CN112852942A - Application of long-chain non-coding RNA in preparation of liver injury biomarker - Google Patents
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Abstract
The invention provides a kit for detecting liver injury, which comprises a reagent for detecting lncRNA; the lncRNA comprises lncRNA of which NONCODE TRANSCRIPT ID is NONRATT 022466. The invention also provides application of the kit in preparation of a preparation for detecting liver injury and application of lncRNA in preparation of a liver injury biomarker. The application can apply the lncRNA to preparation of liver injury biomarkers, especially to detection of liver injury caused by exogenous compounds, has high detection sensitivity 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 single detection or joint evaluation of liver injury by combining other indexes.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of long-chain non-coding RNA 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.
Currently, the conventional liver injury evaluation method, non-clinical drug safety evaluation and the liver injury indexes commonly used in clinic are alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and Total Bilirubin (TBIL), and then are combined with histopathological examination. ALT, AST and TBIL may vary in severity from asymptomatic serum biochemical markers to fulminant liver failure and lead to life-threatening complications of multiple organ injury. ALT is a liver injury detection index widely used at present, and is mainly distributed in liver tissues, and is also distributed in a small amount in kidneys, hearts and skeletal muscles. Because of its low false negative or false positive rate in the case of liver tissue damage, it is considered the current gold standard for liver damage detection, however, ALT activity often increases after liver damage occurs and is not always consistent with the histopathological results of preclinical testing. Especially, ALT cannot predict drug-induced liver injury accurately in time under the condition that clinical histopathological examination is not easy to implement. AST is ubiquitous in heart, brain, skeletal muscle and liver tissues and has poor specificity compared with ALT. And serum ALT and AST may also be significantly elevated in extrahepatic injuries such as muscle injuries. Liver damage cannot be effectively distinguished from extrahepatic damage. TBIL is a product of heme catabolism, and only the liver can clear bilirubin in blood, so that the TBIL can reflect the overall function of the liver more directly than ALT and AST. However, hemolysis and other hematologic diseases also cause an increase in TBIL, and therefore TBIL requires a joint evaluation of liver damage in combination with other indicators.
With the progressive research on liver injury, some new detection indexes of hepatotoxic injury, such as glutamate dehydrogenase (GLDH), Arginase (argnase, ARG), glutathione-S-transferase alpha (α GST), serum F Protein (F-Protein), etc., are discovered, and reported to have higher sensitivity and specificity than ALT and AST. The use of these new biomarkers will improve the ability of traditional indicators to monitor drug-induced liver damage. However, due to the complexity of the detection technology and the expensive reagents, the method has not been widely applied to the clinical evaluation of liver injury and is still in the research stage.
Long non-coding RNAs (lncrnas) are a class of open reading frame-lacking non-coding RNA molecules that are greater than 200 ribonucleotides in length. 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.
Disclosure of Invention
The invention aims to overcome the defects that the 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, accurately predicting drug-induced liver injury in time and the like, and provides the application of long-chain non-coding RNA (lncRNA) in preparing 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 conducts long-term research on liver injury, and through a large number of experiments, unexpectedly discovers that some lncRNA can be used as a specific biomarker of liver injury caused by exogenous compounds, is highly expressed in liver tissues of the liver injury caused by the exogenous compounds, and can exert the application of the lncRNA in preparing the biomarker of the liver injury, thereby detecting the liver injury caused by the exogenous compounds.
In order to solve the above technical problems, the present invention provides in a first aspect a kit for detecting (diagnosing) liver damage, comprising reagents for detecting (the level of) lncRNA; the lncRNA comprises lncRNA of which NONCODE TRANSCRIPT ID is NONRATT 022466.
Preferably, the liver injury is caused by exogenous compounds; more preferably, the exogenous compound is acetaminophen.
Preferably, the incRNA further comprises an incRNA with NONCODE TRANSCRIPT ID being NONRATT007487 and/or an incRNA with NONCODE TRANSCRIPT ID being NONRATT 009739.
Preferably, the detection is a real-time fluorescent quantitative PCR detection.
Preferably, the kit further comprises an RNA extraction reagent, a reverse transcription reagent and/or a real-time fluorescent quantitative PCR reagent.
Preferably, the reagents may be those conventional in the art, such as primers and the like.
In order to solve the above technical problems, the second aspect of the present invention provides a use of the kit according to the first aspect of the present invention in the preparation of a preparation (e.g., a diagnostic agent) for detecting (diagnosing) liver damage.
Preferably, the liver injury is caused by an exogenous compound.
More preferably, the exogenous compound is acetaminophen.
In order to solve the above technical problem, the third aspect of the present invention provides a biomarker combination comprising lncRNA whose none cluster TRANSCRIPT ID is none cluster 022466, lncRNA whose none cluster TRANSCRIPT ID is none cluster 007487 and/or lncRNA whose none cluster TRANSCRIPT ID is none cluster 009739.
In order to solve the technical problem, the fourth aspect of the present invention provides a use of lncRNA in preparing a liver injury biomarker, wherein the lncRNA comprises lncRNA of which nonoODE TRANSCRIPT ID is NONRATT 022466.
Preferably, the incRNA further comprises an incRNA with NONCODE TRANSCRIPT ID being NONRATT007487 and/or an incRNA with NONCODE TRANSCRIPT ID being NONRATT 009739.
Preferably, the liver injury is caused by exogenous compounds. The lncRNA serving as a liver injury biomarker can be used for detecting the liver injury caused by exogenous compounds.
More preferably, the exogenous compound is acetaminophen (APAP).
Preferably, the lncRNA is used alone as the sole biomarker for liver injury.
Preferably, the lncRNA is used in combination with a conventional liver injury biomarker.
More preferably, the conventional liver injury biomarker is alanine aminotransferase, aspartate aminotransferase and/or total bilirubin.
The invention also provides the application of the reagent for measuring the level of the biomarker IncRNA in the preparation of the diagnostic agent for diagnosing liver injury; wherein the nonoODE TRANSCRIPT ID of the lncRNA is NONRATT 022466.
Preferably, the incRNA further comprises an incRNA with NONCODE TRANSCRIPT ID being NONRATT007487 and/or an incRNA with NONCODE TRANSCRIPT ID being NONRATT 009739.
Preferably, the liver injury is caused by exogenous compounds; more preferably, the exogenous compound is acetaminophen.
Preferably, the assay is a real-time fluorescent quantitative PCR assay.
Preferably, the reagents may be those conventional in the art, such as primers and the like.
The invention also provides application of the primer for detecting the lncRNA in preparing a kit for diagnosing liver injury; wherein the nonoODE TRANSCRIPT ID of the lncRNA is NONRATT 022466.
Preferably, the incRNA further comprises an incRNA with NONCODE TRANSCRIPT ID being NONRATT007487 and/or an incRNA with NONCODE TRANSCRIPT ID being NONRATT 009739.
Preferably, the liver injury is caused by exogenous compounds; more preferably, the exogenous compound is acetaminophen.
Preferably, the detection is a real-time fluorescent quantitative PCR detection.
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 application of the invention can apply lncRNA to the preparation of the liver injury biomarker, especially to the detection of liver injury caused by exogenous compounds, has high detection sensitivity and simple detection method and reagent, can be widely applied to the detection of liver injury, has reliable detection result, and can be used for single detection or joint evaluation of liver injury by combining other indexes.
Drawings
Fig. 1 is a graph of the changes in serum ALT, AST, ALP and TBIL activity in rats in the acetaminophen dosed group, where ﹡ represents P <0.05 compared to the vehicle 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. P1395820, available from Chemicals, Inc., national pharmaceutical group);
(2) vehicle control: 0.5% sodium carboxymethylcellulose (0.5% CMC-Na);
ALT, AST, ALP, TBIL 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
20 male SD rats, SPF grade, weight 180-200 g, 7-9 weeks old, purchased from Beijing Wintonlithan laboratory animal technology Co., Ltd [ license number: SCXK (Kyoto) 2016-. Animals are marked by ear tags and cage plates, 5 animals are kept in a cage, the animals are raised in an SPF (specific pathogen free) animal room of Shanghai Yinuo Si biotechnology limited (national Shanghai New drug safety evaluation research center), the temperature of the animal room 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 be freely fed, SPF (specific pathogen free) large and small mouse maintaining feed sterilized by cobalt 60 irradiation is provided by Beijing Australian cooperative feed limited, 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 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 group (0.5% CMC-Na) and administration group (1250mg/kg acetaminophen), detailed in Table 1.
Table 1 experimental dose design
1.1.2.3 administration and observation
The administration routes of the negative control group and the acetaminophen administration group are oral gavage administration, and the administration capacity is 10mg/kg by single administration. The volume administered was calculated from the body weight measured last time.
After 24 hours post-dose, anesthesia with 3% sodium pentobarbital, whole blood was collected from the abdominal aorta and placed in a separator vacuum blood collection tube for serum separation: 3500rpm, 4 deg.C, 5min, sucking serum, subpackaging in EP tube, storing in-80 deg.C refrigerator, and testing liver function index. Collecting liver tissue, dividing into two parts, collecting left and right outer leaves of liver in the first part, and observing liver histopathology and morphology (method is shown as 2.2.3); the second part is to pick the residual liver tissue and to split it into EP tubes, each tube is about 30mg, the sample is stored in a refrigerator at-80 deg.C for lncRNA chip detection.
1.1.2.4 Biochemical detection of serum
The experiment mainly detects the changes of hematology indexes ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase) and TBIL (total bilirubin) which are commonly used for the evaluation of liver injury in preclinical and clinical tests. As shown in the results of figure 1, ALT and AST in the serum of rats in the administration group are remarkably increased compared with the vehicle group 24h after the acetaminophen of 1250mg/kg is administered (P < 0.05).
1.1.2.5 histopathological examination results
Histopathological examination of liver shows that the rats in the vehicle group have no obvious abnormality, and the rats in the administration group have different degrees of hepatocyte necrosis with 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 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, SD rats are orally gavaged with 1250mg/kg of acetaminophen and 0.5% of sodium carboxymethylcellulose, and blood is collected after 24 hours to carry out serum biochemical detection and histopathological examination, and the condition of acetaminophen-induced liver injury is adopted.
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.
1.2 preparation of IncRNA expression profiling chip
1.2.1 sampling
The same species, normal rats with similar age and consistent size and 4 rats with established liver injury models are taken respectively. After the rat is dissected, the liver is immediately weighed, the liver tissue with the size of about 0.5 multiplied by 0.5cm is immediately placed into a freezing tube without ribozyme after weighing, and then is quickly frozen for 1 hour by liquid nitrogen, and then is transferred into an ultra-low temperature refrigerator with the temperature of minus 80 ℃ for storage and standby.
1.2.2 extraction of Total RNA
Total RNA extraction of the samples was performed using TAKARA RNAISo Plus #9109 and following standard procedures provided by the manufacturer, and the total RNA extracted was purified using NucleoSpin RNA Clean-up XS kit (Cat #740903, MN, Germany) and RNase-Free DNase Set (Cat #79254, QIAGEN, GmBH, Germany) after passing the quality control of the electrophoresis by Agilent Bioanalyzer 2100(Agilent technologies, Santa Clara, CA, US).
1.2.3 RNA quality characterization
The initial sample of the chip experiment is total RNA, the total RNA is subjected to quality inspection by a NanoDrop ND-2000 spectrophotometer and an Agilent Bioanalyzer 2100(Agilent technologies, Santa Clara, Calif., US), and the qualified RNA can be subjected to subsequent chip experiments.
1.2.4 amplification and labeling of RNA
Experimental sample RNA Total RNA was amplified and labeled using an Agilent expression profiling chip Kit, Low Input Quick Amp Labeling Kit, One-Color (Cat. # 5190-.
The operation steps are as follows:
1.2.4.1 preparation of single-labeled spike-in: spike-in was diluted with Dilution Buffer (an exogenous reference, with different concentrations to monitor the course of the experiment, e.g., to monitor signal values at different concentrations, etc.) according to different starting amounts of RNA, as detailed in Table 3 below.
TABLE 3
In the above table, "/" indicates absence or no detection.
1.2.4.2 reverse transcription
The reaction solution shown in Table 4 below was prepared.
TABLE 4
| Component name | Volume of |
| 10-200ng of total RNA | 1.5μL |
| Diluted spike in | 2.0μL |
| T7 Promoter Primer | 0.8μL |
| Nuclease-free water(white cap) | 1.0μL |
| Total volume | 5.3μL |
Keeping the temperature of the PCR instrument at 65 ℃ for 10min, and carrying out ice bath for 5 min.
Thirdly, 5 XFirst Strand Buffer is preheated at 80 ℃ for 3min at the same time and is reserved at room temperature.
And fourthly, configuring reverse transcription mix.
TABLE 5
| Component name | Volume of |
| 5×First Strand Buffer | 2.0μL |
| 0.1MDTT | 1.0μL |
| 10mM dNTP mix | 0.5μL |
| AffinityScript RNase Block Mix | 1.2μL |
| Total volume | 4.7μL |
Adding 4.7 microliter mix into the denatured RNA in ice bath, mixing and centrifuging.
Sixth, PCR: reacting for 2 hours at 40 ℃; inactivating at 70 ℃ for 15 minutes; the reaction was carried out at 4 ℃ for 5 minutes.
1.2.4.3 fluorescent labels
Arranging a mark mix.
TABLE 6
| Component name | Volume of |
| Nuclease-free water | 0.75μL |
| 5×Transcription buffer | 3.2μL |
| 0.1M DTT | 0.6μL |
| NTP mix | 1.0μL |
| T7 RNA Polymerase Blend | 0.21μL |
| Cy3-CTP | 0.24μL |
| Total volume | 6.0μL |
Purification of the tagged product
1) Add 84. mu.L of nucleic-free water to a total volume of 100. mu.L.
2) Add 350. mu.L of RLT and mix well.
3) Add 250. mu.L of absolute ethanol and mix well without centrifugation.
4) 700. mu.L of mix was transferred to the column. 13000rpm, centrifuge at 4 ℃ for 30 sec. The flow-through was discarded.
5) Add 500. mu.L of RPE,13000rpm, centrifuge at 4 ℃ for 30 sec. The flow-through was discarded.
6) An additional 500. mu.L of RPE was added, 13000rpm, and centrifuged at 4 ℃ for 60 sec. The flow-through was discarded.
7) The sleeve was replaced, 13000rpm, and idled at 4 ℃ for 30 sec. And the column was transferred to the elution tube.
8) 30 μ L of nucleotide-free water was added, and the mixture was left standing for 1min at 13000rpm and centrifuged at 4 ℃ for 30 sec.
9) The 30. mu.L sample in the elution tube was again transferred back to the column, allowed to stand for 1min, 13000rpm, and centrifuged at 4 ℃ for 30 sec.
10) RNA concentration, Cy3 concentration, 260/280 was measured with NanoDrop.
11) Requirement for probe amount:
TABLE 7
| 1X chip | cRNA>5μg | Cy3>6pmol/μg |
| 2X chip | cRNA>3.75μg | Cy3>6pmol/μg |
| 4X chip | cRNA>1.65μg | Cy3>6pmol/μg |
| 5X chip | cRNA>0.825μg | Cy3>6pmol/μg |
1.2.5 chip hybridization
According to the Hybridization standard protocol and Kit provided by the Agilent Expression profiling chip Kit, Gene Expression Hybridization Kit (Cat. # 5188-. The operation is as follows:
1) partitioning mix according to table 8 below;
TABLE 8
| Component name | 1× | 2× | 4× | 8× |
| Cy3-cRNA | 5μg | 3.75μg | 1.65μg | 600ng |
| 10×Blocking Agent | 50μl | 25μl | 11μl | 5μl |
| Nuclease-free water | To 240. mu.l | To 120. mu.l | To 52.8. mu.l | To 24. mu.l |
| 25×Fragmentation Buffer | 10μl | 5μl | 2.2μl | 1μl |
| Total volume | 250μl | 125μl | 55μl | 25μl |
2) Keeping the temperature at 60 ℃ for 30 min.
3) Ice-bath for 1min, and centrifuging briefly.
4) Add equal volume of 2 XGEx Hybridization Buffer HI-RPM and mix well.
5) Centrifuge at 13,000rpm for 1 min.
6) Placed on ice.
7) The hybridization chamber is placed on a horizontal table top, and a cover glass with a gasket is placed on the hybridization chamber.
8) Samples were added at the volumes given in table 9 below.
TABLE 9
| Component name | 1× | 2× | 4× | 8× |
| Volume of preparation | 500μl | 250μl | 110μl | 50μl |
| Volume for hybridization | 490μl | 240μl | 100μl | 40μl |
9) The chip with "Agilent" side down was covered onto a cover slip.
10) The hybridization chamber is assembled quickly.
11) Hybridization was carried out for 17h at 65 ℃ and 10rpm in a hybridization oven.
1.2.6 chip Wash and Scan
The chip that completed the hybridization was scanned with an Agilent Microarray Scanner (Cat. # G2565CA, Agilent technologies, Santa Clara, CA, US) and the software set up Dye channel: Green, Scan resolution ═ 3 μm, PMT 100%, 20 bit. Data were read using Feature Extraction software 12.0(Agilent technologies, Santa Clara, CA, US) and finally normalized using limma package in R software, using Quantile as algorithm. The operation steps are as follows:
1) wash 1 and wash 2 were added to 2mL of 10% Triton X-102 and wash 2 was preheated at 37 ℃ overnight.
2) The chip after hybridization was taken out of the hybridization oven, the hybridization chamber was disassembled, and the chip was washed as follows.
| Tear-off piece | GE Wash Buffer 1 | At room temperature | |
| Wash 1 | GE Wash Buffer 1 | At room temperature | 1min |
| Wash 2 | GE Wash Buffer 2 | 37℃ | 1min |
3) Loading the washed chip into a film holder, and scanning with a scanner, wherein the scanning parameters are as shown in the following table 11:
TABLE 11
1.2.7 Experimental quality control conditions of chips
The CV values were analyzed by comparison between two sets of data to determine whether the system was stable. In the Agilent expression profiling chip experiments, the CV values of the repeated probe spot (10 repeats) signals were used to calculate the stability of the chip and the stability of the technique. Such probe points vary from 10 to 100, depending on the chip. The result shows that the CV value of most samples except individual samples is controlled within 10 percent, and the result is reliable and can be used for further analysis.
1.3 chip analysis results
1.3.1 overview
Clustering analysis was used to plot 4 incrnas that were statistically significant for differences in samples (P <0.05) and had Fold Change (FC) values > 2-Fold or greater. The results show (fold change of partial lncRNA is listed in table 12): there were 902 for FC (abs, absolute) 2-fold more and p <0.05 in the liver injury group, 566 for up-regulation and 336 for down-regulation compared to the vehicle group; 182 for more than 4-fold change and p <0.05, 125 for up-regulation and 57 for down-regulation; there were 35 with a 10-fold change and p <0.05, 23 with up-regulation and 12 with down-regulation. Suggesting that the lncRNAs with differential expression are closely related to the occurrence, development and molecular regulation of drug-induced liver injury. Wherein the expression level of LncRNA (NONCODE TRANSCRIPT ID: NONRATT022466) in the liver tissue of rats in the administered group was 16.74 times that in the liver tissue of the controls, the expression level of LncRNA (NONCODE TRANSCRIPT ID: NONRATT007487) in the liver tissue of rats in the administered group was 19.15 times that in the liver tissue of the controls, and the expression level of LncRNA (NONCODE TRANSCRIPT ID: NONRATT009739) in the liver tissue of rats in the administered group was 28.29 times that in the liver tissue of the controls.
TABLE 12
1.3.2 GO analysis results
GO analysis of differentially expressed lncRNAs target gene mRNAs was performed using R/bioconductor/clusterirprofiler data package. Molecular criteria for mRNAs incorporated by predictive analysis were fold difference >2, p < 0.05. Functional annotation analysis of GO found that cis-regulated target gene mRNAs of differentially expressed lncRNAs mainly focused on biological processes: positive regulation of fibroblast proliferation, lipid oxidation, fatty acid catabolic processes, energy homeostasis; molecular function: activity of oxidoreductase, flavin adenine dinucleotide binding. Differentially expressed lncRNAs trans-regulated target gene mRNAs are mainly focused on biological processes: the metabolic process of the exogenous substance, the glucuronidation of the exogenous substance, and the reaction of the cell to the stimulation of the exogenous substance.
1.3.3 KEGG analysis results
The differentially expressed lncRNAs are further analyzed by KEGG for predicting target gene mRNAs by using an R/bioconductor/clusterirprofiler data packet. Molecular criteria for mRNAs incorporated by predictive analysis were fold difference >2, p < 0.05. KEGG analysis finds that differentially expressed lncRNAs predict target gene mRNAs mainly concentrated in an organism system: immune system pathways, endocrine system; metabolic pathway: exogenous metabolism, carbohydrate metabolism, energy metabolism, lipid metabolism, metabolism of cofactors and vitamins; cell pathway: protein translation, signaling, cell proliferation, and apoptosis. Suggesting that differentially expressed lncRNAs may be involved in the regulation of these pathways by regulating and predicting target gene mRNAs.
1.3.4 summary
The IncRNA chip results of the liver tissues of the experiment show that the number of IncRNAs differentially expressed in the liver tissues is very large, wherein the expression level of the IncRNA (NONCODE TRANSCRIPT ID: NONRATT022466) in the liver tissues of rats in the administration group is 16.74 times that of the control group, the expression level of the LncRNA (NONCODE TRANSCRIPT ID: NONRATT007487) in the liver tissues of rats in the administration group is 19.15 times that of the control group, the expression level of the LncRNA (NONCODE TRANSCRIPT ID: NONRATT009739) in the liver tissues of rats in the administration group is 28.29 times that of the control group (see Table 12), and the expression fold is very high, which indicates that the IncRNA (NONCODE TRANSCRIPT ID: NONRATT022466) alone or together with one or two of the other IncRNAs (NONCODE TRANSCRIPT ID: NONRATNRATT 007487 and NONRODE TRANSCRIPT ID: NOATT 009739) can be used as an exogenous biological damage detection marker for detecting liver damage.
The inventor verifies by using real-time fluorescent quantitative PCR, and proves that the NONRATT022466, NONRATT007487 and/or NONRATT009739 can be used as a biomarker to detect in time, and the detection result is reliable.
Claims (10)
1. A kit for detecting liver damage, comprising reagents for detecting lncRNA; the lncRNA comprises lncRNA of which NONCODE TRANSCRIPT ID is NONRATT 022466.
2. The kit of claim 1, wherein the liver injury is caused by an exogenous compound, preferably acetaminophen;
and/or the lncRNA also comprises lncRNA of which NONCODE TRANSCRIPT ID is NONRATT007487 and/or lncRNA of which NONCODE TRANSCRIPT ID is NONRATT 009739.
3. The kit of claim 1 or 2, wherein the assay is a real-time fluorescent quantitative PCR assay, and/or wherein the kit further comprises an RNA extraction reagent, a reverse transcription reagent, and/or a real-time fluorescent quantitative PCR reagent.
4. Use of a kit according to any one of claims 1 to 3 in the preparation of a formulation for detecting liver damage;
preferably, the liver injury is caused by exogenous compounds; more preferably, the exogenous compound is acetaminophen.
5. A biomarker combination comprising lncRNA where nonode TRANSCRIPT ID is nononratt 022466, and further comprising lncRNA where nonode TRANSCRIPT ID is nononratt 007487 and/or lncRNA where nonode TRANSCRIPT ID is nononratt 009739.
6. Use of lncrnas in the preparation of a biomarker for liver injury, wherein the lncrnas comprise lncrnas with ninctode TRANSCRIPT ID being ninrtatt 022466.
7. The use of claim 6, wherein the incRNA further comprises an incRNA wherein NONCODE TRANSCRIPT ID is NONRATT007487 and/or an incRNA wherein NONCODE TRANSCRIPT ID is NONRATT 009739.
8. The use according to claim 6 or 7, wherein the liver injury is caused by an exogenous compound; preferably, the exogenous compound is acetaminophen.
9. The use according to any one of claims 6 to 8, wherein the IncRNA is used alone as the sole biomarker for liver injury or in combination with a conventional biomarker for liver injury.
10. The use of claim 9, wherein the conventional liver injury biomarker is alanine aminotransferase, aspartate aminotransferase and/or total bilirubin.
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