CN107460256B

CN107460256B - Application of lncRNA as biomarker in detection of enterovirus EV71

Info

Publication number: CN107460256B
Application number: CN201710913071.4A
Authority: CN
Inventors: 张振杰; 史卫峰; 李娟�; 周宏�
Original assignee: Taishan Medical University
Current assignee: Taishan Medical University
Priority date: 2017-09-30
Filing date: 2017-09-30
Publication date: 2019-12-20
Anticipated expiration: 2037-09-30
Also published as: CN107460256A

Abstract

The invention discloses long-chain non-coding RNA (lncRNA) ENST00000606915, the sequence of which is shown in SEQ ID No.1, wherein the lncRNA can be used as a biomarker for detection and diagnosis of enterovirus EV71, can also be used for early diagnosis of related diseases or for judging stage typing of the diseases, and provides a basis for improving diagnosis, treatment and prognosis of the diseases.

Description

Application of lncRNA as biomarker in detection of enterovirus EV71

The technical field is as follows:

the invention relates to the technical field of biology, in particular to application of lncRNA as a biomarker in detection of enterovirus EV 71.

Background art:

with the rapid development of molecular biology theory and experimental technology, biomarkers become a very popular research field. The quantitative analysis of key biological macromolecules such as long-chain non-coding RNA (LncRNA) is helpful for early diagnosis of diseases or judging the stage typing of the diseases, and provides a basis for improving the diagnosis, treatment and prognosis of the diseases. In the stage of discovering and identifying candidate lncRNA markers, RNA-Seq or fluorescence quantitative technology is an important way to screen differentially expressed genes, and the ability to analyze and identify infectious diseases at the nucleic acid level is the research direction and trend of clinical diagnosis in the future.

The enterovirus EV71 mainly causes hand-foot-and-mouth diseases and also can cause various nervous system diseases such as aseptic meningitis, brainstem encephalitis and poliomyelitis-like paralysis; among them, hand-foot-and-mouth disease and central nervous system infection are two common clinical symptoms caused by EV71 infection. At present, for the detection of enterovirus EV71, the separation and culture are mainly carried out through in vitro cells, and the virus nucleic acid is detected by combining Real-time fluorescent quantitative RT-PCR, which is the 'gold standard' for clinically identifying the enterovirus, but the separation of the virus is difficult to complete in a common laboratory due to the particularity of the hand-foot-and-mouth disease, the limitation of cell culture conditions and the requirement of aseptic operation. Therefore, the search for new biological markers becomes an urgent problem to be solved for the clinical rapid identification of the EV71 infection.

Based on differential expression of marker lncRNA related to EV71 infection in fluorescent quantitative detection, enterovirus separation and culture are not needed, the detection cost is low, the flux is high, the speed is high, hundreds of samples can be detected at one time, the severity of virus infection can be preliminarily determined, lncRNA shows great superiority in the verification link of biomarkers, and serum has the characteristics of safety in collection, stability in biological properties, convenience in storage and the like. Therefore, if valuable specific markers can be found in serum for clinical application, the detection of the enterovirus EV71 and the prevention, early diagnosis and treatment of related diseases are greatly facilitated.

The invention screens lncRNA differentially expressed in cells after EV71 infection by using RNA-Seq, verifies the correlation between the differential expression of the screened marker lncRNA and the EV71 infection stage by combining fluorescent quantitative PCR, and performs specific identification between the differential lncRNA and different types of enterovirus infection, thereby providing a new thought for detection of enterovirus EV71 and diagnosis of related diseases.

The invention content is as follows:

on one hand, the invention discloses a long-chain non-coding RNA (lncRNA), wherein the lncRNA is ENST00000606915, and the sequence of the long-chain non-coding RNA is shown in SEQ ID No.1, and further discloses that the lncRNA can be used as a biomarker for detection and diagnosis of enterovirus EV 71.

On the other hand, the lncRNA is used as a biomarker, the down-regulation of the transcription level of the lncRNA can reflect that the subject is infected with the enterovirus EV71, can be used as an index for detecting EV71, and can also be used for diagnosing whether the subject is infected with EV 71.

On the other hand, the lncRNA of the present invention as a biomarker can be used for detecting and/or diagnosing diseases caused by infection with enterovirus EV71, such as: the clinical application of the traditional Chinese medicine composition can be used for treating the diseases of the hand-foot-and-mouth disease, the central nervous system diseases, the aseptic meningitis, the brainstem encephalitis, the poliomyelitis-like paralysis and other nervous system diseases, preferably can be used for early diagnosis of the diseases or judging the stage typing of the diseases, and provides a basis for improving diagnosis, treatment and prognosis of the diseases.

In another aspect, the invention provides the use of the lncRNA in the preparation of a reagent and/or a kit for detecting and/or diagnosing enterovirus EV71 or a disease caused by enterovirus EV71 infection.

In another aspect, the invention provides a product for detecting and/or diagnosing enterovirus EV71 or a disease caused by infection with enterovirus EV71, said product comprising an agent capable of detecting the expression of said lncRNA.

The reagent capable of detecting the expression of the lncRNA comprises but is not limited to a primer/primer group for amplifying the lncRNA or a probe capable of hybridizing with the lncRNA.

The primer/primer set for amplifying the lncRNA can be designed at the 5 'end and the 3' end of the lncRNA sequence, can also be designed in the sequence, and can also be designed aiming at the sequence overlapping region according to the position of the lncRNA in a chromosome, so long as the sequence obtained by amplification of the primer/primer set can be completely matched with the full-length sequence or partial sequence of the lncRNA; preferably, the primer set is selected from any one or a combination of the following groups:

primers 1-F: 5'-CTTCCTTGGACAGAATCACC-3', R: 5'-CTCACTCTCCCTGCACTGCA-3', respectively;

primer 2-F: 5'-GGGCAATGGGCCGCACGC-3', R: 5'-TTTTTTTTTTTTGAGGTGG-3' are provided.

In another aspect, the invention provides a kit for detecting and/or diagnosing enterovirus EV71 or a disease caused by infection with enterovirus EV71, said kit comprising reagents capable of detecting the expression of said lncRNA.

On the other hand, the long-chain non-coding RNA (lncRNA) disclosed by the invention can also improve the proliferation capacity of the enterovirus EV71, and the up-regulation of the expression level of the lncRNA can improve and/or promote the proliferation of the virus.

On the other hand, the lncRNA of the present invention, as a biomarker, can also be used for typing of enteroviruses, wherein the enteroviruses to be tested infect RD cells, and if the lncRNA can cause down-regulation of the transcription level of the lncRNA, the lncRNA is of the EV71 type, and if the lncRNA cannot cause change of the transcription level of the lncRNA, the lncRNA is of the non-EV 71 type. Preferably, the downregulation is more than 10%, preferably 10-90%, more preferably 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 85% downregulation compared to normal levels. When the lncRNA is used for enterovirus typing, the lncRNA can be used for the purposes of diagnosis and treatment of diseases and can also be used for the purposes of non-disease treatment/diagnosis.

Description of the drawings:

FIG. 1: a detection result graph aiming at the lncRNA expression profile of the infant infected with the enterovirus EV 71; wherein "+" indicates a sample positive for EV71 infection and "-" indicates a sample negative for EV71 infection.

FIG. 2: real-time PCR verifies the differential expression graph of lncRNA 1-9 in different enterovirus EV71 infected infants; wherein "+" indicates an EV71 infection positive sample and "-" indicates an EV71 infection negative sample; the positive and negative differences of the band results shown by respectively amplifying different lncRNA are obvious.

FIG. 3: the effect of differential expression of lncRNA 1-9 on EV71 replication; wherein, the upper graph shows that the transcription level of RD cells is detected by RT-PCR 24 hours after the RD cells are respectively transfected by constructed lncRNA 1-9 overexpression plasmids; the lower panel shows the effect of lncRNA 1-9 overexpression on EV71 viral replication; n-empty plasmid control, 1-9 insert lncRNA 1-9 plasmid.

FIG. 4: effect of different types of enterovirus infection on the level of lncRNA8 transcription.

FIG. 5: effect of infection with other types of enteroviruses in the blood of children with hand-foot-and-mouth disease on the level of lncRNA8 transcription.

The specific implementation mode is as follows:

the present invention is further described below in conjunction with specific embodiments, which are given by way of illustration only and are not intended to limit the scope of the present invention. The test methods in the following examples are, unless otherwise specified, conventional in the art. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified.

Example 1 Collection of samples and preparation of RNA

5ml of peripheral venous blood of 3 children infected with enterovirus EV71 were collected in heparin sodium anticoagulation tubes, 1ml of whole blood was immediately dispensed in enzyme-free EP tubes as clinical samples, the children were not subjected to any intervention treatment before the samples were collected, all patients had known consent, and the consent was obtained by the tissue ethics committee. At the same time, 3 healthy children's sera were collected as negative controls.

The method for extracting total RNA from whole blood by using the RNAprep Pure blood total RNA extraction kit of Tiangen company comprises the following steps: the whole blood was collected on ice and 1mL Trizol was added. Mixing thoroughly with vortex oscillator until no solid exists, and standing at room temperature for 10 min. Centrifuge at 13000rpm and 4 ℃ for 10 min. The supernatant was carefully pipetted into a clean RNase-Free l.5mL centrifuge tube. 0.2mL of methylene chloride was added, and the mixture was shaken with a vortex mixer for 15 seconds and allowed to stand at room temperature for 10 min. Centrifuging at 3000rpm and 4 deg.C for 10 min. Carefully pipette the aqueous phase and transfer to a 1.5mL RNase-free centrifuge tube. Add an equal volume of isopropanol, shake with vortex mixer for 4s to precipitate total RNA, and let stand at room temperature for 30 min. 13000rpm at 4 ℃ centrifugal 10min, discard the supernatant. 1mL of 75% ethanol was added and mixed again. 13000rpm at 4 ℃ centrifugal 10min, discard the supernatant. Drying at room temperature for 10min, adding 12 μ l RNase-Free water to dissolve RNA and placing on ice for use.

RNA degradation is reduced as much as possible in the experimental process, so that RNA with high purity and quality and without protein and other impurity pollution is obtained. RNA integrity was analyzed by 1.5% agarose gel electrophoresis and total RNA optical density values at 280nm and 260nm were determined using a Bio-Red ultraviolet spectrophotometer. Amount of RNA as 1OD_260nmCalculated as OD of 40. mu.g RNA₂₆₀/OD₂₈₀When the value of (A) is 1.8-2.0, the purity of the total RNA is considered to be reliable, and the method can be used for the next experiment.

Example 2 differential expression analysis of Enterovirus EV 71-infected group lncRNA

RNA-Seq detection of the expression profile of IncRNA in the blood of EV71 infection and healthy young children was performed using the Arraystar Human IncRNA Microarray technology from Arraystar corporation, respectively. The Arraystar lncRNA chip is designed with 60nt length oligonucleotide as probe, and these long oligonucleotide probes can obtain ideal experiment result with high sensitivity and specificity under high strict hybridization condition. A plurality of probes are designed for each sequence, so that the reliability of signals is improved.

Chip results are analyzed by using Agilent GeneSpring software, and lncRNAs with significant difference in expression amount (standard is that the difference between the expression amount of lncRNA in a positive infection group and the expression amount of lncRNA in a healthy control group is more than 2 times, and p is less than 0.05) are screened. The expression of lncRNA in peripheral blood of EV71 infected persons and healthy persons is obviously and differentially expressed by lncRNA chip detection analysis, and the cluster analysis result of the differential lncRNA (shown in figure 1) shows that 719 lncRNA (the difference multiple is greater than 2, and P is less than 0.05) are differentially expressed in 19186 independent lncRNA detected in an infected group, wherein the expression comprises 239 up-regulated genes and 480 down-regulated genes. Among them, 9 lncRNAs with the most significant fold up/down regulation (fold difference >6) were expressed as follows. Expression up-regulation: ENST00000561471(lncRNA 1), ENST00000565272(lncRNA 2), ENST00000565955(lncRNA 3), ENST00000592906(lncRNA 4), ENST00000624123(lncRNA 5); down-regulation of expression: ENST00000441379(lncRNA 6), ENST00000173535(lncRNA 7), ENST00000606915(lncRNA 8), ENST00000621571(lncRNA 9).

Detection primers are respectively designed aiming at the 9 lncRNAs, the sequences of the primers are shown in the table 1, GAPDH mRNA is used as an internal reference, and a SYBR Green dye is doped into a relative quantitative method to carry out Real-time PCR to verify the differential expression of the candidate lncRNAs (figure 2). Clustering analysis (figure 2) is carried out on 9 lncRNA with the most obvious up-regulation and down-regulation multiples (the difference multiple is more than 6) to find that the expression difference of the lncRNA of the infection group and the control group can be visually seen; different lncRNA are amplified respectively, and the electrophoresis result shows that the positive difference and the negative difference are obvious; the same group of samples are gathered together, which shows that the gene expression trends among the samples are consistent. Green to red, darker colors represent more significant differences.

TABLE 1 primer sequences for detection of lncRNA 1-9

Example 3 Effect of differential expression of IncRNA 1-9 on EV71 replication

After the lncRNA 1-9 sequence is inserted into eukaryotic expression vector pcDNA3.1, RD cells are transfected by liposome, and EV71 virus (moi is 1.0) is infected after 24 hours in CO₂Culturing for 24 hours in a cell culture box, collecting cell supernatants of an experimental group and a control group, respectively extracting virus total RNA in the supernatants with the same volume according to Trizol reagent (Tiangen) instructions, determining the purity and the concentration of the RNA by an ultramicro spectrophotometer, carrying out reverse transcription on cDNA, freezing and storing at-20 ℃ for later use, and quantitatively detecting the nucleic acid load of EV71 by adopting fluorescence after extracting the RNA.

When lncRNA is inserted into an overexpression vector to be overexpressed in RD cells, the RT-PCR detection result shows that the transcription level of lncRNA 1-9 is obviously increased compared with that of an empty plasmid control (on a figure 3). After infection with EV71, the viral load was quantitatively determined by fluorescence, and it was found that lncRNA 8(ENST00000606915) was overexpressed and significantly enhanced in replication compared to control EV71, with statistical significance (p <0.05), whereas overexpression of the other 8 lncrnas candidate did not affect replication of EV71 (p >0.05) (fig. 3 below). The above results indicate that aberrant expression of lncRNA8 correlates with the level of replication of EV 71; wherein the sequence of lncRNA 8(ENST00000606915) is shown in SEQ ID No. 1.

Example 4 correlation of marker IncRNA 8 differential expression with EV71 and other types of enterovirus infection time

Inoculating enteroviruses EV71, CVA16, CVA6 and CVA10 to the full monolayer RD cells, collecting the cells at 6, 12, 18, 24 and 30 hours after infection, respectively extracting total RNA of the cells of an infected group and a negative control group, quantifying the purity and concentration of the RNA, carrying out reverse transcription on cDNA, taking GAPDH as an internal reference, and carrying out Real-time PCR (polymerase chain reaction) to detect the change of lncRNA in the cells by adopting a SYBR Green method.

The expression of the marker lncRNA8 in the infected cells is detected in a fluorescence quantitative mode at different time points after different types of enteroviruses infect RD cells, and the fact that the transcription level of lncRNA8 is gradually reduced along with the time lapse after EV71 infection, the trend is consistent with the detection result of an RNA-Seq chip, and the transcription level is reduced to 1/5 of a normal level 24 hours after infection (figure 4). While other enteroviruses (CVA16, CVA6 and CVA10) did not detect a significant decrease in IncRNA 8 transcript levels after infection of the cells, and did not differ significantly from the negative control group (P > 0.05). This indicates that the decrease in lncRNA8 transcript levels in vitro infected RD cells is specifically associated with EV71 infection, with longer infection times leading to lower lncRNA8 transcript levels.

Example 5 Effect of infection with other types of Enterovirus in the blood of infants with hand-foot-and-mouth disease on the transcriptional level of IncRNA 8

Collecting the blood of HFMD children patients with high clinical infection rate caused by CVA16, CVA6 and CVA10 viruses, wherein each group contains at least 3 parts, carrying out RNA quantification, carrying out reverse transcription on cDNA, and detecting the transcription level of lncRNA 8.

Collecting blood of children affected with CVA16, CVA6 and CVA10, and quantifying the level of marker lncRNA8, as shown in FIG. 5, the blood of children affected with CVA16, CVA6 and CVA10 has no significant difference in the transcription level of lncRNA8 in the blood of healthy children (p > 0.05); this indicates that the change of the incrna 8 transcript level in the blood of children with hand-foot-and-mouth disease has no detectable correlation with the infection of CVA16, CVA6 and CVA10, and the change of the transcript level can specifically detect the infection of enterovirus EV 71.

Sequence listing

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Claims

1. Use of long non-coding rna (lncRNA) as a biomarker for the preparation of a reagent for the detection and/or diagnosis of enterovirus EV71, wherein the lncRNA is ENST00000606915, the sequence of which is shown in SEQ ID No. 1.

2. Use of long non-coding rna (lncRNA) as a biomarker for the preparation of a reagent for the detection and/or diagnosis of a disease caused by infection with enterovirus EV71, wherein the lncRNA is ENST 00000606915; the sequence is shown in SEQ ID No. 1.

3. Use according to claim 2, wherein the disease is hand-foot-and-mouth disease, central nervous system disease, aseptic meningitis, brainstem encephalitis and polio-like paralytic nervous system disease.

4. The use according to claim 2, wherein the diagnosis is an early diagnosis of a disease or a determination that a disease is EV71 or non-EV 71.

5. Use according to any one of claims 1 to 4, wherein said agent is a product capable of detecting the expression and/or transcription of said IncRNA.

6. The use according to claim 5, wherein the product capable of detecting the expression and/or transcription of IncRNA is a primer or primer set capable of amplifying the full or partial sequence of IncRNA or a probe capable of hybridizing to IncRNA.

7. The use according to claim 6, wherein the primer set is selected from any one or a combination of the following groups:

primer pair 1-F: 5'-CTTCCTTGGACAGAATCACC-3', R: 5'-CTCACTCTCCCTGCACTGCA-3', respectively;

primer pair 2-F: 5'-GGGCAATGGGCCGCACGC-3', R: 5'-TTTTTTTTTTTTGAGGTGG-3' are provided.

8. Use of a product capable of detecting the expression and/or transcription of lncRNA in the preparation of a kit for detecting and/or diagnosing enterovirus EV71, characterized in that said lncRNA is ENST00000606915, the sequence of which is shown in SEQ ID No. 1.

9. Use of a product capable of detecting the expression and/or transcription of lncRNA for the preparation of a kit for the detection and/or diagnosis of a disease caused by infection with enterovirus EV71, wherein lncRNA is ENST00000606915, the sequence of which is shown in SEQ ID No. 1.

10. Use according to claim 9, wherein the disease is hand-foot-and-mouth disease, central nervous system disease, aseptic meningitis, brainstem encephalitis and polio-like paralytic nervous system disease.

11. The use according to claim 9, wherein the diagnosis is an early diagnosis of a disease or a determination that a disease is EV71 or non-EV 71.

12. The use according to any one of claims 8 to 11, wherein the product capable of detecting the expression and/or transcription of incrna is a primer or primer set capable of amplifying the full-length sequence or a partial sequence of the incrna, or a probe capable of hybridizing to the incrna.

13. The use according to claim 12, wherein the primer set is selected from any one or a combination of the following groups:

14. An agent for detecting and/or diagnosing enterovirus EV71 or a disease caused by enterovirus EV71 infection, wherein the agent is a product capable of detecting the expression and/or transcription of lncRNA, and the lncRNA is ENST00000606915, and the sequence of the lncRNA is shown as SEQ ID No. 1.

15. The reagent of claim 14, wherein the product is a primer or a primer set capable of amplifying the full-length sequence or a partial sequence of the incrna, or a probe capable of hybridizing to the incrna.

16. The reagent according to claim 15, wherein the primer set is selected from any one or a combination of the following groups:

17. A kit for detecting and/or diagnosing enterovirus EV71 or a disease caused by infection with enterovirus EV71, comprising the reagents of claim 14.

18. Use of long non-coding rna (lncRNA) as a biomarker for non-therapeutic enterovirus typing, characterized in that the test enterovirus is of type EV71 if it is capable of causing a down-regulation of the transcription level of said lncRNA, and of type non-EV 71 if it is not capable of causing a change in the transcription level of said lncRNA; the lncRNA is ENST 00000606915; the sequence is shown in SEQ ID No. 1.

19. Use of long non-coding rna (lncRNA) for increasing the proliferative capacity of enterovirus EV71, wherein up-regulating the expression level of said lncRNA increases the proliferation of said virus, said lncRNA being ENST 00000606915; the sequence is shown in SEQ ID No. 1.