CN116622705A - circRNA for regulating CVB5 replication and application thereof - Google Patents

circRNA for regulating CVB5 replication and application thereof Download PDF

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CN116622705A
CN116622705A CN202310313659.1A CN202310313659A CN116622705A CN 116622705 A CN116622705 A CN 116622705A CN 202310313659 A CN202310313659 A CN 202310313659A CN 116622705 A CN116622705 A CN 116622705A
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circrna
cvb5
replication
expression
cells
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陈伟
李静
滕培英
杨帆
张继虹
李金伟
李帅鹏
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Kunming University of Science and Technology
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Abstract

The invention discloses circRNA for regulating and controlling CVB5 replication and application thereof, and relates to the technical field of gene biology. The invention discloses a circRNA, which is the circRNA002006, and a sequence table is shown as SEQ ID NO.1; according to the invention, through analyzing the expression profile of the circRNA of CVB5 infected human malignant embryo rhabdomyoma cells (RD), the circRNA002006 is subjected to down-regulation expression in virus infected cells and is dependent on virus infection time and dose; meanwhile, the circRNA002006 can obviously inhibit CVB5 replication; furthermore, circRNA002006 may activate the expression of interferon-stimulating factors (ISGs). The present invention has found that there is a circRNA (circRNA 002006) that significantly inhibits CVB5 replication, thereby providing a new therapeutic regimen for diseases of CVB5 infection.

Description

circRNA for regulating CVB5 replication and application thereof
Technical Field
The invention relates to the technical field of gene biology, in particular to a circRNA for regulating and controlling CVB5 replication and application thereof.
Background
Coxsackie virus B5 (CVB 5) belongs to group B enteroviruses of the family Picornaviridae, which is a non-enveloped single positive-strand RNA virus. The CVB5 genome is about 7.4kb in length and comprises two untranslated regions and an Open Reading Frame (ORF), which can encode a 2,194 amino acid multimeric protein precursor, which is further hydrolyzed by the viral self-encoded protease into three precursor proteins (P1, P2 and P3). CVB5 is involved in outbreaks and epidemic of hand-foot-and-mouth disease, and can cause serious diseases such as aseptic encephalitis, myocarditis, diabetes and the like, and the incidence rate of children under 5 years old is highest, and the diseases are frequently sent out in Yu Xiaqiu seasons. However, currently, no approved vaccine and specific drugs against CVB5 infection are available.
Circular RNA (circRNA) is a particular endogenous non-coding RNA found in recent years and is widely found in mammalian cells. Unlike linear RNA structures, circRNA is a class of covalently closed circular RNA molecules that lack a bondable non-coding end and are not easily degraded by exonucleases, thus having greater stability. The circRNA can be used as competitive endogenous RNA (competing endogenous RNA, ceRNA) for capturing miRNA, can regulate alternative splicing and gene expression, can be used as a scaffold for assembling to form a protein complex, and can also be used for encoding the protein and acting as a regulator of rRNA and tRNA biosynthesis and play roles in various biological processes of cells. Studies have shown that circRNA plays an important role in viral infection and host immunity, and we have therefore deeply mined the function of circRNA after CVB5 infection.
Disclosure of Invention
The invention aims to provide the circRNA for regulating CVB5 replication, which is the circRNA002006 with the function of remarkably inhibiting CVB5 replication, and can provide a new treatment scheme for diseases infected by CVB 5.
In order to achieve the technical purpose and the technical effect, the invention is realized by the following technical scheme:
a circRNA, which is circRNA002006; the nucleotide sequence and splice site of the circRNA are shown in SEQ ID NO. 1.
It is another object of the present invention to provide a circRNA inhibiting CVB5 replication, wherein the circRNA is the circRNA002006 described above.
Further, the circRNA down-regulates expression in CVB 5-infected RD cells.
Further, the circRNA expression was dose dependent with CVB5 infection time.
It is another object of the present invention to provide an overexpression vector sequence of circRNA002006, the sequence of which is shown in SEQ ID NO. 2.
It is another object of the invention to provide the use of circRNA002006 for the preparation of a product for the prevention or treatment of CVB 5.
Furthermore, the product for preventing or treating CVB5 is a hand-foot-and-mouth disease preventing or treating drug.
The invention has the beneficial effects that:
the invention discloses a circRNA, which is circRNA002006, wherein after the circRNA002006 is overexpressed in cells, the replication level of CVB5 is obviously reduced compared with that in control cells, namely, the circRNA002006 provided by the invention has the effect of inhibiting CVB5 virus replication;
the circRNA002006 provided in the present invention regulates the expression of antiviral immune factor ISGs.
The circRNA002006 provided by the invention plays a role in inhibiting CVB5 replication through an interferon pathway, and can be used for preventing or treating hand-foot-mouth disease.
Of course, it is not necessary for any one product to practice the invention to achieve all of the advantages set forth above at the same time.
Drawings
FIG. 1 is a graph showing the difference in the expression level of candidate circRNA between CVB5 infection group and blank control group (left: RNA-seq sequencing result; right: qRT-PCR detection result) according to the embodiment of the present invention;
FIG. 2is a graph showing the expression results of CVB5 infection with different doses of circRNA002006 according to the examples of the present invention;
FIG. 3 is a graph showing the results of expression of circRNA002006 at different times during CVB5 infection according to the examples of the present invention;
FIG. 4 is a schematic diagram of a circRNA002006 over-expression plasmid vector according to an embodiment of the present invention;
FIG. 5 is a graph showing the results of significantly inhibiting the expression level of CVB5 structural protein VP1 after overexpression of circRNA002006 according to the examples of the present invention;
FIG. 6 is a graph showing the results of activating ISGs expression after the overexpression of circRNA002006 according to the embodiment of the invention.
Detailed Description
In order to more clearly describe the technical scheme of the embodiment of the present invention, the embodiment of the present invention will be described in detail below with reference to the accompanying drawings.
The medicines, reagents, instruments, equipment and the like used in the invention can be purchased through commercial approaches unless otherwise specified, and the related experimental methods are all conventional experimental operations in the field or carried out according to the product specifications of the corresponding reagents.
In the early research of the invention, RNA-SEQ analysis is carried out after CVB5 infects RD cells, and the circRNA002006 is screened, and the nucleotide sequence is shown as SEQ ID No.1; to investigate the potential biological functions of circRNA002006, circRNA002006 was overexpressed in cells and the effect of circRNA002006 on viral replication and its mechanism was examined.
The cells and viruses used in the invention are preserved in a laboratory, and the circRNA002006 over-expression vector is synthesized by itself.
The sequence of the circRNA primer related to the invention is shown in table 1, and the sequence of the ISGs primer is shown in table 2;
the map of the circRNA002006 overexpression vector is shown in FIG. 4.
TABLE 1circRNA primer sequences
TABLE 2ISGs primer sequences
Example 1
Screening and characterization of circRNA002006
Analysis of the expression profile of circRNA after CVB5 infection RD
The invention adopts RNA-seq to analyze the expression quantity of the circRNA in the CVB5 infected group and the blank control group, and the RNA-seq result of the circRNA is provided by Beijing NodeB source technology Co. According to the RNA sequencing result, 6 circRNAs are selected for experimental verification.
Expression of circRNA002006
After 24h of CVB5 infection of RD cells, the cell pellet is collected and total RNA is extracted with TRIzol reagent. The qRT-PCR method is used for verifying the expression condition of the selected 6 circRNAs, and the data are analyzed by a 2-delta CT method. Wherein, the expression level of the circRNA002006 is down-regulated, and the RNA-seq is consistent with the qRT-PCR detection result. The nucleotide sequence of the circRNA002006 is shown in SEQ ID NO. 1.
The expression profile of circRNA002006 is associated with CVB5 infection
The above results indicate that the expression of circRNA002006 is closely related to CVB5 infection, and thus the relationship of circRNA002006 expression to CVB5 infection was further examined using qRT-PCR. After RD cells were infected with CVB5 at different MOI (0,0.01,0.1,1,5) for 24h, cell pellet was collected, total RNA was extracted with TRIzol reagent, and expression of circRNA002006 was detected by qRT-PCR method. As shown in FIG. 2, expression of circRNA002006 correlated positively with viral infection dose. After infection of RD cells with 1MOI CVB5, cell pellets were collected at 0h,6h,12h,24h and 36h, respectively, total RNA was extracted with TRIzol reagent, and expression of circRNA002006 was detected by qRT-PCR. As shown in FIG. 3, expression of circRNA002006 was positively correlated with viral infection time. Taken together, circRNA002006 expression is dependent on CVB5 infection dose and infection time, and circRNA002006 plays an important role in CVB5 infection.
Example 2
Functional study of circRNA002006
1. Construction of the circRNA002006 overexpression vector
Based on the sequence of circRNA002006 (SEQ ID No. 1), pcDNA3.1 plasmid vector containing the circRNA002006 sequence, namely pcDNA3.1-circRNA002006-2Flag vector, was constructed (map is shown in FIG. 4). Wherein, ecoRI and BamHI are inserted into the circRNA002006 region.
2. Cell transfection
RD cells in the logarithmic growth phase are inoculated into a 6cm cell culture dish, and when about 60-70% of the cells are grown,
the empty control group (pcDNA3.1) and the overexpressing circRNA002006 group (pcDNA3.1-circRNA 002006-2 Flag) were transfected with the Transindm EL transfection reagent, respectively, and incubated in a 5% CO2 cell incubator at 37℃for 24h.
3. Viral infection
Transfected cells were infected with CVB524h at an infectious dose of 1 MOI.
Western Blot detection of CVB5 VP1 expression
The cells were washed twice with PBS, and RIPA lysate (1 XPMSF, 1 Xphosphatase lysate) was added to collect the cell lysate. The expression of structural protein VP1 of CVB5 was detected by Western Blot experiments.
The results indicate that circRNA002006 significantly inhibited CVB5 replication (as shown in fig. 5).
Example 3
circRNA002006 promotes ISGs expression
The cells were washed twice with PBS and the TRIzol reagent extracted total RNA from the cells. ISGs expression was detected by qRT-PCR.
The results indicate that circRNA002006 overexpression significantly induced ISGs expression (as shown in fig. 6), suggesting that circRNA002006 functions through the interferon pathway.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.

Claims (7)

1. A circRNA, characterized in that: the circRNA is the circRNA002006, and the nucleotide sequence and the splice site of the circRNA are shown in SEQ ID NO. 1.
2. A circRNA that inhibits CVB5 replication, characterized in that: the circRNA is the circRNA002006 of claim 1.
3. A circRNA inhibiting CVB5 replication according to claim 2 where: the circRNA down-regulates expression in CVB 5-infected RD cells.
4. A circRNA inhibiting CVB5 replication according to claim 2 where: the circRNA expression was dose dependent with CVB5 infection time.
An overexpression vector sequence of circrna002006, characterized in that: the sequence is shown as SEQ ID NO. 2.
Use of circrna002006 for the preparation of a product for the prevention or treatment of CVB 5.
7. The use of circRNA002006 according to claim 6 for the preparation of a product for the prevention or treatment of CVB5, wherein: the product for preventing or treating CVB5 is a hand-foot-and-mouth disease preventing or treating drug.
CN202310313659.1A 2023-03-28 2023-03-28 circRNA for regulating CVB5 replication and application thereof Pending CN116622705A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064531A (en) * 2022-09-07 2023-05-05 昆明理工大学 Long-chain non-coding RNA for inhibiting CVB5 virus replication and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064531A (en) * 2022-09-07 2023-05-05 昆明理工大学 Long-chain non-coding RNA for inhibiting CVB5 virus replication and application thereof
CN116064531B (en) * 2022-09-07 2024-04-02 昆明理工大学 Long-chain non-coding RNA for inhibiting CVB5 virus replication and application thereof

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