CN115976022A - Application of inhibitor of METTL3 or METTL14 in preparation of medicine for treating acanthosis nigricans - Google Patents

Abstract

The invention discloses application of an inhibitor of METTL3 or METTL14 in preparation of a medicine for treating acanthosis nigricans, wherein the inhibitor is siRNA. The invention also discloses application of METTL3 or METTL14 as a molecular marker in preparation of an echinodesis melanocortin detection kit. Our findings support: high expression of METTL3 and METTL14 in skin lesions promotes formation of skin lesions of the echinodesis, and targeted inhibition of METTL3 and/or METTL14 molecules can provide a new idea for preparing medicaments for treating the echinodesis.

Description

Application of METTL3 or METTL14 inhibitors in the preparation of drugs for treating acanthosis nigricans

Technical Field

The present invention belongs to the field of biomedicine technology, and specifically relates to the use of an inhibitor of METTL3 or METTL14 in the preparation of a drug for treating AN.

Background Art

Acanthosis nigricans (AN) is a skin disease characterized by hyperkeratosis, pigmentation, papilloma-like hyperplasia, and velvety rash. It mainly occurs in the skin folds of the neck, axilla, external genitalia, and face. It is often associated with obesity, glucose and lipid metabolism disorders, sex hormone disorders, and hyperinsulinemia, and seriously affects the physical and mental health and quality of life of patients. At present, AN is considered to be a specific epidermal marker for metabolic disorders such as insulin resistance and early diabetes. Studies have shown that hyperinsulinemia, persistent low levels of inflammatory factors, and abnormal secretion of adipose cytokines can promote the proliferation of keratinocytes (KCs) and the activation of melanocytes (MCs) in the skin of obese patients, leading to the occurrence of AN. However, the pathogenic mechanism of AN is still unclear, and there is currently no effective treatment.

To date, epigenetic studies have shown that environmental factors can alter the selective expression of target genes through epigenetic regulatory mechanisms. N6-methyladenine (m ⁶ A) is the most abundant dynamic and reversible chemical modification within RNA in eukaryotic cells; it is formed by transferring a methyl group to the sixth nitrogen atom of adenine using S-adenosylmethionine (SAM) as a methyl donor. m ⁶ A has received increasing attention due to its influence on multiple aspects of RNA metabolism, from RNA processing and nuclear export to RNA translation and decay. It has been shown to be involved in regulating a variety of pathophysiological processes, such as cell cycle, cell differentiation, DNA damage, and embryogenesis. At the same time, the dynamic balance of m ⁶ A is maintained by methyltransferases.

It is of great significance to study a new drug or new method for treating AN.

Summary of the invention

The present invention aims to provide a new target that can be used to prepare drugs for treating AN. In this study, we investigated the potential role of m ⁶ A modification in the occurrence and development of AN. We found that methyltransferase 3 (METTL3) and methyltransferase 14 (METTL14) can increase the mRNA and protein expression levels of adrenergic receptor a2A (ADRA2A) and inhibin subunit βA (INHBA) by mediating m ⁶ A levels, while inducing the expression of various biomarkers of KCs and MCs, promoting KCs cell proliferation and MCs activation, and participating in the pathophysiological development of AN.

The technical solution of the present invention is:

Use of METTL3 or METTL14 inhibitors in the preparation of drugs for treating acanthosis nigricans.

Preferably, the inhibitor of METTL3 or the inhibitor of METTL14 is siRNA. Further preferably, the inhibitor of METTL3 is shown as SEQ ID NO.51, SEQ ID NO.52 or SEQ ID NO.53, and the inhibitor of METTL14 is shown as SEQ ID NO.54, SEQ ID NO.55 or SEQ ID NO.56.

The present invention also provides the use of METTL3 or METTL14 as a molecular marker in the preparation of a detection kit for diagnosing acanthosis nigricans.

The present invention shows that the m ⁶ A level detection of skin lesions of patients with acanthosis nigricans and normal human skin tissues found that: compared with normal skin tissue, the overall m ⁶ A methylation level of skin lesions of patients with acanthosis nigricans was significantly increased. Since m ⁶ A modification is mediated by m ⁶ A-related modification enzymes, in order to further explore whether the abnormal m ⁶ A level of skin lesions of acanthosis nigricans is related to the abnormal expression of related catalytic enzymes, we first detected the mRNA expression levels of METTL3, METTL14, WTAP, FTO, ALKBH5, YTHDC1, YTHDC2, YTHDF1, YTHDF2 and YTHDF3 in skin lesions of acanthosis nigricans and normal skin tissues, and found that: compared with normal skin tissue, the mRNA levels of METTL3 and METTL14 in skin lesions of patients with acanthosis nigricans were significantly increased, while the mRNA levels of other enzymes were not significantly different. Then, we used immunohistochemistry to detect the protein expression level and localization of METTL3 and METTL14, and found that METTL3 and METTL14 were mainly expressed in epidermal keratinocytes, and the expression levels of METTL3 and METTL14 in the lesional tissues of patients with acanthosis nigricans were significantly higher than those in normal skin tissues. This study result suggests that high expression of METTL3 and METTL14 may promote the occurrence and development of acanthosis nigricans by increasing the expression level of the methyltransferase complex and regulating the m ⁶ A modification level of a downstream target gene mRNA.

In the present study, we collected skin tissues from 3 patients with acanthosis nigricans and 3 normal people to perform ArrayStar epitranscriptome sequencing. By analyzing the chip results, we found that the mRNA expression and m ⁶ A modification levels of ADRA2A and INHBA were significantly increased. At the same time, through GO/pathway analysis of ADRA2A and INHBA, it was found that it can be significantly enriched in pathways such as inflammatory response, metabolic regulation and pigment production. Subsequently, we collected skin tissues from 10 patients with acanthosis nigricans and 10 normal controls for RT-qPCR detection to confirm the mRNA expression levels of ADRA2A and INHBA and MeRIP-qPCR detection to confirm the m ⁶ A modification level of ADRA2A. Compared with the normal control group, the mRNA expression levels and m ⁶ A modification levels of ADRA2A and INHBAM in the skin tissues of patients with acanthosis nigricans were significantly increased.

The present invention uses in vivo experiments to study the role of knockdown or overexpression of METTL3 and METTL14 in regulating KCs proliferation and apoptosis and MCs pigment synthesis. The results show that METTL3 and METTL14 regulate the expression of ADRA2A and INHBA at the cellular level, and can also change the expression levels of biomarkers of cell proliferation, pigmentation and apoptosis.

In summary, the present invention describes the potential role of ^m6A modification in the pathological development of AN and demonstrates that METTL3 and METTL14 upregulate the expression of ADRA2A and INHBA through ^m6A modification, thereby participating in the pathological development of AN. Our results support the concept that high expression of METTL3 and METTL14 in skin lesions promotes the formation of acanthosis nigricans lesions, and targeting METTL3 and METTL14 molecules can provide new ideas for the preparation of drugs for the treatment of acanthosis nigricans.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an ArrayStar epitranscriptome sequencing analysis of AN lesions and healthy control skin, wherein Figure 1 (A) is a diagram showing that GO function and KEGG pathway enrichment analysis identified three pathways closely related to pathophysiology, and differentially expressed candidate genes in each enriched pathway (control vs. AN); Figure 1 (B) is a diagram showing a Venn diagram describing 7 candidate genes involved in inflammation, hormone regulation, and pigment signaling.

Figure 2 is a diagram showing that METTL3 and METTL14 promote the modification of m ⁶ A, thereby leading to the upregulation of ADRA2A and INHBA in AN lesions, wherein Figure 2 (A) is a diagram comparing the overall m ⁶ A methylation levels in total RNA of AN lesions and normal control skin; Figure 2 (B) is a diagram verifying the expression levels of METTL3 and METTL14 mRNA in AN lesions and normal skin using qRT-PCR (n=10); Figure 2 (C) is a diagram showing that the protein levels of METTL3 and METTL14 are upregulated compared with normal skin by Western Blot results; Figure 2 (D) is a diagram showing that METTL3 and METTL14 are highly expressed in epidermal keratinocytes of AN lesions compared with normal controls by immunohistochemistry (IHC); Figure 2 (E) is a diagram detecting the mRNA and m ⁶ A-IP-PCR of ADRA2A and INHBA in AN lesions by RT-qPCR and m ⁶ A-IP-PCR FIG2 (F): is a graph showing that ADRA2A and INHBA are upregulated in epidermal keratinocytes of AN lesions compared with normal skin by IHC detection; FIG2 (G) is a graph showing that ADRA2A and INHBA protein levels are upregulated in AN lesions by immunoblotting; data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, indicating that there are significant differences between the skin of AN lesions and the control group.

FIG3 is a diagram showing that knocking down METTL3 or METTL14 reduces the expression of ADRA2A, INHBA and the expression of biomarkers of KCs cell proliferation and pigmentation, and increases KCs cell apoptosis, wherein FIG3(A) is a diagram showing the downregulation of mRNA, m6A and protein levels of ADRA2A and INHBA in KCs after knocking down METTL3 or METTL14 by specific siRNA (n=3); FIG3(B) is a diagram showing the downregulation of mRNA and protein levels of keratin 14, cyclin E1, cyclin D1, and cyclinB1 in KCs after knocking down METTL3 or METTL14 genes (n=3); FIG3(C) is a diagram showing the downregulation of mRNA and protein levels of endothelin 14, cyclin E1, cyclin D1, and cyclinB1 in KCs after knocking down METTL3 or METTL14 genes (n=3); Figure 3(D): After knocking down METTL3 or METTL14 in KCs using siRNAs, the mRNA and protein levels of PARP decreased (n=3), but after knocking down METTL3 or METTL14 in KCs, the caspase-3 level increased and apoptosis increased. The data are expressed as mean ± SEM, * indicates comparison with empty vector (EV), *p<0.05, **p<0.01, ***p<0.001.

Figure 4 shows that overexpression of METTL3 or METTL14 enhances the expression of ADRA2A, INHBA and the expression of biomarkers of KCs cell proliferation and pigmentation, while reducing KCs cell apoptosis, wherein Figure 4 (A) shows the upregulation of mRNA, m6A and protein levels of ADRA2A and INHBA in KCs after adenovirus overexpression of METTL3 or METTL14 (n=3); Figure 4 (B) shows the upregulation of mRNA and protein levels of keratin 14, cyclin E1, cyclin D1, and cyclin B1 in KCs after overexpression of METTL3 or METTL14 genes (n=3); Figure 4 (C) shows the upregulation of mRNA and protein levels of endothelin 14, cyclin E1, cyclin D1, and cyclin B1 in KCs after overexpression of METTL3 or METTL14 genes (n=3); 4(D): After knocking down METTL3 or METTL14 in KCs using siRNAs, the mRNA and protein levels of PARP increased (n=3), but after knocking down METTL3 or METTL14 in KCs, the caspase-3 level decreased and apoptosis decreased, the data are expressed as mean ± SEM, * indicates comparison with empty vector (EV), *p<0.05, **p<0.01, ***p<0.001.

Figure 5 is a diagram showing the paracrine effects of METTL3 and METTL14 on human primary KCs on MCs pigment metabolism, wherein Figure 5 (A) shows that knocking down METTL3 or METTL14 inhibits cell viability and pigment metabolism in the KC and MC co-culture system; Figure 5 (B) shows that overexpression of METTL3 or METTL14 stimulates cell proliferation and pigment metabolism in the KC and MC co-culture system; Figure 5 (C) is a hypothesis diagram of the pathophysiological mechanism of AN, and the data are expressed as mean ± SEM, * indicates comparison with siCtrl, *p<0.05, **p<0.01, ***p<0.001.

Figure 6 is a graph showing the detection of mRNA levels of ^m6A RNA methylation regulatory molecules and their potential molecular targets, wherein Figure 6 (A) is a graph showing the detection of WTAP, ALKBH5, YTHDF1, YTHDF2, and YTHDF3 mRNA levels in AN lesions and normal skin using qRT-PCR (n=10); Figure 6 (B) is a graph showing the detection of FGFR1, SFRP1, INHBA, PDGFRB, MEF2C, and EDN3 mRNA levels (n=10).

Figure 7 is a correlation analysis diagram of m6A RNA methylation regulatory factors (METTL3, METTL14) and their potential targets (ADRA2A, INHBA) based on the interactive analysis of gene expression profiles in the GTEx database.

Figure 8 is a diagram of siRNA screening for inhibiting the expression of METTL3 and METTL14, wherein Figure 8 (A) is a diagram of WB detection of METTL3 or METTL14 protein levels in primary KCs after silencing by three different siRNAs; Figure 8 (B) is a diagram based on the results of immunoblotting. All three siRNA sequences have obvious effects, and we selected the siRNA with the best effect for subsequent experiments.

DETAILED DESCRIPTION

The present invention is further explained and illustrated below in conjunction with the accompanying drawings and experimental data.

Table 1: RT-qPCR primer sequences and other sequences in the instruction manual.

1. Materials and methods

qRT-PCR analysis, Western blot (WB) analysis, immunohistochemistry (IHC) assay, tyrosinase and melanin content assay are all existing methods and will not be described here again.

2. Patient tissue specimens

This study used patients with AN diagnosed by pathological histology and normal control patients from the Third Xiangya Hospital of Central South University. Ten AN lesional tissue specimens and corresponding normal control skin tissue specimens were immediately stored at -80°C and used for qRT-PCR and/or Western blotting.

3. ^m6A mRNA and lncRNA transcriptome microarray

Transcriptome microarray (Arraystar) was used for RNA sample preparation and microarray hybridization. Total RNA was extracted from skin tissues of AN group (n=3) or control group (n=3). The threshold was set as greater than 1.5-fold change and statistical significance (p<0.05) to compare the RNA methylation differences between AN group and normal group. Protein-protein interaction (PPI) network analysis was used to determine the interactions between m6A RNA methylation regulators and their target genes using the ^m6A mRNA and lncRNA transcriptome microarray database. KEGG and GO enrichment analysis was performed using the R package cluster analysis.

4. m ⁶ A quantification and MeRIP-qPCR

RNA methylation was measured at a wavelength of 450 nm using a colorimetric ^m6A RNA methylation quantification kit (EpiGentek) after binding to the antibody. For MeRIP-qPCR, total RNA (10-20 μg) was randomly fragmented into 100 nucleotide (nt) fragments. RNA fragments modified with ^m6A were pooled and enriched by co-immunoprecipitation analysis and then quantified by RT-PCR using a MeRIP m6A kit (EpiGentek).

5. Human KCs Culture and Functional Phenotypic Analysis

KCs were isolated and cultured from surgically removed foreskin specimens. For co-culture of KCs (2.5×10 ⁵ cells/well) and MCs, MCs (5×10 ⁴ cells/well) were added to each cell culture medium containing KCs (2/3 + 1/3 melanocyte culture medium) at a cell ratio of 1:5.

For RNA interference experiments, the SiRNA with the best intervention effect was selected from three Si-METTL3 (Si-METTL3-1 (5'-GCCAAGGAACAAUCCAUUGUUTT-3'); Si-METTL3-2 (5'-GCCUUAACAUUGCCCACUGAUTT-3'); Si-METTL3-3 (5'-CCAGUCAUAAACCAGAUGAAATT-3')) or Si-METTL14 (Si-METTL14-1 (5'-GCCGUGUUAAAUAGCAAAGAUTT-3'); Si-METTL14-2 (5'-GCUAAUGUUGACAUUGACUUATT-3'); Si-METTL14-3 (5'-CCAUGUACUUACAAGCCGAUATT-3')), followed by formal functional phenotype verification after knockdown: cells were plated in 24-well plates. KCs were transfected with siRNA using lipo2000 (Invitrogen). For overexpression experiments, adenovirus intervention was used: CMV-MCS-SV40-EGFP adenovirus vector was inserted into the coding sequence of human METTL3 (GenBank: NM_019852, target sequence: 5'-ACATTCCACAGTTAGCTA-GCTATAAATTCTTAGGTTTAGAGATG-3') or human METTL14 (GenBank: NM_020961, target sequence: 5'-GAGGATCCCCGGGTACCGGCGCCACCATGGATAGCCGCTTGCAGGAG-3') gene. For viral infection, cells were cultured overnight in DMEM containing 10% fetal bovine serum and replaced with medium containing HitransGA (GeneChem) the next day. Adenovirus was added to the well at the optimal infection multiplicity (MOI). After 8-16 hours of infection, the medium was replaced with complete medium. The expression levels of protein and mRNA were detected 72 h after infection. Cell apoptosis was detected using Annexin V-FITC/PI apoptosis kit (Elabscience). Flow cytometry analysis was then performed. RT-qPCR and WB were used to detect cell proliferation and apoptosis molecular markers after knockdown or overexpression intervention.

6. Results

6.1 Differentially expressed ^m6A RNA methylation regulatory molecules and potential targets in AN lesions

Normal controls (n = 3) and AN lesions (n = 3) were biopsied and analyzed by m ⁶ A sequencing, and the sequencing data have been submitted to the GEO database (GSE212300). After sequencing, KEGG and GO libraries were used for analysis, and it was found that hormone, pigmentation, and inflammation-related pathways were the most important regulatory pathways (Figure 2A–B). The most significantly differentially expressed (DE) genes in each pathway are shown in Figure 1D. Wayne analysis showed that ADRA2A, FGFR1, SFRP1, INHBA, PDGFRB, MEF2C, and EDN3 were DE candidate proteins related to inflammation, hormone metabolism regulation, and pigment signaling pathways in AN lesions (Figure 1B).

Next, we confirmed that the level of ^m6A mRNA in AN lesions was significantly higher than that in normal skin (Figure 2A). The mRNA and protein levels of METTL3 and METTL14 were significantly upregulated in AN lesions compared with normal skin (Figure 2B-C). METTL3 and METTL14 were highly expressed in epidermal keratinocytes of AN lesions, while they were lowly expressed in normal skin (Figure 2D). The mRNA levels of other ^m6A modification-related enzymes did not change significantly (Figure 6A, p>0.05). We also confirmed that ADRA2A and INHBA showed increased mRNA, ^m6A -modified mRNA, and protein levels in AN lesions compared with normal skin (Figure 2E-G). RT-PCR assays showed that the mRNA levels of other candidate AN lesions, such as FGFR1, SFRP1, PDGFRB, MEF2C, and EDN3, were not significantly different in AN lesions compared with normal skin (Figure 6B, p>0.05).

Correlation analysis of sequencing results from the GTEx database showed that there was a significant positive correlation between the expression level of METTL14 and ADRA2A or INHBA (Figure 7); the level of METTL3 was significantly positively correlated with the level of INHBA (Figure 7). These data suggest that ADRA2A and INHBA are downstream targets of ^m6A RNA methylation and may be regulated by METTL3 and METTL14.

6.2 METTL3 and METTL14 regulate the expression of ADRA2A and INHBA, as well as biomarkers of cell proliferation, pigmentation, and apoptosis

To verify the potential regulatory role of METTL3 and METTL14 in the expression of ADRA2A and INHBA, we screened and identified the siRNA sequences for the expression of METTL3 or METTL14 in KCs (Figure 8). All three siRNA sequences had significant effects on METTL3 or METTL14, and si-METTL3-3 and si-METTL14-3 were subsequently selected for further follow-up experiments. Knockdown of METTL3 or METTL14 resulted in a significant decrease in the mRNA, m ⁶ A, mRNA, and protein levels of ADRA2A and INHBA (Figure 3A). We confirmed that a series of biomarkers of proliferation and differentiation (keratin 14, cyclin E1, cyclin D1, cyclin B1), apoptosis (PARP) and pigmentation (TYRP1) were significantly downregulated (Figure 3B-D), and Cleaved- and pro-caspase-3 were significantly upregulated (Figure 3D), which was also confirmed by the increase in cell apoptosis as shown by Annexin V-FITC detection (Figure 3D).

We then performed functional phenotypic studies by overexpressing METTL3 or METTL14 in KCs (Figure 4). The results showed that overexpression of METTL3 or METTL14 led to increased levels of ADAR2A and INHBAm RNA, m ⁶ A mRNA and protein levels (Figure 4A), and significant upregulation of biomarkers of proliferation and differentiation (keratin 14, cyclin E1, cyclin D1, cyclin B1), apoptosis (PARP) and pigmentation (TYRP1 and ET-1) (Figure 3B-D), and Cleaved-and pro-caspase-3 (Figure 4D). Abnormal proliferation of KCs can activate MCs and stimulate the synthesis and secretion of melanosomes. We confirmed that in a co-culture system of MCs and KCs, tyrosinase activity, melanin content and proliferation were significantly inhibited or increased when METTL3 or METTL14 was knocked out or overexpressed (Figure 5A-B). Based on these data, we proposed a mechanistic hypothesis for the development of AN disease (Figure 5A). METTL3 and METTL14 promoted the expression of ADRA2A and INHBA through ^m6A methylation of mRNA, enhanced the paracrine secretion of KCs and the pigment metabolism of MCs, and thus enhanced the occurrence and development of AN (Figure 5C).

In summary, the present study showed that the m ⁶ A RNA level in acanthosis nigricans (AN) lesions was higher than that in normal skin. In particular, we found that the expression levels of adrenergic receptor 2A (ADRA2A) and inhibin subunit βA (INHBA) were significantly increased in AN lesions compared with normal skin, and their expression was regulated by m ⁶ A methyltransferase 3 (METTL3) and methyltransferase 14 (METTL14). Functional studies showed that METTL3 and METTL14 regulated the expression of various biomarkers of cell proliferation, apoptosis and pigmentation in keratinocytes and melanocytes. In summary, these data indicate that m ⁶ A modification mediated by METTL3 and METTL14 upregulated the expression of ADRA2A and INHBA and promoted the occurrence and development of AN. Therefore, METTL3/METTL14 has the potential to be used as a target site in the preparation of drugs for the treatment of AN, and can also be used as a molecular marker for the diagnosis of AN in clinical detection kits.

Claims (4)

1. Use of inhibitors of METTL3 or METTL14 in the preparation of drugs for the treatment of acanthosis nigricans. 2. The use according to claim 1, wherein the inhibitor of METTL3 or the inhibitor of METTL14 is siRNA. 3. The use according to claim 1, wherein the inhibitor of METTL3 is shown as SEQ ID NO.51, SEQ ID NO.52 or SEQ ID NO.53, and the inhibitor of METTL14 is shown as SEQ ID NO.54, SEQ ID NO.55 or SEQ ID NO.56. 4. Application of METTL3 or METTL14 as molecular markers in the preparation of diagnostic kits for acanthosis nigricans.

Images