Semaphorin-3A as An Immune Modulator Is Suppressed
Semaphorin-3A (SEMA3A) and its receptors are found on some immune cells and act as suppressors of
immune cells over-activation. Considering the role of
Materials and Methods
In this experimental study, we evaluated the effect of miR-145-5p transfection on
Our results showed that miR-145-5p is able to decrease
These results enlightened us about an unknown aspect of
Semaphorins are members of a large and diverse protein family and contain a common conserved cysteinerich domain located in N-terminal region. This domain includes approximately 500 amino acids in length and is termed "sema" domain (1). Semaphorins could be seen as secreted transmembrane or glycosyl phosphatidyl inositol (GPI)-linked proteins and up to now, 20 types of them have been observed in humans (2).
Semaphorins were first found in 1990s as axon guidance molecules which could act as bifunctional signaling molecules to supply chemorepellent or chemoattractant cues in the nervous system (1). Semaphorin 3A (SEMA3A) which belongs to semaphorins family, was the first semaphorin discovered in vertebrate in 1993 (3). Sema3-A is a secreted protein that has prominent roles in regulation of the immune system and has been found to be correlated with some autoimmune diseases.
SEMA3A is able to convey its signal through plexin
A1 or plexin A4 by direct binding to neuropilin-1 with
high affinity. Previously, high expression of
The main function of SEMA3A in the immune system is modulation of immune responses. SEMA3A can suppress B and T cell proliferation and alleviate generation of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-a) and interferon- gamma (IFN-.) by T cells (4). Also, it enhances the regulatory properties of B cells and induces apoptosis in monocyte-derived macrophage colosny-stimulating factor (M-CSF)-differentiated macrophages (6, 10).
Furthermore, microRNAs (MiRNAs) were also discovered in 1993 (11). Amajor part of the human genome is transcribed, but only around 2% of these transcripts are translated into proteins. Many of the remaining transcripts which are not translated into protein, are RNA molecules and have biological functions. These RNA molecules have been classified based on their sizes and non-coding RNAs (ncRNAs) which are less than 200 nucleotides in length are called short non-coding RNAs (sncRNAs) (12).
MiRNAs which belong to sncRNAs have 19-22 nucleotides in length and are posttranscriptional regulatory RNA molecules that participate in the regulation of gene expression through base pairing with 3' untranslated regions (3'UTR) of mRNAs. This binding leads to mRNA instability (13). MiRNAs could participate in the regulation of apoptosis, hematopoiesis, immune regulation and other biological processes (14, 15). Recently, it has been revealed that miRNAs participate in a broad spectrum of human diseases such as neurological disorders.
Moreover, alteration of miR-145-5p was investigated in multiple sclerosis (MS) and it has been demonstrated that miR-145-5p is overexpressed in peripheral blood mononuclear cells (PBMCs) of MS patients. In MS patients, miR-145-5p is overexpressed (3-folds) as compared to controls (21, 22).
As previously described, SEMA3A is produced by PBMCs such as activated lymphocytes and plays anti-inflammatory roles in the immune system. The role and alteration in the level of SEMA3A have been investigated in some autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), psoriasis and systemic sclerosis (SSc). In some of these studies, down-regulation of SEMA3A have been reported (7, 23-25).
Based on our bioinformatic predictions (using
Targetscan and miRwalk softwares), we assumed that
miR-145-5p could have a possible powerful interaction
In this study, we aimed to investigate the relationship
between the inhibitory effect of miR-145-5p and
Materials and Methods
Firstly, Mirwalk 2.0 (http://zmf.umm.uni-heidelberg.
de/apps/zmf/mirwalk2/) was used to make bioinformatic
Cell isolation and culture
In this experimental study, PBMCs were isolated fromhealthy donors using Ficoll-Isopaque (Lymphodex, Germany). The study was approved by the EthicsCommittee of Isfahan University of Medical Sciencesof Iran, Isfahan, Iran. Then, cells were cultured in RPMI 1640 [(Biosera, France), containing 10% fetal bovine serum (FBS, Biosera, France)] and 1% penicillin- streptomycin (Biosera, France) in 6-well U-bottomplates (1×106 cells/well/2 ml) in the presence of 7.5 µL/ml Phytohemagglutinin (PHA, Sigma-Aldrich, Germany). Cells were incubated for 96-144 hours at37°C in a humidified chamber with 5% CO2 (Memert, Germany).
After 72 hours, cultured cells were transfected (50 nM final concentration) with miR-145-5p mimic (Qiagen, Germany) using X-treme gene (Roche, Germany). The culture medium was refreshed and cell culture was continued for 24 hours. Treatment with X-treme gene alone was applied as mock control. PBMCs in some wells were transfected with Label IT® RNAi Delivery Control (Mirus, USA) both as an indicator of transfection efficiency and as a scrambled siRNA (Fig .1,). Transfection efficiency was analyzed by FACS Calibour flow cytometer (Becton Dickinson) and CellQuestTM Pro software.
Quantitative real-time polymerase chain reaction analysis of SEMA3A mRNA level
Total RNA was isolated from PBMCs using RNXTMPLUS (CinnaGen, Iran) and it was reverse transcribed using a first-strand cDNA synthesis kit (Thermo scientific, USA) according to the manufacturer’s instructions. Sense and antisense primers were:
5´-TAGTTGTTGCTGCTTAGTGGAAAG-3´for SEMA3A and
SEMA3A protein assay
Cells were centrifuged and an aliquot of the supernatant was collected for SEMA3A level analysis by ELISA. The level of secreted SEMA3A was evaluated by a commercial ELISA kit (Elabscience, China) according to the manufacturer’s instructions.
Methylthiazole tetrazolium assay
The MTT assay has been used as a rapid and sensitive method for assessment of chemicals’ cytotoxicity. Here, 90 µl cell-containing medium was added to each well of a 96well plate and then, 10 µl MMT solution was added to each well. Cells were incubated for 1 hour at 37°C with 5% CO2.Then, the medium was removed and the plate was frozen for 1 hour at -80°C. Then, 100µl dimethyl sulfoxide (DMSO, Parstous, Iran) was added to each well and incubated for 30 minutes at 37°C while shaking. Finally, the optimal density of each well was measured at 590 nm.
For statistical analyses, SPSS 20.0 software was used. One Way ANOVA was utilized for making comparisons between treated groups. All experiments were performed in triplicate. Data are expressed as mean ± SD, and P<0.05 were considered statistically significant.
The miR-145-5p was predicted as an SEMA3A silencer miRNA
According to the Mirwalk output, miR-145-5p was
predicted to suppress
Efficiency of transfection
Incubation with 50 nM final concentration of Lable IT siRNA Delivery Control-FITC for 24 hour, revealed that 81% of the cells were successfully transfected (Fig .3,).
Decreased expression of
SEMA3A in PBMCs in the presence of miR-145-5p
SEMA3A secretion was down-regulated after miR145- 5p transfection
Based on the ELISA results, the SEMA3A secretion by cells transfected with miR-145-5p mimic was lower than other groups (Fig.5,). There was no significant difference among negative control, mock and scrambled groups. SEMA3A level was considerably lower in miR-145-5ptransfected cells (0.16 ± 0.01 ng/ml) compared to control cells (0.79 ± 0.01 ng/ml) and this decrease was significant (P=0.015).
Cell viability assay
MTT assay was performed in order to assess the cytotoxicity of the transfection process. Our results showed that miR-145-5p mimic has no significant cytotoxic effect on PBMCs (P=0.416) and a viability of 90% was observed in the test group (Fig .6,).
Regulation of the immune system and homeostasis is
vital for prevention of pathological processes that lead to
autoimmune diseases (26). In this context, modulatory
molecules like SEMA3A and miRNAs have a pivotal
role in induction and maintenance of self-tolerance. As
mentioned earlier, SEMA3A acts as a terminator of T cells
and B cell activity and down-regulates the expression of
pro-inflammatory cytokines (23). So, decreased
Some studies have shown that
By applying bioinformatic tools, we observed that
miR-145-5p might be able to silence SEMA3A with a
probability of 99%. Also, information obtained from these
databases indicted that the silencing effect of miR-145-5p
The current study showed that
Based on our literature review, there was no study on
miR-145-5p effect on
As mentioned before, miR-145 expression was up- regulated in MS and primary biliary cirrhosis (22, 29). However, in another study, it was stated that miR-1455p is down-regulated in SLE patients (30). Controversial results reported by these studies may be explained by the differences in immunopathogenesis of these diseases as Th2 cells are more highlighted in SLE.
The significant increase in miR-145 and decrease in
SEMA3A in PBMCs of MS patients might be explained
by our results. In this study, we did not investigate the
exact mechanism underlying this down-regulation, but
we concluded that administration of miR-145 leads
to decreased expression of
It is suggested that more investigations should be done to clear the correlation between immune regulators like SEMA3A and other miRNAs which are altered in T or B cells and these findings can help to open up a new insight about immune regulation.
Our results revealed that miR-145-5pcan down-
This study was partly funded by Multiple Sclerosis and Neuroimmunology Research Center of Isfahan and also received a grant-in-aid of research from Isfahan University of Medical Sciences (Grant No. 394250). The authors have no conflicts of interest to report.
M.R.; Wrote the manuscript, which was revised by M.G.-h. M.G.-h., M.R.; Contributed to study design, data collection and interpretation of data. M.R., S.S.; Contributed to all experimental work. M.M.; Participated in statistical analysis. N.E., M.S., M.E.; Were responsible for overall supervision. All authors read and approved the final manuscript.