CN111826437B - Application of product for detecting CYP46A1 gene expression in preparation of tool for diagnosing non-alcoholic fatty liver disease - Google Patents

Abstract

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

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 nonalcoholic fatty liver disease.

Background

Abnormal fat metabolism can lead to a number of diseases, especially nonalcoholic fatty liver disease. The related genes capable of regulating and controlling lipid droplet aggregation are screened through a latest whole genome CRISPR-Cas9 knockout library screening system. At the same time, transcriptome level analysis was also performed on liver tissues of 7 non-alcoholic fatty liver patients and 9 normal controls. The 43 related genes are found to be 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 fatty liver samples and normal control samples, and the results showed that CYP46A1 expression was reduced in fatty liver. Meanwhile, CYP46A2 was disturbed and overexpressed in normal hepatocytes, and the results showed that non-fatty acid-induced lipid droplet aggregation was increased after the interference with CYP46A 1. Whereas overexpression of CYP46A1 results in reduced lipid droplet aggregation. Further, it was found that after CYP46A1 interference, normal hepatocyte electron microscopy showed abnormal mitochondrial morphology. Mitochondrial respiratory chain function detection revealed impairment of mitochondrial complex function following interference with CYP46 A1. These 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 common worldwide, especially in western countries. NAFLD ranges from fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) cirrhosis. Currently, accurate diagnosis of NASH still requires invasive, costly liver biopsies with potential complications. Furthermore, no effective therapy can reverse or even prevent the progression of the disease.

Therefore, in clinical studies, it is critical to identify and control key events of NAFLD progression. Pathogenic accumulation of lipid droplets within adipocytes is most likely due to increased free fatty acid intake and decreased lipoprotein output and decreased fatty acid oxidation. The pathways involved in the development of hepatic steatosis are intricate. Many studies have revealed a large number of genes associated with lipid droplet formation, including the use of proteomics, transcriptomics in hepatic lipid homeostasis and high throughput imaging-based methods. Recently, in Drosophila cells, some candidate genes were obtained by RNA interference (RNAi) whole genome screening, but a significant portion (about 40%) appeared to be free of human orthologs. The latest effective gene editing technology has been successfully applied to mammalian cells for functional studies of gene deletions. CRISPR/Cas9 is a DNA endonuclease that edits specific DNA sites complementary to guide RNAs. A whole genome-scale CRISPR-Cas9 knockout (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.

Because of the complexity of NAFLD, each patient may carry different inducers (diet, metabolic phenotype, genes, etc.), how to differentiate to different extents for different populations (with overweight, obesity, diabetes or other metabolic diseases) is a key element of future research, and this key element requires the use of systematic research approaches (integrated metabolomics, proteomics, phenotyping, etc.) to reveal molecular characteristics of NAFLD, which can facilitate biomarker discovery and help provide a more accurate therapeutic approach.

Therefore, developing an in vitro molecular diagnosis method for accurately diagnosing NAFLD is a technical problem to be solved 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 preparing a tool for diagnosing non-alcoholic fatty liver disease; the CYP46A1 gene is found to be remarkably reduced in the expression of the nonalcoholic fatty liver disease through research, so that the CYP46A1 gene can be used as a biomarker for diagnosing the nonalcoholic fatty liver disease, and a tool for diagnosing the nonalcoholic fatty liver disease can be developed.

The invention provides an application of a product for detecting CYP46A1 gene expression in preparing 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 in SEQ ID NO: 1.

Among them, sequence information of CYP46A1 gene (SEQ ID NO: 1) can be found in the International public nucleic acid sequence database GeneBank (NC_ 000014.9 (99684262.. 99727321));

SEQ ID NO：1：

ATGAGCCCCGGGCTGCTGCTGCTCGGCAGCGCCGTCCTGCTCGCCTTCGGCCTCTGCTGCACCTTCGTGCACCGCGCTCGCAGCCGCTACGAGCACATCCCCGGGCCGCCGCGGCCCAGTTTCCTTCTAGGACACCTCCCCTGCTTTTGGAAAAAGGATGAGGTTGGTGGCCGTGTGCTCCAAGATGTGTTTTTGGATTGGGCTAAGAAGTATGGACCTGTTGTGCGGGTCAACGTCTTCCACAAAACCTCAGTCATCGTCACGAGTCCTGAGTCGGTTAAGAAGTTCCTGATGTCAACCAAGTACAACAAGGACTCCAAGATGTACCGTGCGCTCCAGACTGTGTTTGGTGAGAGACTCTTCGGCCAAGGCTTGGTGTCCGAATGCAACTATGAGCGCTGGCACAAGCAGCGGAGAGTCATAGACCTGGCCTTCAGCCGGAGCTCCTTGGTTAGCTTAATGGAAACATTCAACGAGAAGGCTGAGCAGCTGGTGGAGATTCTAGAAGCCAAGGCAGATGGGCAGACCCCAGTGTCCATGCAGGACATGCTGACCTACACCGCCATGGACATCCTGGCCAAGGCAGCTTTTGGGATGGAGACCAGTATGCTGCTGGGTGCCCAGAAGCCTCTGTCCCAGGCAGTGAAACTTATGTTGGAGGGAATCACTGCGTCCCGCAACACTCTGGCAAAGTTCCTGCCAGGGAAGAGGAAGCAGCTCCGGGAGGTCCGGGAGAGCATTCGCTTCCTGCGCCAGGTGGGCAGGGACTGGGTCCAGCGCCGCCGGGAAGCCCTGAAGAGGGGCGAGGAGGTTCCTGCCGACATCCTCACACAGATTCTGAAAGCTGAAGAGGGAGCCCAGGACGACGAGGGTCTGCTGGACAACTTCGTCACCTTCTTCATTGCTGGTCACGAGACCTCTGCCAACCACTTGGCGTTCACAGTGATGGAGCTGTCTCGCCAGCCAGAGATCGTGGCAAGGCTGCAGGCCGAGGTGGATGAGGTCATTGGTTCTAAGAGGTACCTGGATTTCGAGGACCTGGGGAGACTGCAGTACCTGTCCCAGGTCCTCAAAGAGTCGCTGAGGCTGTACCCACCAGCATGGGGCACCTTTCGCCTGCTGGAAGAGGAGACCTTGATTGATGGGGTCAGAGTCCCCGGCAACACCCCGCTCTTGTTCAGCACCTATGTCATGGGGCGGATGGACACATACTTTGAGGACCCGCTGACTTTCAACCCCGATCGCTTCGGCCCTGGAGCACCCAAGCCACGGTTCACCTACTTCCCCTTCTCCCTGGGCCACCGCTCCTGCATCGGGCAGCAGTTTGCTCAGATGGAGGTGAAGGTGGTCATGGCAAAGCTGCTGCAGAGGCTGGAGTTCCGGCTGGTGCCCGGGCAGCGCTTCGGGCTGCAGGAGCAGGCCACACTCAAGCCACTGGACCCCGTGCTGTGCACCCTGCGGCCCCGCGGCTGGCAGCCCGCACCCCCACCACCCCCCTGCTGA。

the CYP46A1 gene expression product comprises CYP46A1 protein and partial peptide of the CYP46A1 protein; the partial peptide of the CYP46A1 protein contains a functional domain related to nonalcoholic fatty liver disease. The amino acid sequence of CYP46A1 protein is shown in SEQ ID NO:2 is shown in the figure;

SEQ ID NO：2：

MSPGLLLLGSAVLLAFGLCCTFVHRARSRYEHIPGPPRPSFLLGHLPCFWKKDEVGGRVLQDVFLDWAKKYGPVVRVNVFHKTSVIVTSPESVKKFLMSTKYNKDSKMYRALQTVFGERLFGQGLVSECNYERWHKQRRVIDLAFSRSSLVSLMETFNEKAEQLVEILEAKADGQTPVSMQDMLTYTAMDILAKAAFGMETSMLLGAQKPLSQAVKLMLEGITASRNTLAKFLPGKRKQLREVRESIRFLRQVGRDWVQRRREALKRGEEVPADILTQILKAEEGAQDDEGLLDNFVTFFIAGHETSANHLAFTVMELSRQPEIVARLQAEVDEVIGSKRYLDFEDLGRLQYLSQVLKESLRLYPPAWGTFRLLEEETLIDGVRVPGNTPLLFSTYVMGRMDTYFEDPLTFNPDRFGPGAPKPRFTYFPFSLGHRSCIGQQFAQMEVKVVMAKLLQRLEFRLVPGQRFGLQEQATLKPLDPVLCTLRPRGWQPAPPPPPC。

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

Wherein, the detection of CYP46A1 gene expression by using a high-throughput sequencing technology platform can also realize the detection of CYP46A1 gene expression condition. Along with the development and popularization of high-throughput sequencing technology, the work of constructing gene expression profiles for one person is more and more convenient; by comparing the gene expression profiles of the patient with the disease and the normal population, it is easy to analyze which gene abnormality has a correlation with the disease. Therefore, the analysis of the correlation between the expression level of the CYP46A1 gene and the nonalcoholic fatty liver disease in a high throughput sequencing platform also belongs to the application of the CYP46A1 gene product detection scheme, and is also within the protection scope of the invention.

Further, a product for diagnosing non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by using 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 nonalcoholic 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; products for diagnosing non-alcoholic fatty liver disease using in situ hybridization technology platforms to detect the expression level of the CYP46A1 gene include probes hybridized to the CYP46A1 gene nucleic acid sequence; products for diagnosing non-alcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene using a gene chip technology platform include probes hybridized with the nucleic acid sequence of the CYP46A1 gene; products for diagnosing non-alcoholic fatty liver disease using protein chip technology platforms to detect the expression level of the CYP46A1 gene include antibodies that specifically bind to the CYP46A1 protein; products for diagnosing nonalcoholic fatty liver disease using immunohistochemical technology platform to detect the expression level of the CYP46A1 gene include antibodies that specifically bind to 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 derivative; the length of the probe is not limited, and any length can be used as long as the probe can specifically hybridize and specifically bind to the target nucleotide sequence; the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the probe may be as long as 60, 80, 100, 150, 300 base pairs or more in length, even the entire gene; because different probe lengths have different effects on hybridization efficiency and signal specificity, the probe length is usually at least 14 base pairs, and the maximum length is usually not more than 30 base pairs, and the length complementary to the target nucleotide sequence is optimally 15-25 base pairs; the probe self-complementary sequence is preferably less than 4 base pairs to avoid affecting hybridization efficiency.

The protein chip can be used to detect the expression levels of a plurality of proteins including the CYP46A1 protein (e.g., a plurality of proteins associated with non-alcoholic fatty liver disease).

The specific antibody of the CYP46A1 protein comprises a monoclonal antibody and a polyclonal antibody; specific antibodies 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 is capable of retaining the binding capacity for the CYP46A1 protein, antibody preparation for the protein level is well known to those skilled in the art, and the invention can use any method to prepare the antibodies.

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

Further, the product for diagnosing the nonalcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene by using 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.

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 the CYP46A1 gene in a human sample.

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

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

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

the gene chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe comprises an oligonucleotide probe aiming at CYP46A1 genes and used for detecting the transcription level of the CYP46A1 genes; the protein chip comprises a solid phase carrier and a specific antibody of 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 transcript 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 of the CYP46A1 genes are respectively shown in SEQ ID NO.3, and the forward primer sequences are shown in SEQ ID NO. 4.

Compared with the prior art, the application of the product for detecting CYP46A1 gene expression in the preparation of the tool for diagnosing the nonalcoholic fatty liver disease has the following beneficial effects:

1. conventional non-alcoholic fatty liver disease diagnostic tools, such as imaging diagnostic tools for B-ultrasound, CT, MRI and the like, cannot reflect the presence or absence of inflammation and fibrosis and the cause of the fibrosis; liver biopsy is currently still the gold standard for non-alcoholic fatty liver disease diagnosis and its grading, but belongs to the invasive diagnosis. Compared with the method, the biomarker CYP46A1 gene for diagnosing the non-alcoholic fatty liver disease provides a basis for the preparation of a tool for noninvasively diagnosing the non-alcoholic fatty liver disease in vitro, and has the characteristics of high specificity and high sensitivity.

2. The in vitro diagnosis products comprising the primer, the probe, the reagent and the like for detecting the CYP46A1 gene expression level have the advantages of high accuracy, strong specificity, high sensitivity and the like, and are convenient to use clinically.

Drawings

FIG. 1 shows the immunohistochemical staining differences and CYP46A1 gene expression differences of 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 detection of the difference in CYP46A1 gene expression levels in non-alcoholic fatty liver disease and healthy control populations using real-time fluorescent quantitative PCR.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The experimental procedure, in which no specific conditions are noted in the examples, is generally followed by conventional conditions, such as the molecular cloning by Sambrook et al, the conditions described in the laboratory Manual (New York: cold Spring HarborLaboratory Press), or the conditions suggested by the manufacturer.

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

1. Clinical samples and patient data

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

Normal liver tissue samples of the control group were taken from patients receiving liver metastasis and hemangioma surgical resection. Two experienced pathologists read histological pictures of all liver specimens, they were unaware of all clinical data, and scored histological features using NAFLD Activity Scoring (NAS).

All patients obtained written informed consent. The study protocol was approved by the second national institutes of ethics committee of xiangya, university of south China.

2. RNA-Seq for liver transcriptomic analysis

Total RNA was extracted from liver tissue of patients with NAFLD by using Illustra RNAspin Mini Kit (GE Healthcare), and controls were performed. RNA samples were quantitated 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-ligation, 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) Lentivirus generation of sgRNA library

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

4. Mu.g of GeCKO library (# 1000000048, addgene), 2. Mu.g of pV-SVg (# 8454, addgene) and 6. Mu.g of psPAX2 (# 12260, addgene) 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 at 4℃in DMEM and split into aliquots, which were then stored at-80 ℃.

2) Lentiviral transduction of sgRNA libraries

Human hepatocyte strain L02 was obtained from the national academy of sciences typical culture collection and cultured in RPMI-1640 medium containing 20% FBS and 4mM L-glutamine.

GeCKO library viruses 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. Mu.g/mL Polybrene.

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

3) Sorting and DNA sequencing

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

Subsequently, lipid droplets were stained with BODIPY493/503 (staining. Sorting and collecting 10% of cells with high fluorescence intensity by flow cytometry. Genomic DNA was isolated from the sorted cells using Blood & Cell Culture DNA Midi Kit (Qiagen, germany.) PCR was performed in two steps according to the method described in Zhang Feng doctor.

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

Raw sequencing data was saved in FASTQ format. And analyzed using custom GeCKO screening software.

Briefly, the original sequencing was read and analyzed by using different barcodes in the reverse primer. The processed data were deleted from the beginning to the sequence of the sgRNA primer site primer. The collated data were compared to the GeCKO v2 library a and B data. The reads of sgrnas were quantified by model-based software of model-based whole genome CRISPR-Cas9 knockout (MAGeCK) v 5.6.0.

4) RNA interference

Both the CYP46A1 gene and the non-targeting oligonucleotide were obtained from GenePharma (Shanghai, china).

siRNA was transfected with Lipofectamine 2000 reagent (Invitrogen, USA) according to the experimental procedure provided by the manufacturer. After 48h of siRNA transfection, lipid droplets in the cells were stained and counted with BODIPY 493/503.

5) Transfection

CYP46A1-Flag plasmid was transfected with Lipofectin 2000 reagent.

6) BODIPY immobilization and immunofluorescence staining

Lipid droplets were stained by BODIPY according to the experimental procedure reported previously. After transfection, cells were washed with PBS and incubated at 37℃for 15 min 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 min and washed 3 more times with PBS. Photographs were taken using a confocal fluorescence image microscope (Leica, usa).

7) Immunohistochemistry

Paraffin-embedded sections (5 μm) of each tissue were stained using immunohistochemistry against CYP46A1 (Sigma, usa). Briefly, after dewaxing, 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 and then with primary antibodies. The sections were then incubated with biotinylated diabodies, avidin-biotin-peroxidase complex (Vector laboratories, U.S.) and 3, 3-diaminobenzidine in the presence of H2O 2. All stained sections were photographed under an optical microscope.

8) Mitochondrial isolation and detection of mitochondrial complex activity

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

Cells were then drawn 45 times through a 25g 5/8 needle, centrifuged at 1500g for 10min, and after centrifugation at 13,000g for 20 min, cytosolic extracts were recovered, precipitated as crude mitochondria, and the activity of the mitochondrial OXPHOS complex was determined using BCA assay protein concentration-Kit (Pierce, USA) using mitochondrial complex activity assay Kit (Abcam, UK) and MitoTox OXPHOS complex activity Kit (Abcam, UK) according to the instructions.

3. Results

The RNA-seq result shows that the relative expression level of mRNA of CYP46A1 gene in blood of healthy control population is 1, and the expression level of mRNA of CYP46A1 gene in blood of non-alcoholic fatty liver disease patient is 0.xx+/-0.xx. The above results indicate that the mRNA level of the CYP46A1 gene in blood of non-alcoholic fatty liver disease patients is significantly reduced compared to normal persons, and the difference is statistically significant (P < 0.05).

In particular, referring to FIGS. 1-2, FIG. 1 shows the difference in immunohistochemical staining of healthy control liver tissue and non-alcoholic fatty liver disease tissue samples and CYP46A1 gene expression; figure 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 of healthy control liver tissue and tissue samples of NAFLD, oil red O staining, lipid droplets were observed; part B is a statistic of the relative expression of CYP46A1, the relative levels of CYP46A1 shown by normalization to the control sample, with a statistical difference (< P0.01). After 48h of transfection, part A in FIG. 2, mitochondria were observed by electron microscopy to find fragmentation and swelling of the mitochondrial inner ridge; fraction B was detected as mitochondrial complex activity in CYP46A1 siRNA or control transfected cells, indicating a decrease in mitochondrial complex I and IV activity, citrate synthase was used as a control (< 0.01, < 0.05).

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

1. Study object:

65 patients with non-alcoholic fatty liver disease and 50 normal patients were selected according to the method of example 1.

2. Total RNA extraction from blood

Extraction of total blood RNA using the blood RNA extraction kit from Baitaike:

(1) 250 μl (or 0.25 g) of whole blood was applied to an RNase-Free filter column, centrifuged at 13000rpm for 2 minutes, the lower solution was collected, and 0.75ml of lysate RLS was added.

(2) The homogenized sample was mixed with vigorous shaking and incubated at 15-30℃for 5min to completely decompose the nucleoprotein.

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

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

(5) Centrifugation at 12,000rpm at 4℃for 10 minutes, the sample was divided into three layers: the lower organic phase, the middle and upper colorless aqueous phases, the RNA being present in the aqueous phase. The volume of the aqueous phase layer was about 60% of the volume of the RLS added, and the aqueous phase was transferred to a fresh tube for the next operation.

(6) 1 volume of 70% ethanol was added, mixed upside down (precipitation may occur at this time), and the resulting solution was transferred to the adsorption column RA (the adsorption column was housed in the collection tube) together with the possible precipitation.

(7) Centrifuge at 10,000rpm for 45 seconds, discard the waste liquid and re-insert the column into the collection tube.

(8) Mu.l of deproteinized solution RE was added, and the mixture was centrifuged at 12,000rpm for 45 seconds, and the waste solution was discarded.

(9) Mu.l of rinse RW was added and centrifuged at 12,000rpm for 60 seconds, and the waste liquid was discarded.

(10) Mu.l of rinse RW was added and centrifuged at 12,000rpm for 60 seconds, and the waste liquid was discarded.

(11) The adsorption column RA was put back into the empty collection tube, centrifuged at 12,000rpm for 2 minutes, and the rinse solution was removed as much as possible to prevent the residual ethanol in the rinse solution from inhibiting downstream reactions.

(12) Taking out the adsorption column RA, placing into a centrifuge tube without RNase, adding 50-80 μl RNase-free water at the middle part of the adsorption membrane according to the expected RNA yield, standing at room temperature for 2 min, centrifuging at 12,000rpm for 1 min, and collecting eluate.

3. Measurement of Total RNA concentration and purity

The concentration and purity of the sample RNA were measured using a NanoVue Plus instrument.

4. Reverse transcription synthesis of mRNA cDNA

Mu.g of total RNA was reverse transcribed with reverse transcription buffer to cDNA. Using a 25. Mu.l reaction system, 1. Mu.g total RNA was used as template RNA for each sample, and the following were added to the PCR tube: DEPC water, 5 Xreverse transcription buffer, 10mmol/L dNTPs, 0.1mmol/L DTT, 30. Mu. Mmol/L Oligo dT, 200U/. Mu. L M-MLV, template RNA. Incubate at 42℃for 1h, at 72℃for 10min, briefly centrifuge.

5、QPCR

(1) Primer design

QPCR amplification primers are designed according to the coding sequences of CYP46A1 gene and GAPDH reference gene in Genbank and are synthesized by Shanghai Bioengineering technical service Co. The specific primer sequences are as follows:

CYP46A1 gene:

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

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

GAPDH reference gene:

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

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

(2) Amplification of

The reaction system: 25 μl of reaction system, 3 parallel tubes were set per sample. The following reaction system was prepared: 12.5. Mu.l of SYBR Green polymerase chain reaction system, 1. Mu.l of forward primer (5. Mu.M), 1. Mu.l of reverse primer (5. Mu.M), 2.0. Mu.l of template cDNA and 8.5. Mu.l of enzyme-free water; each operation was performed on ice.

Amplification procedure: 95 ℃ for 5min, (95 ℃ for 5s,60 ℃ for 60 s) 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 by a delta CT method.

6. Statistical method

Experiments were performed in 3 replicates, and the data obtained were expressed as mean ± standard deviation, and were statistically analyzed using SPSS13.0 statistical software, and differences between the different groups were determined using t-test, and were considered 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 healthy Control population (Control), and the difference was statistically significant (P < 0.05), resulting in the same RNA-seq experiment as described above.

The application of the product for detecting CYP46A1 gene expression in the preparation of the tool for diagnosing the nonalcoholic fatty liver disease has the following beneficial effects:

1. conventional non-alcoholic fatty liver disease diagnostic tools, such as imaging diagnostic tools for B-ultrasound, CT, MRI and the like, cannot reflect the presence or absence of inflammation and fibrosis and the cause of the fibrosis; liver biopsy is currently still the gold standard for non-alcoholic fatty liver disease diagnosis and its grading, but belongs to the invasive diagnosis. Compared with the method, the biomarker CYP46A1 gene for diagnosing the non-alcoholic fatty liver disease provides a basis for the preparation of a tool for noninvasively diagnosing the non-alcoholic fatty liver disease in vitro, and has the characteristics of high specificity and high sensitivity.

2. The in vitro diagnosis products comprising the primer, the probe, the reagent and the like for detecting the CYP46A1 gene expression level have the advantages of high accuracy, strong specificity, high sensitivity and the like, and are convenient to use clinically.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent processes or direct or indirect applications in other related arts using the present invention are included in the scope of the present invention.

Sequence listing

<110> Xiangya two hospitals at university of south China

<120> application of product for detecting CYP46A1 gene expression in preparation of tool for diagnosing non-alcoholic fatty liver disease

<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 Ser Val 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 (6)

1. An application of a product for detecting the expression level of CYP46A1 gene in preparing a tool for diagnosing nonalcoholic fatty liver disease, which is characterized in that the sequence of the CYP46A1 gene is shown in SEQ ID NO: 1.

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

3. The use according to claim 2, wherein the product for diagnosing nonalcoholic fatty liver disease by detecting the expression level of the CYP46A1 gene using 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 nonalcoholic 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;

products for diagnosing non-alcoholic fatty liver disease using in situ hybridization technology platforms to detect the expression level of the CYP46A1 gene include probes hybridized to the CYP46A1 gene nucleic acid sequence;

products for diagnosing nonalcoholic fatty liver disease using gene chip technology platform to detect the expression level of the CYP46A1 gene include probes that hybridize to the nucleic acid sequence of the CYP46A1 gene.

4. The use according to claim 1, wherein the tool comprises a kit, a gene chip or a high throughput sequencing technology platform.

5. The use according to claim 4, wherein the kit comprises reagents for detecting the level of CYP46A1 gene transcription;

the gene chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe comprises an oligonucleotide probe aiming at CYP46A1 genes and used for detecting the transcription level of the CYP46A1 genes;

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

6. The use according to claim 5, wherein the reagent for detecting the transcription level of the CYP46A1 gene comprises primers and probes for the CYP46A1 gene.