taking pictures with a digital camera (taking pictures of the whole six-well plate and each well separately);
(6) The number of clonally formed cells was calculated using image J-processed pictures.
5. Experimental results
The experimental results of CCK-8 cell activity detection are shown in FIG. 6, and after circHP BP3 is over-expressed, the cell activity of colorectal cancer cells is obviously inhibited, and the activity of colorectal cancer cells is obviously weakened.
The experimental results of EDU cell proliferation assay are shown in FIG. 7, and after circHP BP3 is over-expressed, the cell proliferation capacity of colorectal cancer cells is obviously inhibited, and the cell division capacity of colorectal cancer cells is obviously weakened.
The experimental results of the cell scratch experiments are shown in fig. 8A and 8B, and compared with the colorectal cancer cell line of the control group of empty vector, the colorectal cancer cell line of the over-expression circHP BP3 has obviously inhibited migration and invasion capacity.
The results of the cloning experiments are shown in fig. 9A and 9B, and the cell strain overexpressing circHP BP3 significantly inhibited the oncologic capacity of colorectal cancer cells compared to the empty vector control cell strain.
The experiment shows that circHP BP3 gene and its expression product can be used in colorectal cancer treatment.
Example 4 Effect of over-expression circHP of 1BP3 on proliferation of colorectal cancer cells in NCG mice
1. Experimental method
The experiment was performed using 6-week male NCG mice, which were purchased from Peking Violet laboratory animals Co., ltd.
After digestion with pancreatin, 300g of the log-phase grown CACO2 cells were centrifuged for 5min for cell count, the cells were resuspended in serum-free DMEM medium at a density of 10 7/100 ul, injected subcutaneously in the left armpit of NCG mice at a volume of 100 ul/only, tumor size was detected after 7 days, and after tumor volume exceeded 100mm 3, the subsequent experiments were performed.
Tumor-forming NCG mice meeting the standard are selected as an evaluation model, pCDNA3.1-vector and pCDNA3.1-circHP1BP3 over-expression nano-particles are injected into the tumor in the NCG mice subcutaneous tumor, tumor volume is measured once a week (V=1/2×a×b2, a is a long axis and b is a short axis), the longest and shortest positions of the tumor are measured by a vernier caliper, and a tumor volume growth curve is drawn.
2. Experimental results
As shown in FIGS. 10A and 10B, the volume of circHP BP3 group into which circHP BP3 was introduced was significantly reduced (see FIG. 10A). NCG mice were sacrificed on day 28, tumors were removed, photographed, and a subcutaneous tumor volume increase curve was drawn for nude mice, which showed that the tumor volume of the pCDNA3.1-circHP1BP3 group overexpressing circHP1BP3 was significantly smaller than that of the control group pCDNA3.1-Vector (see FIG. 10B).
The experiment shows that the over-expression circHP BP3 can obviously inhibit the subcutaneous tumorigenicity of colorectal cancer cells in NCG mice, namely circHP BP3 and an expression product thereof can be effectively applied to the treatment of colorectal cancer.
Claims (10)
1. Use of a cyclic RNA CIRCHP BP3 expression promoter for the manufacture of a medicament for the treatment and/or prophylaxis of colorectal cancer.
2. The use according to claim 1, wherein CircBase ID of the loop RNA CIRCHP BP3 is hsa_circle_ 0005782.
3. The use according to claim 2, wherein the cDNA sequence corresponding to the circular RNA CIRCHP BP3 is shown as SEQ ID NO. 1;
The RNA sequence corresponding to the annular RNA CIRCHP BP3 is shown as SEQ ID NO. 2;
the annular RNA CIRCHP BP3 is an end-to-end annular structure formed by splicing after transcription of the nucleotide sequence shown in SEQ ID NO. 1;
The annular RNA CIRCHP BP3 is of an annular structure formed by connecting the nucleotide sequences shown in SEQ ID NO. 2 end to end.
4. The use of claim 1, wherein the cyclic RNA CIRCHP BP3 expression-promoting agent comprises a natural purification substance, a modified natural purification substance, a semisynthetic substance, a chemically synthetic substance, and/or any combinations thereof capable of promoting expression of cyclic RNA CIRCHP BP 3.
5. The use according to claim 1, wherein the circular RNA CIRCHP BP3 expression promoter comprises circular RNACIRCHP BP3, a recombinant vector comprising circular RNACIRCHP BP3, a nanoparticle comprising circular RNACIRCHP BP3, a protein microsphere comprising circular RNACIRCHP BP3, a liposome comprising circular RNACIRCHP BP3, a PEG-modified protein comprising circular RNACIRCHP BP3, an extracellular vesicle comprising circular RNACIRCHP BP3, and/or any combination thereof.
6. A pharmaceutical composition for the treatment and/or prevention of colorectal cancer, characterized in that it comprises a cyclic RNACIRCHP BP3 expression-promoter as defined in any one of claims 1 to 5.
7. Use of the cyclic RNACIRCHP BP3 as a diagnostic marker in the manufacture of a reagent for diagnosing and/or assessing colorectal cancer, characterized in that the cyclic RNACIRCHP BP3 is the cyclic RNACIRCHP BP3 as claimed in any one of claims 1 to 5.
8. The use according to claim 7, wherein the reagent comprises a primer that specifically amplifies loop RNACIRCHP BP3 and/or a probe that specifically recognizes loop RNACIRCHP BP 3;
the sequence of the primer for specifically amplifying the circular RNACIRCHP BP3 is shown as SEQ ID NO:3-SEQ ID NO: 4.
9. A product for diagnosing colorectal cancer, characterized in that it comprises an agent as claimed in claim 7 or 8;
The product comprises a kit, a chip and/or a test strip;
the product diagnoses colorectal cancer by detecting the expression level of the annular RNACIRCHP BP3 in a sample to be tested.
10. A method of inhibiting colorectal cancer cell proliferation, inhibiting colorectal cancer cell migration, inhibiting colorectal cancer cell invasion and/or inhibiting colorectal cancer tissue formation at a non-therapeutic destination in vitro, the method comprising the steps of: adding an effective amount of a cyclic RNACIRCHP BP3 expression-promoting agent as described in any one of claims 1 to 5 to a system in need thereof.
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