CN111826437A

CN111826437A - Application of product for detecting CYP46A1 gene expression in preparation of tool for diagnosing non-alcoholic fatty liver disease

Info

Publication number: CN111826437A
Application number: CN202010721818.8A
Authority: CN
Inventors: 李杰群; 滕祥云; 谭洁琼; 陈广顺; 李强; 周依; 易军方; 马永强
Original assignee: Second Xiangya Hospital of Central South University
Current assignee: Second Xiangya Hospital of Central South University
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2020-10-27
Anticipated expiration: 2040-07-24
Also published as: CN111826437B

Abstract

The invention relates to application of a product for detecting CYP46A1 gene expression in preparation of a tool for diagnosing non-alcoholic fatty liver disease. The non-alcoholic fatty liver disease diagnosis tool takes the CYP46A1 gene as a diagnosis marker, and realizes the diagnosis purpose by detecting the expression level of the CYP46A1 gene in blood. The research in the embodiment of the invention proves that the CYP46A1 mRNA expression level in the blood of the nonalcoholic fatty liver disease patient is obviously reduced compared with that of a healthy control population. According to the relevance between the CYP46A1 gene and the non-alcoholic fatty liver disease, the kit for diagnosing the non-alcoholic fatty liver disease can be prepared, and can be widely applied to clinical non-invasive in-vitro diagnosis of the non-alcoholic fatty liver disease.

Description

Application of product for detecting CYP46A1 gene expression in preparation of tool for diagnosing non-alcoholic fatty liver disease

Technical Field

The invention relates to the technical field of in-vitro diagnosis, in particular to application of a product for detecting CYP46A1 gene expression in preparation of a tool for diagnosing non-alcoholic fatty liver disease.

Background

Abnormal fat metabolism can lead to a number of diseases, particularly nonalcoholic fatty liver disease. According to the scheme, related genes capable of regulating lipid droplet aggregation are screened through a latest whole genome CRISPR-Cas9 knockout library screening system. Meanwhile, the liver tissues of 7 non-alcoholic fatty liver patients and 9 normal controls were also analyzed for transcriptome level. The research shows that 43 related genes are knocked out in a CRISPR-Cas9 library screening system to cause lipid droplet aggregation, and the expression level is also found to be reduced in non-alcoholic fatty liver. Subsequently, immunohistochemical staining was performed on all the fatty liver samples and the normal control samples, and the results showed that CYP46a1 expression was reduced in the fatty liver. Meanwhile, CYP46A2 was interfered and overexpressed in normal hepatocytes, and the results showed that non-fatty acid-induced lipid droplet aggregation was increased after interference with CYP46A 1. Whereas overexpression of CYP46a1 resulted in a reduction in lipid droplet aggregation. Further, after CYP46A1 interference, normal hepatocyte electron microscope observation shows that the morphology of mitochondria is abnormal. Mitochondrial respiratory chain function tests show that after CYP46A1 is interfered, the function of mitochondrial complexes is damaged. These all suggest that CYP46a1 may regulate lipid metabolism by affecting mitochondrial function.

Abnormalities in liver Lipid Droplets (LDs) are associated with metabolic syndrome, type 2 diabetes, hepatitis c, and alcoholic and non-alcoholic fatty liver disease (NAFLD). NAFLD is becoming more and more prevalent worldwide, especially in western countries. NAFLD ranges from fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) cirrhosis, a range of diseases. Currently, accurate diagnosis of NASH still requires invasive, costly liver biopsies with potential complications. Furthermore, no effective therapy can reverse or even arrest the progression of the disease.

Therefore, in clinical studies, identifying and controlling the key events of NAFLD progression are crucial. The pathogenic accumulation of lipid droplets within adipocytes is most likely due to increased free fatty acid uptake and decreased lipoprotein output and fatty acid oxidation. The pathways involved in the development of hepatic steatosis are intricate. Numerous studies have revealed a large number of genes associated with lipid droplet formation, including the use of proteomics, transcriptomics in hepatic lipid homeostasis and methods based on high-throughput imaging. Recently, in drosophila cells, several candidate genes were obtained by RNA interference (RNAi) genome-wide screening, but a large fraction (about 40%) appeared to have no human orthologs. The latest effective gene editing techniques have been successfully applied to mammalian cells for functional studies of gene deletions. CRISPR/Cas9 is a DNA endonuclease that can edit specific DNA sites complementary to guide RNAs. A whole genome-scale CRISPR-Cas9 knock-out (GeCKO) library to contain all genomic genes. This tool can help assess gene function, identify and validate novel drug targets or study the underlying mechanisms of human disease.

Due to the complexity of NAFLD, each patient may carry different causative factors (diet, metabolic phenotype, genes, etc.), and how to differentiate to varying degrees for different populations (with overweight, obesity, diabetes or other metabolic diseases) is a key to future research that requires the use of systematic research methods (integrated metabolomics, proteomics, phenomics, etc.) to reveal the molecular characteristics of NAFLD, which can facilitate biomarker discovery and help provide more accurate therapies.

Therefore, the development of an in vitro molecular diagnostic method for accurately diagnosing NAFLD is a technical problem to be solved urgently in clinic.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides application of a product for detecting CYP46A1 gene expression in preparation of a tool for diagnosing non-alcoholic fatty liver disease; the research shows that the CYP46A1 gene has a remarkable reduction in the expression of non-alcoholic fatty liver disease, so that the CYP46A1 gene can be used as a biomarker for diagnosing the non-alcoholic fatty liver disease, and a tool for diagnosing the non-alcoholic fatty liver disease is developed.

The invention provides an application of a product for detecting CYP46A1 gene expression in preparation of a tool for diagnosing non-alcoholic fatty liver disease, wherein the tool for diagnosing non-alcoholic fatty liver disease takes the CYP46A1 gene as a diagnostic marker, and the sequence of the CYP46A1 gene is shown as SEQ ID NO: 1 is shown.

Wherein, the sequence information (SEQ ID NO: 1) of the CYP46A1 gene can be inquired in the International public nucleic acid sequence database GeneBank (NC-000014.9 (99684262.. 99727321));

SEQ ID NO：1：

ATGAGCCCCGGGCTGCTGCTGCTCGGCAGCGCCGTCCTGCTCGCCTTCGGCCTCTGCTGCACCTTCGTGCACCGCGCTCGCAGCCGCTACGAGCACATCCCCGGGCCGCCGCGGCCCAGTTTCCTTCTAGGACACCTCCCCTGCTTTTGGAAAAAGGATGAGGTTGGTGGCCGTGTGCTCCAAGATGTGTTTTTGGATTGGGCTAAGAAGTATGGACCTGTTGTGCGGGTCAACGTCTTCCACAAAACCTCAGTCATCGTCACGAGTCCTGAGTCGGTTAAGAAGTTCCTGATGTCAACCAAGTACAACAAGGACTCCAAGATGTACCGTGCGCTCCAGACTGTGTTTGGTGAGAGACTCTTCGGCCAAGGCTTGGTGTCCGAATGCAACTATGAGCGCTGGCACAAGCAGCGGAGAGTCATAGACCTGGCCTTCAGCCGGAGCTCCTTGGTTAGCTTAATGGAAACATTCAACGAGAAGGCTGAGCAGCTGGTGGAGATTCTAGAAGCCAAGGCAGATGGGCAGACCCCAGTGTCCATGCAGGACATGCTGACCTACACCGCCATGGACATCCTGGCCAAGGCAGCTTTTGGGATGGAGACCAGTATGCTGCTGGGTGCCCAGAAGCCTCTGTCCCAGGCAGTGAAACTTATGTTGGAGGGAATCACTGCGTCCCGCAACACTCTGGCAAAGTTCCTGCCAGGGAAGAGGAAGCAGCTCCGGGAGGTCCGGGAGAGCATTCGCTTCCTGCGCCAGGTGGGCAGGGACTGGGTCCAGCGCCGCCGGGAAGCCCTGAAGAGGGGCGAGGAGGTTCCTGCCGACATCCTCACACAGATTCTGAAAGCTGAAGAGGGAGCCCAGGACGACGAGGGTCTGCTGGACAACTTCGTCACCTTCTTCATTGCTGGTCACGAGACCTCTGCCAACCACTTGGCGTTCACAGTGATGGAGCTGTCTCGCCAGCCAGAGATCGTGGCAAGGCTGCAGGCCGAGGTGGATGAGGTCATTGGTTCTAAGAGGTACCTGGATTTCGAGGACCTGGGGAGACTGCAGTACCTGTCCCAGGTCCTCAAAGAGTCGCTGAGGCTGTACCCACCAGCATGGGGCACCTTTCGCCTGCTGGAAGAGGAGACCTTGATTGATGGGGTCAGAGTCCCCGGCAACACCCCGCTCTTGTTCAGCACCTATGTCATGGGGCGGATGGACACATACTTTGAGGACCCGCTGACTTTCAACCCCGATCGCTTCGGCCCTGGAGCACCCAAGCCACGGTTCACCTACTTCCCCTTCTCCCTGGGCCACCGCTCCTGCATCGGGCAGCAGTTTGCTCAGATGGAGGTGAAGGTGGTCATGGCAAAGCTGCTGCAGAGGCTGGAGTTCCGGCTGGTGCCCGGGCAGCGCTTCGGGCTGCAGGAGCAGGCCACACTCAAGCCACTGGACCCCGTGCTGTGCACCCTGCGGCCCCGCGGCTGGCAGCCCGCACCCCCACCACCCCCCTGCTGA。

the products expressed by the CYP46A1 gene comprise CYP46A1 protein and partial peptide of CYP46A1 protein; the partial peptide of the CYP46A1 protein contains a functional domain related to non-alcoholic fatty liver disease. The amino acid sequence of the CYP46A1 protein is shown as SEQ ID NO: 2 is shown in the specification;

SEQ ID NO：2：

MSPGLLLLGSAVLLAFGLCCTFVHRARSRYEHIPGPPRPSFLLGHLPCFWKKDEVGGRVLQDVFLDWAKKYGPVVRVNVFHKTSVIVTSPESVKKFLMSTKYNKDSKMYRALQTVFGERLFGQGLVSECNYERWHKQRRVIDLAFSRSSLVSLMETFNEKAEQLVEILEAKADGQTPVSMQDMLTYTAMDILAKAAFGMETSMLLGAQKPLSQAVKLMLEGITASRNTLAKFLPGKRKQLREVRESIRFLRQVGRDWVQRRREALKRGEEVPADILTQILKAEEGAQDDEGLLDNFVTFFIAGHETSANHLAFTVMELSRQPEIVARLQAEVDEVIGSKRYLDFEDLGRLQYLSQVLKESLRLYPPAWGTFRLLEEETLIDGVRVPGNTPLLFSTYVMGRMDTYFEDPLTFNPDRFGPGAPKPRFTYFPFSLGHRSCIGQQFAQMEVKVVMAKLLQRLEFRLVPGQRFGLQEQATLKPLDPVLCTLRPRGWQPAPPPPPC。

further, the products include, but are not limited to: the product for diagnosing the non-alcoholic fatty liver disease is prepared by detecting the expression level of CYP46A1 gene by adopting real-time fluorescent quantitative PCR, reverse transcription PCR, in situ hybridization, a gene chip, a protein chip, immunohistochemistry or a high-throughput sequencing technology platform.

The product for detecting the CYP46A1 gene expression by using a high-throughput sequencing technology platform can also realize the detection of the expression condition of the CYP46A1 gene. With the development and popularization of high-throughput sequencing technology, the work of constructing a gene expression profile for one person is more and more convenient; by comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the diseases can be easily analyzed. Therefore, the analysis of the correlation between the expression level of the CYP46A1 gene and the non-alcoholic fatty liver disease in a high-throughput sequencing platform also belongs to the application of detecting the CYP46A1 gene product in the scheme, and is also within the protection scope of the invention.

Further, the product for diagnosing the nonalcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting a real-time fluorescent quantitative PCR technology platform comprises at least one pair of primers for specifically amplifying the CYP46A1 gene; the product for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting a reverse transcription PCR technology platform comprises at least one pair of primers for specifically amplifying the CYP46A1 gene; the product for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting an in situ hybridization technology platform comprises a probe hybridized with a CYP46A1 gene nucleic acid sequence; the product for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting a gene chip technology platform comprises a probe hybridized with a CYP46A1 gene nucleic acid sequence; the product for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting a protein chip technology platform comprises an antibody specifically combined with CYP46A1 protein; products for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting an immunohistochemical technology platform comprise antibodies specifically bound with the CYP46A1 protein.

Wherein, the probe hybridized with the nucleic acid sequence of CYP46A1 gene can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives; the length of the probe is not limited, and any length can be used as long as specific hybridization and specific binding with a target nucleotide sequence are achieved; the length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene; since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and at most usually not more than 30 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15 to 25 base pairs; the probe self-complementary sequence is preferably less than 4 base pairs so as not to affect hybridization efficiency.

The protein chip can be used for detecting the expression level of a plurality of proteins including CYP46A1 protein (such as a plurality of proteins related to non-alcoholic fatty liver disease).

The specific antibody of the CYP46A1 protein comprises a monoclonal antibody and a polyclonal antibody; antibodies specific for the CYP46a1 protein include intact antibody molecules, any fragment or modification of an antibody (e.g., chimeric antibodies, scFv, Fab, F (ab') 2, Fv, etc., so long as the fragment retains the ability to bind to the CYP46a1 protein.

In addition, the accuracy of the diagnosis of the non-alcoholic fatty liver disease can be greatly improved by simultaneously detecting a plurality of biomarkers of the non-alcoholic fatty liver disease.

Further, the product for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting a real-time fluorescent quantitative PCR technology platform comprises a pair of primers for specifically amplifying the CYP46A1 gene, wherein the primers are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

SEQ ID NO：3：

5'-CTTGTGGAAAGGACGAAACA-3'。

SEQ ID NO：4：

5'-GCCAATTCCCACTCCTTTCA-3'。

Further, the tool can diagnose non-alcoholic fatty liver disease by detecting the expression level of CYP46A1 gene in a human sample.

Wherein, the sources of CYP46A1 gene and its expression product for diagnosing non-alcoholic fatty liver disease include, but are not limited to, body fluids such as blood, interstitial fluid, urine, saliva, spinal fluid, etc., or tissues; in a specific embodiment of the present invention, the source of the CYP46a1 gene and its expression product used for diagnosing non-alcoholic fatty liver disease is blood, which is peripheral blood taken from patients and normal persons with non-alcoholic fatty liver disease.

Further, the tool comprises a kit, a gene chip, a protein chip, an immunochromatographic test paper or a high-throughput sequencing technology platform.

Further, the kit comprises a reagent for detecting the transcription level of CYP46A1 gene;

the gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise oligonucleotide probes for detecting the CYP46A1 gene transcription level and aiming at the CYP46A1 gene; the protein chip comprises a solid phase carrier and an antibody specific to CYP46A1 protein fixed on the solid phase carrier; the immunochromatographic test paper comprises a specific antibody of CYP46A1 protein; the high throughput sequencing technology platform comprises reagents for detecting the transcription level of the CYP46A1 gene.

Further, the reagent for detecting the transcription level of the CYP46A1 gene comprises a primer and a probe aiming at the CYP46A1 gene.

Further, the primer sequences for the CYP46A1 gene are respectively shown as SEQ ID NO.3 for the forward primer sequence and SEQ ID NO.4 for the reverse primer sequence.

Compared with the prior art, the application of the product for detecting CYP46A1 gene expression in the preparation of a tool for diagnosing non-alcoholic fatty liver disease provided by the invention has the following beneficial effects:

1. conventional non-alcoholic fatty liver disease diagnostic tools, such as B-ultrasound, CT, MRI and other imaging diagnostic tools, cannot reflect the existence and cause of inflammation and fibrosis; liver biopsy is still the gold standard for non-alcoholic fatty liver disease diagnosis and staging thereof at present, but belongs to traumatic diagnosis. Compared with the prior art, the CYP46A1 gene serving as the biomarker for diagnosing the non-alcoholic fatty liver disease provides a basis for preparing a tool for in-vitro non-invasive diagnosis of the non-alcoholic fatty liver disease, and has the characteristics of high specificity and high sensitivity.

2. The in vitro diagnosis products such as the primer, the probe and the reagent which comprise the CYP46A1 gene expression level detection provided by the invention have the advantages of high accuracy, strong specificity, high sensitivity and the like, and are convenient for clinical use.

Drawings

FIG. 1 shows immunohistochemical staining differences and CYP46A1 gene expression differences for healthy control liver tissue and non-alcoholic fatty liver disease tissue samples.

Figure 2 shows the difference in mitochondrial function between healthy control hepatocyte siRNA and CYP46a1 siRNA.

FIG. 3 shows the real-time fluorescent quantitative PCR detection of the difference in CYP46A1 gene expression levels between nonalcoholic fatty liver disease and healthy control populations.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example as described in molecular cloning, A laboratory Manual (New York: Cold Spring harbor laboratory Press) by Sambrook et al, or according to the conditions recommended by the manufacturer.

Example 1 screening of genes differentially expressed in non-alcoholic fatty liver disease patients and healthy control population

1. Clinical specimen and patient data

The study included 7 histologically confirmed NAFLD adult patients and 9 healthy control populations. NAFLD is defined according to the american association for liver disease research (AASLD) guidelines.

Normal liver tissue samples from the control group were taken from patients who received liver metastases and surgical resection of hemangiomas. Two experienced pathologists read histological pictures of all liver specimens, they had no knowledge of all clinical data, and scored histological features using NAFLD Activity Scoring (NAS).

All patients received written informed consent. The study protocol was approved by the ethical committee of xiangya second hospital, university in south China.

2. RNA-Seq for liver transcriptomics analysis

Total RNA was extracted from liver tissue of patients with NAFLD by using the illumina RNAspin Mini Kit (GE Healthcare) and subjected to control. RNA samples were quantified using Nanodrop and identified by agarose gel electrophoresis. The Illumina kit includes procedures for RNA fragmentation, random hexamer initiated first strand cDNA synthesis, dUTP based second strand cDNA synthesis, end repair, a-labeling, adaptor ligation and library PCR amplification for RNA-seq library preparation.

Finally, the prepared RNA-seq library was identified using an Agilent 2100 bioanalyzer and quantified by qPCR absolute quantification method. Sequencing was performed using Illumina Hiseq 4000. Filtered raw sequencing data was generated by Illumina HiSeq4000 for the following analysis.

1) Lentiviral production of sgRNA library

Lentiviral packaging and purification of the sgRNA library was as described previously. Briefly, HEK293T cells were cultured in DMEM with 10% Fetal Bovine Serum (FBS).

Mu.g of GeCKO library (#1000000048, Addge), 2. mu.g of pV-SVg (#8454, Addge) and 6. mu.g of psPAX2(#12260, Addge) packaging plasmids were co-transfected in 10cm dishes using Lipofectamine 2000.

After 6 hours of transfection, the cell culture medium was changed to complete medium. After 48 hours, the medium was collected and centrifuged at 3,000rpm for 20 minutes at 4 ℃ to remove cell debris. The supernatant (pore size 0.45 μm) was filtered and concentrated by ultracentrifugation at 24,000rpm for 2h at 4 ℃.

Finally the virus preparation was resuspended overnight in DMEM at 4 deg.C, aliquoted and then stored at-80 deg.C.

2) Lentiviral transduction of sgRNA libraries

Human liver cell line L02 was obtained from the national academy of sciences type culture Collection and cultured in RPMI-1640 medium containing 20% FBS and 4mM L-glutamine.

The GeCKO library virus infected 3x 108L 02 cells at a multiplicity of infection (MOI) of 0.3 to ensure that most cells received only 1 virus in the presence of 10 μ g/mL Polybrene.

48 hours after infection, the medium was changed to fresh complete DMEM medium containing 1. mu.g/mL puromycin and antibiotic selection was performed for 7 days.

3) Sorting and DNA sequencing

After 7 days of puromycin screening, cells were treated with sodium oleate (OA, 200. mu.M) and sodium palmitate (PA, 100. mu.M) for 12 hours.

Subsequently, the lipid droplets were subjected to BODIPY493/503 (staining. 10% of the cells with high fluorescence intensity were sorted and collected by flow cytometry. genomic DNA was isolated from the sorted cells using the Blood & Cell Culture DNA Midi Kit (Qiagen, Germany).

Amplicons were added by second step PCR and sequenced using HiSeq 2500). The forward primer was 5'-CTTGTGGAAAGGACGAAACA-3'. The reverse primer was 5'-GCCAATTCCCACTCCTTTCA-3'.

The raw sequencing data is saved in FASTQ format. And analyzed using custom GeCKO screening software.

Briefly, raw sequencing is read and analyzed by using different barcodes in the reverse primer. The processed data was deleted from the start to the sequence of the sgRNA primer site primer. The collated data was compared to the GeCKO v2 library A and B data. Reads of sgrnas were quantified by model-based whole genome CRISPR-Cas9 knockout (MAGeCK) v5.6.0 model-based software.

4) RNA interference

The CYP46a1 gene and the non-targeted oligonucleotide were both obtained from GenePharma (shanghai, china).

siRNA was transfected with Lipofectamine 2000 reagent (Invitrogen, USA) according to the experimental procedure provided by the manufacturer. Lipid droplets in cells were stained with BODIPY493/503 and counted 48h after siRNA transfection.

5) Transfection

CYP46A1-Flag plasmid was transfected with Lipofectinmin 2000 reagent.

6) BODIPY immobilization and immunofluorescence staining

Lipid droplets were stained by BODIPY according to previously reported experimental procedures. After transfection, cells were washed with PBS and incubated at 37 ℃ for 15 minutes in BODIPY493/503 staining solution.

Cells were then washed twice with PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.1% Triton X-100. After blocking with 5% BSA for 30 min, cells were incubated with Flag (M2) antibody (Sigma, usa) and detected with Alexa-conjugated secondary antibody (Invitrogen, usa). Nuclei were stained with DAPI (Invitrogen, USA) for 2 minutes and washed 3 times with PBS. Photographs were taken using a confocal fluorescence imaging microscope (Leica, usa).

7) Immunohistochemistry

Paraffin-embedded sections (5 μm) of each tissue were stained using immunohistochemistry against CYP46a1(Sigma, usa). Briefly, after deparaffinization, sections were incubated with 10% normal goat serum in 50. mu. mmol/L Tris-HCl (pH 7.4) containing 150. mu. mmol/L NaCl (TBS) at room temperature, followed by primary antibody incubation. The sections were then incubated with biotinylated antibodies, avidin-biotin-peroxidase complex (Vector laboratories, usa) and 3, 3-diaminobenzidine in the presence of H2O 2. All stained sections were taken under an optical microscope.

8) Mitochondrial isolation and mitochondrial Complex Activity detection

Cells were washed twice in PBS and incubated in frozen lysis buffer (68mM sucrose, 200mM mannitol, 50mM kcl, 1mM EDTA, 1mM EGTA, 1mM dithiothreitol and protease inhibitor cocktail (Sigma, usa) for 30 minutes.

The cells were then pulled 45 times through a 25G 5/8 needle, centrifuged at 1500G for 10 minutes, and 13,000G for 20 minutes before recovering the cytoplasmic extract, precipitated as crude mitochondria, and the protein concentration was determined using BCA assay Kit (Pierce, USA) the activity of mitochondrial OXPHOS complex was determined using the mitochondrial Complex Activity assay Kit (Abcam, UK) and the MitoTox OXPHOS Complex Activity Kit (Abcam, UK) according to the instructions.

3. Results

The RNA-seq results show that the relative mRNA expression level of the CYP46A1 gene in the blood of the healthy control population is 1, and the mRNA expression level of the CYP46A1 gene in the blood of the nonalcoholic fatty liver disease patient is 0.xx +/-0. xx. The results show that the mRNA level of CYP46A1 gene in blood of nonalcoholic fatty liver disease patients is remarkably reduced compared with normal people, and the difference has statistical significance (P < 0.05).

Specifically, referring to fig. 1-2, fig. 1 shows immunohistochemical staining differences and CYP46a1 gene expression differences of healthy control liver tissue and non-alcoholic fatty liver disease tissue samples; fig. 2 shows the difference in mitochondrial function between healthy control hepatocyte siRNA and CYP46A1 siRNA. Wherein, part a in fig. 1 is H & E staining, oil red O staining, and lipid droplets are observable on healthy control liver tissue and NAFLD tissue samples; part B is a statistical result of the relative expression of CYP46a1, and the relative levels of CYP46a1 were shown by normalization to control samples, which were statistically different (./P < 0.01). Part A in figure 2 is mitochondria observed by an electron microscope after transfection for 48h, and mitochondrial inner ridge is broken and swollen; part B is the activity of the mitochondrial complex detected in CYP46a1siRNA or control transfected cells, showing a decrease in mitochondrial complex I and IV activity, citrate synthase was used as a control (. P <0.01,. P < 0.05).

Example 2 verification of genes differentially expressed in non-alcoholic fatty liver disease patients and healthy control populations

1. Study subjects:

65 patients with nonalcoholic fatty liver disease, 50 normal patients were selected according to the method of example 1.

2. Blood Total RNA extraction

The extraction of total RNA from blood was performed using a blood RNA extraction kit from fortek:

(1) mu.l (or 0.25g) of whole blood was applied to RNase-Free filter column, centrifuged at 13000rpm for 2 minutes, the supernatant was collected, and 0.75ml of lysis buffer RLS was added.

(2) The homogenate was vigorously shaken and mixed, and incubated at 15-30 ℃ for 5 minutes to completely decompose the nucleoprotein body.

(3) Optional steps are as follows: centrifugation was carried out at 12,000rpm for 10 minutes at 4 ℃ and the supernatant was carefully removed and transferred to a new RNase-free centrifuge tube.

(4) 0.2ml of chloroform was added per 1ml of RLS. The sample tube cap was closed, shaken vigorously for 15 seconds and incubated for 3 minutes at room temperature.

(5) After centrifugation at 12,000rpm for 10 minutes at 4 ℃ the sample will separate into three layers: the lower organic phase, the middle layer and the upper colorless aqueous phase, RNA is present in the aqueous phase. The volume of the aqueous layer was about 60% of the volume of the added RLS, and the aqueous layer was transferred to a fresh tube for further processing.

(6) 70% ethanol is added in 1 volume, the mixture is inverted and mixed (precipitation may occur), and the resulting solution is transferred to an adsorption column RA (which is sleeved in a collection tube) together with the possible precipitation.

(7) Centrifuge at 10,000rpm for 45 seconds, discard the waste liquid, and re-wrap the adsorption column back to the collection tube.

(8) Add 500. mu.l deproteinized solution RE, centrifuge at 12,000rpm for 45 seconds, and discard the waste.

(9) 700. mu.l of the rinsing solution RW was added, and centrifuged at 12,000rpm for 60 seconds, and the waste liquid was discarded.

(10) 500. mu.l of the rinsing solution RW was added, centrifuged at 12,000rpm for 60 seconds, and the waste liquid was discarded.

(11) The adsorption column RA was returned to the empty collection tube and centrifuged at 12,000rpm for 2 minutes to remove the rinse as much as possible so as not to inhibit downstream reactions due to residual ethanol in the rinse.

(12) The adsorption column RA was taken out, put into an RNase-free centrifuge tube, 50 to 80. mu.l of RNase-free water was added to the middle part of the adsorption membrane according to the expected RNA yield, left at room temperature for 2 minutes, centrifuged at 12,000rpm for 1 minute, and the eluate was collected.

3. Measurement of Total RNA concentration and purity

The concentration and purity of the sample RNA was measured with a NanoVue Plus instrument.

4. Reverse transcription to synthesize mRNA cDNA

Mu.g of total RNA was reverse transcribed with reverse transcription buffer to synthesize cDNA. A25-mu-l reaction system is adopted, 1 mu g of total RNA is taken from each sample as template RNA, and the following components are respectively added into a PCR tube: DEPC water, 5 Xreverse transcription buffer, 10mmol/L dNTP, 0.1mmol/L DTT, 30. mu. mmol/L Oligo dT, 200U/. mu. L M-MLV, template RNA. Incubate at 42 ℃ for 1h, 72 ℃ for 10min, and centrifuge briefly.

5、QPCR

(1) Primer design

QPCR amplification primers were designed based on the coding sequences of CYP46A1 gene and GAPDH reference gene in Genbank and synthesized by Shanghai Bioengineering services, Inc. The specific primer sequences are as follows:

CYP46a1 gene:

the forward primer is 5'-CTTGTGGAAAGGACGAAACA-3' (SEQ ID NO. 3);

the reverse primer is 5'-GCCAATTCCCACTCCTTTCA-3' (SEQ ID NO. 4);

GAPDH reference gene:

the forward primer is 5'-TTTAACTCTGGTAAAGTGGATAT-3' (SEQ ID NO. 5);

the reverse primer was 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO. 6).

(2) Amplification of

Reaction system: 25 μ l reaction, 3 parallel channels per sample. The following reaction system was prepared: SYBRGreen polymerase chain reaction system 12.5. mu.l, forward primer (5. mu.M) 1. mu.l, reverse primer (5. mu.M) 1. mu.l, template cDNA 2.0. mu.l, 8.5. mu.l without enzyme water; all operations were performed on ice.

And (3) amplification procedure: 95 ℃ 5min, (95 ℃ 5s, 60 ℃ 60s) 45 cycles. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent real-time quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, and relative quantification is carried out through a delta CT method.

6. Statistical method

The experiments were performed in 3 replicates, the results were expressed as mean ± sd, statistically analyzed using SPSS13.0 statistical software, and the differences between the different groups were determined by t-test to be statistically significant when P < 0.05.

7. Results

As shown in fig. 3, the mRNA level of CYP46a1 gene in blood of non-alcoholic fatty liver disease (NAFLD) patients was significantly decreased compared to the healthy Control population (Control), and the difference was statistically significant (P <0.05), and the results were the same as the above RNA-seq experiment.

The application of the product for detecting CYP46A1 gene expression in the preparation of a tool for diagnosing non-alcoholic fatty liver disease provided by the invention has the following beneficial effects:

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent flow transformations made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Sequence listing

<110> Xiangya II Hospital of Zhongnan university

<130>2020

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>1503

<212>DNA

<213>Homo sapiens

<400>1

atgagccccg ggctgctgct gctcggcagc gccgtcctgc tcgccttcgg cctctgctgc 60

accttcgtgc accgcgctcg cagccgctac gagcacatcc ccgggccgcc gcggcccagt 120

ttccttctag gacacctccc ctgcttttgg aaaaaggatg aggttggtgg ccgtgtgctc 180

caagatgtgt ttttggattg ggctaagaag tatggacctg ttgtgcgggt caacgtcttc 240

cacaaaacct cagtcatcgt cacgagtcct gagtcggtta agaagttcct gatgtcaacc 300

aagtacaaca aggactccaa gatgtaccgt gcgctccaga ctgtgtttgg tgagagactc 360

ttcggccaag gcttggtgtc cgaatgcaac tatgagcgct ggcacaagca gcggagagtc 420

atagacctgg ccttcagccg gagctccttg gttagcttaa tggaaacatt caacgagaag 480

gctgagcagc tggtggagat tctagaagcc aaggcagatg ggcagacccc agtgtccatg 540

caggacatgc tgacctacac cgccatggac atcctggcca aggcagcttt tgggatggag 600

accagtatgc tgctgggtgc ccagaagcct ctgtcccagg cagtgaaact tatgttggag 660

ggaatcactg cgtcccgcaa cactctggca aagttcctgc cagggaagag gaagcagctc 720

cgggaggtcc gggagagcat tcgcttcctg cgccaggtgg gcagggactg ggtccagcgc 780

cgccgggaag ccctgaagag gggcgaggag gttcctgccg acatcctcac acagattctg 840

aaagctgaag agggagccca ggacgacgag ggtctgctgg acaacttcgt caccttcttc 900

attgctggtc acgagacctc tgccaaccac ttggcgttca cagtgatgga gctgtctcgc 960

cagccagaga tcgtggcaag gctgcaggcc gaggtggatg aggtcattgg ttctaagagg 1020

tacctggatt tcgaggacct ggggagactg cagtacctgt cccaggtcct caaagagtcg 1080

ctgaggctgt acccaccagc atggggcacc tttcgcctgc tggaagagga gaccttgatt 1140

gatggggtca gagtccccgg caacaccccg ctcttgttca gcacctatgt catggggcgg 1200

atggacacat actttgagga cccgctgact ttcaaccccg atcgcttcgg ccctggagca 1260

cccaagccac ggttcaccta cttccccttc tccctgggcc accgctcctg catcgggcag 1320

cagtttgctc agatggaggt gaaggtggtc atggcaaagc tgctgcagag gctggagttc 1380

cggctggtgc ccgggcagcg cttcgggctg caggagcagg ccacactcaa gccactggac 1440

cccgtgctgt gcaccctgcg gccccgcggc tggcagcccg cacccccacc acccccctgc 1500

tga 1503

<210>2

<211>500

<212>PRT

<213>Homo sapiens

<400>2

Met Ser Pro Gly Leu Leu Leu Leu Gly Ser Ala Val Leu Leu Ala Phe

1 5 10 15

Gly Leu Cys Cys Thr Phe Val His Arg Ala Arg Ser Arg Tyr Glu His

20 25 30

Ile Pro Gly Pro Pro Arg Pro Ser Phe Leu Leu Gly His Leu Pro Cys

35 40 45

Phe Trp Lys Lys Asp Glu Val Gly Gly Arg Val Leu Gln Asp Val Phe

50 55 60

Leu Asp Trp Ala Lys Lys Tyr Gly Pro Val Val Arg Val Asn Val Phe

65 70 75 80

His Lys Thr SerVal Ile Val Thr Ser Pro Glu Ser Val Lys Lys Phe

85 90 95

Leu Met Ser Thr Lys Tyr Asn Lys Asp Ser Lys Met Tyr Arg Ala Leu

100 105 110

Gln Thr Val Phe Gly Glu Arg Leu Phe Gly Gln Gly Leu Val Ser Glu

115 120 125

Cys Asn Tyr Glu Arg Trp His Lys Gln Arg Arg Val Ile Asp Leu Ala

130 135 140

Phe Ser Arg Ser Ser Leu Val Ser Leu Met Glu Thr Phe Asn Glu Lys

145 150 155 160

Ala Glu Gln Leu Val Glu Ile Leu Glu Ala Lys Ala Asp Gly Gln Thr

165 170 175

Pro Val Ser Met Gln Asp Met Leu Thr Tyr Thr Ala Met Asp Ile Leu

180 185 190

Ala Lys Ala Ala Phe Gly Met Glu Thr Ser Met Leu Leu Gly Ala Gln

195 200 205

Lys Pro Leu Ser Gln Ala Val Lys Leu Met Leu Glu Gly Ile Thr Ala

210 215 220

Ser Arg Asn Thr Leu Ala Lys Phe Leu Pro Gly Lys Arg Lys Gln Leu

225 230 235 240

Arg Glu Val Arg Glu Ser Ile Arg Phe Leu Arg Gln Val Gly Arg Asp

245 250 255

Trp Val Gln Arg Arg Arg Glu Ala Leu Lys Arg Gly Glu Glu Val Pro

260 265 270

Ala Asp Ile Leu Thr Gln Ile Leu Lys Ala Glu Glu Gly Ala Gln Asp

275 280 285

Asp Glu Gly Leu Leu Asp Asn Phe Val Thr Phe Phe Ile Ala Gly His

290 295 300

Glu Thr Ser Ala Asn His Leu Ala Phe Thr Val Met Glu Leu Ser Arg

305 310 315 320

Gln Pro Glu Ile Val Ala Arg Leu Gln Ala Glu Val Asp Glu Val Ile

325 330 335

Gly Ser Lys Arg Tyr Leu Asp Phe Glu Asp Leu Gly Arg Leu Gln Tyr

340 345 350

Leu Ser Gln Val Leu Lys Glu Ser Leu Arg Leu Tyr Pro Pro Ala Trp

355 360 365

Gly Thr Phe Arg Leu Leu Glu Glu Glu Thr Leu Ile Asp Gly Val Arg

370 375 380

Val Pro Gly Asn Thr Pro Leu Leu Phe Ser Thr Tyr Val Met Gly Arg

385 390 395 400

Met Asp Thr Tyr Phe Glu Asp Pro Leu Thr Phe Asn Pro Asp Arg Phe

405 410 415

Gly Pro Gly Ala Pro Lys Pro Arg Phe Thr Tyr Phe Pro Phe Ser Leu

420 425 430

Gly His Arg Ser Cys Ile Gly Gln Gln Phe Ala Gln Met Glu Val Lys

435 440 445

Val Val Met Ala Lys Leu Leu Gln Arg Leu Glu Phe Arg Leu Val Pro

450 455 460

Gly Gln Arg Phe Gly Leu Gln Glu Gln Ala Thr Leu Lys Pro Leu Asp

465 470 475 480

Pro Val Leu Cys Thr Leu Arg Pro Arg Gly Trp Gln Pro Ala Pro Pro

485 490 495

Pro Pro Pro Cys

500

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

cttgtggaaa ggacgaaaca 20

<210>4

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gccaattccc actcctttca 20

<210>5

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

tttaactctg gtaaagtgga tat 23

<210>6

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ggtggaatca tattggaaca 20

Claims

1. The application of a product for detecting CYP46A1 gene expression in preparing a tool for diagnosing non-alcoholic fatty liver disease is characterized in that the tool for diagnosing non-alcoholic fatty liver disease uses the CYP46A1 gene as a diagnostic marker, and the sequence of the CYP46A1 gene is shown in SEQ ID NO: 1 is shown.

2. The use according to claim 1, wherein the products include but are not limited to: the product for diagnosing the non-alcoholic fatty liver disease is prepared by detecting the expression level of CYP46A1 gene by adopting real-time fluorescent quantitative PCR, reverse transcription PCR, in situ hybridization, a gene chip, a protein chip, immunohistochemistry or a high-throughput sequencing technology platform.

3. The use of claim 2, wherein the product for detecting the expression level of CYP46A1 gene for diagnosing non-alcoholic fatty liver disease using a real-time fluorescent quantitative PCR technology platform comprises at least one pair of primers for specifically amplifying CYP46A1 gene;

the product for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting a reverse transcription PCR technology platform comprises at least one pair of primers for specifically amplifying the CYP46A1 gene;

the product for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting an in situ hybridization technology platform comprises a probe hybridized with a CYP46A1 gene nucleic acid sequence;

the product for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting a gene chip technology platform comprises a probe hybridized with a CYP46A1 gene nucleic acid sequence;

the product for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting a protein chip technology platform comprises an antibody specifically combined with CYP46A1 protein;

products for diagnosing the non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by adopting an immunohistochemical technology platform comprise antibodies specifically bound with the CYP46A1 protein.

4. The use of claim 3, wherein the product for diagnosing non-alcoholic fatty liver disease by detecting the expression level of CYP46A1 gene using real-time fluorescence quantitative PCR technology platform comprises a pair of primers for specifically amplifying CYP46A1 gene as set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

5. The use of claim 1, wherein said means is capable of diagnosing non-alcoholic fatty liver disease by detecting the level of CYP46a1 gene expression in a human sample.

6. The use of claim 5, wherein the kit comprises a kit, a gene chip, a protein chip, an immunochromatographic strip, or a high-throughput sequencing platform.

7. The use according to claim 6, wherein the kit comprises reagents for detecting the level of CYP46A1 gene transcription;

the gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise oligonucleotide probes for detecting the CYP46A1 gene transcription level and aiming at the CYP46A1 gene;

the protein chip comprises a solid phase carrier and an antibody specific to CYP46A1 protein fixed on the solid phase carrier;

the immunochromatographic test paper comprises a specific antibody of CYP46A1 protein;

the high throughput sequencing technology platform comprises reagents for detecting the transcription level of the CYP46A1 gene.

8. The use of claim 7, wherein the reagent for detecting the transcript level of CYP46A1 gene comprises primers and probes for CYP46A1 gene.

9. The use according to claim 8, wherein the primer sequences for CYP46A1 gene are respectively shown as SEQ ID No.3 and SEQ ID No. 4.

10. Use according to claim 5, wherein the sample is blood.