CN111617096A

CN111617096A - Application of lncRNA AK124826 and related molecules thereof in diagnosis of polymyositis/dermatomyositis

Info

Publication number: CN111617096A
Application number: CN202010532347.6A
Authority: CN
Inventors: 蒋廷旺; 薛建中; 韩志君; 龚燕萍; 高明珠
Original assignee: Changshu No2 People's Hospital
Current assignee: Changshu No2 People's Hospital
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2020-09-04

Abstract

The invention discloses an application of a medicament or a diagnostic reagent or a kit for preventing or treating polymyositis/dermatomyositis. The invention also discloses application of the lncRNA AK124826 lentiviral over-expression vector in preparation of a medicament or a diagnostic reagent or a kit for preventing or treating polymyositis/dermatomyositis. The invention also discloses application of the RNAi interference vector of the lncRNA AK124826 in preparation of a medicament or a diagnostic reagent or a kit for preventing or treating polymyositis/dermatomyositis.

Description

Application of lncRNA AK124826 and related molecules thereof in diagnosis of polymyositis/dermatomyositis

Technical Field

The invention belongs to the field of biotechnology and treatment, and particularly relates to application of lncRNA AK124826 and related molecules thereof in diagnosis of polymyositis/dermatomyositis.

Background

Polymyositis/dermatomyositis (PM/DM) is a common autoimmune disease, CD4+ T cells play an important role in the pathogenesis process, and the number and the function of Th17/Treg cells are unbalanced, but the specific pathogenesis is not completely clear.

Polymyositis and dermatomyositis (PM/DM) are typical autoimmune diseases which are mainly characterized by muscle tissue lymphocyte infiltration and striated muscle involvement, and positive serum characteristic anti-Jo-1 antibody, the incidence rate of PM/DM is increased year by year in recent years, and no effective radical treatment therapy is available at present. The pathogenesis of PM/DM is complex, and relates to a plurality of factors such as heredity, infection, immune function, microRNA abnormal expression and the like, in recent years, the pathogenesis of the disease is continuously and deeply researched, and the abnormal activation of the T cell and the abnormal response of the Th17 cell factor are considered at present, but the specific mechanism is not completely clarified. Th17 expression abnormalities are present in PM/DM patients. Th17 cells are a subset of newly discovered CD4+ T cells. Tournandrea et al found that the muscle tissue of PM/DM patients had CD4⁺T cell positive. Tournadre A et al found CD4 in muscle tissue of PM/DM patients⁺T cells are positive, IL-17 expression is positive, and the number of Th17 cells is obviously increased.

Interleukin-27 (IL-27) plays an important role in regulating the Th17 cellular response. IL-27 is a new IL-6/IL-12 family cytokine, mainly acts on various immune cells to play a wide regulation role, in autoimmune diseases play a role in. IL-27 can inhibit the differentiation of Th17 cells by down-regulating the expression of IL-6 and TGF-beta, and IL-27 can also regulate Th17 cell response by inhibiting the phosphorylation of STAT1 and STAT 3. The research finds that the level of IL-27 in the peripheral blood of patients in the acute phase of the multiple sclerosis is obviously reduced, the level of IL-17 and the number of Th17 cells are obviously increased, and the expression of IL-27 and IL-17 is obviously and negatively correlated, and initially suggests that IL-27 can be involved in the pathogenesis of the multiple sclerosis by inhibiting the secretion of IL-17. In the study of an autoimmune encephalomyelitis mouse model, the IL-27 receptor EBI3 gene is knocked out, the inflammatory response of the mouse is aggravated, and the number of Th17 cells in inflammatory tissues is increased.

Disclosure of Invention

The purpose of the invention is as follows: the research of the invention discovers that the PM/DM patients have peripheral blood CD4⁺The percentage of Th17 cells in T cells is increased remarkably, and the initial result indicates that the expression of Th17 cells of PM/DM patients is disordered, and the mechanism is not clear. The research of the invention finds that the expression level of IL-27 in the peripheral blood of PM/DM patients is obviously reduced compared with that of healthy controls, but how the expression of IL-27 in the PM/DM patients is regulated and the balance relation between the expression and Th17/Treg cells is not clear.

The research of the invention finds that lncRNA AK124826 is highly expressed in CD4+ T cells of PM/DM patients, IL-27 is lowly expressed in PM/DM patients, and a chip network regulation diagram predicts that IL-27 is an action target of lncRNA AK 124826.

Therefore, the lncRNA AK124826 is suggested to affect the balance of Th17/Treg through the target gene IL-27 and participate in the pathogenesis of PM/DM. Firstly, observing the expression characteristics of lncRNA AK124826 in PM/DM patients; secondly, researching the action mechanism of lncRNA AK124826 on the differentiation of the Th17/Treg cells of the PM/DM patient by adopting a lentivirus overexpression and interference technology; thirdly, the IL-27 is verified to be a functional target of lncRNA AK 124826. The invention provides a new idea for diagnosing and treating PM/DM.

The invention takes PM/DM patients as disease groups; patients with motor neuron disease, myasthenia gravis, progressive muscular dystrophy, polymyalgia rheumatica, etc. are disease control groups; the physical examination patients with normal liver and kidney function index are normal control group. Peripheral blood CD4+ T cells and serum samples of the subjects were isolated.

The invention finds that the expression level of the IL-27 in the peripheral blood of a PM/DM patient is obviously reduced compared with that of a healthy contrast person, finds out a new diagnosis and immunotherapy target point in the clear self-immune regulation mechanism of the PM/DM and provides a theoretical basis for developing a new therapy means.

The present disclosure also includes Fluorescence In Situ Hybridization (FISH) detection of co-expression of CD4+ T cells lncRNAAK124826 and IL-17 from each study group.

The present disclosure also includes analysis of expression of Th17/Treg cells, lncRNAAK124826, IL-27 gene and protein differences in CD4+ T cells of each study group.

The invention also comprises the detection of the expression of IL-17, TGF-beta and Creatine Kinase (CK) in the serum of each research group.

The invention also comprises the steps of analyzing the relation between lncRNA AK124826 and diseases by integrating the indexes and clinical data so as to verify whether the lncRNA AK124826 participates in the attack of PM/DM.

The technical scheme is as follows: in order to solve the technical problem, the invention provides application of lncRNA AK124826 in preparing a medicament or a diagnostic reagent or a kit for preventing or treating polymyositis/dermatomyositis.

The invention also provides application of the lncRNA AK124826 lentivirus overexpression vector in preparation of a medicament or a diagnostic reagent or a kit for preventing or treating polymyositis/dermatomyositis.

The invention also provides application of the RNAi interference vector of the lncRNA AK124826 in preparation of a medicament or a diagnostic reagent or a kit for preventing or treating polymyositis/dermatomyositis.

Wherein, the lncRNA AK124826 targets IL-27 gene.

Wherein the lncRNA AK124826 regulates the expression level of IL-27, TGF-beta and/or creatine kinase.

Has the advantages that: compared with the prior art, the invention has the advantages that:

1. the research of the invention finds that the expression of TGF-beta and CK in the peripheral blood of PM/DM patients is obviously higher than that of a healthy control group, the percentage of Th17 cells in the peripheral blood CD4+ T cells of the PM/DM patients is (3.24 soil 1.47)%, the percentage is obviously higher than that of the healthy control group (0.82 +/-0.24)%, the percentage of peripheral blood CD4+ T cell Treg cells of the PM/DM patients is (3.27 soil 0.45)%, and the percentage is obviously lower than that of the control group (6.78 +/-0.92)%;

2. the lncRNA AK124826 lentivirus over-expression vector and the RNAi interference vector are transferred into the peripheral blood initial CD4+ CD45RA + T cells of the PM/DM patient, immunofluorescence shows that the transfection efficiency reaches 80%, the over-expression and interference effects are detected by fluorescence quantitative PCR, and the results show that the expression is respectively increased and decreased by 5.6 and 2.2 times. The overexpression of lncRNAAK124826 in CD4+ CD45RA + T cells can obviously activate JAK-STAT and MAPK/P38 signal channels, the reduction of the expression of lncRNA AK124826 can obviously inhibit the signal channels, the proportion of Th17 cells in an lncRNA AK124826 overexpression group is obviously increased, and Treg cells are obviously reduced;

3. the lncRNA AK124826 can obviously reduce the reporter gene activity of a wild type IL-273' UTR, the expression is reduced by about 30 percent, and an RNA co-immunoprecipitation experiment verifies that IL-27 is a functional target of the lncRNA AK 124826. After the lncRNAAK124826 is interfered, Th17 cells are obviously reduced (1.00 +/-0.03)%, Treg cells are obviously increased (6.13 +/-0.64)% (P is less than 0.01); however, after lncRNAAK124826 and IL27 were interfered with each other, it was found that Th17 cells were significantly increased (3.12 ± 0.19)%, and Treg cells were significantly decreased (3.92 ± 0.25)% (P < 0.01).

Drawings

FIG. 1 expression levels of IL-27, TGF- β and creatine kinase in the sera of healthy groups and PM/DM patients;

FIG. 2 lncRNA chip differential clustering map;

FIG. 3 shows the expression of lncRNA AK124826, IL-17 and FoxP3 genes in the sera of healthy and PM/DM patients;

FIG. 4 is a combination of the percentages of Th17 and Treg cells in CD4+ T cells in healthy and PM/DM patients;

FIGS. 5A and 5B show the expression of lncRNA AK124826 detected by transforming lentiviral over-expression vector and RNAi interference vector into PM/DM patient peripheral blood primary CD4+ CD45RA + T cells and performing fluorescence quantitative PCR; FIG. 5C shows the change in the pathway of differentiation signals of the lentiviral overexpression vector and RNAi interference vector into CD4+ CD45RA + T cells in the peripheral blood of PM/DM patients and Th17/Treg cells; FIG. 5D Lentiviral overexpression vector, RNAi interference vector into PM/DM patients peripheral blood of CD4+ CD45RA + T cells, flow cytometry detection of each group of Th17 and Treg cell percentage; con represents a blank control, vector represents an empty vector control, and shNC represents a negative control;

FIG. 6A, PM/DM patient lncRNA AK124826 controls the target gene network map; FIG. 6B, expression levels of IL-27 in peripheral blood of healthy and PM/DM patients; FIG. 6C, lentivirus overexpression of IL-27 significantly inhibited the JAK2/STAT3 and MAPK/P38 signaling pathways; con represents a blank control and vector represents an empty vector control;

FIG. 7A, lncRNA AK124826 lentivirus over-expression vector, RNAi interference vector transferred into PM/DM patients peripheral blood primary CD4+ CD45RA + T cells, compare its effect graph on luciferase activity of luciferase reporter gene vector containing wild type IL-273' UTR full length; FIG. 7B, RNA co-immunoprecipitation assay confirmed that IL-27 is a functional target of lncRNA AK 124826; FIG. 7C, lncRNAAK124826 shows that lentiviral interference vectors are transferred into primary CD4+ CD45RA + T cells in the peripheral blood of PM/DM patients, and the siRNA technology is adopted to reduce the expression of IL-27; FIG. 7D, lncRNAAK124826 shows that lentiviral interference vectors are transferred into primary CD4+ CD45RA + T cells in the peripheral blood of PM/DM patients, the expression of IL-27 is reduced by siRNA technology, and the percentages of Th17 and Treg cells in each group are detected by flow cytometry; IgG represents a protein negative control, input represents a positive control, and shNC represents a negative control.

Detailed Description

The present invention is further illustrated by the following specific examples, it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified.

Example 1 analysis of lncRNA AK124826 expression in PM/DM patients

In this example, PM/DM patients were taken as disease groups, healthy examinees with normal liver and kidney function indexes were taken as normal control groups, samples of 50 PM/DM patients and 50 healthy control groups from the second hospital in the city of tin-free were collected, peripheral blood CD4+ T cells of the above-mentioned study subjects were isolated, and serum samples were retained.

1. The expression of IL-27, TGF-beta and Creatine Kinase (CK) in the serum of the disease group and the control group is analyzed.

ELISA technology is used for detecting the expression levels of IL-27, TGF-beta and Creatine Kinase (CK) in the blood serum of a healthy control group and a PM/DM patient respectively. The operation steps are respectively carried out according to the specifications of ELISA kits of IL-27, TGF-beta and CK, an enzyme-labeling instrument measures the absorbance of each hole at the wavelength of 450nm, a standard curve is drawn by taking the concentration of a standard substance as an abscissa and the OD value of the standard substance as an ordinate, and the concentration of each sample is calculated by a standard curve formula. The results show that the expression of TGF-beta and CK is significantly increased (P < 0.01) in PM/DM patients compared with healthy control group (FIG. 1).

2. The expression difference of lncRNA AK124826, IL-27 and FoxP3 genes and proteins and the proportion of Th17/Treg cells in CD4+ T cells of the control group and the research group are analyzed.

The method comprises the steps of screening lncRNA expression profile chips on peripheral blood CD4+ T cells of a PM/DM patient to obtain original data, preprocessing the original data, removing a background from a signal value, and then performing median normalization. Through normalization, influence of experimental random errors on data analysis is effectively eliminated.

lncRNA criteria were: the P value is less than 0.05, and at least two groups of the three groups of ratios are more than 2 times; the screening down-regulated lncRNA criteria were: p value is less than 0.05, and at least two of the three groups of ratios are less than 0.5 times, the lncRNA chip differential clustering chart is shown in figure 2. The results showed that lncRNA AK124826 expression was significantly increased (fig. 2).

We used fluorescent quantitative PCR to detect the expression of the genes of CD4+ T cell lncRNAAK124826, IL-17 and FoxP3 in PM/DM patients. Total RNA was extracted from the cultured cells using Trizol, and reverse transcription was performed using TaqMan-miRNA reverse transcription kit. Transcribing the RNA into cDNA by using a reverse transcription kit reaction system, and then carrying out PCR primer synthesis, wherein the primer sequence is as follows:

operating according to the manufacturer specification of a Real-time quantitative PCR (RT-PCR) kit, and constructing a Real-time PCR reaction system under the reaction conditions of pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s for 40 cycles, using β -actin as an internal reference gene, and adopting 2-^ΔΔCtAnd calculating the relative expression quantity of the gene to be detected.

The percentage of Th17 and Treg cells in peripheral blood CD4+ T cells was determined by flow cytometry.

(1) Detection of Th17 cells by flow cytometry

The cultured Peripheral Blood Mononuclear Cells (PBMCs) were removed from the incubator, the cell suspension aspirated and placed in two flow tubes and resuspended, and the test and control tubes labeled. Washing with 1ml PBS, centrifuging at 2000rpm for 5min, and discarding supernatant. mu.L of PE-Cy5.5-labeled mouse anti-human CD4 antibody (purchased from eBioscience, USA) was added to specifically stain the CD4 molecule on the cell membrane surface, and the cell membrane was incubated at room temperature in the dark for 30 min. Then, 1mL of PBS was added for washing, and the mixture was centrifuged at 2000rpm for 5min, and the supernatant was discarded. Add 100. mu.L of IL-17A breaker (Fix & Perm) solution A (from eBioscience, USA) and mix gently, incubate for 15min at room temperature in the dark. After washing once with 1ml of PBS, 100. mu.L of IL-17A breaker (Fix & Perm) solution B (from eBioscience, USA) was added and mixed gently, 5. mu.L of PE-labeled mouse anti-human IL-17 antibody (from eBioscience, USA) was added to the test tube, 5. mu.L of anti-mouse PE-labeled anti-mouse IgG1 antibody (from eBioscience, USA) was added to the control tube, and the tubes were incubated at room temperature for 30min in the absence of light. Then, 1ml PBS was added for washing, and the mixture was centrifuged at 2000 rpm. times.5 min, and the supernatant was discarded. And the washing was repeated once. And finally adding 400 mu LPBS (low density polystyrene) to resuspend the cells, uniformly mixing, and detecting the percentage of IL-17A + in CD4+ T cells by using a flow cytometer to show the percentage of Th17 cells and the percentage of FoxP3 to show the proportion of Treg cells.

(2) Flow cytometry detection of Treg cells

Cultured Peripheral Blood Mononuclear Cells (PBMC) were removed from the incubator, washed with 1ml PBS, centrifuged at 2000rpm for 5min, and the supernatant was discarded. The cell membrane surface CD4 molecules were specifically stained by adding 5. mu.L PE-Cy5.5 labeled mouse anti-human CD4 antibody (available from eBioscience, USA) and simultaneously adding 5. mu.L PE labeled anti-human CD25 antibody (available from eBioscience, USA), and incubated at room temperature in the absence of light for 30 min. Then, 1mL of PBS was added for washing, and the mixture was centrifuged at 2000rpm for 5min, and the supernatant was discarded. Adding 500 mu L of the special membrane-rupturing liquid for FoxP3, uniformly mixing, and incubating for 30min at room temperature in the dark. The mixture was washed by adding 1mL of PBS, centrifuged (1500 rpm. times.5 min), the supernatant was discarded, and the washing was repeated once. Then, 5. mu.L of mouse anti-human Foxp3 antibody (ex eBioscience, USA) labeled with LFITC was added to the experimental tube, mixed gently, and 5. mu.L of anti-mouse PE-Cy5 anti-mouse IgG1 antibody (ex eBioscience, USA) was added to the control tube, and incubated at 4 ℃ for 30min in the absence of light. Then, 1ml PBS was added for washing, and the mixture was centrifuged at 2000 rpm. times.5 min, and the supernatant was discarded and the washing was repeated once. And finally adding 400 mu of LPBS (low density polystyrene) to resuspend the cells, uniformly mixing, and detecting the percentage of Treg cells in CD4+ T cells by using a flow cytometer.

The results showed that PM/DM patients expressed lncRNAAK124826 significantly (P < 0.01), while IL-17 gene expression significantly increased (P < 0.01) and FoxP3 gene expression significantly decreased (P < 0.01) compared to healthy controls (fig. 3); the differentiation ratio of Th17/Treg cells in healthy control group and study group was analyzed by flow cytometry, and the results showed that the percentage of Th17 cells in peripheral blood CD4+ T cells of PM/DM patients was (3.24 + -1.47)%, significantly higher than (0.82 + -0.24)% (P < 0.01) of healthy control group, and the percentage of peripheral blood CD4+ T cells Treg cells of PM/DM patients was (3.27 + -0.45)%, significantly lower than (6.78 + -0.92)% (P < 0.01) of control group (FIG. 4).

Example 2 study of the mechanism of action of lncRNA AK124826 on the modulation of Th17/Treg cell differentiation in PM/DM patients

(1) Construction of lncRNA AK124826 overexpression vector

Firstly cloning the full length of lncRNA AK124826 gene, taking the genome of healthy human peripheral blood cells as a template to amplify to obtain a cDNA chain of the NcRNA AK124826, wherein the reaction system comprises 1 muL of an upstream primer, 1 muL of a downstream primer, 1 muL of template DNA, 10 muL of 5 × PBS buffer solution and 4 muL of dNTP Mix,

HS DNA polymerase (2.5U/. mu.L) 0.5. mu.L (purchased from Dalibao Bio Inc.), ddH₂O32.5. mu.L. Reaction conditions are as follows: 98 ℃, 10S, 55 ℃ (10S), 72 ℃ (90S) for 30 cycles. The sequence of the upstream primer is as follows: 5'-GCAAGCTTTATGAACTTTCTGCTGTCTTG-3', the sequence of the downstream primer is: 5'-GCTCTAGATTACCGCCTCGGCCTGTCACATC-3' are provided. The cDNA of the lncRNA AK124826 obtained by amplification was ligated between the cleavage sites of the expression vector pcDNA3.1(+) cleaved with the same EcoRI and XbaI to obtain the overexpression vector pcDNA3.1(+) -lncRNA AK 124826.

(2) Construction of lncRNA AK124826 RNAi interference vector

Following the design principle of RNA interference sequence, 2 interference targets are designed, and the interference sequences are as follows: 5'-GCCGCAGGCGTATACCATAGA-3' are provided. Meanwhile, the negative control insert was designed to be 5'-GCAAGCTGACCCTGAAGTTCAT-3'. An oligonucleotide sequence is designed and synthesized according to an interference target sequence, an interference vector is obtained by respectively inserting restriction enzyme sites of an EcoRI and XbaI restriction enzyme eukaryotic expression vector pcDNA3.1(+) between the 5 'end and the 3' end of the oligonucleotide sequence, each pair of oligonucleotide sequences can form a short hairpin structure after annealing, wherein a hairpin loop is a small fragment DNA double strand consisting of 9 bases.

(3) Study of lncRNA AK124826 on differentiation of Th17/Treg cells of PM/DM patients

The over-expression vector pcDNA3.1(+) -lncRNA AK124826 is introduced into a CD4+ CD45RA + T cell to analyze a signal path playing an important role in regulating and controlling Th17/Treg cell differentiation, meanwhile, RNAi interference technology is applied to knock down the lncRNA AK124826 in the CD4+ CD45RA + T cell and the expression level of the lncRNA AK124826 is reduced to further verify the regulating and controlling effect of the lncRNAAK124826 on Th17/Treg cell differentiation. Wherein the gene knockdown procedure is as follows: according to the lncRNAAAK 124826 gene sequence, selecting a target sequence (5'-GCCGCAGGCGTATACCATAGA-3') and a negative control sequence (5'-GCAAGCTGACCCTGAAGTTCAT-3') interfering the lncRNAAAK 124826 gene sequence, transiently transfecting the cells into the cells in the initial CD4+ CD45RA + T cell logarithmic growth phase of the PM/DM patient peripheral blood according to a Lipofectamine 2000 kit, and preparing the CD4+ CD45RA + T cells stably infecting lncRNAAAK 124826 short hairpin RNA.

Over-expressing and knocking lncRNAAK124826 in initial CD4+ CD45RA + T cells of PM/DM patients in peripheral blood, performing differentiation culture by utilizing IL-6, TGF-beta and PM/DM autoantigen Jo-1 co-stimulation after 24h, and analyzing the proportion of Th17/Treg cells after CD4+ T cell differentiation of each research group by using flow cytometry.

The lncRNA AK124826 lentivirus over-expression vector and the RNAi interference vector are transferred into peripheral blood primary CD4+ CD45RA + T cells of a PM/DM patient, and immunofluorescence shows that the transfection efficiency reaches 80% (FIG. 5A); the over-expression and interference effects were detected by fluorescent quantitative PCR, respectively, and the results showed that expression was increased and decreased by 5.6 and 2.2 times, respectively (FIG. 5B).

The overexpression of lncRNA AK124826 in CD4+ CD45RA + T cells can obviously activate JAK-STAT and MAPK/P38 signal channels, and the signal channels can promote the differentiation of CD4+ T cells to Th17, reduce the proportion of Treg cells and induce the generation and development of PM/DM; similarly, decreasing the expression of lncRNA AK124826 significantly inhibited the signaling pathway described above (fig. 5C). IL-6, TGF-beta and PM/DM autoantigen Jo-1 are respectively added into CD4+ CD45RA + T of over-expression and knock-down lncRNA AK124826 for co-stimulation, and then differentiation culture is carried out, and the percentages of Th17 and Treg cells are detected by flow cytometry. The result shows that the proportion of the LncRNAAK124826 over-expression group Th17 cells is obviously increased, and Treg cells are obviously reduced; while, after reducing the expression of lncRNAAK124826, the proportion of Th17 cells was significantly reduced and Treg cells were significantly increased (fig. 5D).

The experimental results show that the lncRNAAK124826 has a regulating effect on the differentiation of Th17/Treg cells of PM/DM patients, promotes the differentiation of CD4+ CD45RA + T cells to Th17 cells and inhibits the differentiation of the T cells to the Treg cells.

Example 3 correlation of lncRNA AK124826 with IL27 Gene and protein expression

One of the action modes of lncRNA is to regulate the expression of coding genes at the adjacent chromosome position, and we carry out the prediction analysis of cis action and trans action target genes on lncRNA AK124826 through bioinformatics to form a network regulation map (FIG. 6A) taking lncRNAAK124826 as the center, and as a result, it is found that IL-27 is positioned at the adjacent chromosome position and possibly is regulated by the target genes. The level of PM/DM and the level of IL-27 in peripheral blood of a healthy control group are detected by an ELISA method, the operation steps are carried out according to the specification of an ELISA kit of the IL-27, an ELISA reader measures the absorbance of each hole at the wavelength of 450nm, a standard substance concentration is used as an abscissa, the OD value of the standard substance is used as an ordinate, a standard curve is drawn, and the concentration of each sample is calculated by a standard curve formula. The results showed that IL-27 expression in peripheral blood of PM/DM patients was achieved at a level of 13.42. + -. 3.73pg/ml, significantly lower than 36.3. + -. 8.09pg/ml (P < 0.01) in healthy controls (FIG. 6B). To further investigate the mechanism of action of IL-27 in PM/DM patients, the use of lentiviral overexpression technology further up-regulated IL-27 expression in CD4+ CD45RA + T cells initially present in PM/DM patients, and as a result, as shown in FIG. 6C, up-regulated IL-27 expression levels significantly inhibited the activation of the JAK-STAT and MAPK/P38 signaling pathways (FIG. 6C).

Example 4 verification that IL-27 is a functional target of lncRNA AK124826

On the basis of confirming that lncRNAAK124826 participates in the generation and development of PM/DM and is negatively related to the expression of IL-27 protein, whether IL-27 is a functional target of lncRNAAK124826 is further verified, and research is carried out from the following three aspects:

through a dual-luciferase reporter gene experiment, the direct inhibition effect of lncRNA AK124826 on the luciferase activity of a luciferase reporter gene vector containing the full length of a wild type IL-273' UTR is observed. And (3) amplifying a 3' UTR region gene fragment of the lncRNA AK124826 binding site on the IL-27 by using an RT-PCR method. The method comprises the following steps: taking the genome of the peripheral blood cells of the healthy human as a template, and according to the sequence of a primer: forword: 5'-ATGGACTATCATATGCTTACCGTA-3', respectively; reverse: 5'-CTGCACTGTACCCCCCAATC-3', reaction system: premixtaq25 μ L (available from Dalibao Bio Inc.), H₂O₂2. mu.L, 1uL of the forward primer, 1. mu.L of the reverse primer, and 1. mu.L of the template DNA. Reaction conditions are as follows: pre-uncoiling at 95 deg.C for 5min, pre-uncoiling at 95 deg.C (30S), pre-uncoiling at 60 deg.C (30S), pre-uncoiling at 72 deg.C (3min), and pre-uncoiling at 72 deg.C (10min) for 32 cycles, and performing electrophoresis on the pre-uncoiled product with 1.0% agarose gel for 30min to recover target fragment. This fragment was inserted into pGL3 promoter luciferase reporter vector to construct IL-273 'UTR wild-type (IL-273' UTR-WT) plasmid. Then, the gene mutation technology is used for mutating partial nucleotides of the binding sites, and the specific mutation site sequences are as follows: forword: 5'-GGTGCAAAGTGGGAACTGTG-3', respectively; reverse: 5'-CGTGCGACGGATGAGTTAACG-3' are provided. Thus, an IL-273 'UTR mutant (IL-273' UTR-Mut) plasmid was constructed. All plasmids were synthesized by Shanghai bioengineering, and verified by DNA sequencing. IL-273 ' UTR-WT plasmid or IL-273 ' UTR-Mut plasmid was co-transfected with IncRNA AK12482 mimic (available from Shanghai Jikai pharmaceutical technology, Inc., SEQ ID NO: 5'-GGAGAGACTTAGTT-3') into primary CD4+ CD45RA + T cells in PM/DM patients ' peripheral blood, and the transfection procedure was performed according to the Lipofectamine 2000 kit instructions. Luciferase activity was measured in the absence of light after 48h incubation according to the instructions of the dual luciferase reporter kit.

lncRNA AK124826 significantly reduced the reporter gene activity of the wild type IL-273' UTR (P < 0.01), and the expression was down-regulated by about 30% (FIG. 7A). The result shows that the lncRNA AK124826 has the target regulation function on the IL-27 and can inhibit the expression of the IL-27. RNA co-immunoprecipitation experiments verified that IL-27 is a functional target of lncRNA AK124826 (FIG. 7B).

The lncRNAAK124826 lentiviral interference vector was transferred into primary CD4+ CD45RA + T cells in PM/DM patients' peripheral blood, the expression of IL-27 was down-regulated by siRNA technique, and the expression of IL27 was detected in each group by ELISA (FIG. 7C). The expression of Th17 and Treg cells in each group of CD4+ T cells was detected by flow cytometry in cultures co-stimulated with IL-6, TGF-. beta.and the PM/DM autoantigen Jo-1. The result shows that after lncRNAAK124826 is interfered, Th17 cells are obviously reduced (1.00 +/-0.03)%, Treg cells are obviously increased (6.13 +/-0.64)% (P is less than 0.01); however, after lncRNAAK124826 and IL27 were simultaneously interfered with, a significant increase (3.12 ± 0.19)%, and a significant decrease (3.92 ± 0.25)% (P < 0.01) of Treg cells were found in Th17 cells (fig. 7D).

The experimental results show that the lncRNAAK124826 participates in the regulation of Th17/Treg cell differentiation by negatively regulating IL27, thereby participating in the generation of PM/DM.

Sequence listing

<110> second people hospital in the normal market

Application of <120> ncRNA AK124826 and related molecules thereof in diagnosis of polymyositis/dermatomyositis

<160>15

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> lncRNA AK124826 upstream primer (Artificial Sequence)

<400>1

cagaccgttg aaaagacccc 20

<210>2

<211>21

<212>DNA

<213> lncRNA AK124826 downstream primer (Artificial Sequence)

<400>2

ccatcgtcgt tgtgactctc g 21

<210>3

<211>17

<212>DNA

<213> IL-27 upstream primer (Artificial Sequence)

<400>3

caggcgacct tggctgg 17

<210>4

<211>20

<212>DNA

<213> IL-27 downstream primer (Artificial Sequence)

<400>4

ggtacaggtt cactcctggc 20

<210>5

<211>22

<212>DNA

<213> FoxP3 upstream primer (Artificial Sequence)

<400>5

acagcacatt cccagagttc ct 22

<210>6

<211>21

<212>DNA

<213> FoxP3 downstream primer (Artificial Sequence)

<400>6

cattgagtgt ccgctgcttc t 21

<210>7

<211>29

<212>DNA

<213> upstream primer (Artificial Sequence)

<400>7

gcaagcttta tgaactttct gctgtcttg 29

<210>8

<211>31

<212>DNA

<213> downstream primer (Artificial Sequence)

<400>8

gctctagatt accgcctcgg cctgtcacat c 31

<210>9

<211>21

<212>DNA

<213> interference lncRNAAK124826 target Sequence (Artificial Sequence)

<400>9

gccgcaggcg tataccatag a 21

<210>10

<211>22

<212>DNA

<213> interference negative control Sequence (Artificial Sequence)

<400>10

gcaagctgac cctgaagttc at 22

<210>11

<211>24

<212>DNA

<213> IL-27 upstream primer (Artificial Sequence)

<400>11

atggactatc atatgcttac cgta 24

<210>12

<211>20

<212>DNA

<213> IL-27 downstream primer (Artificial Sequence)

<400>12

ctgcactgta ccccccaatc 20

<210>13

<211>20

<212>DNA

<213> IL-27 mutation upstream primer (Artificial Sequence)

<400>13

ggtgcaaagt gggaactgtg 20

<210>14

<211>21

<212>DNA

<213> IL-27 mutant downstream primer (Artificial Sequence)

<400>14

cgtgcgacgg atgagttaac g 21

<210>15

<211>13

<212>DNA

<213> lncRNA AK12482 mimic (Artificial Sequence)

<400>15

ggagagactt agt 13

Claims

Application of lncRNA AK124826 in preparing medicine or diagnostic reagent or kit for preventing or treating polymyositis/dermatomyositis.
Application of lncRNA AK124826 lentivirus overexpression vector in preparation of medicament or diagnostic reagent or kit for preventing or treating polymyositis/dermatomyositis.
3, application of RNAi interference vector of lncRNA AK124826 in preparing medicament or diagnostic reagent or kit for preventing or treating polymyositis/dermatomyositis.
4. The use of claim 1, 2 or 3, wherein the lncRNA AK124826 target gene is the IL-27 gene.
5. The use according to claim 1 or 2 or 3, wherein the lncRNA AK124826 regulates the expression level of IL-27, TGF- β and/or creatine kinase.