CN104056279B - 885 224 applications of the 5p in medicine preparation of 5p, miR of miR - Google Patents

Abstract

The invention belongs to biomedicine field, more particularly to 885 224 applications of the 5p in medicine preparation of 5p, miR of miR.The present invention is demonstrated under height inflammatory environment in vitro, after the principal causative cell socket of the eye of Graves oculopathy, fibroblast directly produces opposing to glucocorticoid, and miR 224 5p of 885 5p and miR can increase the protein level of glucocorticoid receptor (GR) in fibroblast after socket of the eye, recover sensitivity of the fibroblast to glucocorticoid after socket of the eye, therapeutic effect of the glucocorticoid to Graves oculopathy is improved, and theoretical foundation and experimental data is provided for clinical treatment Graves oculopathy.Baseline serum miR 885 5p, miR 224 the model that constitutes together of 5p, TRAb and palpebral fissure width can effectively predict the patient insensitive for glucocorticoid treatment.

Description

The application of miR-885-5p, miR-224-5p in medicine preparation

Technical field

The invention belongs to biomedicine field, more particularly to miR-885-5p, miR-224-5p answering in medicine preparation With.

Background technology

Graves oculopathy (GO) is the outer pathological changes of Graves diseases (GD) thyroid the most prominent, Tanda in 2013 et al. roots Just with GO when 26% GD patient's onset is found according to EUGOGO standards in the initial patient of 346 hyperthyroidisms.Into fiber finer after socket of the eye Born of the same parents (OF) are the pathogenic antigens carrier of GO and effector lymphocyte.It is different with the fibroblast at other positions, except OF specificity tables Outside the antigens such as the IGFR-1R for reaching, OF also expresses thyroid cell specifically expressing molecule, and such as TSHR, Tg, TPO and NIS are claimed Rebuild for simulation of the thyroid in eye socket.Thus OF is also hit for thyroid antibody in GD patient's circulation, along with other For the antibody of OF, stimulate its propagation, to Adipose Differentiation and a large amount of secretion inflammatory factor and glycosaminoglycans (GAG).Its secretion Soluble intercellular adhesion molecule-1-1 (sICAM-1) recruit and activated lymphocyte, promote angtigen presentation, the lymph of activation is thin Born of the same parents further promote OF secretion inflammatory factors, aggravate active stage inflammatory reaction.And the GAG based on hyaluronic acid (HA) is in frame A large amount of afterwards to assemble, because its hydrophilic, osmotic pressure effect absorbs tissue edema after moisture causes ball, extraocular muscles edema paralysis to be caused Exophthalmos and diplopia, intraocular pressure increase compressing optic nerve, and the intrinsic pressure compressing venous return that increases of socket of the eye makes palpebral conjunctiva edema, fiber Change, cause eyelid contracture, cause stravismus and exposure keratitises, in addition it is blind, greatly reduce the quality of life of patient.Therefore, After suppressing 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 the fiest-tire medication for treating severe GO in activeness at present.GC passes through glucocorticoid The transcripting suppressioning action that receptor (GR) is mediated directly suppresses the synthesis of the numerous inflammatory factors of OF cells, adhesion factor and HA, together When, also directly or indirectly suppress the immunocytes such as the activation of T cell, the balance of Th1/Th2, activation of macrophage so as to suppress Inflammatory reaction.Through comparison (the vein effective percentage 77%Vs oral availabilities of randomized clinical controlled trial effectiveness and safety 51%), vein GC curative effects are better than other routes of administration such as oral, vein, retrobulbar injections.However, the response of vein GC treatments has Larger individual variation：The patient of 75-80% can obtain relatively satisfactory clinical remission, and 6.5% patient has newly send out disease, 0.6% death.Serious side effects such as induce that cardiovascular disease and liver dysfunction etc. be main and the medical history of patient or Therapeutic dose is relevant.Thus GC resistance mechanisms are illustrated, and drag-resistance marker is found, so as to be estimated to patient in advance, prediction swashs Plain curative effect is particularly important.

In recent years, multinomial research prompting microRNA (miRNA) lowers the important gene of GC paths so as to affect GC curative effects. The GR α that response of the septic patient to GC is mediated by miR-124 are lowered and are weakened；In Neuroscreen cells, miR-124a and MiR-18 has not only lowered GR protein levels, also have impact on the transcriptional events in GR downstreams.However, whether miRNA joins in GO patient With GC drug resistances, and how which to participate in there is no research to GC opposings by pathogenic cell OF.

This research builds the GC mdr cell models of OF first, finds and confirms to regulate and control the miRNAs of OF drug resistances, finally divide Above-mentioned miRNAs is analysed for the predictive value of GC drug resistances.

The content of the invention

The purpose of the present invention be after principal causative cell socket of the eye to Graves oculopathy fibroblast directly to vein sugar skin The drug-fast research of matter hormone, and miR-885-5p and miR-224-5p can recover its sensitivity, improve vein glucocorticoid Therapeutic effect to Graves oculopathy, provides theoretical foundation and experimental data for clinical treatment Graves oculopathy.

The present invention relates to the application of miR-885-5p, miR-224-5p in treatment Graves ocular disease drugs are prepared.

The invention further relates to miR-885-5p, miR-224-5p are in recovery vein glucocorticoid sensitivity medicine is prepared Application.

The invention further relates to miR-885-5p, miR-224-5p glucocorticoid in increase vein glucocorticoid is prepared Application in the protein level medicine of receptor.

In this research, it has been found that after socket of the eye, fibroblast (OF) is in vitro under high inflammatory factor environmental stimuluses to vein sugar 17-hydroxy-11-dehydrocorticosterone (GC) drug resistance, and miR-885-5p and miR-224-5p can increase the albumen water of glucocorticoid receptor (GR) (GR) in OF Equal and improve sensitivity of the OF to GC.In Graves oculopathy (GO) patient, the low-level miR-885-5p of serum is GC insensitive Independent hazard factor, and baseline serum miR-885-5p, miR-224-5p, TRAb and palpebral fissure width combine can be successful GC is insensitive for prediction, and negative predictive value is up to 100%.

In testing in vitro, under the stimulation of high concentration TNF ɑ, the inhibitory action that Dex secretes sICAM-1 to which is obvious for OF Decline, form opposings of the OF to GC, simulate clinically similar phenomenon.This experimental result can be by inflammatory factor and sugared skin The balance for mutually suppressing before matter hormone and regulating and controlling is explaining.In this research, serum miR-885-5p and miR-224-5p may quilts The pathogenic cell of GO is OF intakes, so as to mediate the regulation and control to GC responses.The antiinflammatory action of GC is mainly by GR to NF- κ B's What Transcription inhibition was realized.Therefore, on the basis of the OF models of our the GC opposings under TNF ɑ inductions, further have evaluated miRNA For NF- κ B promoter activities, NF- κ B controlling genes and the effect for GR expression.In OF the and 293T cells of GC opposings Middle overexpression miR-885-5p and miR-224-5p has recovered the transcripting suppressioning action of GC.And this process can use GR albumen water Flat increase is explaining, and the increase of GR protein levels is not due to the increase of its transcriptional level, but as GSK-3 β are reduced GR protein degradation is caused to reduce.

We have found that the patient baseline TRAb insensitive to GC treatments is more notable than sensitive patients to increase.However, the moon of TRAb Property predictive value NPV only reaches 55.55%.Palpebral fissure width is that another GC treats insensitive independent correlative factor, and the factor is used NPV when prediction reaches 60.86%.And there is no difference after glucocorticoid treatment between two groups of patients in this two indexs It is different, point out them not to be the factor for determining curative effect.

In sum, present invention demonstrates under high ira vitro inflammatory environment, the principal causative cell OF of GO is directly controlled to GC Treat high-caliber miR-885-5p, the miR-224-5p produced in drug resistance, but patient's body and can recover sensitivities of the OF to GC.And Baseline circulation miR-885-5p, miR-224-5p add TRAb and palpebral fissure width and combine as mark, can predict GO patient Carry out the curative effect (NPV=100%) of GC treatments.This is that first utilization is circulated microRNAs to assess the research of GC curative effects, and First there is provided effective serology information before GC treatments.

Prior art is compared, the beneficial effects of the present invention is：

1st, the present invention is demonstrated under height inflammatory environment in vitro, and the principal causative cell OF of Graves oculopathy is directly to GC Opposing is produced, and miR-885-5p and miR-224-5p can recover its sensitivity；Therapeutic effect of the GC to GO is improved, is clinical Upper treatment Graves oculopathy provides theoretical foundation and experimental data.2nd, baseline serum miR-885-5p, miR-224-5p, TRAb The patient insensitive for glucocorticoid treatment can effectively be predicted with the model that palpebral fissure width is constituted together.3rd, the present invention grinds Study carefully and be found that miR-885-5p and miR-224-5p can increase the protein level of glucocorticoid receptor (GR) (GR) in OF and improve OF Sensitivity to GC.

Description of the drawings

Fig. 1 is that (wherein, A is miR-885-5p at two groups to expression figure of difference microRNA in sensitive group and opposing group In there were significant differences, B be miR-224-5p in two groups, there were significant differences, C does not show for miR-155-5p differences in two groups Write).

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

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

Specific embodiment

With reference to embodiment, the invention will be further described：

Embodiment 1

1st, test of the primary OF cells under the stimulation of high concentration inflammatory factor to glucocorticoid drug resistance

The primary OF cells of 3 severe GO patients are utilized under base state and TNF α two concentration of height by the present invention The stimulation of (the 5ng/ml and 20ng/ml) inflammatory environment different degrees of to simulate local, 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 αs can increase sICAM-1's Secretion, and 20ng/ml no longer dramatically increases sICAM-1 concentration.However, Jing after 1 μM of dexamethasone (Dex) is processed, for 20ng/ Ml TNF ɑ stimulate the inhibitory action of lower sICAM-1 secretions from 5ng/ml when 69% be reduced to 28%, prompt referring now to Dex Opposing.

2nd, the checking of curative effect correlation miRNAs

(1), the screening of curative effect correlation miRNAs

In order to further look into the causes, we have selected most significant 6 patients of GC curative effects and 7 refractory patients, totally 13 Patient baseline's serum sample that curative effect has extreme differences is used for pcr chip (miScript PCR array) and screens difference microRNA。

MiScript PCR array have found 8 difference miRNA (p<0.05).As shown in table 1, the major part in 8 exists It is to lower in opposing patient.That wherein fold differences are maximum is miR-885-5p6.099 times of (p=0.04), miR- respectively 224-5p5.8893 (p=0.03), miR-155-5p4.7209 times (p=0.03) again.

The screening of 1 curative effect relevant difference microRNA of table

According to the above results, we further in all specimen including above-mentioned sample carry out real-time PCR and test Card.As shown in figure 1, the Δ Ct values of miR-885-5p and miR-224-5p are dramatically increased in opposing group, respectively p=0.0013 and P=0.0054 (Figure 1A, Figure 1B).And miR-155 is not significantly different from (Fig. 1 C) between two groups.

We further analyze impact of the GC treatments to the two serum miRNAs itself.Before and after treatment, miR-885- The serum levels of 5p and miR-224-5p the two miRNAs are significantly changed.

(2) miRNAs increases sensitivity of the GC to OF

For further seek candidate miRNA whether can direct regulation and control GC responses, our overexpression in primary OF MiR-885-5p or miR-224-5p, the change of the soluble intercellular adhesion factor (sICAM-1) in study condition culture medium. We have found that when the dosage of TNF ɑ increases to 20ng/ml from 5ng/ml, dexamethasone (Dex) lower sICAM- for TNF ɑ stimulate The inhibitory action of 1 secretion is reduced to 28% from 69%, and inhibitory action after miR-885-5p has been transfected, in 20ng/ml 60% (p=0.03) is returned to significantly,.The inhibitory action of Dex can be recovered after miR-224-5p transfections, it is concrete as shown in Figure 2.

Due to GC antiinflammatory action mainly by GR to the Transcription inhibition of NF- κ B realizing, we have evaluated miR- Impacts of the 885-5p and miR-224-5p to GR Transcription inhibition NF- κ B reporter genes.As shown in figure 3, Dex reports base to NF- κ B Because inhibitory action successively decrease with being incremented by for TNF ɑ stimulating doses, suppression ratio from 1ng/ml TNF ɑ when 26% drop to 5ng/ml During TNF ɑ 19%, then to only 0.9% suppression ratio during 20ng/ml TNF ɑ.We have found that miR-224-5p has effectively recovered Dex Suppression to NF- κ B transcriptional activities.In 5ng/ml TNF ɑ, suppression ratio reaches 29% (29%vs.19%, p=0.03), During 20ng/ml TNF ɑ, suppression ratio reaches 25% (25%vs.0.9%, p=0.05).It is observed that miR-885-5p also has phase Same trend.

(3) miRNAs is the independent hazard factor of GC opposings and has the value for predicting opposing

The evidence of summary baseline clinical index and experiment in vitro, single factor analysis obtain miR-885-5p, miR- 224-5p, TRAb are related to GC opposings.Correcting age, sex, tobacco smoking status, baseline CAS, sTSH, TRAb and TPOAb Afterwards, miR-885-5p and palpebral fissure width are still all the independent correlative factors of GC opposings.

We further assess the index that there were significant differences between two groups, i.e. miR-885-5p, miR-224-5p, TRAb and eye Width is split for the predictive value that GC is resisted.Area (AUC), positive predictive value (PPV), negative predictive value (NPV) under ROC curve For the various models of comparison and the predictive efficiency of combination.When miR-885-5p, miR-224-5p, TRAb and palpebral fissure width are used as only During vertical prediction index, AUC be respectively 0.8092 (p=0.0018), 0.7845 (p=0.0042), 0.7204 (p=0.026) and 0.7155 (p=0.030).The Combined model forecast best results of comprehensive 4 indexs, take point of contact for -1.566 when, AUC reaches 0.8717 and negative predictive value NPV=100%.

Claims (1)

1. Applications of the 1.miR-885-5p or miR-224-5p in treatment Graves ocular disease drugs are prepared.