CN111450250A

CN111450250A - Application of enhancer or inhibitor of ENST00000606533 in preparation of medicine for treating pigmentary dermatosis

Info

Publication number: CN111450250A
Application number: CN202010172284.8A
Authority: CN
Inventors: 曾庆海; 陈静; 蒋玲; 黄进华; 鲁建云; 雷厉; 胡怡博; 童晓亮
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2020-03-12
Filing date: 2020-03-12
Publication date: 2020-07-28

Abstract

The invention discloses application of an enhancer or an inhibitor of ENST00000606533 in preparing a medicament for treating pigmentary dermatosis. The experimental result of the invention shows that the expression level of tyrosinase is obviously reduced after the expression of ENST00000606533 is inhibited in melanocyte. ENST00000606533 regulates the expression of tyrosinase through a cerRNA mechanism of sponge absorbing miR-1291, and further influences the melanin synthesis function of melanocytes. The research result indicates that ENST00000606533 is a potential therapeutic target for regulating melanin synthesis, and an enhancer or an inhibitor for regulating the expression level of ENST00000606533 is a potential new method for treating pigmentary dermatosis.

Description

Application of enhancer or inhibitor of ENST00000606533 in preparation of medicine for treating pigmentary dermatosis

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of an enhancer or an inhibitor of ENST00000606533 in preparation of a medicine for treating pigmented dermatosis.

Background

The pigmented dermatosis includes leukoderma, pigment-free nevus, etc. skin diseases with reduced pigment, and chloasma, freckle, etc. skin diseases with increased pigment. Pigmentary dermatosis does not cause organic damage, but seriously affects the beauty and physical and psychological health of patients due to the characteristics of the pigmentary dermatosis and the damage. At present, various methods such as laser, antioxidant, Chinese herbal medicine, exfoliating agent or color compound agent and the like are used for treating the pigmentary dermatosis, but the effect is not good enough. At present, the pathogenesis of the pigmentary dermatosis is explained from the aspects of signal transduction, transcription factor regulation, DNA methylation, histone modification and the like, and corresponding treatment medicines are developed according to the current basic research, but the current situations of poor curative effect and high recurrence rate of the pigmentary dermatosis cannot be improved at present. Therefore, there is a need to further explore new mechanisms affecting skin pigmentation and develop new therapeutic agents.

Skin pigmentation is determined most primarily by the activity of melanocyte function. The more active the melanocyte melanin synthesis function, the more skin pigmentation; conversely, if the melanocyte melanin synthesis function is impaired or inhibited, skin pigmentation will be reduced. Melanin synthesis is a multi-step enzymatic biochemical reaction, wherein Tyrosinase (TYR) is a key rate-limiting enzyme for regulating melanin synthesis, so TYR can be used as a key molecular marker for melanin synthesis and also can be used as an index for evaluating the activity degree of melanocyte functions.

Long non-coding RNA (incrna) is a non-coding RNA with a length of more than 200 nucleotides. Researches show that lncRNA participates in regulation of multiple activities such as proliferation, differentiation, migration, apoptosis and the like of cells, and can participate in regulation of multiple physiological and pathological processes such as tumorigenesis and development, nervous system diseases, cardiovascular diseases, embryonic development and the like through regulation of signal pathways, transcription factors, DNA methylation, histone modification and the like. Researches show that the lncRNA is rich in binding sites of microRNA (miRNA), can adsorb the miRNA through a sponge, and further relieves the inhibition effect of the miRNA on downstream target genes of the miRNA. This mechanism of action is known as the competitive endogenous rna (cerna) mechanism. Research shows that lncRNA can participate in the regulation and control of physiological pathology such as embryonic development, tumor progression, cardiovascular diseases and the like through a cerRNA mechanism. However, the role and mechanism of lncRNA in skin pigmentation has not yet been fully elucidated. ENST00000606533 is one of the members of the lncRNA family.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to explore the regulation and control effect and mechanism of ENST00000606533 on TYR in melanocytes and provide a novel medicament for treating pigmentary dermatosis.

The technical scheme of the invention is as follows:

the application of the enhancer or the inhibitor of ENST00000606533 in preparing the medicament for treating pigmentary dermatosis.

Preferably, the pigmentary skin diseases include pigmentary reduction skin diseases and pigmentary increase skin diseases.

Preferably, the pigmentation-reducing dermatosis includes vitiligo, naevus pigmentosus, pityriasis simplex, hypopigmentation after inflammation, and the like.

Preferably, the pigmentation-increasing skin diseases include chloasma, freckles, coffee spots, post-inflammatory pigmentation, and the like.

The experimental result of the invention shows that the expression level of tyrosinase is obviously reduced after the expression of ENST00000606533 is inhibited in melanocyte. ENST00000606533 regulates the expression of tyrosinase through a cerRNA mechanism of sponge absorbing miR-1291, and further influences the melanin synthesis function of melanocytes. The research result indicates that ENST00000606533 is a potential therapeutic target for regulating melanin synthesis, and an enhancer or an inhibitor for regulating the expression level of ENST00000606533 is a potential new method for treating pigmentary dermatosis.

The detailed structure of the present invention will be further described with reference to the accompanying drawings and the detailed description.

Drawings

FIG. 1 shows that α -MSH can promote the expression of ENST00000606533, (A) α -MSH at 0, 50, 100 and 150nM treats primary melanocytes, and RT-PCR detects TYR expression, (B) α -MSH at 150nM treats primary melanocytes, and RT-PCR detects the expression of ENST 00000606533.

FIG. 2: ENST00000606533 positively regulates the expression of TYR. (A) After the transfection of melanocytes by SiRNAs, the expression of ENST00000606533 is detected by RT-PCR. After transfection of melanocytes with si-ENST00000606533mix, the expression level of TYR mRNA was measured by RT-PCR (B) and the expression level of TYR protein was measured by WB (C).

FIG. 3: ENST00000606533 can bind miR-1291. (A) Schematic graphical models of Wild Type (WT) or mutant (mut) transcripts of ENST00000606533 luciferase reporter gene. (B) The dual-luciferase reporter gene detection kit is used for detecting the combination condition of miR-1291 and ENST 00000606533. (C) After si-ENST00000606533mix transfects melanocytes, the expression level of miR-1291 is detected by RT-PCR.

FIG. 4: ENST00000606533 promotes the expression of TYR by absorbing miR-1291 by sponge. After transfection of miR-1291mimics in melanocytes: (A) detecting the expression of miR-1291 by RT-PCR; (B) detecting the expression of TYR mRNA by RT-PCR; (C) WB detected the expression of TYR protein. (D) After miR-1291inhibitor is transfected in melanocytes, the expression of miR-1291 is detected by RT-PCR. After co-transfection of si-ENST00000606533mix and miR-1291inhibitor in melanocytes: (E) detecting the expression of miR-1291 by RT-PCR; (F) RT-PCR was used to detect the expression of TYR mRNA.

Detailed Description

The experimental method comprises the following steps:

1. cell culture

Melanocytes (MC) of human skin are extracted from foreskin. Foreskin tissues were donated by adolescent males of circumcision in urinary surgery in yasan hospital, central southern university (with informed consent from the patients and approval from the ethical committee of yasan hospital, central southern university). MC were cultured in 254 medium (# M-254-500, Gibco, USA) containing 5% fetal bovine serum (FBS, Biological Industries, Israel) and 1% HMGS (# S0025, Gibco, USA). The cells were cultured at 37 ℃ and 5% CO₂In an incubator.

2. Cell transfection

3 siRNAs targeting ENST00000606533 (GenePharma, Shanghai, China) were used to down-regulate the expression of ENST 00000606533. mimics (mimics) and inhibitors (inhibitors) of MiR-1291 were purchased from GenePharma Inc. the corresponding non-silencing siRNA oligonucleotides were used as Negative Control (NC) cells were transfected with L ipofectamine2000(Invitrogen, CA, USA) and the subsequent experiments were performed 48 hours after transfection.

3、RT-PCR

Total RNA was extracted by TRIzol reagent (Invitrogen, Thermo Fisher Scientific, USA), miRNA was extracted by E.Z.N.A. Micro RNA Kit (R6842-01, Omega Bio-Tek, USA) for qRT-PCR analysis.

4. Western Blot (WB) analysis

Total protein of cells was extracted using RIPA lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail (Roche, Mannheim, Germany.) Total protein was separated by 10% SDS-PAGE electrophoresis and transferred to PVDF membrane after blocking with 0.1% BSA, the membrane was incubated with primary anti-TYR (# ab180753, Abcam, Cambridge, UK) overnight at 4 ℃ and then with secondary antibodies (1: 10000, L I-COR Biosciences, USA) for 1 hour at room temperature, protein content was detected by enhanced L I-COR Odyssey infrared imaging system (L I-COR Biosciences, NE, USA) GAPDH was used as an internal control.

5. Luciferase Activity assay

180ng luciferase reporter vector (empty pmirG L O-NC, pmirG L O-ENST00000606533-WT or pmirG L O-ENST00000606533-mut), 18ng Renila luciferase reporter vector (pR L-TK) (Genechem, Shanghai, China) and miR-1291 mics (GenePharma) were co-transfected into melanocytes after 48 hours, the activity was measured using a multifunctional enzyme microplate reader (Perkin Elmer EnVision Xcite, UK) after processing according to the instructions of the Dual luciferase reporter assay kit (D L101-01, Vazyme, Nanjing).

6. Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0.2 software, and when comparing two experimental groups, analysis of variance was performed first, followed by T-test. P <0.05 is considered to have statistical significance, indicating P <0.05, indicating P < 0.01.

The experimental results are as follows:

1.α melanocyte-stimulating hormone (α -MSH) can promote expression of ENST00000606533

α -MSH is one of the main factors for promoting melanogenesis of melanocyte, and can induce the expression of TYR in melanocyte and activate the melanin synthesis function of melanocyte, RT-PCR results show that α -MSH with 50nM, 100 nM and 150nM can dose-dependently promote the expression of TYR in primary melanocyte (FIG. 1A), wherein the promotion effect of 150nM α -MSH is the best, therefore, after treating melanocyte with 150nM α -MSH, we detect the expression of ENST00000606533, and consequently, α -MSH can obviously promote the expression of ENST00000606533 (FIG. 1B), thus suggesting that ENST00000606533 may play an important role in the melanin synthesis function of melanocyte.

2. Inhibiting expression of ENST00000606533 can inhibit expression of TYR

In order to further explore the functions of ENST00000606533, 3 siRNAs (si-ENST 000006065331, si-ENST 000006065332 and si-ENST000006065333) which can target and inhibit the expression of ENST00000606533 are designed in the experiment. Control group siRNA (NC) and 3 siRNAs for targeted inhibition of ENST00000606533 expression were transferred into melanocytes using liposomes. RT-PCR detection is carried out on extracted RNA, and the result shows that 3 single siRNAs have no obvious inhibition effect on the expression of ENST00000606533, but the mixture (mix) of 3 siRNAs can obviously inhibit the expression of ENST00000606533 (figure 2A), so that the subsequent experiments are carried out by using the mixture (si-ENST00000606533 mix) of 3 siRNAs. RT-PCR results further found that si-ENST00000606533mix inhibited the expression of TYR mRNA (FIG. 2B). After transfection of melanocytes with si-ENST00000606533mix, protein was extracted, and as a result of WB assay, si-ENST00000606533mix was found to inhibit the expression of TYR protein (FIG. 2C). The above experimental results suggest that ENST00000606533 can positively regulate the expression of TYR.

3. ENST00000606533 can combine miR-1291

The MiRanda database predicts that the ENST00000606533 and the miR-1291 have a binding site, and a simulation graph of the binding site is shown in figure 3A. We further constructed a plasmid for the luciferase reporter gene comprising the wild type of ENST00000606533 transcript and a mutant form of the predicted binding site (fig. 3A). Luciferase reporter gene analysis showed that there was no significant change in luciferase activity in the control reporter or the mutated luciferase reporter after transfection with the miR-1291mimic (mimics), whereas the luciferase activity of the wild type ENST00000606533 was significantly reduced (fig. 3B). The results suggest that ENST00000606533 can bind miR-1291. In order to further explore the effect of ENST00000606533 on miR-1291, after si-ENST00000606533mix is transfected into melanocytes, RT-PCR results show that the inhibition of the expression of ENST00000606533 can remarkably promote the expression of miR-1291 (FIG. 3C). Therefore, ENST00000606533 is supposed to adsorb miR-1291 on the sponge, and further negatively regulate the expression of miR-1291.

4. ENST00000606533 adsorbs miR-1291 through a sponge to promote expression of TYR, and then miR-1291mimics are transfected in melanocytes, and RT-PCR detection finds that miR-1291mimics can remarkably promote expression of miR-1291 (figure 4A). Further, RT-PCR detection shows that miR-1291mimics can inhibit the expression of TYR mRNA and protein (fig. 4B-C). The results indicate that miR-1291 can negatively regulate the expression of TYR. As ENST00000606533 can negatively regulate the expression of miR-1291, we further verify whether ENST00000606533 promotes the expression of TYR by adsorbing miR-1291 through a sponge by a functional recovery experiment. miR-1291inhibitor is transfected in melanocytes, and RT-PCR detection finds that miR-1291inhibitor can obviously inhibit expression of miR-1291 (figure 4D). The co-transfection of si-ENST00000606533mix and miR-1291inhibitor in melanocytes resulted in the finding that the miR-1291inhibitor can antagonize the promoting effect of si-ENST00000606533mix on miR-1291 (figure 4E) and the inhibiting effect of si-ENST00000606533mix on TYR (figure 4F). In conclusion, ENST00000606533 promotes the expression of TYR by absorbing miR-1291 by sponge.

And (4) conclusion:

1.ENST00000606533 positively regulated TYR expression

2. ENST00000606533 uses sponge to adsorb miR-1291 to promote expression of TYR

3. An enhancer or inhibitor targeting ENST00000606533 can affect the melanin synthesis function of melanocytes by regulating TYR.

Claims

Use of an enhancer or inhibitor of ENST00000606533 in the manufacture of a medicament for the treatment of pigmentary dermatosis.
2. The use of claim 1, wherein the pigmentary skin disorders include hypopigmentation and hyperpigmentation skin disorders.
3. The use according to claim 2, wherein the pigmentary-reducing skin disorders comprise vitiligo, naevus pigmentosus, pityriasis simplex, hypopigmentation after inflammation.
4. The use according to claim 2, wherein the pigmentation-increasing skin disorders comprise chloasma, freckles, coffee spots, post-inflammatory pigmentation.