CN115261462A - Application of exosome lncRNA in preparation of liver injury biomarker - Google Patents
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Abstract
The invention discloses application of exosome lncRNA in preparation of a liver injury biomarker. The lncRNA comprises lncRNA with NONCODE TRANSCRIPT ID NONRATT 004188.2. The lncRNA disclosed by the invention can be applied to preparation of liver injury biomarkers, especially used for detecting liver injury caused by exogenous compounds, has high detection specificity and simple detection method and reagent, can be widely applied to detection of drug-induced liver injury, has reliable detection results, and can be used for singly 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 an exosome lncRNA in preparation of a 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 an organism is exposed to an exogenous toxicant, the liver reacts more often to toxicity 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 Injury (DILI), also known as Drug Induced Liver Injury, refers to Liver Injury 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, and 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 damage, and although the reported incidence rate of DILI is in the range of 1/10000 to 1/100000, the actual incidence rate of DILI is much higher than the above range due to misdiagnosis or untimely reporting 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 pharmaceutical enterprises to comprehensively and deeply evaluate DILI risks in the process of developing 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 bile duct epithelial cells, and elevation of TBIL may reflect impairment of 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. These 2 biomarkers have all been supported by the U.S. food and drug administration and the european drug administration as liver-specific candidate biomarkers.
Exosomes are one of 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 is finally fused to the plasma membrane via MVB, secreted in exosomes with diameters of 40-160 nm via exocytosis. The density of exosomes was 1.15-1.19g/mL. The tetrameric transmembrane proteins CD63, CD9 and CD81 found on their surface are commonly used 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 a protein 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: regulate 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 to date), and lncRNA which is closely related to liver damage, especially liver damage 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 specificity, too complex in detection method, expensive in reagent, and incapable of jointly evaluating liver injury by combining with other indexes, predicting drug-induced liver injury timely and accurately and the like, and provides application of long-chain non-coding RNA (lncRNA) from exosome in preparation of the liver injury biomarker, particularly the drug-induced liver injury biomarker. The application prepares the lncRNA as a liver injury biomarker, particularly the liver injury caused by an exogenous compound, has high detection specificity and simple detection method and reagent, can be widely applied to timely detection of the drug-induced liver injury, can timely find the drug-induced liver injury in the 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 conducts long-term research on liver injury, and through a large number of experiments, unexpectedly discovers that some lncRNA derived from exosome can be used as a specific biomarker of drug-induced liver injury caused by exogenous compounds, the lncRNA is highly expressed in plasma of drug-induced liver injury caused by the exogenous compounds, and can exert the application of the lncRNA in preparation of the biomarker of the liver injury, so that the liver injury caused by the exogenous compounds can be detected.
The invention solves the technical problems through the following technical scheme.
The invention provides an application of exosome lncRNA in preparing a product for detecting liver injury, wherein the lncRNA comprises lncRNA with NONCODE TRANSCRIPT ID NONRATT 004188.2.
In some embodiments of the invention, the lncRNA is used as the sole biomarker for detecting liver damage, or as a first biomarker for detecting liver damage to be used in combination with a second biomarker.
In the present invention, the second biomarker is selected from the group consisting of lncRNA with NONRODE TRANSCRIPT ID NONRATT018001.2, alanine aminotransferase and aspartate aminotransferase.
In some embodiments of the invention, the detecting is detecting the mRNA level of the incrna in the sample.
In the invention, the mRNA level detection can be RNA whole transcriptome sequencing, real-time fluorescence quantitative PCR detection or expression profiling chip.
In some embodiments of the invention, the sample is plasma.
In the invention, the liver injury is drug-induced liver injury caused by an exogenous compound.
In some embodiments of the invention, the exogenous compound is acetaminophen (APAP).
In some embodiments of the invention, the product is a reagent.
In a second aspect, the invention provides a combination of biomarkers of liver injury, the combination comprising a first biomarker and a second biomarker; the first biomarker is lncRNA with nonocode TRANSCRIPT ID no nratt 004188.2.
In some embodiments of the invention, the second biomarker is as described in the first aspect.
In a third aspect, the invention provides a use of a reagent for detecting an expression level of a biomarker of liver injury in the preparation of a product for diagnosing liver injury; the biomarker is lncRNA with a nonoode trans ID of nonirtatt 004188.2 or a combination as described in the second aspect.
In some embodiments of the invention, the liver injury is a drug-induced liver injury.
In some embodiments of the invention, the liver injury is a drug induced liver injury caused by acetaminophen.
The fourth aspect of the invention provides a kit for detecting liver damage, which comprises a reagent for detecting the expression level of lncRNA; the lncRNA is a NON TRANSCRIPT ID NONRATT004188.2 lncRNA or a combination as described in the second aspect.
In some embodiments of the invention, the agent is an agent that detects the mRNA level of the incrna in the sample.
In some embodiments of the invention, the liver injury is as described in the third aspect.
In some embodiments of the invention, the kit further comprises an RNA extraction reagent.
The fifth aspect of the invention provides a chip for detecting liver injury, wherein a reagent for detecting the expression level of lncRNA is arranged on the chip; the lncRNA is a NON TRANSCRIPT ID NONRATT004188.2 lncRNA or a combination as described in the second aspect.
In some embodiments of the invention, the agent is as described in the fourth aspect; the liver injury is as described in the third aspect.
A sixth aspect of the present invention provides a system for assessing risk of liver injury, the system comprising a detection module and a determination module; the detection module is used for detecting the expression level of the biomarker in the sample to be detected and inputting the detection result into the judgment module; the judging module judges according to the judging condition and outputs the judging result; the biomarker is a NONCODE transit lrap with nnrna ID no rtatt 004188.2 or a combination as described in the second aspect.
In some embodiments of the invention, the expression level is mRNA level.
In some embodiments of the present invention, the determination condition is whether the expression level is higher than a predetermined threshold.
The seventh aspect of the present invention provides a use of the kit according to the fourth aspect, the chip according to the fifth aspect, or the system according to the sixth aspect for preparing 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 using lncRNA (long non-ribonucleic acid) from exosomes, 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 used for detecting liver injury, has reliable detection result, and can be used for singly detecting or jointly evaluating the liver injury by combining other indexes.
Drawings
FIG. 1 is a graph showing the change of ALT and AST activities in rat serum in the acetaminophen administration group, wherein 65121represents P <0.05 compared with the vehicle group.
FIG. 2 is a graph showing 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 aladine biochem technologies, ltd);
(2) Vehicle control: 0.5% (w/w) sodium carboxymethylcellulose (0.5% CMC-Na);
ALT and 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, body weight 180-320g, 6-9 weeks old, purchased from beijing vintongli laboratory animal technology limited [ license number: SCXK (Jing) 2016-0006]. Animals are marked by chips and cages, 5 animals are per cage, the animals are raised in an SPF 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 Beijing Cork Australian cooperative feed GmbH, 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 3.75g of acetaminophen was weighed, transferred to a glass bottle, and an appropriate amount of 0.5% CMC-Na (w/w) was added thereto. Stirring with glass rod, opening homogenizer, fully dispersing, shutting down, adding 0.5% CMC-Na to required volume, mixing, and making into 1250mg/kg suspension, ensuring upper and lower uniformity, standing at room temperature in dark place for use. The preparation process needs to be protected from light.
1.1.2.2 animal test dose settings
Using a randomized block design grouping, 20 rats were randomly assigned to 2 groups according to body weight: the vehicle control group (0.5% CMC-Na) and the administration group (1250 mg/kg acetaminophen) are shown in Table 1.
Table 1 experimental dose design
In the table, F is female and M is male.
1.1.2.3 administration and visual inspection
The administration routes of the solvent control group and the acetaminophen administration group are oral gavage administration, and the administration capacity is 10mL/kg by single administration. The volume administered was calculated from the body weight measured last time.
After 24 hours after administration, anesthesia is carried out by adopting 40mg/mL sutai +5mg/mL xylazine injection mixed preparation, a small part of whole blood collected from abdominal aorta is placed in a separation gel vacuum blood collection tube for serum separation: 3500rpm,4 deg.C, 5min, sucking serum, subpackaging in EP tube, and storing in-80 deg.C refrigerator for liver function index detection. Most of the EDTA-K is placed in 2 For separating plasma in vacuum blood collection tubes: 800g,4 ℃,10min, sucking the upper plasma layer, subpackaging in an EP tube, storing in a refrigerator at-80 ℃ for exosome RNA complete transcriptome sequencing.
1.1.2.4 serum Biochemical assay results
The experiment mainly detects the changes of hematology indexes ALT (alanine aminotransferase) and AST (aspartate aminotransferase) which are commonly used in preclinical and clinical liver injury evaluation. As shown in the result of figure 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 of acetaminophen (P: 0.05).
1.1.2.5 histopathological examination results
Histopathological examination of liver shows that rats in the vehicle control group have no obvious abnormality, and liver cells necrosis with different degrees, inflammatory cell infiltration, vacuole degeneration of cells around lobules and even necrosis are observed in the administration group. The detailed scores are shown in table 2.
TABLE 2 histopathological morphological observation of liver 24h 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 hepatocellular necrosis 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 inspected.
In the serum biochemical test, ALT and AST of animals in the 1250mg/kg APAP group 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.
1.2 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 500 mu L of plasma sample at-80 ℃, and unfreezing in water bath at 25 ℃;
2) 13000g, centrifugation at 4 ℃ for 10 minutes;
3) Buffer XBP was added according to the sample volume of 1, and inverted 5 times upside down;
4) Transferring the mixture of the sample and Buffer XBP to exo easy spin column, and centrifuging at 500g and 4 ℃ for 1min; discarding the bottom waste liquid;
5) 3.5mL of Buffer XWP was added to exoEasy spin column and centrifuged at 5000g for 5 minutes at 4 ℃; discarding the bottom waste liquid;
6) Transferring spin columns to a new collection tube;
7) Adding 200. Mu.L Buffer XE, centrifuging at 5000g and 4 ℃ for 5 minutes, and collecting bottom exosome 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 of 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
1.2.3RNA quality identification
The initial sample of the sequencing experiment is total RNA, the exosome total RNA is obtained by a QIAGEN exoRNeasy Midi Kit (Cat No./ID: 77144), the quality inspection is carried out by Agilent 4200TapeStation, 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
Separating exosome and extracting the obtained total RNA, establishing a library by using an ultra-high sensitive micro sample strand specific kit aiming at exosome RNA, and using the constructed library
2.0Fluorometer assay concentration, agilent2100 assay library size.
1.2.5 machine sequencing
The qualified library was tested and prepared for Illumina sequencing, with the sequencing strategy being PE150. The basic principle of Sequencing is Sequencing By Synthesis (SBS): and (2) loading a flow cell with a 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 every time one fluorescence-labeled dNTP is added when each sequencing cluster extends a complementary chain, and converting an optical signal into a sequencing peak through a sequencer by capturing a fluorescence signal and computer software so as to obtain sequence information of the fragment to be detected.
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 (clear 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 the mass Q of the 3' end lower than 20, namely the error rate of the bases is 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 a global and local search method, can efficiently map, can effectively compare spliced reads in RNA Seq sequencing data, sets default parameters for the parameters, and has a reference genome version of Rnor-6.0.95 and a BAM file result.
1.2.9 lncRNA quantitation and differential analysis
1.2.9.1 prediction of New 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 human and mouse), pfam, CPC and CNCI predictions, intersection sets are taken for 4 (or 3) predictions, and transcripts of PholoCSF score <0 &Pfamvs. insignificant & CPC score <0 &CNCIscore 0 are selected 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 predicted novel lncRNA and NONCODE databases (version: NONCODE 2016, http:// www. NONCode. Org /), as well as 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 lncRNA differential expression analysis
Differential 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 such mRNA interacting with lncRNA is called a 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.
1.3 Whole 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 004188.2) in the plasma exosome of the rat in the administration group is 6.25 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 performing Fisher accurate test calculation to obtain p-value, and performing multiple hypothesis test correction to obtain q-value. 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
KEGG analysis was further performed on differentially expressed lncRNAs predicted target gene mRNAs. Similar to GO classification statistics, the number of differentially expressed genes on each pathway major class of KEGG was counted. KEGG analysis found that differentially expressed lncRNA predicted target gene mRNA to be mainly concentrated in the body system: immune system, endocrine system; metabolic pathway: an overall metabolic pathway; cell pathway: and (6) conducting signals. Suggesting that differentially expressed lncRNA may be involved in the regulation of these pathways by regulating the predicted target gene mRNA.
1.4 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:
quantitative PCR instrument: ABI 7500 real-time fluorescence quantitative PCR system
1.4.3 Experimental procedures
1.4.3.1 First Strand cDNA Synthesis
1) The RNA was taken out from a-80 ℃ refrigerator, thawed at 4 ℃ and then prepared into a reaction solution 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
| Total RNA | 1μg |
| 5×RT Buffer | 2μL |
| RT Enzyme Mix | 0.5μL |
| Primer Mix | 0.5μL |
| H 2 O | X 1 μL |
| RNA template | X 2 μL |
| Total volume | 20μL |
2) Place the PCR tube in the PCR instrument, run the program: incubation at 37 ℃ for 15min, denaturation at 98 ℃ for 5min, and heat preservation at 4 ℃.
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 (b) 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×SYBR Green PCR buffer | 10μL |
| Forward primer (10. Mu.M) | 0.5μL |
| Reverse primer (10. Mu.M) | 0.5μL |
| Form panel | 10ng |
| ddH 2 O | X 3 μL |
| Total volume | 20μL |
2) The reaction 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 ℃ for 2min; at 95 ℃ for 10min;40 cycles: 95 deg.C, 15 seconds, 60 deg.C, 1min.
1.5 ROC curve
An ROC (Receiver operating characteristics curves) curve was plotted using a spread (version 24.0).
As a result, as shown in fig. 2, AUC =0.806, sensitivity was 83%, and specificity was 67%, and it was found that this incrna had 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.);
ALT, AST detection kit was 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, body weight 180-320g, 6-9 weeks old, purchased from beijing vintongli laboratory animal technology limited [ license number: SCXK (Jing) 2016-0006]. Animals are marked by chips and cages, 5 animals are per cage, the animals are raised in an SPF 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 Beijing Cork Australian cooperative feed GmbH, 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 (0.25 mg/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 test dose settings
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), detailed in table 6.
Table 6 experimental dose design
In the table, F is female and M is male.
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 10mL/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: 3500rpm,4 deg.C, 5min, sucking serum, subpackaging in EP tube, and storing in-80 deg.C refrigerator for liver function index detection. Most were placed in EDTA-K2 vacuum blood collection tubes for separation of plasma: 800g,4 deg.C, 10min, aspirate supernatant plasma into EP tubes and store in-80 deg.C refrigerator for subsequent PCR validation.
1.6.2.4 serum Biochemical assay results
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. In addition, the CTn1 level in the serum of rats in the administration group was significantly increased (p < 0.05) compared to the vehicle control group, which suggests that the modeling of the myocardial injury model was successful.
1.6.2.5 PCR validation
The specific process is the same as 1.4, and the conclusion is shown below.
1.7 summary
The sequencing result of lncRNA of the experimental rat plasma exosome shows that the number of lncRNA which is differentially expressed in the exosome is large, wherein the expression level of lncRNA (NONCODE TRANSCRIPT ID: NONRAT T004188.2) in the rat plasma exosome of an administration group is 6.25 times that in a solvent control group, and the expression differential multiple is very high, which indicates that lncRNA (NONCODE TRANSCRIPT ID: NON RATT 004188.2) can be used as a liver injury biomarker for detecting liver injury caused by an exogenous compound.
And (3) performing verification by using real-time fluorescent quantitative PCR, wherein the verification result shows that: lncRNA (NONCOD E TRANSCRIPT ID: NONRATT 004188.2) was expressed in 2.07 times of the amount in the rat plasma exosomes of the administration group compared with that of the vehicle control group (p < 0.05). This proves that when NONRATT004188.2 is used as the biomarker, the detection can be carried out in time, the detection result is reliable, and the detection of the liver injury caused by the exogenous compound by using NONRATT004188.2 as the biomarker has good specificity.
Using ROC curve, it was examined whether lncRNA (NONCODE TRANSCRIPT ID: NONRATT 004188.2) could discriminate the rats in the administration group from the rats in the control group. Area under the curve AUC =0.806, sensitivity was 83%, specificity was 67%, showing that the incrna has a certain discrimination ability.
The specificity of lncRNA (NON CODE TRANSCRIPT ID: NONRATT 004188.2) 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 (10)
1. An application of an exosome lncRNA in preparing a reagent or a product for detecting liver injury, wherein the lncRNA comprises lncRNA with NONCODE TRANSCRIPT ID NONRATT 004188.2.
2. The use according to claim 1, wherein the lncrnas are used as the sole biomarker for detecting liver damage or as a first biomarker for detecting liver damage in combination with a second biomarker;
preferably, the second biomarker is selected from the group consisting of lncRNA with a nonode TRANSCRIPT ID of nononratt 018001.2, alanine aminotransferase and aspartate aminotransferase.
3. The use of claim 2, wherein the detection is detecting the mRNA level of the incrna in the sample;
preferably, the sample is plasma.
4. The use of any one of claims 1 to 3, wherein the liver injury is a drug induced liver injury caused by an exogenous compound;
preferably, the exogenous compound is acetaminophen.
5. A combination of biomarkers of liver injury, wherein the combination comprises a first biomarker and a second biomarker; the first biomarker is lncRNA with NONCODE TRANSCRIPT ID NONRATT 004188.2;
preferably, the second biomarker is selected from the group consisting of lncRNA having a nonoode TRANSCRIPT ID of nononratt 018001.2, alanine aminotransferase and aspartate aminotransferase.
6. Use of a reagent for detecting the expression level of a biomarker of liver injury in the manufacture of a product for diagnosing liver injury; the biomarker is a IncRNA with NONRODE TRANSCRIPT ID NONRATT004188.2 or a combination according to claim 5;
preferably, the liver injury is a drug-induced liver injury;
more preferably, the liver injury is acetaminophen-induced drug induced liver injury.
7. A kit for detecting liver damage, comprising reagents for detecting the expression level of lncRNA; the lncRNA is a NONCODE TRANSCRIPT ID NONRATT004188.2 or the combination of claim 5;
preferably, the reagent is a reagent for detecting the mRNA level of the lncRNA in the sample; and/or, the liver injury is a drug-induced liver injury;
more preferably, the liver injury is acetaminophen-induced drug induced liver injury.
8. A chip for detecting liver injury is characterized in that a reagent for detecting the expression level of lncRNA is arranged on the chip; the lncRNA is a NONCODE TRANSCRIPT ID NONRATT004188.2 or the combination of claim 5;
preferably, the reagent is a reagent for detecting the mRNA level of the lncRNA in the sample; and/or, the liver injury is a drug-induced liver injury;
more preferably, the liver injury is acetaminophen-induced drug induced liver injury.
9. A system for assessing the risk of liver injury, comprising a detection module and a judgment module; the detection module is used for detecting the expression level of the biomarker in the sample to be detected and inputting the detection result into the judgment module; the judgment module judges according to the judgment condition and outputs the judgment result; the biomarker is a IncRNA with NONRODE TRANSCRIPT ID NONRATT004188.2 or a combination according to claim 5;
preferably, the expression level is mRNA level; and/or, the judgment condition is whether the expression level is higher than a preset threshold value.
10. Use of a kit according to claim 7, a chip according to claim 8 or a system according to claim 9 for the preparation of a product for the detection of liver damage.
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