CN116926073A - CircVRK1 and application thereof in preparation of colorectal cancer treatment drugs - Google Patents
CircVRK1 and application thereof in preparation of colorectal cancer treatment drugs Download PDFInfo
- Publication number
- CN116926073A CN116926073A CN202310681050.XA CN202310681050A CN116926073A CN 116926073 A CN116926073 A CN 116926073A CN 202310681050 A CN202310681050 A CN 202310681050A CN 116926073 A CN116926073 A CN 116926073A
- Authority
- CN
- China
- Prior art keywords
- circvrk1
- colorectal cancer
- seq
- gene
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010009944 Colon cancer Diseases 0.000 title claims abstract description 44
- 208000001333 Colorectal Neoplasms Diseases 0.000 title claims abstract description 38
- 239000003814 drug Substances 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 229940079593 drug Drugs 0.000 title abstract description 11
- 230000014509 gene expression Effects 0.000 claims description 28
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 239000013598 vector Substances 0.000 claims description 15
- 239000002502 liposome Substances 0.000 claims description 12
- 239000002105 nanoparticle Substances 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 230000002018 overexpression Effects 0.000 claims description 7
- 239000004480 active ingredient Substances 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 3
- 108091028075 Circular RNA Proteins 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 7
- 125000004122 cyclic group Chemical group 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 36
- 230000006907 apoptotic process Effects 0.000 description 13
- 230000035755 proliferation Effects 0.000 description 13
- 238000013508 migration Methods 0.000 description 12
- 230000005012 migration Effects 0.000 description 10
- 238000003197 gene knockdown Methods 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 7
- 208000029742 colonic neoplasm Diseases 0.000 description 6
- 230000009545 invasion Effects 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000004055 small Interfering RNA Substances 0.000 description 5
- 101710159080 Aconitate hydratase A Proteins 0.000 description 4
- 101710159078 Aconitate hydratase B Proteins 0.000 description 4
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 4
- 101710105008 RNA-binding protein Proteins 0.000 description 4
- 108091027967 Small hairpin RNA Proteins 0.000 description 4
- 108020004459 Small interfering RNA Proteins 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000034994 death Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 230000012292 cell migration Effects 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 238000010008 shearing Methods 0.000 description 3
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010017842 Telomerase Proteins 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 230000009702 cancer cell proliferation Effects 0.000 description 2
- 230000004709 cell invasion Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000006567 cellular energy metabolism Effects 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000001351 cycling effect Effects 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000001808 exosome Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000037451 immune surveillance Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 108091070501 miRNA Proteins 0.000 description 2
- 239000002679 microRNA Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 230000010473 stable expression Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 208000003200 Adenoma Diseases 0.000 description 1
- 206010001233 Adenoma benign Diseases 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 206010052358 Colorectal cancer metastatic Diseases 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 101000649929 Homo sapiens Serine/threonine-protein kinase VRK1 Proteins 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 208000037062 Polyps Diseases 0.000 description 1
- 102100028235 Serine/threonine-protein kinase VRK1 Human genes 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 101150070651 Vrk1 gene Proteins 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012059 conventional drug carrier Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000005414 inactive ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/50—Physical structure
- C12N2310/53—Physical structure partially self-complementary or closed
- C12N2310/532—Closed or circular
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
Abstract
The application relates to the technical field of biological medicines, and particularly provides a brand-new cyclic RNA molecule circVRK1 (circBase ID: hsa_circ_ 0000566) and application of the circVRK1 in preparation of colorectal cancer treatment medicines. The application has the beneficial effects that: the circVRK1 plays an important role in inhibiting the occurrence and development of tumors, can be used as a new potential treatment target of colorectal cancer, and provides a scientific and accurate treatment scheme for colorectal cancer patients.
Description
Technical Field
The application relates to the technical field of biological medicines, in particular to a brand-new cyclic RNA molecule circVRK1 (circBase ID: hsa_circ_ 0000566) and application thereof in preparing colorectal cancer treatment medicines.
Background
Global cancer data according to 2020 published by the national cancer institute of the world health organization showed that: the global colorectal cancer (CRC) incidence number in 2020 is 193.2 ten thousand, accounting for 10% of the global total cancer incidence; the incidence rate of the standardization is 19.5/10 ten thousand, the death number is 93.5 ten thousand, and the death number of the standardization accounts for 9.4% of the total cancer death number in the world; the normalized mortality rate is 9/10 ten thousand. Colorectal cancer occurs and progresses through a relatively long evolution process of normal intestinal epithelial cells-adenoma (polyp) -cancer, and the disease development is hidden, so that the early detection rate of colorectal cancer is low, most patients are diagnosed with advanced colorectal cancer, and advanced colorectal cancer and metastatic colorectal cancer are main causes of colorectal cancer death. Therefore, the key molecules in colorectal cancer occurrence and development are identified, and research on the key molecules as colorectal cancer treatment targets has great practical significance and clinical value.
The circRNAs are in a closed loop structure formed by reversely shearing the precursor mRNA, are often present in cytoplasm, have stronger expression specificity compared with linear RNA, and have very stable expression in exosomes, saliva, blood plasma and tissues. Currently, the mechanistic study of circRNAs has focused mainly on three directions: 1. acting as miRNA sponge, regulating the expression of a downstream target gene through a circRNA-miRNA-mRNA shaft, interfering with a signal pathway, and acting as competitive endogenous RNA (ceRNA) to play an important role in the occurrence and development of cancers; 2. binding to an RNA Binding Protein (RBP), regulating gene expression; 3. has coding function, can be used as a transcription template, and can be translated into peptide fragments or proteins. Functionally, many studies have found that circRNAs are involved in cancer cell proliferation, migration, invasion, apoptosis, and cycling processes; in addition, circRNAs play an important role in participation in angiogenesis, immune surveillance, affecting telomerase activity, and cellular energy metabolism. And based on the biological functions of the circRNAs, the circRNAs have strong potential as therapeutic drugs or therapeutic targets.
Chinese patent document CN108611420A discloses the application of circ-0000423 in preparing or screening a medicine for colon cancer; the high expression of circ-0000423 is closely related to the stage, differentiation degree and prognosis of the colorectal cancer, and the higher the expression degree is, the later the disease stage is, the worse the differentiation and prognosis is, which suggests that circ-0000423 is closely related to the malignant behavior of the colorectal cancer. The circ-0000423 can adsorb an oncogene miR-519 to promote invasion and metastasis of colorectal cancer through a ceRNA mechanism. The circ-0000423 can be used as a therapeutic target in preparing or screening medicaments for treating colon cancer or inhibiting invasion and migration of colon cancer. The circ-0000423 can be used as a detection target spot in the preparation of colorectal cancer diagnosis reagents. The circ-0000423 can be used as a detection target in the preparation of a reagent for predicting colorectal cancer stage, differentiation degree or prognosis. Chinese patent document CN109706245A discloses a small interfering RNA aiming at hsa_circ_0000478 gene and application thereof; the application successfully obtains the siRNA of hsa_circ_0000478, and the transient transfection LOVO cells can effectively interfere the expression of hsa_circ_0000478 genes, and the interference efficiency reaches (90.25+/-0.96 percent); the result of transfecting colon cancer LOVO cells shows that the hsa_circ_0000478 gene can obviously reduce the proliferation rate of the LOVO cells, promote early apoptosis of the cells and inhibit invasion and migration of the cells after being interfered; the direct correspondence between the knockdown of hsa_circ_0000478 expression and proliferation, invasion and migration of colon cancer cells is proved for the first time, and the hsa_circ_0000478 expression knockdown can be used as a treatment target of colon cancer. However, there is no report on circVRK1 and its application in colorectal cancer treatment.
Disclosure of Invention
The application aims to provide a novel cyclic RNA molecule circVRK1 and an application of the circVRK1 in preparing colorectal cancer therapeutic drugs.
In a first aspect of the application, a cyclic RNA molecule circVRK1 is provided, the nucleotide sequence of which is shown as SEQ ID NO. 1.
Furthermore, the application also provides the reverse splice connection site of the circVRK1 (figure 1A).
In a second aspect of the present application, there is provided a recombinant vector comprising the circVRK1 gene as described above.
Furthermore, the recombinant vector is an over-expression plasmid constructed by inserting the circVRK1 sequence into the PLCDH-ciR vector.
In a third aspect of the present application, there is provided a primer set for amplifying circVRK1, comprising: a Convergent primer group and/or a divrgent primer group, wherein the nucleotide sequence of the Convergent primer group is shown as SEQ ID NO. 2 and SEQ ID NO. 3; the nucleotide sequences of the Divergent primer group are shown as SEQ ID NO. 4 and SEQ ID NO. 5;
convergent primer set:
F:5’-CTGTGAGTCTTGCGAGGTGG-3’(SEQ ID NO:2)；
R:5’-ATCACAAACACACGGCTTTGG-3’(SEQ ID NO:3)；
divergent primer set:
F:5’-ACGTGACATTCTTTTGCAAGGAC-3’(SEQ ID NO:4)；
R:5’-AGCTGCTTTTACACGAGGCA-3’(SEQ ID NO:5)。
in a fourth aspect of the application, there is provided the use of circVRK1 in the manufacture of a medicament for the treatment of colorectal cancer.
Furthermore, the colorectal cancer treatment drug takes the circVRK1 as a treatment target.
Furthermore, the colorectal cancer treatment drug improves the expression quantity of the circVRK 1.
In a fifth aspect, the application provides the use of an agent that increases the expression level of circVRK1 in the manufacture of a medicament for the treatment of colorectal cancer.
Further, the reagent for improving the expression level of the circVRK1 comprises any one of the following components:
a) The circVRK1 gene;
b) A recombinant vector containing the circVRK1 gene;
c) Recombinant viruses containing the circVRK1 gene.
Furthermore, the recombinant vector containing the circVRK1 gene is an over-expression plasmid constructed by inserting the circVRK1 sequence into a PLCDH-ciR vector.
Furthermore, the agent for improving the expression level of the circVRK1 is liposome nano-particles containing the circVRK1 gene.
Furthermore, the agent for improving the expression level of the circVRK1 is used for colorectal cancer treatment by over-expressing the circVRK 1.
Furthermore, the medicament for treating colorectal cancer is a pharmaceutical composition prepared from an agent for improving the expression level of the circVRK1 serving as an active ingredient and a conventional pharmaceutical carrier.
Further, the dosage form of the medicament for treating colorectal cancer comprises, but is not limited to, tablets, capsules, granular preparations, aerosols or injection. The mode of treatment includes, but is not limited to, the drug alone or in combination with any other active or inactive ingredient. Routes of administration include, but are not limited to, oral, intravenous, nasal inhalation, subcutaneous or intramuscular injection, buccal, and the like.
In a sixth aspect of the present application, there is provided a medicament for treating colorectal cancer, comprising an agent for increasing the expression level of circVRK1 as an active ingredient.
Furthermore, the medicament for treating colorectal cancer also comprises a pharmaceutically acceptable carrier or auxiliary material.
In the application, the following components are added:
circrna: the circRNAs are in a closed loop structure formed by reversely shearing the precursor mRNA, are often present in cytoplasm, have stronger expression specificity compared with linear RNA, and have very stable expression in exosomes, saliva, blood plasma and tissues. Currently, the mechanistic study of circRNAs has focused mainly on three directions: (1) Acting as miRNA sponge, regulating the expression of a downstream target gene through a circRNA-miRNA-mRNA shaft, interfering with a signal pathway, and acting as competitive endogenous RNA (ceRNA) to play an important role in the occurrence and development of cancers; (2) Binding to an RNA Binding Protein (RBP), regulating gene expression; (3) Has coding function, can be used as a transcription template, and can be translated into peptide fragments or proteins. Functionally, many studies have found that circRNAs are involved in cancer cell proliferation, migration, invasion, apoptosis, and cycling processes; in addition, circRNAs play an important role in participation in angiogenesis, immune surveillance, affecting telomerase activity, and cellular energy metabolism.
2. Knocking down: also called knockdown, refers to the effect of reducing or weakening gene expression by degrading mRNA of a target gene having a homologous sequence. The double-stranded small RNA is utilized to efficiently and specifically degrade homologous mRNA in cells, so that the expression of target genes in the body is weakened, and the cells have the phenotype of target gene deletion.
3. Overexpression: the expression cassette containing the target gene is transferred into the target cell by using the gene expression vector, the target gene in the vector is expressed by using the related tool enzyme of the gene expression of the cell, and the result is that the obtained cell expresses the target gene in a higher quantity than the starting cell. Common over-expression vectors include vectors such as viruses, plasmids and the like, including cloning a target gene into the genome of a cell by means of gene editing; wherein the viral vectors include but are not limited to each serotype of lentiviruses, adenoviruses, adeno-associated viruses, and the therapeutic vectors include but are not limited to pCDNA series, pLCDH-ciR series, pBK series, and the like.
The circVRK1 found in the present application is transcribed from the exon of vaccinia related kinase (VRK 1) and then alternatively spliced into a circular structure. We performed PCR experiments and Sanger sequencing and clearly observed reverse splice sites. After Rnase R treatment, the abundance of circVRK1 changes less, while the level of the corresponding VRK1 linear region drops significantly. All these results indicate that circVRK1 is a true circRNA. After the liposome nanoparticle form is adopted to overexpress the circVRK1, the proliferation and migration capacity of SW620 cells and HCT116 cells are weakened, and the apoptosis is increased; and after the circVRK1 is knocked down, the proliferation and migration capacity of SW620 cells and HCT116 cells are enhanced, and the apoptosis is reduced. Therefore, the application provides that the circVRK1 can be used as a new potential treatment target, and the targeting treatment is carried out on patients through the circVRK1 liposome nano-particles and other forms, so that a scientific and accurate treatment scheme is provided for colorectal cancer patients.
The application has the advantages and beneficial effects that:
1. the application provides a brand new cyclic RNA molecule circVRK1, and provides a primer group for reverse shearing a connecting site and amplifying the circVRK1, wherein the primer group comprises a Convergent primer group and/or a Divergent primer group;
2. the application provides therapeutic uses of circVRK1 as a targeted drug. Overexpression of circVRK1 resulted in reduced proliferation and migration of colorectal cancer cells and increased apoptosis. Thus, circVRK1 may be used as a potential drug for the treatment of colorectal cancer.
Drawings
FIG. 1. Sequence of circVRK1 and its reverse splice junction site. A: the splice site of circVRK1 was verified by sanger sequencing; b: carrying out amplification after RNAase enzyme treatment, and proving the stability of the circular RNA; c: PCR detection, both the Convergent primer and the Divergent primer in the cDNA group can be amplified to obtain fragments, while Divergent in the genome can not be amplified to obtain fragments.
Fig. 2: schematic of the construction of the vector from the circVRK1 overexpressing plasmid.
Fig. 3: schematic of the construction of the vector from the circVRK1 knockdown plasmid.
FIG. 4. Influence of circVRK1 on cell proliferation capacity. Liposome nano-particles which overexpress and knock down the circVRK1 are led into SW620 and HCT116 cells, and the proliferation capacity of the cells is detected by using a CCK8 reagent, and after the circVRK1 is overexpressed, the proliferation capacity of the SW620 and HCT116 cells is obviously reduced; after the circVRK1 is knocked down, the proliferation capacity of SW620 and HCT116 cells is obviously enhanced.
FIG. 5. Transfer well experiments were performed after introducing liposome nanoparticles overexpressing and knockdown circVRK1 into HCT116 cells, and HCT116 cells were significantly reduced in migration and proliferation capacity after overexpressing circVRK 1; after the circVRK1 is knocked down, the migration and proliferation capacity of HCT116 cells is obviously enhanced.
FIG. 6. Influence of circVRK1 on apoptosis. After liposome nano-particles which are used for over-expressing and knocking down the circVRK1 are led into SW620 and HCT116 cells, apoptosis detection is carried out, and after the circVRK1 is over-expressed, apoptosis is obviously increased; apoptosis of cells was significantly reduced after knockout of circVRK 1.
Detailed Description
The following provides a detailed description of embodiments of the present application with reference to examples.
Example 1: effect of circVRK1 on cell proliferation potency
The circVRK1 sequence was inserted into the PLCDH-ciR vector according to EcoRI and BamHI cleavage sites, and the 1073bp sequence of hsa_circ_0000566 was PCR amplified and ligated into pLCDH-ciR In-fusion, thereby constructing the circVRK1 over-expression plasmid. The cloning vector selected is shown in FIG. 2. The sequence of the target gene cloning primer is as follows:
Primer-F:
CGGAATTCTGAAATATGCTATCTTACAGTGAAAATGCCTCGTGTAAAAGC(SEQ ID NO:6)
Primer-R:CGGGATCCTCAAGAAAAAATATATTCACCTTTGTTATTGTTT TTGCTTTCAAACCTCCA(SEQ ID NO:7)
according to the siRNA sequence, the shRNA sequence is designed and cloned to an interference vector, so that the aim of knocking down the circVRK1 is fulfilled. The cloning vector is shown in FIG. 3.
The siRNA sequence and shRNA sequence of circVRK1 are as follows:
siRNA1 sequence: CAAAGTGAAAATGCCTCGT (SEQ ID NO: 8)
shRNA 1/primer sequence:
Top strand:
GATCCGCAAAGTGAAAATGCCTCGTTTCAAGAGAACGAGGCATTTTC ACTTTGTTTTTTG(SEQ ID NO:9)
Bottom strand:
AATTCAAAAAACAAAGTGAAAATGCCTCGTTCTCTTGAAACGAGGCA TTTTCACTTTGCG(SEQ ID NO:10)
siRNA2 sequence: AAACAATAACAAAGTGAAAAT (SEQ ID NO: 11)
shRNA 2/primer sequence:
Top strand:
GATCCGAAACAATAACAAAGTGAAAATTTCAAGAGAATTTTCACTTTG TTATTGTTTTTTTTTG(SEQ ID NO:12)
Bottom strand:
AATTCAAAAAAAAACAATAACAAAGTGAAAATTCTCTTGAAATTTTCA CTTTGTTATTGTTTCG(SEQ ID NO:13)
the liposome nanoparticle is formed after the plasmid is wrapped by liposome. Liposome nano-particles which overexpress and knock down the circVRK1 are led into SW620 and HCT116 cells, and the proliferation capacity of the cells is detected by using a CCK8 reagent, and after the circVRK1 is overexpressed, the proliferation capacity of the SW620 and HCT116 cells is obviously reduced; after the circVRK1 is knocked down, the proliferation capacity of SW620 and HCT116 cells is obviously enhanced. P <0.05, P <0.01, P <0.001, P < 0.0001) (see fig. 4).
Example 2: effect of circVRK1 on cell migration and invasiveness
The liposome nano-particles which are used for over-expressing and knocking down the circVRK1 and constructed in the example 1 are introduced into HCT116 cells, and after the circVRK1 is over-expressed, the migration and invasion capacity of the cells are obviously reduced; after knockdown of circVRK1, the migration and invasion capacity of the cells was significantly increased (see FIG. 5).
Example 3: influence of circVRK1 on apoptosis
The liposome nano-particles which are used for over-expressing and knocking down the circVRK1 and constructed in the example 1 are introduced into SW620 and HCT116 cells, and after the circVRK1 is over-expressed, the apoptosis is obviously weakened; after knockdown of circVRK1, apoptosis of the cells increased (see FIG. 6).
While the preferred embodiments of the present application have been described in detail, the present application is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.
Claims (9)
1. A circular RNA molecule circVRK1 is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. A recombinant vector comprising the circVRK1 gene according to claim 1.
3. A primer set for amplifying circVRK1, comprising: a Convergent primer group and/or a divrgent primer group, wherein the nucleotide sequence of the Convergent primer group is shown as SEQ ID NO. 2 and SEQ ID NO. 3; the nucleotide sequences of the Divergent primer group are shown as SEQ ID NO. 4 and SEQ ID NO. 5.
Use of circvrk1 in the preparation of a medicament for the treatment of colorectal cancer.
5. Use of an agent for increasing the expression level of circVRK1 in the preparation of a medicament for treating colorectal cancer.
6. The use according to claim 5, wherein the agent for increasing the expression level of circVRK1 comprises any one of the following:
a) The circVRK1 gene;
b) A recombinant vector containing the circVRK1 gene;
c) Recombinant viruses containing the circVRK1 gene.
7. The use according to claim 5, wherein the agent for increasing the expression level of circVRK1 is a liposome nanoparticle comprising the circVRK1 gene.
8. The use according to claim 5, wherein the agent that increases the expression level of circVRK1 is used for colorectal cancer treatment by overexpression of circVRK 1.
9. A medicament for treating colorectal cancer, which is characterized in that the active ingredient is an agent for improving the expression quantity of circVRK 1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2023104581084 | 2023-04-26 | ||
| CN202310458108 | 2023-04-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116926073A true CN116926073A (en) | 2023-10-24 |
Family
ID=88379623
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310681050.XA Pending CN116926073A (en) | 2023-04-26 | 2023-06-09 | CircVRK1 and application thereof in preparation of colorectal cancer treatment drugs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116926073A (en) |
-
2023
- 2023-06-09 CN CN202310681050.XA patent/CN116926073A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hou et al. | Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma | |
| EP3594348B1 (en) | C/ebp alpha short activating rna compositions and methods of use | |
| US9163261B2 (en) | Adeno-associated virus 2/8—micro RNA-101 therapy for liver cancer | |
| US10704044B2 (en) | Albumin production and cell proliferation | |
| CN113817823B (en) | Long non-coding RNA LETN as tumor marker and therapeutic target | |
| EP3733213A1 (en) | Tumor-targeting trans-splicing ribozyme and use thereof | |
| Liao et al. | CD38 is highly expressed and affects the PI3K/Akt signaling pathway in cervical cancer | |
| AU2023200960A1 (en) | Compositions and Methods for the Treatment of a Beta-Catenin-Associated Disease or Disorder | |
| US11078487B2 (en) | Cancer-specific trans-splicing ribozymes and use thereof | |
| Li et al. | Downregulation of NOB1 suppresses the proliferation and tumor growth of non-small cell lung cancer in vitro and in vivo Retraction in/10.3892/or. 2020.7483 | |
| WO2017009837A1 (en) | Micro-rna for the treatment of malignant solid tumors and metastasis | |
| Zhao et al. | pH low insertion peptide mediated cell division cycle-associated protein 1-siRNA transportation for prostatic cancer therapy targeted to the tumor microenvironment | |
| CN116926073A (en) | CircVRK1 and application thereof in preparation of colorectal cancer treatment drugs | |
| Meng et al. | Adenovirus-mediated siRNA targeting NOB1 inhibits tumor growth and enhances radiosensitivity of human papillary thyroid carcinoma in vitro and in vivo | |
| CN114317539B (en) | hsa_circ_0001137 circular RNA and application thereof in cancer diagnosis and treatment | |
| CN104031917B (en) | A kind of siRNA and its Liposomal formulation for HBx | |
| CN111265658A (en) | Novel hypoxia signal regulating molecule and application thereof | |
| CN114712507B (en) | Application of TRIM4 in preparing sensitizer or reverse tolerance agent of ovarian cancer treatment drug | |
| KR20200052294A (en) | Method of treating cancer by inhibition of SETD2 | |
| CN115212308B (en) | Application of GASDERMIN E pathway targeting agent in treating pancreatic cancer | |
| CN113774137B (en) | Application of reagent for detecting biomarker expression in preparation of kit for identifying leukemia drug resistance and/or adverse prognosis | |
| CN101474413A (en) | Use of DRR1 gene in preparing anti-tumor medicament | |
| WO2022206812A1 (en) | Rna delivery system for treating cancer | |
| JP7037160B1 (en) | A prophylactic or therapeutic agent for at least one cancer selected from the group consisting of pancreatic cancer, lung cancer, colon cancer, bile duct cancer and liver cancer, and the prophylactic or therapeutic agent for the cancer in combination with the agent. A method for screening a therapeutic agent, a combination drug containing the agent, and a preventive or therapeutic agent for cancer. | |
| WO2023040828A1 (en) | Sirna conjugate targeting fap-positive cells, and pharmaceutical composition and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |