miR-146a modulates TLR1/2 and 4 induced inflammation and links it with proliferation and lipid production via the indirect regulation of GNG7 in human SZ95 sebocytes

Activation of Toll-like receptors (TLR) 1/2 and 4 are central in inducing inflammation in sebocytes by regulating the expression of protein coding mRNAs, however the microRNA (miRNA) profile in response to TLR activation and thus the possible role of miRNAs in modulating sebocyte functions has not been elucidated. In this work we identified miR-146a to have the highest induction in the TLR1/2 and 4 activated SZ95 sebocytes and found that its increased levels led to the down-regulation of IL-8 secretion, decreased the chemoattractant potential and stimulated the proliferation of sebocytes. Assessing the gene expression profile of SZ95 sebocytes treated with a miR-146a inhibitor, the induction of GNG7 was one of the highest, while when cells were treated with a miR-146a mimic, the expression of GNG7 was down-regulated. These findings correlated with our in situ hybridization results, that compared with control, miR-146a showed an increased, while GNG7 a decreased expression in sebaceous glands of acne samples. Further studies revealed, that when inhibiting the levels of GNG7 in SZ95 sebocytes, cells increased their lipid content and decreased their proliferation. Our findings suggest, that miR-146a could be a potential player in acne pathogenesis by regulating inflammation, inducing proliferation and, through the indirect down-regulation of GNG7, promoting the lipid production of sebocytes.

shaping the inflammatory environment [19][20][21] , in which their activation through TLRs 14,22,23 might play a central role. Supporting this pustulate, our previous genome wide gene expression study showed that sebocytes are able to rapidly gain and prioritize an immune-competent status in response to TLR1/2 and/or TLR4 activation at the level of mRNA expression 22 .
MicroRNAs (miRNA) are small non-coding RNAs also selectively transcribed from the genome under various conditions. In contrast to mRNAs they do not encode proteins but control gene expression by binding and destabilizing their target mRNA 24 . Therefore, they regulate transcriptional and post-transcriptional gene expression. In human sebocytes, by transfecting SZ95 sebocytes with siRNAs directed against DICER, a key enzyme of miRNA biogenesis, miRNA presence was confirmed and was proven essential for lipogenesis, but without providing any disease-specific conclusions on their possible role 25 . Moreover using whole tissue samples, miR-NAs were also linked to sebaceous gland associated tumors [26][27][28][29] , however the cellular source of the differentially expressed miRNAs was not assessed.
In the present study, we aimed to extend our knowledge on the gene expression regulation and profile of TLR-activated sebocytes and investigated the role of miRNAs in it. We found that in TLR1/2-and 4-activated sebocytes, miRNAs had altered expression levels, with miR-146a showing the most prominent upregulation. Confirming that sebaceous glands of acne samples also exhibited high expression levels of miR-146a, we aimed to define a possible pathophysiological role for miR-146a in sebocytes. Our results suggest that miR-146a may not only regulate TLR-induced inflammation in sebocytes but could be a missing link in connecting it with hyperproliferation and increased sebum production by indirectly regulating the expression of G protein gamma 7 (GNG7), which may have both pathological and therapeutic implications in sebaceous gland-associated diseases, such as acne.
Results miR-146a shows the most prominent induction in TLR1/2 and 4-stimulated SZ95 sebocytes. Sebocytes are able to sense and respond to different TLR stimuli 22 , making them an active player in a pathogen-associated inflammatory environment. To investigate the change in their miRNA profile in such a response, we applied two different TLR activators, PAM3CSK4 (TLR1/2 activator) and LPS (TLR4 activator), to treat human SZ95 sebocytes. Performing genome wide expression studies in samples treated for 24 h, 23 miR-NAs responded to PAM3CSK treatment, while 54 miRNAs were significantly upregulated after LPS treatment (Supplementary Table 1A-B). Both TLR activators induced common significant elevation of 14 miRNA expression levels (Fig. 1a), of which miR-146a showed the most abundant values (Fig. 1b). This significant increase in the levels of miR-146a could also be detected by in situ hybridization in TLR1/2-and 4-activated SZ95 sebocytes (Fig. 1c). miR-146a is elevated in sebaceous glands of human acne tissue samples. To provide a biological relevance for our finding, we performed in situ hybridization against miR-146a in 5 acne vulgaris and 5 normal skin samples from the back of young male adults. While in normal skin, miR-146a was detectable with lowintensity homogeneous staining, a more intense, mainly granular staining was observed in sebaceous glands of acne tissue samples (Fig. 2). miR-146a decreases IL-8 secretion and negatively regulates the chemoattractant potential of SZ95 sebocytes. To assess the possible role of miR-146a, we focused on the functional analysis of SZ95 sebocytes transfected with hsa-miR-146a mimic, hsa-miR-146a inhibitor, or their negative controls for 72 h.
As IL-8 is an important chemokine in immune cell migration, we examined the migratory capacity of peripheral blood monocytes towards supernatants of transfected SZ95 sebocytes. We observed a lower migration to hsa-miR-146a mimic-transfected and an increased migration towards hsa-miR-146a inhibitor treated SZ95 sebocyte supernatants, showing that miR-146a may have a negative regulatory role on monocyte chemoattraction under inflammatory conditions (Fig. 3b). miR-146a regulates cell proliferation of SZ95 sebocytes. To examine the role of miR-146a in sebocyte proliferation and lipid production of cells treated with hsa-miR-146a mimic, hsa-miR-146a inhibitor, or their negative controls for 72 h, we analyzed the cell numbers in different cell cycle phases using DNA content histograms and applied Oil Red O staining to detect lipids.
When incubated with miR-146a mimic sequence, the proportion of cells in G2 and M phases increased (56.02% versus 36.78% of the cells) with a significant decrease in S phase (8.69% versus 21.51% of cells, p < 0.05) and a slight decrease in G0 and G1 phases (35.28% versus 41.71% of cells) in comparison with control ( Fig. 3c), showing that cell proliferation is stimulated by higher miR-146a levels. Transfecting SZ95 sebocytes with miR-146a inhibitor, the cell proportion in the S phase increased (32.62% versus control 25.95% of cells) and the G0/ G1 population decreased (30% versus 38.93%), suggesting that the mitotic machinery itself was activated but might be blocked in S phase. According to the DNA histogram, a peak of apoptotic cells was also observed following miR-146a inhibitor treatment.
Lipid measurements found a slight, but statistically not significant decrease in the lipid content of miR-146a inhibitor-treated cells and no change in cells treated with the mimic sequence (Fig. 3d).  Fig. 4a. Functional gene clustering confirmed that the altered levels of miR-146a in sebocytes may lead to changes in immune response, angiogenesis and signaling pathways such as Wnt or G protein mediated ones (Fig. 4b).
miR-146a levels in sebocytes lead to changes in the expression of GNG7. According to the transcriptome analysis, GNG7 had one of the highest upregulation following miR-146a inhibition (Fig. 5a). The miR-146a mediated expression changes of GNG7 could be further confirmed by qRT-PCR and in situ hybridization in SZ95 sebocyte cultures transfected with mimic, inhibitor and control sequences (Fig. 5b,c), suggesting a central role for GNG7 in miR-146a-induced signaling.
To assess if GNG7 mRNA could be also detected in vivo, in situ hybridization was performed on human acne and normal skin samples. The presence of GNG7 mRNA could be visualized in sebaceous glands of normal skin, while in acne samples, in which elevated miR-146a levels were shown, it could not be detected (Fig. 5d). in sebocytes, next we performed RNA silencing experiments, using siRNA against GNG7 mRNA. According to our findings, down-regulation of GNG7 in SZ95 sebocytes did not affect IL-8 secretion ( Fig. 5e) but caused notable changes in cell proliferation (Fig. 5f). The cell population transfected with siRNA, showed elevated proportion of G0/G1 cells (73% vs control 47%, p-value: 0.1) and decreased proportion of G2/M cells (5% vs control 30%, p-value: 0.1) compared to cell populations transfected with negative control sequences showing that the mitotic cascade is less active, the majority of the cells are not proliferating. Performing lipid measurements, we found a tendentious elevation in the lipid content of GNG7 siRNA-treated SZ95 sebocytes compared to negative control ones (p-value: 0.15, Fig. 5g). www.nature.com/scientificreports/

Discussion
The application of a system-based approach of whole genome sequencing of SZ95 sebocytes, treated with specific and selective TLR1/2 and TLR4 activators, provided evidence that miRNAs are selectively induced in sebocytes   www.nature.com/scientificreports/ upon TLR activation. Moreover, identifying miR-146a as the miRNA with the most abundant induction with an increased expression also in the sebaceous glands of acne samples, our work is the first to identify a miRNA, which is increased in sebaceous glands in a non-malignant, inflammatory disease setting. Characterizing sebocytes with altered levels of miR-146a, we showed that miR-146a is not only a marker for activation, but could have a regulatory role on cell proliferation and on the immune-competence of sebocytes. Our further findings, that GNG7 was found to be oppositely regulated both in SZ95 sebocytes as well as in sebaceous glands of acne samples, suggests a cascade of events in which the induction of miR-146a leads to proliferation, however the consecutive down-regulation of GNG7 promotes lipid production of sebocytes.
In line with our previous results, showing that TLR1/2 and TLR4 pathways induced a similar change in the mRNA profile of sebocytes, miRNAs also changed similarly in response to the used activating agents. This finding further supports our previously raised hypothesis that these pathways and the related changes are not stimulus-/pathogen-specific in sebocytes as these receptors can be activated with a wide range of stimuli both of pathogenic and of non-pathogenic origin 22 . In other words, sebocytes use these receptors to sense changes in their environment, such as an altered microbiome or the presence of lipids, which activation needs to be further modulated to gain its disease specific role. Based on our results miR-146a may be a potent regulator, just as it is observed in various cell types of lymphoid, myeloid and of non-immune origin, where miR-146a decreases the production of inflammatory cytokines 31 . Indeed, regarding dermatological diseases, increased levels of miR-146a was already confirmed in keratinocytes of acne just as in psoriasis and atopic dermatitis samples, with a suggested role to regulate inflammation. Moreover, in psoriasis, its genetic alterations even showed an association with disease severity [32][33][34][35] . Higher levels of miR-146a was also detected in keratinocytes treated with LTA (TLR2 activator), where it may down-regulate C. acnes-induced production of IL-6, -8, and TNF-α by inhibiting the TLR2/IRAK1/TRAF6/NF-κB and MAPK pathways 36 . Our findings that miR-146a was also highly expressed in sebaceous glands of acne samples, confirms that miR-146a may be involved in acne also at the level of sebocytes and adds further important details on the immune-competence of this cell type. Therefore, one of the most interesting findings is that the induction of the TLR-miR-146a axis in sebocytes may result in a decreased production of IL-8, a cytokine characteristic in acne-related inflammation, and in a decreased chemoattractant potential of sebocytes, a feature that was recently reported by our group 19 . Speculating on the in vivo relevance of this finding, it is reasonable to put forward that the increased levels of miR-146a in sebocytes could serve as a negative regulator of inflammation in acne lesions with an impact on the production of inflammatory cytokines and the number of infiltrating immune cells.
Further results showed that the complex changes induced by miR-146a may go beyond changing the inflammatory properties of sebocytes. While the proliferation and apoptosis of sebocytes were dependent on the levels of miR-146a, the increased levels led to an increased proliferation while decreased ones to apoptosis, our unbiased strategy of whole transcriptome analysis performed on sebocytes treated with a specific miR-146a inhibitor, revealed that miR-146a may also influence the gene expression profile of sebocytes. Although the exact mechanisms remain to be elucidated, in inhibitor-treated sebocytes, pathways with pivotal roles in sebocyte functions, such as Wnt or G protein mediated signalling, might be altered at the level of transcription 37 .
Based on the fold-change values, the functional clustering of the differentially expressed genes in miR-146 inhibitor treated SZ95 sebocytes and the in situ hybridization studies, GNG7 came into the focus showing an opposite regulation with miR-146a both in sebocytes and in sebaceous glands of acne samples, in which the expression of miR-146a increased while GNG7 was not detectable compared to control skin samples. Our findings revealed, that such decrease in the levels of GNG7 may promote sebocyte differentiation and as a result to an increased sebum secretion. Interestingly, although GNG7 has not been detected in the skin yet, in the nervous tissue where it is known to be predominantly expressed 38 , GNG7 inhibited cell proliferation, promoted cell differentiation and induced cell death by inhibiting mTOR signalling 39,40 . The findings that the activation of mTOR pathway is also involved in the regulation of sebocyte proliferation and maturation, and its induction by various agents is central in the development of acne [41][42][43][44] , make GNG7 an interesting candidate for further studies in sebocyte biology (Fig. 6).
In summary, our findings may bring us closer to understand the morphological and functional changes of sebaceous glands observed in acne, where the characteristic changes of increased sebum production assumes that both the proliferation and the lipid production of sebocytes are stimulated at the same time. Based on a recent publication 45 reporting that the pathogenetic basis of acne is the alteration in sebocyte differentiation, the less differentiated and with that the more proliferating the sebocytes are the more they are responding to regulatory stimuli, therefore our results point on the therapeutic relevance which the modulation of miR-146a, and with that GNG7 levels, may deliver to acne therapy by targeting inflammation, sebocyte hyperproliferation and sebum secretion at the same time.

Methods
Cells, transfection and treatment. Immortalized human SZ95 sebocytes were maintained as adherent culture at 37 °C in a humidified chamber containing 5% (v/v) CO 2 in Sebomed basal medium (Sigma-Aldrich, St. Louis, MO, USA) as previously described 46 .
For microRNA profiling, SZ95 cells were treated with 1 μg/ml PAM3CSK4 (TLR1/2  To obtain global transcriptome data high throughput mRNA sequencing analysis was performed on Illumina sequencing platform. Total RNA sample quality was checked on Agilent BioAnalyzer using Eukaryotic Total RNA Nano Kit according to the manufacturer's protocol. Samples with RNA integrity number value > 7 were accepted for library preparation process. RNA-Seq libraries were prepared from total RNA using Ultra II RNA Sample Prep kit (New England BioLabs) according to the manufacturer's protocol. Briefly, poly-A RNAs were captured by oligo-dT conjugated magnetic beads then the mRNAs were eluted and fragmented at 94 °C for 15 min. First strand cDNA was generated by random priming reverse transcription and after second strand synthesis step double-stranded cDNA was generated. After repairing ends and adapter ligation steps, adapter-ligated fragments were amplified in enrichment polymerase chain reaction and finally, sequencing libraries were generated. The sequencing run was executed on Illumina NextSeq500 instrument using single-end 75 cycle sequencing.

RNA-Seq data analysis.
Raw sequencing data was aligned to human reference genome version GRCh37 using HISAT2 algorithm and BAM files were generated. Downstream analysis was performed using StrandNGS Changes of relative GNG7 mRNA levels measured by qRT-PCR in SZ95 sebocytes normalized to PPIA. Note that GNG7 mRNA levels decreased in SZ95 sebocytes transfected with miR-146a mimic (mimic) and increased in the inhibitor treated cells (a-miR) when compared to their control sequences (mimic CTR and a-miR CTR respectively). (c) in situ hybridization for the detection of GNG7 mRNA in SZ95 sebocytes. Note, that when compared to miR-146a mimic (A), a more intense blue hybridization signal was seen in the cytoplasm of the miR-146a inhibitor treated SZ95 sebocytes (B). (C) Negative control staining. (d) in situ hybridization for the detection of GNG7 in sebaceous glands of normal (A) and acne vulgaris (B) FFPE human tissue samples. Note that while in acne samples, GNG7 mRNA could not be detected, hybridization signals were observed in healthy skin samples. Arrows show GNG7 mRNA-positive sebocytes. Negative control staining of normal (C) and of acne vulgaris samples (D) as described in "Methods". Representative photomicrograph, n = 5. Chromogenic in situ hybridization, NBT/BCIP blue chromogen with nuclear Fast Red background staining. Original magnification × 400. (e) Silencing of GNG7 mRNA does not affect IL-8 production (e), but promotes SZ95 sebocyte differentiation according to our cell cycle analysis (f) and lipid content measurements (g).    www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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