Research on Hotair and 7SL-RNA Gene Expression Levels in Psoriasis Vulgaris

Abstract

Backgrounds:

Mutation of protein-coding genes and non-coding genes is a factor in psoriasis etiology. Non-coding RNA (ncRNA), which does not have protein-coding capacity, is available in the human genome. HOTAIR (HOX Antisense Intergenic RNA) and 7SL-RNA are known as ncRNA. They may play a role in psoriasis pathogenesis.

Aims:

In our study, we aimed to investigate the level of HOTAIR and 7SL-RNA gene expression in the lesional and perilesional healthy skin of psoriasis patients.

Methods:

Total RNA isolation from the skin samples was achieved by modifying the RNeasy Mini Kit (Qiagen, Cat No: 74104) protocol. Real Time Polymerase Chain Reaction (qPCR) phase was performed in accordance with the protocol of the relevant brand (WizPure qPCR).

Results:

7SL-RNA gene expression decreased in the skin with psoriatic lesions (FC: 0.01; p: 0.028), and this decrease was statistically significant. HOTAIR gene expression decreased (FC: 0.92; p: 0.218), but this decrease was not statistically significant.

Conclusions:

lncRNAs may play a role in the pathogenesis of psoriasis disease.

Introduction

Psoriasis is a chronic inflammatory skin disease caused by the immune system. It is thought that mutation of protein-coding genes and non-coding genes is a factor in psoriasis etiology in addition to environmental factors.[1] A single gene locus is not responsible for psoriasis pathogenesis. As a result of the conducted gene analysis studies, 15 psoriasis susceptibility gene loci (PSORS1-15, Psoriasis Susceptibility) were identified in the human genome.[2] PSORS1 is the main susceptibility locus for psoriasis, and genome studies suggest that the PSORS1 gene is responsible for 50% of psoriasis patients.[3,4]

There are gene sequences encoding 20,000–25,000 proteins in the human genome sequence, and these genes make up less than 2% of the total genome sequence. Transcription continues along the mammalian genome as well as protein-coding domains. Non-coding RNA (ncRNA), which does not have protein-coding capacity, is available in the human genome.[5] In humans, ncRNAs constitute 98% of the transcriptional efficiency. They have tissue-specific expression patterns of ncRNAs and are potential biomarkers. Therefore, they can be used as clinical diagnosis and prognostic indicators.[6] ncRNAs are divided into two as small non-coding RNAs (snRNA) and long non-coding RNAs (lncRNA).[7] As a result of the transcription studies carried out in the past 10 years, it has been revealed that lncRNAs are involved in the regulation of the inflammatory reaction in psoriatic skin.[8]

HOTAIR (HOX Antisense Intergenic RNA) is a lncRNA, and its expression level is a potential biomarker for diagnosis and treatment in many types of cancers.[9] 7SL-RNA, which is also a lncRNA, is a component of the signal recognition particle (SRP) complex in the ribonucleoprotein structure involved in intracellular protein regulation, and the decrease in its expression causes a malfunction in the antiviral response.[7] In addition, 7SL-RNA is overexpressed in many tumors and contributes to the proliferation of tumor cells by suppressing p53 translation.[10] Based on this, we aimed to analyze the level of HOTAIR and 7SL-RNA gene expression in the lesional and perilesional healthy skin of psoriasis patients to investigate whether these genes play a role in psoriasis pathogenesis in our study.

Materials and Methods

Study population

In this study, approval was obtained from Sivas Cumhuriyet University interventional clinical research ethics committee and was carried out according to the Declaration of Helsinki principles. Fifteen patients admitted to the Department of Dermatological and Venereal Diseases of Cumhuriyet University Medical Faculty Hospital and diagnosed (clinically or histopathologically) with psoriasis were included in the study. A total of 30 skin samples (two punch biopsies from 15 psoriasis patients' psoriatic lesions and perilesional healthy skin) were taken. Psoriasis patients who were 18–65 years old, not pregnant, had not received systemic treatment in the past 6 months (this period is 3 months for acitretin), had tested negative for hepatitis B, C, and HIV, did not have an active infection, a chronic disease, or a metabolic syndrome were included in the study.

Skin samples were placed in tubes containing RNA later as soon as they were taken and then stored at -20°C until the study.

RNA isolation and real time polymerase chain reaction (qPCR)

Total RNA isolation from the skin samples was achieved by modifying the RNeasy Mini Kit (Qiagen, Cat No: 74104) protocol. Complementary DNA (cDNA) synthesis was performed in accordance with the kit protocol using the WizScript cDNA Synthesis Kit (W2211-1) for the reverse transcription of RNAs. The qPCR synthesis phase was started upon the completion of cDNA synthesis. HOTAIR (F-190729-1-49, R-190729-1-50), 7SL-RNA (F-190729-1-51, R-190729-1-52) and optimized primers of the reference gene Beta Actin (ACTB) (160527) were used, and the qPCR phase was performed in accordance with the protocol of the relevant brand (WizPure qPCR).

Statistical analysis

The power of the study was calculated as 93% when the prevalence was taken as 2% in identifying the sample size for the skin samples to be used in this study.

Data obtained as a result of qPCR were uploaded on the software “GeneGlobe Data Analysis Center” (https://www.qiagen.com/de/shop/genes-and-pathways/data-analysis-center overview-page/). The analysis was carried out according to the △△Cq method. The P value was calculated based on the Student's t-test for each gene studied from the healthy and lesional skin sample of psoriasis patients. A P value of less than 0.05 was considered significant.

Results

Fifteen patients diagnosed with psoriasis were included in the study. Nine (60%) of the patients were male and six (40%) were female. The mean age was 28. Two punch biopsy samples were taken from the lesional skin and perilesional healthy skin of psoriasis patients, and HOTAIR and 7SL-RNA gene expression levels were studied in a total of 30 skin samples, including 15 perilesional healthy skin samples and 15 psoriatic lesional samples.

The fold change graphs of HOTAIR and 7SL-RNA in the patient group compared to the control group are shown in Figure 1.

Figure 1.

The fold change graphs of HOTAIR and 7SL RNA in the patient group compared to the control group

In patients with psoriasis, 7SL-RNA gene expression decreased in the skin with psoriatic lesions (FC: 0.01; p: 0.028), and this decrease was statistically significant [Table 1]. HOTAIR gene expression also decreased (FC: 0.92; p: 0.218), but this decrease was not statistically significant.

Table 1.

Findings of comparison of gene expressions in lesions and healthy skin of psoriasis patients

Parameter	Gene	Fold Change	P
Skin with Lesion Healthy Skin	7SL-RNA	0.01	0.028
	HOTAIR	0.92	0.218

When a group of 28-year old and young patients was compared, it was observed that HOTAIR expression increased (FC: 2.50; p: 0.182) and 7SL-RNA expression decreased (FC: 0; p: 0,123) in the group with patients older than 28, but none of the values was statistically significant.

HOTAIR expression decreased (FC: 0.86; p: 0.812); and the expression of 7SL-RNA increased in smokers compared to non-smokers (FC: 65.99; p: 0.272); however, none of the values was statistically significant.

When disease durations were compared, HOTAIR expression decreased (FC: 0.49; p: 0.351) and the expression of 7SL-RNA increased (FC: 214.4; p: 0.101) in patients with disease durations of 6–10 years; however, none of the values was statistically significant.

Discussion

Our study is the first study investigating the level of HOTAIR and 7SL-RNA gene expression in the lesional and perilesional healthy skin of psoriasis patients. The studies investigating the relationship between lncRNAs and psoriasis are limited in the literature.

Psoriasis is a chronic inflammatory skin disease that affects 2% of the population.[11] Many studies have shown that environmental factors, immune system, and genetic factors play a role in the pathogenesis of psoriasis. The emergence of the disease in the first- and second-degree relatives of individuals with psoriasis disease shows that psoriasis has a genetic basis. The heritage pattern is complex; more than one chromosomal locus is associated with the disease. Various antigens stimulate T cells in genetically susceptible individuals, resulting in keratinocyte proliferation, epidermal differentiation, and cutaneous inflammation.[12,13]

Non-coding RNAs (ncRNA) constitute a new class of transcripts that are encoded by the genome but are mostly not converted into proteins. They play an important role in many cellular and physiological events. The lncRNAs (>200 nt ncRNAs) in particular play a key role in regulating chromatin dynamics, gene expression, growth, differentiation, and development. Therefore, it has been suggested that they can be used as a therapeutic target and biomarker for many diseases.[14] There are studies in the literature showing that lncRNAs play a role in cardiovascular diseases such as cancer, hypertension, myocardial infarction, atherosclerosis, and chronic inflammatory diseases, bacterial and viral infections such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis; monoclonal gammopathies, chronic kidney diseases, and neurodegenerative diseases.[15,16]

As an lncRNA, HOTAIR (Gene ID: 100124700) is located in the 12q13.13 region of the human genome and plays an important role in the pathological process of endocrine system diseases, cardiovascular diseases, and tumoral diseases such as cervical cancer and breast cancer.[16] In a study by Zhang et al., the overexpression of the HOTAIR gene has been shown to suppress inflammation by causing a decrease in proinflammatory cytokines such as IL-17 and IL-23, and the HOTAIR overexpression has been identified to have a proliferative and anti-inflammatory effect in vivo by rheumatoid arthritis (RA) mouse experiments. There are Th17 cells and pro-inflammatory cytokines such as IL-17 and IL-23 in the pathogenesis of RA.[17] Based on this information, studies have been conducted regarding the assumption that the HOTAIR gene may be a new marker for RA.[17,18,19] We may think that the proliferative effect of HOTAIR gene expression may be a secondary development to suppress inflammation. HOTAIR gene expression level was examined in skin samples taken from psoriatic lesion and adjacent healthy skin for this purpose. In the psoriatic lesion, HOTAIR gene expression level had decreased by 0.92 times compared to perilesional healthy skin, but this was not statistically significant.

The signal recognition particle (SRP) consists of the 7SL-RNA molecule and six polypeptides that have molecular weights of 72 kd, 68 kd, 54 kd, 19 kd, 14 kd, and 9 kd. It regulates the translocation of proteins along the endoplasmic reticulum membrane during protein synthesis. Anti-SRP autoantibodies were first described in 1980 in myositis patients.[20,21] In subsequent studies, patients with autoimmune diseases such as Dermatomyositis (DM), RA, inflammatory bowel disease (IBM), systemic sclerosis (SSc), and systemic lupus erythematosus had shown anti-SRP antibody positivity.[22,23] In addition, in a study by Wang et al., it was stated that 7SL-RNA, a component of SRP, has been shown to play a role as a cofactor in the natural antiviral function of cytidine deaminases.[24] There are studies showing that skin microbiota can stimulate immune reactions in individuals with genetic predisposition to psoriasis. The cutaneous microbiome of patients with psoriasis is different from that of healthy individuals. Although a wide range of microorganisms are associated with psoriasis (Beta hemolytic streptococcus, Staphylococcus aureus, Porphyromonas gingivalis, Candida albicans, Chlamydia psittaci, human immunodeficiency virus, and hepatitis C virus, etc.), evidence on antimicrobial treatment preventing exacerbations of psoriasis is not sufficient.[25] Similarly, human papilloma virus (HPV) type 5 sequence was found positive in the lesions of psoriasis patients and this resulted in the hypothesis that HPV type 5 might play a role in the pathogenesis of psoriasis, and this virus might cause antigen stimulation that induces replication in psoriatic keratinocytes, epidermal hyperproliferation, and also autoimmune reactions.[26]

Based on these functions defined in relation to 7SL-RNA, we wanted to investigate the level of 7SL-RNA gene expression in psoriatic lesions and perilesional healthy skin of psoriasis patients. As a result of our study, 7SL-RNA gene expression was observed to decrease by 0.01 times in psoriatic lesion compared to perilesional healthy skin, and this decrease was found to be statistically significant. This reduction in the 7SL-RNA level may be related to a possible antibody development. Developed antibodies can contribute to inflammatory reactions in psoriasis. When considered from another point of view, decreased 7SL-RNA expression in psoriasis lesion may cause a decrease in antiviral activity and consequently changes in skin microbiota. Further studies are required to clarify this issue.

This study had certain limitations. We thought that IL-17 and IL-23 levels may have increased as a result of decreased HOTAIR gene expression, but we could not evaluate the levels of these interleukins in the skin sample.

We anticipated that a possible antibody presence for 7SL-RNA may contribute to inflammation. However, we could not evaluate proinflammatory and anti-inflammatory cytokines at tissue level with the 7SL-RNA antibody.

As a result, lncRNAs may play a role in the pathogenesis of psoriasis disease, but further studies on correlations between IL17/IL23 and lncRNAs need to performed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.