阅读说明：本技术 一种非酒精性脂肪性肝炎的miRNA标志物的制备方法 (Preparation method of miRNA marker of non-alcoholic steatohepatitis ) 是由 梁廷明 沈璐璐 马敏娟 段瑞 贾琳 于 2020-12-01 设计创作，主要内容包括：一种非酒精性脂肪性肝炎的miRNA标志物的制备方法,本发明涉及非酒精性脂肪性肝炎研究技术领域,从GEO数据库中筛选出2个符合条件的人NASH相关模型miRNA测序数据集和7个符合条件的小鼠NASH相关模型的miRNA测序数据集,下载这些数据集然后分别进行miRNA差异表达分析并取交集；构建蛋氨酸胆碱缺乏饮食诱导的小鼠NASH模型,并记录小鼠体重；小鼠肝组织总RNA进行反转录；将得到的反转录产物为模板,检测得到的人和小鼠NASH中都差异表达的miRNA；将miRNA表达结果进行比对,选取表达趋势一致,且表达差异最明显的2种miRNA作为判断非酒精性脂肪性肝炎的标志物。(The invention relates to a preparation method of miRNA markers of non-alcoholic steatohepatitis, in particular to a preparation method of miRNA markers of non-alcoholic steatohepatitis, which comprises the steps of screening 2 qualified miRNA sequencing data sets of a human NASH-related model and 7 qualified miRNA sequencing data sets of a mouse NASH-related model from a GEO database, downloading the data sets, respectively carrying out miRNA differential expression analysis, and taking an intersection; constructing a methionine choline deficiency diet induced mouse NASH model, and recording the weight of the mouse; carrying out reverse transcription on the total RNA of the liver tissue of the mouse; detecting miRNAs which are differentially expressed in the NASH of the obtained human and the NASH of the mouse by taking the obtained reverse transcription product as a template; comparing the miRNA expression results, and selecting 2 miRNAs with consistent expression trends and most obvious expression differences as markers for judging the non-alcoholic steatohepatitis.)

1. A preparation method of miRNA markers of non-alcoholic steatohepatitis is characterized by comprising the following steps: the operation steps are as follows:

screening 2 qualified human nonalcoholic steatohepatitis from a GEO database of NCBI (national center of health), namely, miRNA sequencing data sets of a NASH (national advanced standards institute) related model and 7 miRNA sequencing data sets of a mouse NASH related model meeting the conditions, downloading the data sets, respectively carrying out miRNA differential expression analysis, and taking intersection;

step two, respectively carrying out differential miRNA expression analysis on the 9 miRNA sequencing data sets obtained in the step one to obtain 9 groups of differential expression miRNAs; taking the intersection of 9 groups of differential expression miRNAs to obtain miRNAs which are differentially expressed in human and mouse NASH;

constructing a methionine choline deficiency diet induced mouse NASH model according to literature data, and recording the weight of the mouse; after the NASH model of the mouse is successfully constructed, blood is taken from eyeballs of the mouse, the mouse is killed by a cervical vertebra dislocation method, a tissue sample is dissected and taken, one part of the tissue sample is fixed by formaldehyde, and the other part of the tissue sample is stored in a refrigerator at the temperature of-80 ℃ for standby; photographing the liver of the mouse during dissection;

step four, detecting triglyceride and total cholesterol in the serum obtained in the step three to obtain serological indexes in the mouse NASH model, and confirming that the mouse model is successfully constructed again;

fixing a part of the mouse liver tissue obtained in the step (III) with formaldehyde to perform hematoxylin eosin staining and oil red staining, and detecting the lipid accumulation condition in the liver of the NASH model mouse;

step six, storing a part of the liver tissues of the mice obtained in the step three in a refrigerator at-80 ℃ for later use, and respectively extracting total RNA;

step seven, carrying out reverse transcription on the mouse liver tissue total RNA obtained in the step six;

step (eight), the reverse transcription product obtained in the step (seven) is used as a template, and miRNA which is differentially expressed in the NASH of the human and the mouse obtained in the step (two) is detected;

comparing the miRNA expression result obtained in the step (eighth) with the expression result obtained in the step (second), and selecting 2 miRNAs with consistent expression trends and most obvious expression differences as markers for judging the non-alcoholic steatohepatitis.

2. The method for preparing the miRNA marker for nonalcoholic steatohepatitis according to claim 1, wherein the miRNA marker comprises: the NASH miRNA sequencing data set in the step (one) is obtained by screening conditions from a data set searched in a GEO database, wherein the screening conditions are as follows: HFD, NAFLD, NASH, miRNA, tissue; each data set contains a minimum of 5 sample normal groups and 5 sample treatment groups; each dataset has a data matrix with row names for miRNA and column names for sample names.

3. The method for preparing the miRNA marker for nonalcoholic steatohepatitis according to claim 1, wherein the miRNA marker comprises: in the step (two), the obtained 9 miRNA sequencing data sets are subjected to differential miRNA expression analysis, the limma package in the R language is used for performing differential miRNA expression data analysis, and the threshold conditions of the differential miRNA expression analysis are set as follows: | log₂FC|>1, and FDR<0.05; FC is difference multiple, FDR is false discovery rate; the genes satisfying the threshold condition are differentially expressed genes; 9 groups of miRNA with differential expression are respectively obtained.

4. The method for preparing the miRNA marker for nonalcoholic steatohepatitis according to claim 1, wherein the miRNA marker comprises: the miRNAs which are obtained in the step (II) and are differentially expressed in the human and mouse NASH comprise miR-210, miR-222, miR-301a-3p and miR-532-5 p.

5. The method for preparing the miRNA marker for nonalcoholic steatohepatitis according to claim 1, wherein the miRNA marker comprises: the mouse NASH model in the step (three) is constructed by MCD diet induction, the period is 2-3 weeks, and the used mouse is a C57BL/6J mouse of 6-8 weeks; the blood sample is obtained by blood sampling of mouse eyeball and is divided into two parts, wherein one part is used for detecting triglyceride TG, total cholesterol content TC, aspartate aminotransferase and alanine aminotransferase content in the blood sample; the other was used to detect miRNA in the blood sample.

6. The method for preparing the miRNA marker for nonalcoholic steatohepatitis according to claim 1, wherein the miRNA marker comprises: the expression trend of the miRNA detected in the step (nine) in the NASH mouse model liver is consistent with the expression trend of the miRNA differentially expressed in the human and mouse NASH obtained in the step (two), and the miRNA are miR-301a-3p and miR-532-5 p.

Technical Field

The invention relates to the technical field of research on non-alcoholic steatohepatitis, and in particular relates to a preparation method of a miRNA marker of non-alcoholic steatohepatitis.

Background

Non-alcoholic steatohepatitis (NASH) is an extreme development form of non-alcoholic steatohepatitis (NAFLD), defined as the occurrence of steatosis accompanied by inflammation and hepatocyte injury, and mainly shows that liver fat accumulation of people without alcoholism causes inflammation and fibrosis, and part of patients finally develop into liver cirrhosis and even liver cancer. The incidence of NAFLD is increasing at home and abroad, and has become a major problem threatening public health on a global scale. The highest prevalence rate of NAFLD of Chinese adult population is 27%, and NAFLD has replaced chronic hepatitis B and becomes the first chronic liver disease in China.

NASH involves abnormalities in many aspects, such as nutritional metabolism, immune inflammation, genetic and cellular stress repair. Currently, NAFLD's pathogenesis is widely accepted as "multiple hits", with insulin resistance and intracellular lipid accumulation as the first hit, followed by triggering of a series of cytotoxic events, such as secondary inflammatory responses, oxidative stress, endoplasmic reticulum stress, cell death, etc. as the second hit. Although the widely recognized "multiple warfare theory" can partially reveal the pathogenesis of NAFLD, its etiology and pathogenesis are very complex and have not yet been fully elucidated. NASH histologically exhibits lobular inflammation and hepatocellular ballooning characteristics with a faster rate of fibrosis progression than NAFLD. The diagnosis of the liver fatty degeneration NAFLD requires the evidence of histology or image technology to show the liver cell fatty degeneration, namely, the histology examination shows the liver cell fatty degeneration of more than or equal to 5 percent or the Proton Density Fat Fraction (PDFF) measured by the magnetic resonance technology is more than or equal to 5.5 percent. micrornas (mirnas) are involved in the development of NAFLD through various pathways, such as regulation of carbohydrate metabolism, lipid catabolism in hepatocytes, and the like. Since miRNAs in circulation are very stable, with a high degree of reproducibility and consistency in plasma or serum, they are ideal substitutes for liver damage and liver damage indicative of different liver diseases. The strong heterogeneity of NASH may be the main reason for the low and inconsistent diagnostic efficacy of single gene markers, and the diagnostic efficacy is expected to be improved by multigene combination. Genetic, epigenetic and omics approaches not only reveal the mechanisms by which NAFLD develops, but also provide many potential new markers. New markers and combinations need to be explored continuously and large-scale verification of existing markers or marker combinations is performed. The multiple non-coding RNA genes are carried out on the NASH marker, and particularly the combined analysis research of miRNA is particularly important in combination with animal experiment verification from the perspective of omics.

Disclosure of Invention

The invention aims to provide a preparation method of a miRNA marker of non-alcoholic steatohepatitis with reasonable design aiming at the defects and shortcomings of the prior art, provides a new method for developing non-alcoholic steatohepatitis clinically on a molecular level, and provides a new drug target for treating non-alcoholic steatohepatitis.

In order to achieve the purpose, the invention adopts the following technical scheme: the operation steps are as follows:

screening 2 qualified miRNA sequencing data sets of a human nonalcoholic steatohepatitis (NASH) related model and 7 qualified miRNA sequencing data sets of a mouse NASH related model from a Gene Expression Omnibus (GEO) database of a National Center for Biotechnology Information (NCBI), downloading the data sets, performing miRNA differential Expression analysis respectively, and taking intersection;

step two, respectively carrying out expression analysis of differential miRNA on the 9 miRNA sequencing data sets obtained in the step one to obtain 9 groups of differential expression miRNA; taking the intersection of 9 groups of differential expression miRNAs to obtain miRNAs which are differentially expressed in human and mouse NASH;

step three, constructing a mouse NASH model induced by Methionine Choline deficiency (Methionine and Choline deficiency L-Amino Acid Diet, MCD) Diet according to literature data, and recording the weight of the mouse; after the NASH model of the mouse is successfully constructed, blood is taken from eyeballs of the mouse, the mouse is killed by a cervical vertebra dislocation method, a tissue sample is dissected and taken, one part of the tissue sample is fixed by formaldehyde, and the other part of the tissue sample is stored in a refrigerator at the temperature of-80 ℃ for standby; photographing the liver of the mouse during dissection;

step four, detecting triglyceride and total cholesterol in the serum obtained in the step three to obtain serological indexes in the mouse NASH model, and confirming the success of the mouse model construction again;

fifthly, fixing a part of the liver tissues of the mice obtained in the third step by formaldehyde for Hematoxylin and Eosin (HE) staining and oil red staining, and detecting the lipid accumulation condition in the liver of the NASH model mouse;

sixthly, storing a part of the liver tissues of the mice obtained in the third step in a refrigerator at-80 ℃ for later use, and respectively extracting total RNA;

seventhly, carrying out reverse transcription on the mouse liver tissue total RNA obtained in the sixth step;

step eight, detecting miRNA differentially expressed in the NASH of the human and the mouse obtained in the step two by taking the reverse transcription product obtained in the step seven as a template;

step nine, comparing the miRNA expression result obtained in the step eight with the expression result obtained in the step two, and selecting 2 miRNAs with consistent expression trends and most obvious expression differences as markers for judging the non-alcoholic steatohepatitis.

Further, the NASH miRNA sequencing dataset described in the first step is a dataset searched from a GEO database, and is obtained by screening conditions, where the screening conditions are as follows: HFD, NAFLD, NASH, miRNA, tissue; each data set contains a minimum of 5 sample normal groups and 5 sample treatment groups; each dataset has a data matrix with row names for miRNA and column names for sample names.

Further, in the second step, the obtained 9 miRNA sequencing data sets are respectively subjected to differential miRNA expression analysis, the differential miRNA expression data analysis is performed by using limma package in the R language, and the threshold conditions of the differential miRNA expression analysis are set as: | log₂FC|>1, and FDR<0.05; FC is difference multiple, FDR is false discovery rate; the genes satisfying the threshold condition are differentially expressed genes; 9 groups of miRNA with differential expression are respectively obtained.

Further, the miRNAs which are obtained in the second step and are differentially expressed in both human and mouse NASH comprise miR-210, miR-222, miR-301a-3p and miR-532-5 p.

Further, the mouse NASH model in the third step is constructed by MCD diet induction, the period is 2-3 weeks, and the used mouse is C57BL/6J mouse of 6-8 weeks; the blood sample is obtained by blood sampling of mouse eyeball and is divided into two parts, wherein one part is used for detecting triglyceride TG, total cholesterol content TC, aspartate aminotransferase and alanine aminotransferase content in the blood sample; the other was used to detect miRNA in the blood sample.

Further, the expression trend of the miRNA detected in the ninth step in the NASH mouse model liver is consistent with the expression trend of the miRNA differentially expressed in the human and mouse NASH obtained in the second step, and the miRNA are miR-301a-3p and miR-532-5 p.

After the method is adopted, the invention has the beneficial effects that: the invention provides a preparation method of a miRNA marker of non-alcoholic steatohepatitis, provides a new method for developing non-alcoholic steatohepatitis clinically on a molecular level, and provides a new drug target for treating non-alcoholic steatohepatitis.

Description of the drawings:

FIG. 1 is an anatomical diagram of a NASH model mouse of the present invention (normal mouse on the left, NASH model mouse on the right).

FIG. 2 is a graph showing the measurement of ALT and AST contents in serum of mice of the NASH model of the present invention.

FIG. 3 is a photograph of the oil red staining and HE staining of liver of mice of the NASH model of the present invention.

FIG. 4 is a graph of the differential expression of miRNA in liver of mice model NASH screened for both human and mouse according to the present invention.

FIG. 5 is a table of the NASH-associated model human and mouse miRNA sequencing datasets used in the present invention.

The specific implementation mode is as follows:

the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The specific implementation mode adopts the following technical scheme: the operation steps are as follows:

step one, screening 2 qualified miRNA sequencing data sets of a human nonalcoholic steatohepatitis (NASH) related model from a GEO database (https:// www.ncbi.nlm.nih.gov/gds) of NCBI (conditions: species are human, samples are human liver tissues, the data types are miRNA high-throughput sequencing data, and normal samples and NASH related disease matched samples) and 7 qualified miRNA sequencing data sets of a mouse NASH related model (conditions: species are mouse, samples are mouse liver tissues, the data types are high-throughput sequencing data, and contain normal samples and NASH related model matched samples), and downloading the data sets (9 data sets are shown in figure 5);

step two, respectively carrying out expression analysis of differential miRNA on the 9 miRNA sequencing data sets obtained in the step one, wherein the method is a limma package in an R language, and screening to obtain 9 groups of differential expression miRNA; taking the intersection of 9 groups of miRNAs with differential expression to obtain miRNAs (including miR-210, miR-222, miR-301a-3p and miR-532-5p) with differential expression in both human NASH and mouse NASH; the threshold conditions for the differential gene expression analysis were set as follows: | log₂FC|>1, and FDR<0.05; FC is difference multiple, FDR is false discovery rate; the genes satisfying the threshold condition are differentially expressed genes;

step three, according to literature data, constructing a Methionine Choline deficiency (Methionine and Choline deficiency L-Amino Acid Diet, MCD) Diet-induced mouse NASH model, wherein 20 mice C57BL/6J with 6-8 weeks are used, 10 mice are used as controls, 10 mice are used for constructing the NASH model, the mice are raised in SPF-level animal centers, the weight of the mice is recorded every 3 days, MCD feed used by the model mice is purchased from Nantong Telofey feed science and technology limited company, and the model construction time is 2-3 weeks; after the NASH model of the mouse is successfully constructed, blood is taken from eyeballs of the mouse, the mouse is killed by a cervical vertebra dislocation method, tissue sampling is carried out through dissection, one part of the tissue sampling is fixed by 4% formaldehyde, and the other part of the tissue sampling is stored in a refrigerator at the temperature of-80 ℃ for standby; photographing mouse liver when dissecting (as figure 1);

step four, placing the serum obtained in the step three in a refrigerator at 4 ℃ for overnight, then detecting the contents of triglyceride TG, total cholesterol TC, aspartate aminotransferase and alanine aminotransferase to obtain a serological index (shown as a figure 2) in the mouse NASH model, and confirming the success of the mouse model construction again; in the part, a triglyceride determination kit and a total cholesterol determination kit for determining the blood index detection of a mouse NASH model, an aspartate aminotransferase (glutamic-oxaloacetic transaminase/AST) test box and an alanine aminotransferase (glutamic-pyruvic transaminase ALT) test box are purchased from Nanjing to build bioengineering Co., Ltd;

fifthly, performing Hematoxylin and Eosin (HE) staining and oil red staining on a part of the mouse liver tissues fixed by 4% formaldehyde to detect the lipid accumulation condition in the liver of the NASH model mouse (as shown in figure 3);

step six, storing the other part of the liver tissues of the mice obtained in the step three in a refrigerator at the temperature of-80 ℃ for later use, and respectively extracting the total RNA of the liver tissues by using a Trizol reagent purchased from Bao bioengineering (Dalian) Co., Ltd;

seventhly, carrying out reverse transcription on the mouse liver tissue total RNA obtained in the sixth step; the reverse transcription of the step is carried out by using a miRNA 1st Strand CdnaSynthesis Kit (by stem-loop) reverse transcription Kit purchased from Nanjing Novowed Biotechnology GmbH, and operating according to the Kit instruction; the miRNA reverse transcription primer is synthesized by Shanghai Czeri biological company;

step eight, detecting miRNAs (mmu-miR-122(has-miR-122), mmu-miR-210(has-miR-210), mmu-miR-222(has-miR-222), mmu-miR-301a (has-miR-301a-3p) and mmu-miR-532-5p (has-miR-532-5p) which are differentially expressed in the NASH of the human and the mouse obtained in the step two by taking the reverse transcription product obtained in the step seven as a template; carrying out qRT-PCR detection on related miRNA; the used qRT-PCR kit miRNA Universal SYBR Qpcr Master Mix was purchased from Nanjing Novowed Biotech Co., Ltd, and U6 snRNA is used as an internal reference during detection, and the expression quantity of the screened miRNA is detected (as shown in figure 4).

Step nine, enabling the expression trend of the miRNA detected in the step eight in the NASH mouse model liver to be consistent with the expression trend of the miRNA differentially expressed in the human and mouse NASH obtained in the step two; the miRNA is miR-301a-3p and miR-532-5p, and the two miRNAs can be used as markers for judging the non-alcoholic steatohepatitis.

After the method is adopted, the beneficial effects of the embodiment are as follows: the invention provides a preparation method of miRNA markers of non-alcoholic steatohepatitis, and the miRNA markers miR-301a-3p and miR-532-5p are found to be used for judging the progress and severity of the non-alcoholic steatohepatitis for the first time; tests prove that miR-301a-3p and miR-532-5p can effectively distinguish a non-alcoholic steatohepatitis sample from a normal sample; on the basis, miR-301a-3p and miR-532-5p can also be used for preparing medicines for inhibiting non-alcoholic steatohepatitis, so that a new method is provided for developing the non-alcoholic steatohepatitis clinically on a molecular level, and a new medicine target is provided for treating the non-alcoholic steatohepatitis.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.