CN104056279A - Application of miR-885-5p and miR-224-5p to medicament preparation - Google Patents

Abstract

The invention belongs to the field of biological medicine and particarly relates to application of miR-885-5p and miR-224-5p to medicament preparation. The invention verifies that under an in-vitro high inflammation environment, main pathogenic cell postorbital fibroblast of Graves eye disease directly generates resistance on glucocorticoid, but miR-885-5p and miR-224-5p can increase protein level of a glucocorticoid receptor in the postorbital fibroblast, can recover sensitivity of the postorbital fibroblast on glucocorticoid, can improve treatment effect of the glucocorticoid on Graves eye disease, so that theoretical basis and experimental data are provided for clinically treating Graves eye disease. A model consisting of baseline serum miR-885-5p, miR-224-5p, TRAb and palpebral fissure width can effectively predict a patient insensitive to glucocorticoid treatment.

Description

MiR-885-5p, the miR-224-5p application in medicine preparation

Technical field

The invention belongs to biomedicine field, particularly miR-885-5p, the miR-224-5p application in medicine preparation.

Background technology

Graves oculopathy (GO) is the outer pathological changes of the most outstanding thyroid of Graves sick (GD), when the people such as Tanda in 2013 find GD patient's onset of 26% according to EUGOGO standard in 346 routine hyperthyroidism onset patients just with GO.Fibroblast after socket of the eye (OF) is pathogenic antigens carrier and the effector lymphocyte of GO.Different with the fibroblast at other positions, except the specific expressed antigens such as IGFR-1R of OF, OF also expresses thyroid cell specifically expressing molecule, and for example TSHR, Tg, TPO and NIS, be called as thyroid and rebuild in the simulation of eye socket.GD patient also hits OF for thyroid antibody in circulating thus, adds other for the antibody of OF, stimulates its propagation, to fat differentiation and secrete in a large number inflammatory factor and glycosaminoglycans (GAG).Recruit and activated lymphocyte soluble intercellular adhesion molecule-1-1 of its secretion (sICAM-1), promotes angtigen presentation, and the lymphocyte of activation further promotes OF secretion inflammatory factor, makes active stage inflammatory reaction aggravation.And take hyaluronic acid (HA) as main GAG gathering in a large number after frame, because its hydrophilic, osmotic pressure effect absorbs moisture and causes tissue edema after ball, extraocular muscles edema paralysis to cause exophthalmos and diplopia, increased intraocular pressure compressing optic nerve, the intrinsic pressure compressing venous return that increases of socket of the eye makes palpebral conjunctiva edema, fibrosis, causes eyelid contracture, causes stravismus and exposure keratitis, even blind, greatly reduce quality of life of patient.Therefore, after inhibition socket of the eye, fibroblastic a large amount of secretion inflammatory factors and a large amount of propagation become the key point for the treatment of GO.

Vein glucocorticoid (GC) is a line medication for the treatment of at present severe GO in activeness.GC directly suppresses the synthetic of the numerous inflammatory factors of OF cell, adhesion factor and HA by the transcripting suppressioning action of glucocorticoid receptor (GR) (GR) mediation, meanwhile, thus the also directly or indirectly immunocyte such as the activation of suppressor T cell, the activation of the balance of Th1/Th2, macrophage compacting inflammatory reaction.Through the comparison (the oral effective percentage 51% of vein effective percentage 77%Vs) of random controlled clinical trial effectiveness and safety, other routes of administration such as that vein GC curative effect is better than is oral, vein, retrobulbar injection.Yet the patient who replys larger individual variation: 75-80% of vein GC treatment can obtain more satisfied clinical remission, 6.5% patient has new disease, 0.6% death.Serious side effects is as main relevant with patient's medical history or therapeutic dose in brought out cardiovascular disease and liver dysfunction etc.Thereby illustrate GC drug resistance mechanism, and to find drug resistance label, thereby in advance patient is assessed, prediction hormone therapy is particularly important.

In recent years, multinomial research prompting microRNA (miRNA) thus lowering the important gene of GC path affects GC curative effect.The GR α downward that septic patient is mediated by miR-124 replying of GC weakens; In Neuroscreen cell, miR-124a and miR-18 have not only lowered GR protein level, have also affected the event of transcribing in GR downstream.Yet whether miRNA participates in GC drug resistance in GO patient, with and how by pathogenic cell OF, participate in the opposing to GC and there is no research.

First this research build the GC mdr cell model of OF, finds and confirm the miRNAs of regulation and control OF drug resistance, and the above-mentioned miRNAs of ultimate analysis is for the predictive value of GC drug resistance.

Prior art is compared, and beneficial effect of the present invention is:

1, the present invention has verified that in vitro under height inflammation environment, the main pathogenic cell OF of Graves oculopathy directly produces opposing to GC, and miR-885-5p and miR-224-5p can recover its sensitivity; Improve the therapeutic effect of GC to GO, for treating clinically Graves oculopathy, provide theoretical foundation and experimental data.2, the model that baseline serum miR-885-5p, miR-224-5p, TRAb form together with palpebral fissure width can effectively be predicted for the insensitive patient of glucocorticoid treatment.3, the present invention studies and has found that miR-885-5p and miR-224-5p can increase the protein level of glucocorticoid receptor (GR) in OF (GR) and improve the sensitivity of OF to GC.

Summary of the invention

The object of the invention is fibroblast after the main pathogenic cell socket of the eye of Graves oculopathy directly to the drug-fast research of vein glucocorticoid, and miR-885-5p and miR-224-5p can recover its sensitivity, improve the therapeutic effect of vein glucocorticoid to Graves oculopathy, for treating clinically Graves oculopathy, provide theoretical foundation and experimental data.

The present invention relates to miR-885-5p, the miR-224-5p application in preparation treatment Graves oculopathy medicine.

The invention still further relates to miR-885-5p, miR-224-5p and recover the application in vein glucocorticoid sensitivity medicine in preparation.

The invention still further relates to miR-885-5p, miR-224-5p and increase the application in the protein level medicine of glucocorticoid receptor (GR) in vein glucocorticoid in preparation.

In this research, we find after socket of the eye fibroblast (OF) in vitro under high inflammatory factor environmental stimulus to vein glucocorticoid (GC) drug resistance, and miR-885-5p and miR-224-5p can increase the protein level of glucocorticoid receptor (GR) in OF (GR) and improve the sensitivity of OF to GC.In Graves oculopathy (GO) patient, the low-level miR-885-5p of serum is the insensitive independent hazard factor of GC, and baseline serum miR-885-5p, miR-224-5p, TRAb and palpebral fissure width combine and can successfully predict that GC is insensitive, negative predictive value is up to 100%.

In experiment, OF is under the stimulation of high concentration TNF ɑ in vitro, and Dex obviously declines to the inhibitory action of its secretion sICAM-1, forms the opposing of OF to GC, has simulated clinically similarly phenomenon.This experimental result can be explained by the balance mutually suppressing before inflammatory factor and glucocorticoid and regulate and control.In this research, serum miR-885-5p and miR-224-5p may be that OF absorbs by the pathogenic cell of GO, thus the regulation and control that mediation is replied GC.The antiinflammatory action of GC is mainly by GR, transcribing of NF-κ B to be suppressed to realize.Therefore,, on the basis of our the OF model of GC opposing under TNF ɑ induction, further assessed miRNA for NF-κ B promoter activity, NF-κ B controlling gene and the effect expressed for GR.In the OF resisting at GC and 293T cell, excessively express the transcripting suppressioning action that miR-885-5p and miR-224-5p have recovered GC.And this process can be explained with the increase of GR protein level, and the increase of GR protein level is not due to the increase of its transcriptional level, but causes GR protein degradation to reduce because GSK-3 β reduces.

We find that GC is treated to insensitive patient's baseline TRAb to be increased than sensitive patients is remarkable.Yet the negative predictive value NPV of TRAb only reaches 55.55%.Palpebral fissure width is the insensitive independent correlative factor of another GC treatment, and NPV when this factor is used for predicting reaches 60.86%.And these two indexs not there are differences after glucocorticoid treatment between two groups of patients, pointing out them is not the factor that determines curative effect.

In sum, the present invention confirmed under external height inflammation environment, and directly to GC, treatment produces drug resistance to the main pathogenic cell OF of GO, but high-caliber miR-885-5p, miR-224-5p in patient body can recover the sensitivity of OF to GC.And baseline circulation miR-885-5p, miR-224-5p add the as a token of thing combination of TRAb and palpebral fissure width, can predict that GO patient carries out the curative effect of GC treatment (NPV=100%).This is that first utilization circulation microRNAs assesses the research of GC curative effect, and provides first GC treatment front effectively serology information.

Accompanying drawing explanation

The expression figure that Fig. 1 is difference microRNA in responsive group and opposing group (wherein, A be miR-885-5p in two groups, there were significant differences, B be miR-224-5p in two groups, there were significant differences, C is that miR-155-5p difference in two groups is not remarkable).

Fig. 2 is miR-885-5p and the inhibition result figure of miR-224-5p to curative effect relevant cell factor sICAM-1.

Fig. 3 is miR-885-5p and the inhibition result figure of miR-224-5p to NF-κ B.

The specific embodiment

Below in conjunction with embodiment, the invention will be further described:

Embodiment 1

1, primary OF cell test to glucocorticoid drug resistance under high concentration inflammatory factor stimulates

The present invention is by 3 severe GO patients' primary OF cell, utilization under base state and the stimulation of two concentration of TNF α height (5ng/ml and 20ng/ml) simulate local inflammation environment in various degree, in testing conditions culture supernatant, GO causes a disease and the concentration of curative effect relevant cell factor sICAM-1.Than base state, the stimulation of 5ng/ml TNF α can increase the secretion of sICAM-1, and 20ng/ml no longer significantly increases sICAM-1 concentration.Yet after 1 μ M dexamethasone (Dex) is processed, the 69% when inhibitory action of sICAM-1 secretion is from 5ng/ml under stimulating for 20ng/ml TNF ɑ is reduced to 28%, there is the opposing for Dex in prompting.

2, the checking of the relevant miRNAs of curative effect

(1), the screening of the relevant miRNAs of curative effect

In order further to look into the causes, we have selected the most significant 6 routine patients of GC curative effect and 7 routine invalid patients, and totally 13 routine curative effects have patient's baseline serum sample of extreme differences for pcr chip (miScript PCRarray) screening difference microRNA.

MiScript PCR array finds 8 difference miRNA (p<0.05).As shown in table 1, the major part in 8 is lowered in opposing patient.Wherein that difference multiple maximum is respectively doubly (p=0.04), miR-224-5p5.8893 times (p=0.03), miR-155-5p4.7209 times (p=0.03) of miR-885-5p6.099.

The screening of table 1 curative effect relevant difference microRNA

According to the above results, we further carry out real-time PCR checking in comprising all specimen of above-mentioned sample.As shown in Figure 1, the Δ Ct value of miR-885-5p and miR-224-5p significantly increases in opposing group, is respectively p=0.0013 and p=0.0054 (Figure 1A, Figure 1B).And miR-155 does not have significant difference (Fig. 1 C) between two groups.

We have further analyzed the impact of GC treatment on these two serum miRNAs itself.Before and after treatment, the serum levels of these two miRNAs of miR-885-5p and miR-224-5p does not occur significantly to change.

(2) miRNAs increases the sensitivity of GC to OF

Whether for further seeking candidate miRNA, can reply by direct regulation and control GC, we cross and have expressed miR-885-5p or miR-224-5p, the variation of adhesion factor (sICAM-1) between soluble cell in study condition culture medium in primary OF.We find when the dosage of TNF ɑ is increased to 20ng/ml from 5ng/ml, dexamethasone (Dex) stimulates the inhibitory action of lower sICAM-1 secretion to be reduced to 28% from 69% for TNF ɑ, and when transfection after miR-885-5p, inhibitory action when 20ng/ml returns to 60% (p=0.03) significantly.After miR-224-5p transfection, can recover the inhibitory action of Dex, specifically as shown in Figure 2.

Because the antiinflammatory action of GC is mainly by GR, transcribing of NF-κ B to be suppressed to realize, we have assessed miR-885-5p and miR-224-5p transcribes the impact that suppresses NF-κ B reporter gene on GR.As shown in Figure 3, Dex to the inhibitory action of NF-κ B reporter gene with the increasing progressively and successively decrease of TNF ɑ stimulating dose, 19% when suppression ratio 26% during from 1ng/ml TNF ɑ drops to 5ng/ml TNF ɑ, then 0.9% suppression ratio only during to 20ng/ml TNF ɑ.We find the miR-224-5p inhibition of Dex to NF-κ B transcriptional activity that had efficient recovery.When 5ng/ml TNF ɑ, suppression ratio reaches 29% (29%vs.19%, p=0.03), and during 20ng/ml TNF ɑ, suppression ratio reaches 25% (25%vs.0.9%, p=0.05).We observe miR-885-5p also identical trend.

(3) miRNAs is the independent hazard factor of GC opposing and the value that has prediction opposing

The evidence of comprehensive above-mentioned baseline clinical indices and experiment in vitro, it is all relevant with GC opposing that single factor analysis obtains miR-885-5p, miR-224-5p, TRAb.After having proofreaied and correct age, sex, smoking situation, baseline CAS, sTSH, TRAb and TPOAb, miR-885-5p and palpebral fissure width are all still the independent correlative factors of GC opposing.

Our the index that further between two groups of assessments, there were significant differences, miR-885-5p, miR-224-5p, TRAb and palpebral fissure width are for the predictive value of GC opposing.ROC area under curve (AUC), positive predictive value (PPV), negative predictive value (NPV) are used for the predictive efficiency of more various models and combination.When miR-885-5p, miR-224-5p, TRAb and palpebral fissure width are during as independent prediction index, AUC is respectively 0.8092 (p=0.0018), 0.7845 (p=0.0042), 0.7204 (p=0.026) and 0.7155 (p=0.030).The built-up pattern prediction effect of comprehensive 4 indexs is best, and getting point of contact is-1.566 o'clock, and AUC reaches 0.8717 and negative predictive value NPV=100%

Claims (3)

1.miR-885-5p, the miR-224-5p application in preparation treatment Graves oculopathy medicine.

2.miR-885-5p, miR-224-5p recover the application in vein glucocorticoid sensitivity medicine in preparation.

Application in the protein level medicine of 3.miR-885-5p, miR-224-5p glucocorticoid receptor (GR) in preparation increase vein glucocorticoid.