CN112941150A

CN112941150A - Method for screening activity index of ulcerative colitis disease

Info

Publication number: CN112941150A
Application number: CN202110322018.3A
Authority: CN
Inventors: 夏先明; 沈兵; 宣佶; 魏娟; 黄媛媛; 李静
Original assignee: Anhui Medical University
Current assignee: Anhui Medical University
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2021-06-11

Abstract

The invention discloses a method for screening an ulcerative colitis disease activity index, which adopts an isolated ulcerative colitis colon biopsy sample and a cancer tissue beside a colon cancer operation patient to carry out transcriptome sequencing analysis and screen genes which are not reported by domestic and foreign documents and are related to the ulcerative colitis. Thirdly, verifying by using a colon biopsy sample of ulcerative colitis and the tissues beside the cancer of a colon cancer operation patient by using real-time fluorescent quantitative RT-PCR; and verifying the protein expression level of the gene codes screened by the transcriptome sequencing by using an immunoblotting method, and finally screening the genes with consistent expression. And finally, carrying out clinical verification and screening out the index of the activity degree of the ulcerative colitis. The invention overcomes the defects of the traditional method that documents are searched, genes are screened and then the genes are verified step by step, and the method is also suitable for screening the activity indexes of diseases such as Crohn's disease, eosinophilic gastroenteritis and the like and the stage indexes of diseases such as polyp, tumor and the like in colon parts.

Description

Method for screening activity index of ulcerative colitis disease

Technical Field

The invention relates to the field of transcriptome sequencing of colon tissues, in particular to a method for screening an activity index of ulcerative colitis.

Background

Ulcerative colitis is chronic inflammation and ulcerative lesion of large intestinal mucosa with unknown etiology, is better in young people of 20-40 years old, and is clinically manifested by chronic diarrhea with mucopurulent bloody stool, which seriously affects the life quality and labor capacity of patients. Patients with severe ulcerative colitis may develop toxic megacolon, intestinal perforation and large intestinal hemorrhage, which endangers the life of the patient. The incidence of ulcerative colitis in China tends to increase year by year, the pathogenesis of ulcerative colitis is not completely clarified, and the ulcerative colitis is probably caused by the combined action of factors such as environment, heredity, immunity and the like. The damage of the barrier function of the colonic epithelial cells is probably an important link for the occurrence and the development of the ulcerative colitis, the permeability of the intestinal mucosa is increased, and bacteria, toxins and macromolecular substances easily pass through the mucosa and enter the submucosa, so that the local immune disorder is caused, and the pathological damage of the colonic epithelial cells is aggravated. There are many factors that cause the barrier function of the intestinal mucosa to be damaged, including inflammatory factors, integrin families, calcium channel regulatory proteins, intestinal bacteria, butyrate, zinc and the like. Whether the factors are related to the activity of the ulcerative colitis disease or not can be used as an index for clinically evaluating the activity of the disease or as a basis for clinical differential diagnosis, and further evidence is lacked.

Transcriptome sequencing differential analysis and functional enrichment analysis can comprehensively and systematically analyze differentially expressed genes in the disease progression process and the relationship between the differentially expressed genes and related signal paths. Through transcriptome sequencing differential analysis and functional enrichment analysis of pathological specimens and normal controls and comparison of databases, it is possible to find some differentially expressed genes and signal transduction pathways thereof which are not reported in literatures but are closely related to diseases, and the method has important clinical significance for discussion of clinical disease pathogenesis and evaluation of disease severity.

Disclosure of Invention

In order to achieve the above purpose, the invention adopts the technical scheme that:

the invention aims to provide a method for screening an activity index of ulcerative colitis, which has simple experimental process and accurate and effective experimental result.

The technical scheme for realizing the aim of the invention is as follows:

a method for screening an activity index of ulcerative colitis disease comprises the following specific steps:

1) collecting in-vitro colon biopsy specimens of ulcerative colitis patients and tissues beside carcinoma of colon cancer patients, soaking in RNA sample preservation solution for 5 minutes, and then preserving in liquid nitrogen;

2) in vitro ulcerative colitis colon biopsy samples and tissues beside cancer excised from colon cancer surgical patients are adopted, 6 cases of each group are extracted, tissue mRNA is extracted for reverse transcription, and transcriptome sequencing analysis is carried out. Respectively screening the first 20 genes of each group according to FC value ascending and descending sorting; further screening new genes which are not reported by domestic and foreign documents and are related to the ulcerative colitis;

3) adopting isolated ulcerative colitis colon biopsy samples and the para-cancer tissues excised by colon cancer operation patients, extracting colon tissue mRNA for reverse transcription in 20 cases of each group, and verifying the transcription difference of the screened genes by transcriptome sequencing by using real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction); extracting colon tissue protein, verifying protein expression level of screened gene codes by transcriptome sequencing by using an immunoblotting method, and finally screening out genes with consistent expression;

4) and screening genes with consistent expression, and performing clinical verification. 50 colon biopsy samples of patients with isolated ulcerative colitis are collected, the protein level coded by the genes is detected by an immunoblotting method, and the protein level is related to the disease activity of the patients, and finally, the index of the activity of the ulcerative colitis is screened out.

Furthermore, the genes screened in the step two are not reported to be related to the ulcerative colitis by domestic and foreign documents: ASS1, CAPRIN 1.

The present invention provides Argininosuccinate Synthase 1 (ASS 1), which is an index of the activity of ulcerative colitis screened by the above method.

The invention has the beneficial effects that:

the index for judging the activity degree of the ulcerative colitis screened by the method of the invention is as follows: argininosuccinate Synthase 1 (ASS 1) has strong positive linear correlation with the activity of ulcerative colitis Mayo score, indicating that ASS1 can be used as an index for judging the activity of ulcerative colitis.

The invention overcomes the defects of slow speed and insufficient novelty of the traditional method that documents are searched, genes are screened and then the genes are verified step by step. The method is also suitable for screening disease activity indexes such as Crohn's disease, eosinophilic gastroenteritis and the like, and screening disease stage indexes such as colon polyp, tumor and the like.

Drawings

FIG. 1 is a transcriptome sequencing gene expression descending map (first 20);

FIG. 2 is a Western immunoblot of ASS 1;

figure 3 is a western blot of CAPRIN 1.

Wherein PC is the abbreviation of paracancerous tisssues, namely the tissue beside cancer; UC is short for an ulcerogenic colitis, i.e. ulcerative colitis.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

1) Preparation of Experimental materials

RNA/protein sample preservation solution (Biyuntian Biotechnology Co., Ltd.), cryovial (Corning), RNA extraction kit (TRizol), reverse transcription kit (Qiagen), and real-time quantitative PCR kit (Qiagen). Total protein extraction kit (sermeranfo), protein primary antibody (sermeranfo), and secondary antibody (shanghai anabolic).

2) Sample retention method for ulcerative colitis patient

When enteroscopy is performed on patients with ulcerative colitis, 3 tissues at the lesion position are taken by biopsy forceps and placed in a freezing storage tube (containing RNA/protein sample storage solution), the frozen tissue is placed in a liquid nitrogen tank for quick freezing within 15 minutes, and the frozen tissue is placed in a refrigerator at the temperature of-80 ℃ for storage within one week and used for transcriptome sequencing, real-time quantitative RT-PCR verification and western blot detection.

3) Method for retaining tissue sample beside carcinoma of colon cancer patient

Tissue excised in colon cancer operation is separated for 15 min, a small amount of para-cancer tissue (about 100 mg) is obtained by an operating scissors and is placed in a freezing storage tube (containing RNA/protein sample preservation solution), the para-cancer tissue is placed in a liquid nitrogen tank for quick freezing within 15 min, and the para-cancer tissue is placed in a refrigerator at minus 80 ℃ for storage within one week and is used for transcriptome sequencing, real-time quantitative RT-PCR verification and western blot detection.

4) Tissue RNA extraction, reverse transcription, transcriptome sequencing analysis

Extracting colon tissue RNA by an RNA extraction kit (TRizol), then carrying out reverse transcription by using a reverse transcription kit (Qiagen), carrying out transcriptome sequencing by using an illumina NovaSeq 6000 sequencer, respectively sorting the upper 20 genes of each group according to the ascending and descending order of an FC value; and further screening new genes ASS1 and CAPRIN1 (shown in figure 1) which are not reported to be related to ulcerative colitis by domestic and foreign literatures.

5) Tissue RNA extraction, real-time fluorescence quantitative RT-PCR verification

20 colon tissue mRNA is extracted from 20 colon tissue biopsy samples of ulcerative colitis and cancer tissues of colon cancer operation patients, reverse transcription is carried out, real-time fluorescence quantitative RT-PCR is used for detecting the transcription difference of ASS1 and CAPRIN1 genes of two groups of samples, and the result shows that the mRNA expression difference of ASS1 and CAPRIN1 of the two groups of samples is obvious (see table 1).

TABLE 1 ulcerative colitis biopsy and cancer-associated tissue Gene expression Difference (chi. + -.s) in Colon cancer surgery

6) Extracting total protein of tissue, and verifying by immunoblotting method

Adopting 20-25 cases of colon tissue biopsy samples of ulcerative colitis and paracarcinoma tissues of colon cancer operation patients, extracting total protein of colon tissues, performing electrophoresis and membrane transformation, and adding a protein primary antibody (Samerafei) and a secondary antibody (Shanghai Kangsheng organisms). The difference between the protein levels of ASS1 and CAPRIN1 in the two groups of samples was observed, and as a result, the difference between the protein expression of ASS1 in the two groups was found to be significant, while the protein expression of CAPRIN1 in the two groups was not significant (see Table 2, FIG. 2, and FIG. 3).

TABLE 2 difference in expression of proteins in tissue adjacent to ulcerative colitis biopsy and colon cancer surgery (chi. + -.s)

7) Screening expression consistent gene for clinical verification

50 colon biopsy specimens of patients with ulcerative colitis were collected and subjected to immunoblotting to detect ASS1 protein levels and to perform correlation analysis with the disease activity of the patients. The ulcerative colitis activity Mayo score includes (a) stool frequency: 0 is normal, 1 is 1-2 times more stool than normal per day; 2, the stool is 3 to 4 times more than normal every day; (b) rectal bleeding: 0-none, 1-half of the time that stool is visible with blood, 2-half or more of the time that stool is visible with blood, 3-continuous stool with blood or bloody stool; (c) endoscopic mucosal appearance: 0-normal or disease-inactive, 1-mild disease (erythema, diminished vascular morphology, mild fragility), 2-moderate disease (marked erythema, avascular morphology, fragility, erosion), 3-severe disease (spontaneous bleeding, ulceration); (d) physician scoring of disease activity: normal 0, mild 1 and moderate 2. The sum of the total points is <2 points for symptom relief; performing mild activities for 3-5 minutes; 6-10 minutes of moderate activity; the activity is classified into 11-12 parts.

TABLE 3 Association analysis (χ + -s) of ulcerative colitis activity score with tissue ASS1 protein expression

And (4) surface note: ASS1 protein was expressed as a relative amount (% of control group), and the relative expression amount of ASS1 in colon tissues of 50 patients with ulcerative colitis was observed in relation to the Mayo score, using 6 mixed colon cancer tissues as a control. # Person correlation coefficient 0.965, P ═ 0.000; at the 0.01 level, the expression level of ASS1 protein is strongly and linearly correlated with the score of the activity degree of ulcerative colitis.

The index ASS1 of the activity degree of ulcerative colitis screened by the method has strong correlation with the Mayo score of the activity degree of ulcerative colitis (see Table 3), and shows that ASS1 can be used as an index for judging the activity degree of ulcerative colitis.

The invention overcomes the defects of slow speed and insufficient novelty of the traditional method that documents are searched, genes are screened and then the genes are verified step by step. The method is also suitable for screening disease activity indexes such as Crohn's disease, eosinophilic gastroenteritis and the like, and screening disease stage indexes such as polyp, tumor, inflammation and the like in colon parts. The technology of the invention has obvious effects on judging the activity degree of the ulcerative colitis, guiding clinical medication and follow-up visits.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A method for screening an index of disease activity of ulcerative colitis is characterized by comprising the following steps:

collecting in-vitro colon biopsy specimens of ulcerative colitis patients and tissues beside cancer excised from colon cancer surgical patients, soaking in RNA sample preservation solution for 3-8min, and then preserving in liquid nitrogen;

secondly, performing transcriptome sequencing analysis by adopting an isolated ulcerative colitis colon biopsy sample and the para-carcinoma tissues excised by a colon cancer surgical patient, wherein 6 cases of each group are subjected to sequencing analysis; screening genes which are not reported by domestic and foreign documents and are related to the ulcerative colitis according to FC value ascending and descending sequences respectively;

thirdly, adopting isolated ulcerative colitis colon biopsy samples and the tissues beside the cancer excised by colon cancer surgical patients, carrying out verification on the mRNA and protein levels of the genes screened by the sequencing of the transcription group by using a real-time fluorescent quantitative RT-PCR and immunoblotting method for 20 cases in each group, and further screening the genes with consistent expression;

step four, carrying out clinical verification on the screened genes; collecting colon biopsy samples of patients with ulcerative colitis in vitro, detecting the protein level coded by the genes by an immunoblotting method in 50 cases of each group, carrying out correlation analysis on the protein level and the disease activity of the patients, and finally screening out the index of the activity of the ulcerative colitis.

2. The method of screening for an indicator of ulcerative colitis disease activity of claim 1, wherein: genes which are screened in the step two and are not reported to be related to the ulcerative colitis by domestic and foreign documents: ASS1, CAPRIN 1.

3. The method of screening for an indicator of ulcerative colitis disease activity of claim 1, wherein: the screened index of the activity degree of the ulcerative colitis disease is as follows: argininosuccinate Synthase 1(Argininosuccinate synthsase 1, ASS 1).