CN114854851A - Application of exosome lncRNA (long chain ribonucleic acid) derived from plasma in preparation of drug-induced liver injury biomarker - Google Patents
Application of exosome lncRNA (long chain ribonucleic acid) derived from plasma in preparation of drug-induced liver injury biomarker Download PDFInfo
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Abstract
The invention discloses application of plasma-derived exosome lncRNA in preparation of a drug-induced liver injury biomarker, wherein the lncRNA comprises lncRNA of which NONCODE TRANSCRIPT ID is NONRATT 018001.2. The invention discloses a method for preparing a biomarker for liver injury by using lncRNA, in particular to a method for detecting liver injury caused by exogenous compounds, which has high detection sensitivity and simple detection method and reagent, can be widely applied to detection of drug-induced liver injury, has reliable detection result, and can be used for independently detecting or jointly evaluating the liver injury by combining other indexes.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of plasma-derived exosome lncRNA in preparation of a drug-induced liver injury biomarker.
Background
The liver is the most important metabolic organ in the body and is also one of the main target organs of exogenous toxicants in the body. The susceptibility of the liver to chemical damage is determined by its particular anatomical location and physiological and biochemical functions. Since the liver is anatomically close to the blood supply from the digestive tract, it is the primary target for poisons absorbed through the digestive tract to exert their toxic effects; the liver has the ability to concentrate and transform exogenous chemical substances, and plays an important role in the process of excreting exogenous chemical substances and metabolites thereof into bile. Thus, when a body is exposed to an exogenous toxicant, the liver is more often subjected to toxic reactions than other organs. Therefore, it is urgently needed to establish a new liver injury biomarker which can be found and confirmed, can detect liver injury early, can reflect improvement or aggravation of liver injury, and even can reflect an injury mechanism.
Drug Induced Liver Damage (DILI), also known as Drug Induced Liver damage, refers to Liver damage caused by drugs and/or their metabolites, which can occur in healthy people who have no previous history of Liver disease or in patients who have had a serious disease, to varying degrees after the use of certain drugs. DILI is clinically the leading cause of acute liver failure and death, and is also a major factor in the withdrawal of marketed drugs. Statistically, at least 13% of acute liver failure in the united states is caused by DILI. In addition, at least over 1100 of the currently clinically used drugs are associated with liver injury, and although the reported incidence rate of DILI is in the range of 1/10000-1/100000, the actual incidence rate of DILI is far higher than the above range due to misdiagnosis or non-timely report in some cases.
In response to the potential occurrence of serious Clinical adverse events caused by DILI, the European Drug administration (EMEA) issued the "Non-Clinical guidelines on Drug-Induced Hepatotoxicity" in 2008, and the U.S. FDA issued "Drug-Induced Liver approach in 2009: the premarking Clinical Evaluation' guiding principle helps medical enterprises to comprehensively and deeply evaluate the risk of DILI in the research and development process of new drugs.
For nearly half a century, traditional biomarkers such as alanine transferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), and Total Bilirubin (TBD) have been used clinically for detection of DILI. ALT and AST are present in hepatocytes and during drug-induced liver injury, serum levels of ALT and AST are elevated, which correlates with hepatocyte death and its released content. Elevation of serum ALP is associated with damage to biliary epithelial cells, and elevation of TBIL may reflect impaired hepatocyte function or be associated with bilirubin production and processing. However, while existing liver injury biomarkers such as alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), etc. can identify liver injury or altered function, these indicators have failed to meet the early and accurate assessment of DILI risk due to lack of specificity (other organ or tissue injury can also increase their activity) and poor compliance with tissue morphology data.
With the progressive research on liver injury, a plurality of new detection indexes of hepatotoxic injury are discovered. Recently 2 new DILI biomarkers were discovered: glutamate dehydrogenase (GLDH) and miRNA-122, 2 biomarkers, have been supported by the U.S. food and drug administration and the European drug administration as liver-specific candidate biomarkers.
Exosomes are one of the extracellular vesicles, which are divided into three types: apoptotic bodies, microvesicles and exosomes. The process of exosome production involves double invagination of the plasma membrane and the formation of intracellular vesicles (MVBs) containing intraluminal vesicles (ILVs). ILV finally fuses to a plasma membrane through MVB, and is secreted in an exocrine form with the diameter of 40-160 nm through exocytosis. The density of exosomes was 1.15-1.19 g/mL. The tetrameric transmembrane proteins CD63, CD9 and CD81 found on their surfaces generally serve as surface biomarkers for exosomes. Meanwhile, most cells secrete exosomes, which are widely present in various body fluids, such as blood, saliva, urine, cerebrospinal fluid, and the like. Exosomes transport various molecules from parent cells to other cells, including proteins, DNA, mRNA/miRNA, lncRNA, and the like. Exosomes are similar in composition to the parental cells and therefore are able to provide information specific to the parental cells that can be traced. Scanning Electron Microscopy (SEM), transmission electron microscopy, dynamic light scattering and nanoparticle tracking analysis are widely used to measure physical characteristics of exosomes, such as the size, distribution and concentration of vesicles.
Long non-coding RNAs (lncrnas) are a class of open reading frame-lacking non-coding RNA molecules that are over 200 ribonucleotides in length, and either have no capacity to encode proteins or have limited coding function. It was initially recognized as "noise" in genome transcription, a byproduct of RNA polymerase II transcription, and has no biological effect. Through intensive research, lncRNA is found to be involved in important regulation and control processes in cells, such as: regulating cell differentiation, aging, proliferation, apoptosis, necrosis and tumor development. It was found that although lncRNA tends to be expressed at lower levels compared to messenger RNA, lncRNA expression is more cell specific than mRNA expression, suggesting lncRNA may be a key regulator of cell fate. Meanwhile, research shows that lncRNA is closely related to liver-related diseases and injuries. However, the lncRNA database is very large (172,216 are total so far), and the lncRNA which is closely related to liver injury, especially liver injury caused by exogenous compounds (such as acetaminophen) has not been developed in clinical tests.
At present, a great deal of research reports that the expression level of a plurality of exosomes lncRNA is obviously different from that of a normal control group under pathological conditions, and the exosomes can selectively package, secrete and transport the lncRNA and specifically exert biological functions. Exosomes can protect lncrnas from rnases and therefore they can be stably present in body fluids.
Disclosure of Invention
The invention aims to overcome the defects that a drug-induced liver injury biomarker in the prior art is low in sensitivity, too complex in detection method, expensive in reagent, and incapable of jointly evaluating liver injury by combining with other indexes, and cannot timely and accurately predict drug-induced liver injury, and provides application of long-chain non-coding RNA (lncRNA) from exosomes in preparation of the liver injury biomarker and a kit containing the same. The application can apply the lncRNA to preparation of a liver injury biomarker, particularly to detection of liver injury caused by an exogenous compound, has high detection sensitivity and simple detection method and reagent, can be widely applied to timely detection of drug-induced liver injury, can timely find drug-induced liver injury in early stage, has reliable detection result, and can be used for independently detecting or jointly evaluating the liver injury by combining other indexes.
The inventor of the invention has long-term research on liver injury, and a large number of experiments show that some of lncRNA derived from exosome can be used as a specific biomarker of drug-induced liver injury caused by exogenous compounds, is highly expressed in plasma of drug-induced liver injury caused by exogenous compounds, and can exert the application of the lncRNA in preparing the biomarker of liver injury, thereby detecting the liver injury caused by exogenous compounds.
In order to solve the technical problem, the first aspect of the present invention provides an application of lncRNA markers in the preparation of kits and/or chips for detecting liver injury (e.g. drug-induced liver injury), wherein the lncRNA markers include lncRNA of which nonoODE TRANSCRIPT ID is NONRATT 018001.2.
The use according to the first aspect of the present invention, wherein the incrna marker is used alone as the sole biomarker for detecting liver injury, or the incrna marker is used in combination with a conventional biomarker for detecting liver injury.
The conventional biomarker for detecting liver injury may be selected from alanine aminotransferase and aspartate aminotransferase as is conventional in the art.
The use according to the first aspect of the present invention, wherein said detecting is detecting the level of transcriptional level expression of said incrna marker in the sample.
The detection of the expression level of the transcription level according to the present invention can be RNA whole transcriptome sequencing, real-time fluorescent quantitative PCR detection or expression profiling chip as is conventional in the art.
In a preferred embodiment of the invention, the detection is RNA whole transcriptome sequencing.
In a preferred embodiment of the invention, the sample is plasma.
The use according to the first aspect of the present invention, wherein the liver injury is caused by an exogenous compound.
In a preferred embodiment of the invention, the exogenous compound is acetaminophen (APAP).
In order to solve the above technical problems, the second aspect of the present invention provides a kit for detecting liver damage, comprising a reagent for detecting lncRNA; the lncRNA comprises lncRNA of which NONCODE TRANSCRIPT ID is NONRATT 018001.2.
The kit according to the second aspect of the present invention, wherein the reagent is a reagent for detecting the level of expression of the lncRNA marker at the transcriptional level in the sample.
In a preferred embodiment of the invention, the detecting comprises RNA whole transcriptome sequencing; and/or, the sample is plasma; and/or the kit also comprises an exosome RNA extraction reagent.
The kit according to the second aspect of the present invention, wherein the liver injury is liver injury caused by an exogenous compound.
In a preferred embodiment of the invention, the exogenous compound is acetaminophen.
In order to solve the above technical problems, a third aspect of the present invention provides a chip for detecting liver damage; the chip is provided with a reagent for detecting lncRNA; the lncRNA comprises lncRNA of which NONCODE TRANSCRIPT ID is NONRATT 018001.2.
The chip according to the third aspect of the present invention, wherein the reagent is a reagent for detecting the expression level of the transcription level of the lncRNA marker in the sample.
In a preferred embodiment of the invention, the detecting comprises RNA whole transcriptome sequencing; and/or, the sample is plasma.
The chip according to the third aspect of the present invention, wherein the liver damage is liver damage caused by an exogenous compound.
In a preferred embodiment of the invention, the exogenous compound is acetaminophen.
In order to solve the above technical problems, the fourth aspect of the present invention provides a reagent for detecting the expression level of nnrna transcript level of nonoode TRANSCRIPT ID which is nononratt 018001.2, a kit according to the second aspect of the present invention, or a chip according to the third aspect of the present invention, for use in the preparation of a product for detecting liver damage.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the invention discloses a method for preparing a liver injury biomarker by applying lncRNA (long-chain ribonucleic acid) from exosome, which is particularly used for detecting liver injury caused by exogenous compounds, has high detection sensitivity and simple detection method and reagent, can be widely applied to detecting liver injury, has reliable detection result, and can be used for independently detecting or jointly evaluating the liver injury by combining other indexes.
Drawings
Fig. 1 is a graph of the changes in serum ALT, AST activity in rats in the acetaminophen dosed group, where ﹡ represents P <0.05 compared to the vehicle group.
FIG. 2 shows ROC curves of rats in the administration group and rats in the control group.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 search for biomarkers of liver injury
1.1 establishment of liver injury model
1.1.1 Experimental reagents and instruments, laboratory animals
(1) Positive drugs: acetaminophen (batch No. D2114266, available from shanghai alatin biochem technologies, ltd);
(2) vehicle control: 0.5% sodium carboxymethylcellulose (0.5% CMC-Na);
ALT, AST detection kits were produced by Japan and Wako pure chemical industries, Ltd.;
the HITACHI 7060 model full-automatic biochemical analyzer is available from Hitachi industries, Ltd.
(3) Laboratory animal
10 male SD rats, 10 female rats, SPF grade, weight 180-320 g, 6-9 weeks old, purchased from Beijing Wintonlithan laboratory animal technology Limited [ license number: SCXK (Kyoto) 2016-. Animals are marked by chips and cage plates, 5 animals are fed in a cage, the animals are raised in an SPF (specific pathogen free) animal house of Shanghai Yinuo Si biotechnology GmbH, the temperature of the animal house is controlled to be 22-26 ℃, the humidity is controlled to be 40-70%, the ventilation frequency per minute is more than or equal to 15 times, the light and dark illumination period of 12 hours/12 hours is adopted, the animals can eat freely, SPF rat maintenance feed sterilized by cobalt 60 irradiation is provided by the Kyowa feed GmbH of Beijing, and the animals can freely drink self-made deionized water through drinking bottles.
1.1.2 Experimental methods
1.1.2.1 preparation of Positive drugs
Suspension formulation of acetaminophen (APAP): each 3.75 g of acetaminophen was weighed, transferred to a glass bottle and added with an appropriate amount of 0.5% CMC-Na (W/W). Stirring with a glass rod, opening a homogenizer for operation, fully dispersing, shutting down, adding 0.5% CMC-Na to the required volume, mixing to obtain 1250mg/kg suspension, ensuring upper and lower uniformity, standing at room temperature in dark place, and standing. The preparation process needs to be protected from light.
1.1.2.2 animal trial dose setting
Using a randomized block design grouping, 20 rats were randomly assigned to 2 groups according to body weight: vehicle control group (0.5% CMC-Na) and administration group (1250 mg/kg acetaminophen), detailed in Table 1.
Table 1 experimental dose design
1.1.2.3 administration and observation
The administration routes of the solvent control group and the acetaminophen administration group are oral gavage administration, and the administration capacity is 10 mL/kg by single administration. The volume administered was calculated from the body weight measured last time.
After 24 hours after the administration, the mixed preparation of 40mg/mL sutai and 5mg/mL xylazine injection is adopted for anesthesia, a small part of the whole blood collected from the abdominal aorta is placed in a separation gel for vacuum collectionSeparation of serum in blood vessels: 3500 rpm, 4 deg.C, 5 min, sucking serum, subpackaging in EP tube, storing in-80 deg.C refrigerator, and testing liver function index. Most of the EDTA-K is placed in 2 For separating plasma in vacuum blood collection tubes: 800g, 4 ℃, 10min, sucking upper plasma, subpackaging in an EP tube, storing in a refrigerator at-80 ℃ and carrying out exosome RNA complete transcriptome sequencing.
1.1.2.4 Biochemical detection of serum
The experiment mainly detects the changes of hematology indexes ALT (alanine aminotransferase) and AST (aspartate aminotransferase) which are commonly used for evaluating the liver injury in preclinical and clinical tests. As shown in the results of FIG. 1, ALT and AST in the serum of rats in the administration group are remarkably increased compared with the vehicle control group after 24 hours of administration of 1250mg/kg acetaminophen (P is 0.05).
1.1.2.5 histopathological examination results
Histopathological examination of liver shows that the rats in the vehicle control group have no obvious abnormality, and the rats in the administration group have different degrees of hepatocyte necrosis accompanied by inflammatory cell infiltration, lobular peripheral cell vacuole degeneration and even necrosis. The detailed scores are shown in table 2.
TABLE 2 histopathological morphological observation of liver 24 h post-exposure to acetaminophen
"N" represents no significant abnormality.
In the column of "hepatocyte necrosis with inflammatory cell infiltration", "0" represents no hepatocyte necrosis with no inflammatory cell infiltration; "1" represents less hepatocyte necrosis with less inflammatory cell infiltration; "2" represents a few hepatocyte necroses with mild inflammatory cell infiltration; "3" represents moderate hepatocyte necrosis with moderate inflammatory cell infiltration; "4" represents severe hepatocyte necrosis with severe inflammatory cell infiltration.
In the column of "leaflet peripheral vacuole degeneration", "0" represents that the leaflet peripheral vacuoles are not degenerated; "1" represents a slight denaturation of the leaflet peripheral vacuoles; "2" represents slight degeneration of the leaflet peripheral vacuoles; "3" represents moderate degeneration of the leaflet peripheral vacuoles; "4" represents severe degeneration of the perilobular vacuoles.
1.1.2.6 conclusion
In the experiment, an SD rat is orally gavaged with 1250mg/kg of acetaminophen and 0.5% of sodium carboxymethylcellulose, blood is collected after 24 hours for serum biochemical detection and histopathological examination, and the acetaminophen-induced liver injury condition is examined.
In the serum biochemical test, ALT and AST of animals in the APAP group of 1250mg/kg are remarkably increased after 24 hours after the administration. Different degrees of hepatocyte necrosis can be seen mainly in the pathological histological examination, and the necrosis is accompanied by inflammatory cell infiltration, lobular peripheral cell vacuole degeneration and necrosis.
In conclusion, after the SD rat is administrated with the acetaminophen through oral gavage, the liver injury of the SD rat is successfully induced, so that an acetaminophen-induced liver injury model is established and can be used for screening and verifying liver injury biomarkers.
Exosome RNA whole transcriptome sequencing
1.2.1 exosome extraction
Exosomes were isolated from the samples using the exoRNeasy Serum/Plasma Maxi kit (Qiagen) kit, according to standard protocols provided by the manufacturer.
The method comprises the following steps:
1) taking out 500uL of plasma sample at-80 ℃, and unfreezing in water bath at 25 ℃;
2) 13000g, centrifuged at 4 ℃ for 10 minutes
3) Adding Buffer XBP according to the sample volume of 1:1, and reversing the sample volume for 5 times;
4) transferring the mixture of the sample and Buffer XBP to exoEasy spin column, and centrifuging at 500g and 4 ℃ for 1 min; discarding the bottom waste liquid;
5) adding 3.5m Buffer XWP to exoEasy spin column, and centrifuging at 5000g and 4 ℃ for 5 minutes; discarding the bottom waste liquid;
6) transferring spin columns to a new collection tube;
7) adding 200uL Buffer XE, centrifuging at 5000g and 4 ℃ for 5 minutes, and collecting bottom exosomes into a 1.5mL centrifuge tube;
8) subpackaging the exosome and storing at-80 ℃.
1.2.2 exosome-associated index identification
1.2.2.1 exosome marker WB identification
And (3) taking a certain amount of PBS (phosphate buffer solution) resuspension of the exosome, adding an equal volume of RIPA (strong) lysate for cracking, extracting protein, and then performing protein concentration determination and WB (white blood cell) detection on an exosome marker.
1.2.2.2 exosome Nanoparticles Tracking Assay (NTA)
1) Taking a frozen sample, thawing in a water bath at 25 ℃, and placing on ice;
2) exosome samples were diluted with 1 × PBS and used directly for NTA detection.
3) Information on instruments used for testing
The instrument name: nano-particle size particle tracking analyzer
Production company: PARTICLE METRIX
The instrument model is as follows: ZetaVIEW S/N17-310
Software version for analysis: ZetaView 8.04.02
1.2.3 RNA quality characterization
The initial sample of the sequencing experiment is total RNA, the exosome total RNA is obtained by a QIAGEN exoRNeasy Midi Kit (Cat number/ID: 77144), the quality inspection is carried out by Agilent 4200 TapeStation, and the RNA qualified by the quality inspection can be subjected to the subsequent whole transcriptome sequencing.
1.2.4 library construction and quality control
And (3) separating the exosomes, extracting the obtained total RNA, constructing a library by using an ultra-high sensitive trace sample strand specific kit aiming at the exosome RNA, detecting the concentration of the constructed library by using the Qubit 2.0 Fluorometer, and detecting the size of the library by using the Agilent 2100.
1.2.5 machine sequencing
The qualified library was tested and prepared for Illumina sequencing, with the sequencing strategy being PE 150. The basic principle of Sequencing is Sequencing By Synthesis (SBS): and (2) loading a flow cell with cluster on a computer, adding four kinds of fluorescence-labeled dNTPs, DNA polymerase and a joint primer into the flow cell for amplification, releasing corresponding fluorescence when each fluorescence-labeled dNTP is added when each sequencing cluster extends a complementary chain, and converting an optical signal into a sequencing peak by a sequencer through capturing a fluorescence signal and computer software so as to obtain sequence information of the to-be-detected fragment.
1.2.6 data quality control
After obtaining sequencing original Data (Raw Data), firstly, the Data needs to be filtered, the connector sequences are removed, low-quality reads are processed, then the sequencing quality is evaluated, and repeated inspection is carried out to obtain high-quality Data (Clean Data). Comparing clear Data with a reference genome, and quantitatively analyzing known mRNA and lncRNA; unaligned reads were analyzed for circular RNA using ACFS.
1.2.7 data preprocessing
The original sequencing data contains sequencing joint sequences, some reads contain bases with lower quality, the quality of the tail ends of the reads is low and other unqualified conditions which can affect the reliability of the subsequent analysis result, so the original data is preprocessed to remove the joint sequences and the low-quality reads. The data pre-processing software uses Seqtk.
The method mainly comprises the following steps:
1) removing the linker sequence contained in the reads;
2) removing bases with a mass Q of less than 20 at the 3' end, i.e., a base error rate of less than 0.01, wherein Q = -10log (error _ ratio);
3) removing reads with the length less than 25;
4) the ribosome RNA reads of the species to which they belong were removed.
And (4) after the raw data is preprocessed, obtaining clean reads used for subsequent analysis.
1.2.8 alignment analysis
The pretreated sequencing sequences were subjected to genomic mapping analysis using HISAT2 software. Typically we aligned clean no RNA reads to the genome, which is also the basis for subsequent analysis. HISAT2 adopts global and local search methods, mapping can be efficiently carried out, and spliced reads in RNA Seq sequencing data can be effectively compared, parameters are set as default parameters, a reference genome version is Rnor _6.0.95, and the result is a BAM file.
1.2.9 lncRNA quantitation and differential analysis
1.2.9.1 prediction of novel lncRNA
Comparing annotation information obtained by mapping with reference annotations (NONCODE and Ensembl databases) by applying cuffcompare in cufflinks (version: 2.1.1) to obtain a new transcript which cannot be matched with known annotation genes, extracting three transcripts of { i, u and x } for lncRNA prediction, and specifically comprising the following steps:
1) the transcription length is more than 200bp and exon > 2;
2) overlay fragment counts > 3;
3) predicted ORF < 300;
4) PholoCSF (currently limited to humans and mice), Pfam, CPC, CNCI prediction, intersection of 4 (or 3) predictions, and selection of transcripts with PholoCSF score <0& Pfam versus no significant & CPC score <0& CNCI score <0 as potential lncRNA.
5) The same sequence as known lncRNA was removed compared to known lncRNA.
1.2.9.2 lncRNA expression quantification
Expression quantification of the predicted novel lncRNA and the NONCODE database (version: NONCODE 2016; http:// www.noncode.org /), as well as the known lncRNA in the Ensembl database, was performed using Stringtie (version: 1.3.0). Wherein the ID at the beginning of MSTRG is novel lncRNA, the ID at the beginning of NON is known lncRNA in a database, and the ID at the beginning of ENS is known lncRNA in an Ensembl database.
1.2.9.3 IncRNA differential expression analysis
The different lncRNA analysis between samples was performed using edgeR. And performing volcano graph and Heatmap graph drawing display on the different lncRNA.
1.2.9.4 prediction and enrichment analysis of lncRNA target gene
The interaction relationship between lncRNA and mRNA is classified into cis (cis) and trans (trans), and the mRNA that interacts with lncRNA is called the target gene of lncRNA.
The high-throughput sequencing can obtain more different genes, and the number of the different genes is from hundreds to thousands. To better understand the biological functions that these differential genes may perform in cells or the signaling pathways that may be perturbed, we performed annotation and functional enrichment of the Gene Ontology and KEGG databases on the differential genes.
Total transcriptome sequencing results
1.3.1 overview
Mapping of statistical significance of differences in samples Using Cluster analysis (P)<0.05) and log 2 FC (FC) value>More than 2 times of variation and at least one group of count minimum values>10 lncRNA. The results show (fold change of partial lncRNA is listed in table 3): log of the administered group compared with the vehicle control group 2 FC (abs, absolute value) 2-fold or more and p<There were 24 in 0.05, with 18 in the up-regulation and 6 in the down-regulation; more than 4 times of change and p<There were 2 of 0.05, with 1 in the up-regulation and 1 in the down-regulation. The suggested that the lncRNA with differential expression is closely related to the occurrence, development and molecular regulation of drug-induced liver injury. Wherein the expression level of lncRNA (NONCODE TRANSCRIPT ID: NONRATT 018001.2) in the plasma exosomes of rats in the administration group is 159.7 times that in the vehicle control group.
TABLE 3 fold change of part of lncRNA
1.3.2 GO analysis results
Differentially expressed genes GO analysis using Fisher's exact test. And obtaining p-value by Fisher accurate test calculation, and obtaining q-value by multiple hypothesis test correction. GO entries with q-value less than 0.05 were screened as significantly enriched GO entries. GO functional annotation analysis found that target gene mRNAs of differentially expressed lncRNAs were mainly focused on biological processes: positive regulation of chromosome segregation; molecular function: ketosteroid monooxygenase activity, alditol: NADP + 1-oxidoreductase activity.
1.3.3 KEGG analysis results
The differentially expressed lncRNAs were further analyzed for predicting target gene mRNAs by KEGG. Similar to GO classification statistics, the number of differentially expressed genes on each pathway major class of KEGG was counted. KEGG analysis finds that differentially expressed lncRNAs predict target gene mRNAs mainly concentrated in an organism system: immune system, endocrine system; metabolic pathway: an overall metabolic pathway; cell pathway: and (4) conducting signals. Suggesting that differentially expressed lncRNAs may be involved in the regulation of these pathways by regulating and predicting target gene mRNAs.
Real-time quantitative PCR validation
1.4.1 reagents
Reverse transcription reagent TOYOBO ReverTra Ace qPCR RT Kit
Quantitative PCR reagent ABI Power SYBR Green PCR Master Mix
Primer synthesis: shanghai Bioengineering Co., Ltd
1.4.2 Instrument:
ABI 7500 real-time fluorescent quantitative PCR system
1.4.3 Experimental procedures
1.4.3.1 first Strand cDNA Synthesis
1) The RNA was removed from the-80 ℃ freezer, thawed at 4 ℃ and then the reaction solution was prepared in a 0.2ml PCR tube as shown in Table 4, wherein X 1 And X 2 The value of (d) is different depending on the concentration of RNA extracted from different samples, and the volume to be used is converted depending on the concentration.
TABLE 4 reaction solution System
2) The PCR tube was placed in a PCR instrument, and the program was run: incubating at 37 deg.C for 15 min, denaturing at 98 deg.C for 5 min, and holding at 4 deg.C.
1.4.3.2 SYBR Green qPCR
1) The reaction solution was prepared in a 0.2ml PCR tube as shown in Table 5, wherein X 3 The value of (A) is different according to the cDNA concentration of different samples, and the volume taken is converted according to the concentration.
TABLE 5 reaction solution System
2) The reaction solution was added to a 96-well plate
3) The 96-well plate was placed in an ABI 7500 real-time fluorescent quantitative PCR instrument. And (3) running a program: incubating at 50 deg.C for 2 min; at 95 ℃ for 10 min; 40 cycles: 95 deg.C, 15 seconds, 60 deg.C, 1 min.
ROC curve
The plotting of ROC (receiver operating characteristics curves) curve was performed using MedCalc (version 19.1.2).
As shown in fig. 2, AUC =0.750, sensitivity was 100%, and specificity was 66.67%, and it can be seen that this incrna has some significance in distinguishing rats in the administration group from rats in the control group.
1.6 specificity verification
1.6.1 Experimental reagents and instruments, laboratory animals
(1) Positive drugs: isoproterenol hydrochloride (batch: WXBD4216V, available from Shanghai Allantin Biotechnology Ltd.);
(2) vehicle control: 0.9% sodium chloride injection (batch No. 21111304B, available from Agrimonia pilosa, Inc., Anhui);
ALT, AST detection kits were produced by Japan and Wako pure chemical industries, Ltd.;
the HITACHI 7060 model full-automatic biochemical analyzer is available from Hitachi industries, Ltd.
(3) Laboratory animal
10 male SD rats, 10 female rats, SPF grade, weight 180-320 g, 6-9 weeks old, purchased from Beijing Wintonlithan laboratory animal technology Limited [ license number: SCXK (Kyoto) 2016-. Animals are marked by chips and cage plates, 5 animals are fed in a cage, the animals are raised in an SPF (specific pathogen free) animal house of Shanghai Yinuo Si biotechnology GmbH, the temperature of the animal house is controlled to be 22-26 ℃, the humidity is controlled to be 40-70%, the ventilation frequency per minute is more than or equal to 15 times, the light and dark illumination period of 12 hours/12 hours is adopted, the animals can eat freely, SPF rat maintenance feed sterilized by cobalt 60 irradiation is provided by the Kyowa feed GmbH of Beijing, and the animals can freely drink self-made deionized water through drinking bottles.
1.6.2 Experimental methods
1.6.2.1 preparation of Positive drugs
2.5mg/kg (0.25mg/mL) of isoproterenol hydrochloride solution, a positive dosing formulation was prepared in a volume of 60 mL: weighing 15mg of isoproterenol hydrochloride; transfer to a beaker and add appropriate amount of 0.9% sodium chloride injection. Initially stirring with a glass rod, adding a rotor, turning on a magnetic stirrer, fully dispersing, then shutting down, adding 0.9% sodium chloride injection to a required volume (60 mL), uniformly mixing to ensure the upper and lower uniformity, and standing at room temperature in a dark place for later use. The preparation process needs to be protected from light and aseptic operation is needed.
1.6.2.2 animal trial dose setting
Using a randomized block design grouping, 20 rats were randomly assigned to 2 groups according to body weight: vehicle control group (0.9% sodium chloride injection) and administration group (2.5 mg/kg isoproterenol hydrochloride), as detailed in table 6.
Table 6 experimental dose design
1.6.2.3 administration and visual inspection
The administration routes of the solvent control group and the isoproterenol hydrochloride administration group are tail vein injection, and the administration volume is 10 mL/kg by single administration. The volume administered was calculated from the body weight measured last time.
4 hours after administration, anesthesia is carried out by adopting 40mg/mL sutai +5mg/mL xylazine injection mixed preparation, a small part of abdominal aorta collected whole blood is placed in a separation gel vacuum blood collection tube for serum separation: 3500 rpm, 4 deg.C, 5 min, sucking serum, subpackaging in EP tube, storing in-80 deg.C refrigerator, and testing liver function index. Most were placed in EDTA-K2 vacuum blood collection tubes for plasma separation: 800g, 4 ℃, 10min, upper plasma was aspirated and aliquoted into EP tubes and stored in a-80 ℃ freezer for subsequent PCR validation.
1.6.2.4 serum biochemical test result
The experiment mainly detects the changes of hematology indexes ALT (alanine aminotransferase) and AST (aspartate aminotransferase) which are commonly used for evaluating the liver injury in preclinical and clinical tests. After 2.5mg/kg of isoproterenol hydrochloride is administered for 4 hours, ALT and AST in the serum of rats in the administration group are slightly increased compared with those in the solvent control group, but no significant difference exists.
1.6.2.5 PCR validation
The specific process is the same as 1.4.
Summary of the invention
The sequencing result of lncRNA of the experimental rat plasma exosomes shows that the number of lncRNA which is differentially expressed in the exosomes is large, wherein the expression level of lncRNA (NONCODE TRANSCRIPT ID: NONRATT 018001.2) in the rat plasma exosomes of an administration group is 159.7 times that of a vehicle control group, and the expression difference is very high, so that the lncRNA (NONCODE TRANSCRIPT ID: NONRATT 018001.2) can be used as a liver injury biomarker for detecting the liver injury caused by an exogenous compound.
And (3) carrying out verification by using real-time fluorescent quantitative PCR, and displaying a verification result: lncRNA (NONCODE TRANSCRIPT ID: NONRATT 018001.2) was expressed in the plasma exosomes of rats in the administered group 2.72 times as much as in the vehicle control group (p < 0.05). This demonstrates that when norlatt 018001.2 is used as the biomarker, the detection can be performed in time, and the detection result is reliable, which indicates that when norlatt 018001.2 is used as the biomarker, the detection of the liver injury caused by the exogenous compound has good specificity.
Using the ROC curve, it was examined whether lncRNA (NONCODE TRANSCRIPT ID: NONRATT 018001.2) could discriminate the rats of the administration group from the rats of the control group. The area under the curve, AUC =0.750, sensitivity 100%, specificity 66.67%, shows that the incrna has certain discrimination ability.
The specificity of lncRNA (NONCODE TRANSCRIPT ID: NONRATT 018001.2) was examined by establishing a rat myocardial injury model using isoproterenol hydrochloride. The PCR validation result showed that the lncRNA was not elevated in the myocardial injury model, and thus, it was shown that it has good specificity.
Claims (13)
1. The application of plasma-derived exosome lncRNA in preparing a drug-induced liver injury biomarker is characterized in that the lncRNA comprises lncRNA of which NONCODE TRANSCRIPT ID is NONRATT 018001.2.
2. The use of claim 1, wherein the lncRNA is used alone as the sole biomarker for detecting liver injury or the lncRNA is used in combination with a conventional biomarker for detecting liver injury.
3. The use of claim 2, wherein the conventional biomarker for detecting liver injury is selected from the group consisting of alanine aminotransferase and aspartate aminotransferase.
4. The use of claim 2, wherein said detection is the detection of the level of transcription level expression of said incrna in the sample.
5. The use of claim 4, wherein the detection is RNA whole transcriptome sequencing; and/or, the sample is plasma.
6. The use of any one of claims 1 to 5, wherein the liver injury is caused by an exogenous compound, and the exogenous compound is acetaminophen.
7. A kit for detecting liver damage, comprising reagents for detecting lncRNA; the lncRNA comprises lncRNA of which NONCODE TRANSCRIPT ID is NONRATT 018001.2.
8. The kit of claim 7, wherein the reagents are reagents for detecting the level of transcriptional level expression of the incrna in the sample.
9. The kit of claim 7, wherein the liver injury is caused by an exogenous compound, and wherein the exogenous compound is acetaminophen.
10. A chip for detecting liver injury is characterized in that a reagent for detecting lncRNA is arranged on the chip; the lncRNA comprises lncRNA of which NONCODE TRANSCRIPT ID is NONRATT 018001.2.
11. The chip of claim 10, wherein said reagents are reagents for detecting the level of transcription level expression of said incrna in said sample.
12. The chip of claim 10, wherein the liver damage is liver damage caused by an exogenous compound, and the exogenous compound is acetaminophen.
13. Use of a reagent for detecting the lncRNA transcript level expression level of nonoODE TRANSCRIPT ID NONRATT018001.2, a kit according to any one of claims 7 to 9 or a chip according to any one of claims 10 to 12 for the preparation of a product for detecting liver damage.
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