Hsa_circ_0105040 promotes Cutbacterium acnes biofilm induced inflammatory via sponge miR-146a in human keratinocyte

Hsa_circ_0105040 promotes Cutbacterium acnes biofilm induced inflammatory via sponge miR-146a in human keratinocyte | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hsa_circ_0105040 promotes Cutbacterium acnes biofilm induced inflammatory via sponge miR-146a in human keratinocyte Yuzhen Liu, Meng Zhou, Nana Zheng, Haoxiang Xu, Xu Chen, Zhimin Duan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2825777/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Circular RNAs (circRNAs) are thought to play a crucial function in controlling gene expression, according to expanding findings. However, the importance of circRNAs in the regulation of acne inflammation is unclear. Methods Microarray analysis has been carried out to investigate circRNAs/miRNAs/mRNAs that express abnormally in acne. RNase R digestion assay is used for confirmation of the hsa_circ_0105040 characteristic. The functional roles of hsa_circ_0105040 on inflammatory response induced by Cutibacterium acnes ( C. acnes ) biofilm in human primary keratinocytes were revealed by Fluorescence in situ hybridization (FISH), Reverse transcription quantitative (PCR), Western blotting analysis, Immunoprecipitation, Luciferase reporter assay, Biotin-labeled miRNA pull-down assay, RNA immunoprecipitation (RIP). Results We first evaluate the human circRNA expression patterns in acne tissues and find that hsa_circ_0105040 expression is considerably reduced in acne tissues. Moreover, we discover that the majority of hsa_circ_0105040 is found to be localized in the cytoplasm of primary human keratinocytes. Hsa_circ_0105040 overexpression significantly enhances the production of proinflammatory factors (interleukin-8, interleukin-6, and tumor necrosis factor-α). Mechanistic research reveals that the microRNA miR-146a binds to hsa_circ_0105040, which then actively sponges miR-146a to prevent the level of IRAK1 and TRAF6. Conclusions These findings point to hsa_circ_0105040 as a critical circRNA that function as "microRNA sponges" for the controlled inflammatory response in the development of acne. Our findings may provide valuable insights into the progression of acne. acne vulgaris circRNA ceRNA miR-146a inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Acne is a pervasive, persistent, relapsing inflammatory skin disease that affects 90% teenagers and likely to be negative physical and psychological repercussions [ 1 ]. The complicated skin disease known as acne is brought on by various causes, such as hyperkeratosis of the sebaceous units, infection with Cutibacterium acnes ( C. acnes ), increased sebum production, and inflammation [ 2 , 3 ]. However, studies on the molecular processes by which C. acnes produce acne are scarce [ 4 ]. Therefore, a better comprehension of the potential functions of circRNAs underlying the occurrence and development of acne will may shed fresh light on the potential pathogenic mechanism causing acne. Eukaryotic genomes can present different transcriptional features, which generate diverse RNA transcripts and most of them are non-coding RNAs (ncRNAs). However, the primary reports on the pathogenesis of acne are confined to protein-coding genes [ 5 ]. Only a few studies have suggested that ncRNAs including long non-coding RNAs (lncRNA) and microRNAs are strongly related to acne [ 6 – 8 ]. Apparently, little research has been focused on the functions of ncRNAs in acne. CircRNAs is a novel type of non-coding RNA that is created when linear RNA is back-spliced in eukaryotes [ 9 ]. It is significant to note that circRNAs may function as competitive endogenous RNAs (ceRNAs) that regulate and sponge miRNAs on their target genes. According to reports, circRNAs has significant regulatory functions in several illnesses and are connected to the pathophysiology and development of inflammatory skin conditions through diverse molecular pathways [ 10 – 12 ]. For example, Lower-level expression of hsa_circ_0045272 in T-cells of patients with systemic lupus erythematosus, which contains miR-6127 binding sites and regulates downwards DTX4 and PAX8 [ 13 ]. Furthermore, 538 differently expressed circRNAs are linked to the emergence and development of severe acne in severe acne tissues and adjacent normal skin tissues [ 14 ]. However, circRNAs in acne has not yet been fully comprehended in terms of their underlying biochemical roles and molecular processes. In our study, we employ in circRNA microarray analysis to identify the differential expression of circRNAs, miRNAs and mRNAs in acne and normal tissues from six pairs of volunteers. Furthermore, we find that hsa_circ_0105040, which is associated with miR-146a and positively controls IRAK1 and TRAF6, leads to elevated levels of inflammation in acne, is dramatically downregulated in acne. Our research suggests that hsa_circ_0105040 can be a critical indicator for acne patients. Materials And Methods Clinical specimen acquisition The clinical and demographic properties are performed in Table 1 . This experiment follows the Declaration of Helsinki's criteria. This study is approved by the Medical Ethics Council of the Skin Diseases Hospital of the Chinese Academy of Medical Sciences (CAMS) (approval number:2018-KY-012), and all patients provide in forms consent. Table 1 Demographic characteristics and clinical sampling information of the acne patients and nomal group. Nomal1 Nomal2 Nomal3 Nomal4 Nomal5 Nomal6 Acne1 Acne2 Acne3 Acne4 Acne5 Acne6 Age(year) 25 22 29 19 27 23 21 23 19 26 24 27 Gender Male Male Male Male Male Male Male Male Male Male Male Male skin region Cheek Forehead Cheek Cheek Cheek Chin Forehead Cheek Cheek Cheek Forehead Cheek Microarray Analysis Of Circrna The samples (six pairs of normal and acne-prone skin tissues) are hybridized and pretreated with Aksomics (Shanghai, China). The amply circular RNAs are then converted into fluorescent circRNA using the Arraystar Super RNA Labeling Kit after digestion with RNase R (Epicentre Technologies, Madison, WI, USA) to remove linear RNAs (Arraystar). NanoDrop ND-1000 is used to detect the concentration and specific activity of labeled cRNA (Pmol Cy3/g cRNA), follow by fragmentation in 5µl 10 × block and 1µl 25 fragment buffer. Samples are then heat at 60°C for 30 min, and dilute with 25µl 2 × hybridization buffer. After filling the gasket slides with the 50µl of hybridization solution. The Slides are cultivated in an Agilent hybridization chamber at 65°C for 17 h. The hybrid array is detected by Agilent Scanner G2505C. Microarray Analysis Of Mirna The total RNAs are extracted by TRIzol® Reagent (Invitrogen life technologies) and then labeled using miRCURYTM Array Power Labeling Kit (Cat #208032-A, Exiqon). Briefly, total RNAs are incubated at 65°C for 15 min, followed by hybridization at 95°C for 2 min. Carefully placed the miRCURYTM Array on top of the coverslip and spacer, where the printed side of the array should face the coverslip to form the hybrid assembly. After hybridization, the slides were washed with washing buffer at 56°C for 2min. Finally, scans were performed using an Axon GenePix 4000B microarray scanner. Microarray Analysis Of Mrna RNA concentration is detected by Agilent ND-1000. Hybridization is carried out by Arraystar RNA Flash Labeling Kit. Briefly, the slides are washed and scanned using an Agilent DNA Microarray Scanner. The probe signal is collected by Agilent Feature Extraction software (V11.0.1.1). Agilent GeneSpring GX V12.1 software is used to measure differentially expressed mRNA in acne and normal skin tissues. Cell Cultures And Stimulation Infant foreskin tissue served as the source of the human primary keratinocyte, and the detail experimental approach was detailed in [ 13 ]. At 80°C for 30 min, the C. acne biofilm was rendered inactive. C.acne biofilm is introduced and cultured for six or twenty-four hours when the cell density was above 90%. Our earlier research has followed an explicit protocol. Rna Extraction, Rnase R Treatment And Quantitative Reverse Transcriptase-polymerase Chain Reaction (Qrt-pcr) TRIzol reagent was used to isolate total RNA (Invitrogen, USA). Additionally, 3 U/mg of RNase R (Geneseed, China) is added to 2g of total RNA for 15 min at 37°C. After that, cDNA was produced using First Strand cDNA Synthesis Kit (Thermo Scientific in the United States), and RT-PCR is performed using SYBR Green SuperMix (YEASEN, China). As an internal reference, U6 or GAPDH are applied. The primers are listed in Table S1 . Plasmids, Short Interfering Rnas (Sirnas) And Cell Transfection The circRNA overexpression plasmid (pcDNA3.1 CircRNA Mini) is provided by MiaolingBio (Wuhan, China). The hsa_circ_0105040 sequence is cloned into pcDNA3.1 circRNA vector (Miaoling, China) by EcoRI and NotI cleavage sites, and then obtained hsa_circ_0105040 overexpression plasmid. Human primary keratinocyte is seeded into 6-well plates and transfection with hsa_circ_0105040 or control siRNA (50nM, General Biosystems, China) using jetPRIME (Polyplus, France) following the manufacturer’s instructions. The siRNA sequences are shown in Table S2. Western Blotting (Wb) RIPA lysis buffer (Beyotime, China) with 1× protease inhibitors and phosphorylase inhibitors are used to obtain cell lysates (Thermo Fisher, USA). The WB is carried out as previously described [ 15 ]. On Bio-Rad gel imaging system, immunoreactive findings are anticipated using the immobilon upgraded chemiluminescence kit (YEASEN, Shanghai, China). The antibodies used are listed in Table S3. RNA fluorescence in situ hybridization (FISH) 5’-FAM-labeled hsa_circ_0102678 and control probe sequence are created by Gene Pharma (Shanghai, China). The FISH kit (Gene Pharma) is utilized for hybridization research. Briefly, 4% paraformaldehyde (PFA) is used to fix human primary keratinocytes or tissues. Slices are hybridized at 37°C in a moist, dark environment with specific probes. And then covered in DAPI-containing paraffin to seal slices. The pictures are taken using Olympus FV1000 confocal laser scanning microscope. Immunofluorescence Human primary keratinocytes are grown using confocal dish (Corning, United States) and then fixed with 4% PFA for 20min at 37°C. Following permeabilization in 0.1% TritonX-100, the cell is washed three times with PBS. After blocking for 1 hour at 37°C with 5% normal goat serum, the primary antibody is incubated for overnight at 4°C. The next day, cells are incubated with secondary antibodies for 1 hour at 37°C after been rinsed three times with PBS. Nuclear staining is carried out using DAPI. A confocal laser scanning microscope is used to take fluorescent micrographs (Olympus FV1000). Luciferase Reporter Assay pGL3 Basic luciferase reporter vector is provided by Tsingke (Nanjing, China), which contain miR-146a binding from hsa_circ_0105040 wild-type (WT) or mutant sequences. These recombinant luciferase reporter plasmids are co-transfected with miR-146a mimics, inhibitors, or controls into human primary keratinocytes. A Dual-luciferase assay equipment (Promega, E2920, USA) is used to measure the level of luciferase activity inside transfected cells. Rna Immunoprecipitation Assay (Rip) RNA immunoprecipitation (RIP) analysis is performed using the EZ Magna RNA immunoprecipitation Kit (Millipore, USA). In brief, human primary keratinocytes are lysed in RIP lysis buffer 24 hours after transfection. After incubation with anti-Ago2 antibody or IgG in magnetic beads. The beads are then washed and incubated with cell lysates for 12 hours at 4°C. After being purified, the coprecipitated RNAs are discovered using qRT-PCR. The RNA sample preparation variations in each group are normalized using the input fraction, and the background fraction is using the negative controls (IgG). Biotin-labeled Mirna Pull-down Assay Firstly, the 3′-end biotinylated miR-146a mimics or the miRNA control (50nM, General Biosystems, Anhui, China) are transfected into human primary keratinocytes, and then cell lysates are harvested 48 hours post transfection. After centrifugation at 12000 rpm for 10 min at 4°C, 30µl of cell lysates are used as an input group. Followed by enrichment of the biotin-linked RNA complex with the Dynabeads MyOne Streptavidin C1 kit (Invitrogen, United States). qRT-PCR is applied to evaluate efficacy. All sequences are listed in Table S4. Statistical analysis The results of each experiment are provided as mean ± the standard error of the mean after being replicated at least three times using biological. One-way ANOVA or Student's t-tests statistical analysis is used to compare the difference. Statistical significance is defined as a value of P < 0.05. Results Different expression profiles of circRNAs-miRNAs-mRNAs between acne patients and normal controls To identify the significance expression of genes involved in acne, we first show by circRNA microarray analyses that differentially expressed circRNAs exist in acne compared to normal skin tissues. The circRNA expression profile is statistically assessed using hierarchical clustering (Figure 1a). Subsequent analysis of scatter plots and volcano plots shows a total of 1,594 significant circRNAs expressions, of which 605 are upregulated and 989 circRNAs are downregulated in acne (Figure 1b-c). We then use an unsupervised hierarchical classification to evaluate differential miRNA expression patterns in acne and normal skin tissues (Figure 2a). As indicated in (Figure 2b), differential miRNA profiles are exposed in acne versus normal skin tissues. A total of 582 mRNAs are expressed differentially significantly (fold change >2.0, P < 0.05), of which 384 mRNAs are elevated and 198 mRNAs are downregulated (Figure 2c). It is well acknowledged that miRNA regulates mRNA expression by suppressing its activity in biological processes [9, 15, 16]. To explain whether mRNA is significantly expressed in acne in comparison with normal skin tissue, we show by hierarchical clustering and volcano plot analysis that a total of 13075 mRNA significantly differentiated expressed (fold-change >2.0, P < 0.05), among these 4955 upregulated and 8120 are downregulated (Figure 3a-b). These results indicate that circRNAs-miRNAs-mRNAs may be involved in the occurrence and development of acne. Functional Analysis Of Differentially Expressed Mrna Between Acne Patients And Normal Controls Further investigate the differential function of mRNA in acne and normal skin tissues. KEGG signaling pathway enrichment demonstrates that these associated genes are strongly involved in herpes simplex virus 1 infection, Fc gamma R-mediated phagocytosis, endocytosis, apoptosis, pyrimidine metabolism, Notch signaling pathway, Fc epsilon RI signaling pathway, sphingolipid signaling pathway, and AGE-RAGE signaling pathway in diabetic complications (Fig. 4 a). GO enrichment examination of the relationship mRNAs reveal that they are predominantly enriched in biological processes, including the regulation of cellular (GO: 0050794), biological (GO: 0050789), cellular metabolic (GO: 0031323), primary metabolic (GO: 0080090), cellular macromolecule metabolic (GO: 0044260), and nitrogen compound metabolic processes (GO: 0051171), in addition to biological regulation (GO: 0065007) (Fig. 4 b). Hsa_circ_0105040 Is Downregulated In Acne Patients, And Primarily Located In The Cytoplasm Our previous studies have shown that miR-146a is overexpressed and regulates inflammatory reactions in acne [ 8 ]. Therefore, we use Cytoscape software (version 3.4.0) to assess possible circRNAs to find out whether circRNAs regulate the expression of miR-146a and three circRNAs (hsa_circ_0105040, hsa_circ_0102678, hsa_circ_0102680) are predicted. To assess potential circRNAs in acne, we first choose hsa_circ_0105040 to further study. We show by histological analysis that expression of hsa_circ_0105040 expression shapely decreases in acne tissues compared to corresponding adjacent normal tissues (Fig. 5 a). Hsa_circ_0105040 is produced from the head-to-tail splicing of the exon17 (170bp) within the filamin A (FLNA) locus (Fig. 5 b). To clarify the molecular characteristics of hsa_circ_0105040, we first use divergent and convergent primers to generate the FLNA mRNA and hsa_circ_0105040 by qRT-PCR analyses. Electrophoresis on an Agarose gel assay shows that hsa_circ_0105040 is only detected in cDNA and not in gDNA (Fig. 5 c). Next, the RNase R degradation analysis demonstrates that hsa_circ_0105040 is not degraded, while the quantity of FLNA mRNA decreases significantly. (Fig. 5 d). Additionally, nuclear and cytoplasmic fractionation and FISH assay indicate that the majority of hsa_circ_0105040 is found in the cytoplasm (Fig. 5 e-f). These results show that hsa_circ_0105040 may be involved in acne progression and is a stable circRNA expressed in the cytoplasm of keratinocytes. Hsa_circ_0105040 promotes inflammatory response induced by C. acnes biofilm in human primary keratinocytes It is generally accepted that the development of acne is correlated with inflammation caused by C. acnes [ 17 , 18 ]. To determine whether hsa_circ_0105040 affects acne by modulating the inflammatory response, we establish an accessible biofilm model of C. acnes in vitro. Our previous study found that the 8th day can be the most effective stimulation period to construct a model of acne [ 8 , 19 ]. We then transfect with hsa_circ_0105040 siRNA (si-circ_0105040) and the control siRNA (si-NC), or overexpression hsa_circ_0105040 plasmid (pcDNA-circ_0105040) and the empty control plasmid (pcDNA-circ-NC) in human primary keratinocytes (Figure S1 a-b). To assess whether hsa_circ_0105040 is involved in acne inflammatory reaction, we suggest by qRT-PCR and enzyme-linked immunosorbent assay (ELISA) analysis that knockdown of hsa_circ_0105040 significantly reduces the expression of TNF-α, IL-6 and IL-8 (Fig. 6 a-b), while hsa_circ_0105040 overexpression elevates the expression of these inflammatory genes (Figure S2a-b). Meanwhile, si-circ_105040 transfection reduces the positive effects of C. acnes biofilm on the expression of p-P38, p-IκBα, and p-ERK1/2 (Fig. 6 c). Consistently, the level of these inflammatory genes is remarkably elevated with the transfection of pcDNA-circ_105040 (Figure S2c). Furthermore, immunofluorescence examination revealed that hsa_circ_0105040 positively affects the ability of C. acnes biofilm to induce NF-κB-p65 nuclear translocation (Fig. 6 d; S2d). Together, these results show that hsa_circ_0105040 promotes inflammation of human primary keratinocytes via MAPK and NF-κB pathways. Hsa_circ_0105040 Acts As A Cerna To Competitively Sponge Mir-146a In Human Primary Keratinocytes This is clearly recognized that cytoplasmic circRNAs function as ceRNAs to sponges for microRNAs which have precisely reduced the synthesis of target mRNAs [ 20 , 21 ]. We then investigate whether hsa_circ_0105040 has similar effects in keratinocytes. Our previous investigation reveals that miR-146a suppressed the expression of IL-6, IL-8 and TNF-α in acne [ 8 ]. Hence, we further explore the interaction between hsa_circ_0105040 and miR-146a. Indeed, bioinformatics instruments predicts that hsa_circ_0105040 may bind to miR-146a (Fig. 7 a). Meanwhile, luciferase experiment shows that hsa_circ_0105040 can bind directly to miR-146a (Fig. 7 b). Furthermore, RNA immunoprecipitation (RIP) assay indicates that hsa_circ_0105040 exists in Ago2-containing miRNA complexes with ribonucleoprotein via binding to miR-146a (Fig. 7 c). Moreover, biotin-labelled miRNA pulldown assays detect that hsa_circ_0105040 expression improves transfect with biotin-labelled miR-146a as opposed to Bio-NC control (Fig. 7 d). These results indicate that hsa_circ_0105040 is acting as a ceRNA for miR-146a in human primary keratinocytes cells. To evaluate these findings in vivo, we transfect human primary keratinocytes with si-circ_0105040 and si-NC or pcDNA-circ_105040 and pcDNA-circ-NC. We discover that knockdown of hsa_circ_0105040 dramatically increased the expression of miR-146a, while overexpression of hsa_circ_0105040 decreased the expression of miR-146a (Fig. 7 e-f). In addition, hsa_circ_0105040 expression is reduced with miR-146a transfection, but elevated with anti-miR-146a transfection (Fig. 7 g-h), suggesting that hsa_circ_0105040 may be directly regulating to miR-146a. Hsa_circ_0105040 Sponge Mir-146a To Alleviate Targeted Irak1 And Traf6 It is essential for miR-146a to bind directly to IRAK1 and TRAF6 and inhibit expression of IRAK1 and TRAF6 in human primary keratinocytes cells [ 8 ]. To test whether hsa_circ_0105040 is upstream of miR-146a to regulate IRAK1 and TRAF6, we find by qRT-PCR and WB analyses that the expression of IRAK1 and TRAF6 was significantly suppressed upon hsa_circ_0105040 siRNA compared with the control (Fig. 8 a-b). Coincidentally, we observe that both IRAK1 and TRAF6 expressions are increased upon transfection with anti-miR-146a, while silence hsa_circ_0105040 can significantly reduce the positive impact of anti-miR-146a on IRAK1 and TRAF6 (Fig. 8 c). Furthermore, TNF-, IL-8 and IL-6 expression induced by C. acnes biofilm is elevated with anti-miR-146a, but reduced with si-circ_105040 (Fig. 8 d-e). Taken together, these data indicate that hsa_circ_0105040 decoy miR-146a to promote the expression of IRAK1 and TRAF6 , and an increase inflammation in human primary keratinocytes trigger by C. acnes biofilm. Discussion It is widely accepted that the development and occurrence of acne are caused by cellular inflammation [ 18 , 22 , 23 ]. During this time, circRNAs are emerging as an important moderator controlling cell inflammation in many human diseases [ 11 , 12 , 24 , 25 ]. However, circRNAs has not been investigated in the pathophysiology of acne Here, we unexpectedly identify numerous differential expression circRNAs/mRNAs/mRNAs in acne and normal skin tissues. In addition, we suggest that hsa_circ_0105040 can increase inflammation levels upon C. acnes biofilm in human primary keratinocytes. We first show mechanically that, during the pathology of acne, hsa_circ_0105040 inhibits miR-146 expression; Given that TRAF6 and IRAK1 are co-targets for miR-146, hsa_circ_0105040 attenuates the negative effect of TRAF6 and IRAK1 on miR-146a, which increase cellular inflammation by C. acnes (Fig. 9 ). These results demonstrate that hsa_circ_0105040 may be an indicator gene for controlling the development of acne inflammation. CircRNAs are single stranded covalently RNA molecules, which connect with occurrence and development of different diseases [ 26 – 28 ]. Moreover, it is supposed that dysregulation of circRNAs may be a factor in inflammatory skin illnesses [ 11 – 13 ]. Clinically, the C. acnes infection, which causes skin inflammation, is one of the causes of acne. It is not clear, however, if circRNAs has physiologic effects on acne. The only relevant information comes from RNA sequencing study of the circRNA expression profile in severe acne, with circRNAs being associated with inflammatory and immunological responses [ 14 ]. Here, circRNAs microarray analysis demonstrates that hsa_circ_0105040 affects acne inflammation, which may provide additional evidence for this study and enhance the comprehending of the novel functional relationships between circRNA and the pathophysiology of acne. As previously mentioned, circRNAs may act as ceRNA to bind miRNAs, thereby inhibiting their activity. For instance, hsa_circRNA_0088036 can interact with miR-140-3p and function as a miRNA sponge to regulate the expression of FOXQ1, which promotes bladder cancer [ 29 ]. Similar to this, circRAB3B sponge miR-1228-3p and negatively regulated miR-1228-3p, resulting in reduced psoriasis [ 30 ]. Parallel to these observations, our research reveals that hsa_circ_0105040 expression within acne is negatively connected with miR-146a expression, but positively correlated with IRAK1 and TRAF6, suggesting that hsa_circ_0105040 may sponge miR-146a to control inflammation in acne. Our results here offer a fresh perspective on how the circRNA/miRNA/mRNA axis regulates inflammation in acne. In summary, we first identify the regulating mechanism by which hsa_circ_0105040, exerts its inhibition of miR-146a on TRAF6 and IRAK1, leading to increased levels of inflammation. Our study, which reveals the clinical significance of the hsa_circ_0105040/miR-146a/TRAF6/IRAK1 axis diagnosis of acne, perhaps gives novel perspectives on the pathological mechanism of acne. Abbreviations Circular RNAs (circRNAs) Differentially expressed genes (DEGs) Gene Ontology (GO) Kyoto Encyclopedia of Genes and Genomes (KEGG) Competitive endogenous RNA (ceRNA) Fluorescence in situ hybridization (FISH) Quantitative real-time polymerase chain reaction (qRT-PCR) Western blotting (WB) Paraformaldehyde (PFA) RNA immunoprecipitation assay (RIP) Genomic DNA (gDNA) Short interfering RNAs (siRNAs) Interleukin (IL) Tumor necrosis factor (TNF) Enzyme-linked immunosorbent assay (ELISA) Interleukin 1 receptor-associated kinase 1 (IRAK1) tumor necrosis factor receptor-associated factor 6 (TRAF6) Nuclear Factor Kappa B (NF-κB) Mitogen-Activated Protein Kinase (MAPK) Filamin A (FLNA) Declarations Funding This study is supported by National Natural Science Foundation of China (81773338, 82103749); CAMS Innovation Fund for Medical Sciences (2017-I2M-1-017, CIFMS-2021-I2M-1-001); Science and Technology Program of Nanjing, China (2019060001); Jiangsu Provincial “Double Innovation Doctors” Program (grant number JSSCBS20211610); Science and Technology development foundation of Nanjing Medical University (NMUB2020154). Competing Interests The authors have no conflict of interest to declare. Acknowledgements We are very grateful to patients for their participation and cooperation in providing us with important clinical evidence. Author Contributions ML, YZL and MZ conceived the study. YZL, MZ, NNZ, XC, HXX performed the study. MZ, HXX and RZ analyzed RNA sequencing data. ML, YZL and ZMD provided fundings. MZ, RZ and TL wrote the manuscript. References Tan JK, Bhate K. A global perspective on the epidemiology of acne. Br J Dermatol 2015; 172 Suppl 1:3-12. Gollnick HP. From new findings in acne pathogenesis to new approaches in treatment. J Eur Acad Dermatol Venereol 2015; 29 Suppl 5:1-7. Tilles G. Acne pathogenesis: history of concepts. Dermatology 2014; 229:1-46. Gu H, An HJ, Gwon MG, Bae S, Leem J, Lee SJ, et al. Bee Venom and Its Major Component Melittin Attenuated Cutibacterium acnes- and IGF-1-Induced Acne Vulgaris via Inactivation of Akt/mTOR/SREBP Signaling Pathway. Int J Mol Sci 2022; 23. 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An atlas of cortical circular RNA expression in Alzheimer disease brains demonstrates clinical and pathological associations. Nat Neurosci 2019; 22:1903-1912. Yang J, Qi M, Fei X, Wang X, Wang K. Hsa_circRNA_0088036 acts as a ceRNA to promote bladder cancer progression by sponging miR-140-3p. Cell Death Dis 2022; 13:322. Lu J, Xu X, Li Y, Yu N, Ding Y, Shi Y. CircRAB3B suppresses proliferation, motility, cell cycle progression and promotes the apoptosis of IL-22-induced keratinocytes depending on the regulation of miR-1228-3p/PTEN axis in psoriasis. Autoimmunity 2021; 54:303-312. Additional Declarations No competing interests reported. Supplementary Files supplement.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2825777","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":192717809,"identity":"ebaf4d81-f3d0-49cc-ae9f-29b65ba13b80","order_by":0,"name":"Yuzhen Liu","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuzhen","middleName":"","lastName":"Liu","suffix":""},{"id":192717810,"identity":"9a0232f6-8a0a-4e45-966a-ba16dbed0771","order_by":1,"name":"Meng Zhou","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Zhou","suffix":""},{"id":192717811,"identity":"4ef12929-ba18-41f5-a548-a0bf3740e2d1","order_by":2,"name":"Nana Zheng","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Zheng","suffix":""},{"id":192717812,"identity":"03314948-e85d-421c-b188-d2494b566aa7","order_by":3,"name":"Haoxiang Xu","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haoxiang","middleName":"","lastName":"Xu","suffix":""},{"id":192717813,"identity":"e10d9b06-8cd7-4b71-8c59-d70153a0af94","order_by":4,"name":"Xu Chen","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Chen","suffix":""},{"id":192717814,"identity":"1e404ec1-4f42-4f58-8266-974b7541985b","order_by":5,"name":"Zhimin Duan","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhimin","middleName":"","lastName":"Duan","suffix":""},{"id":192717815,"identity":"68a695fd-5adb-48b4-a441-99fd755f30c9","order_by":6,"name":"Tong Lin","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Lin","suffix":""},{"id":192717816,"identity":"865c3d8e-768a-4713-a612-9bc25dc2a8d1","order_by":7,"name":"Rong Zeng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYDADfmbGhgMfG0BMxsYDRGmRbG9ufDizgUECqKWBOC0GZ443G/OCtTAw4NUi3957+HVFxR27hhuJbdK2O2zqdNsPA22psYnGpYWx51ya5Zkzz5IbZwC15J5JkzA7kwjUciwttwGHFmaJHDPDxrbDycwSIC1thyXMDgC1MDYcxqmFTf4NRAsbSIslSMv5h/i18EjwGD8EarHj4TnYbMwI0nKDgC0SPDlmjA1nDidIsDc2PuxtS5PcdgNoSwIev8i3nzH+2FBx2N7+MPuDAz/bbPjNzqc/fPChxganFpB3QFGRiKogAbdyEGD+ACTs8asZBaNgFIyCEQ0Awj9m7LwLKmUAAAAASUVORK5CYII=","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Zeng","suffix":""},{"id":192717817,"identity":"eb12175d-2f29-47be-8771-cb3883ca99fc","order_by":8,"name":"Qing Chen","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Chen","suffix":""},{"id":192717818,"identity":"d843796a-e84e-40b7-a685-b31c90134618","order_by":9,"name":"Min Li","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-04-17 07:29:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2825777/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2825777/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36005948,"identity":"1b48854d-cbeb-462d-91b5-9549536d45d1","added_by":"auto","created_at":"2023-04-19 14:20:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4512963,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent expression profiles of circRNAs between acne and normal controltissues\u003c/p\u003e\n\u003cp\u003eHierarchically clustered heat map (a), Scatter plot (b), and Volcano plot map (c) are drawn out differential circRNAs between acne and control skin tissues. Each row of colored boxes represented a circRNA, and each column represented a sample. Red and green indicated higher expression level and lower expression level, respectively. The gray dots in the middle indicated that there was no difference between the two groups. The red dots on the left indicate downregulated circRNAs and the red dots on the right indicated upregulated circRNAs.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/d7b3df519411635d81e3ab20.png"},{"id":36004508,"identity":"42322563-24c5-4e1f-acd0-b8590e92c831","added_by":"auto","created_at":"2023-04-19 14:12:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4814505,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent expression profiles of miRNAs between acne and normal control tissues\u003c/p\u003e\n\u003cp\u003eHierarchically clustered heat map (a), Scatter plot (b), and Volcano plot map (c) are performed differential miRNAs between acne and control skin tissues. Each row of colored boxes represents a miRNA, and each column represent a sample. Red and green indicate higher expression level and lower expression level, respectively. The gray dots in the middle indicate that there is no difference between the two groups. The red dots on the left indicate downregulated miRNAs and the red dots on the right indicate upregulated miRNAs.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/e9b691dec2250003e52da105.png"},{"id":36004510,"identity":"8a87fd60-17e1-4306-9d84-eb0c1fdd78eb","added_by":"auto","created_at":"2023-04-19 14:12:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4298320,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent expression profiles of mRNAs between acne and normal control tissues\u003c/p\u003e\n\u003cp\u003eHierarchically clustered heat map (a) and Volcano plot map (b) are showed differential mRNAs between acne and control skin tissues. Each row of colored boxes represents a mRNA, and each column represent a sample. Red and green indicate higher expression level and lower expression level, respectively. The gray dots in the middle indicate that there is no difference between the two groups. The red dots on the left indicate downregulate mRNAs and the red dots on the right indicate upregulate mRNAs.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/4f3ce712752954737f08a5fb.png"},{"id":36005951,"identity":"411ec7b1-3eea-469b-ae63-ef7ff6f489c7","added_by":"auto","created_at":"2023-04-19 14:20:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6027574,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis of differentially expressed mRNA between acne and normal control tissues\u003c/p\u003e\n\u003cp\u003e(a) Statistical analysis of top 10 different expression genes (DEGs) pathway enrichments between acnes compare to normal control skin tissues. The q-value of each signaling pathway is indicate by the point colors. The number of genes in each pathway is indicated by the point's size. (b) Gene ontology (GO) analysis of DEGs in acne compare to normal control tissues. The number of genes is shown on the X-axis. The GO term is presented the Y-axis.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/6a59dd3157a722c1ae875586.png"},{"id":36004516,"identity":"d27a5f5e-0f07-43f4-9ebd-5891357cdee9","added_by":"auto","created_at":"2023-04-19 14:12:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8270843,"visible":true,"origin":"","legend":"\u003cp\u003eHsa_circ_0105040 is downregulated in acne patients, and primarily located in the cytoplasm\u003c/p\u003e\n\u003cp\u003e(a) RNA-FISH analysis of hsa_circ_0105040 expression in acne and correspondence to adjacent normal tissues patients. Hsa_circ_0105040 (red); DAPI stain to the nucleus (blue). Scale bars: 10 μm. (b) Diagrammatic representation of hsa_circ_0105040 genome locus in FLNA exon region. (c) Relative levels of hsa_circ_0105040 in cDNA and genomic DNA (gDNA). GAPDH serve as negative control. (d) Relative FLNA and hsa_circ_0105040 are digested with or without RNase R. (e) qRT-PCR analysis of the subcellular localization ofhsa_circ_0105040 in human primary keratinocytes. (f) FISH analysis of hsa_circ_0105040 expression in human primary keratinocytes. Scale bars:50μm. Student’s \u003cem\u003et\u003c/em\u003e-test: ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/61a9f59d207493f5c5343681.png"},{"id":36004514,"identity":"08449ff7-2b71-4cee-9c52-1851d3ec365c","added_by":"auto","created_at":"2023-04-19 14:12:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6825554,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing hsa_circ_0105040 inhibits inflammatory response induced by \u003cem\u003eC. acnes\u003c/em\u003e biofilm in human primary keratinocytes\u003c/p\u003e\n\u003cp\u003eRelative IL6, IL8 and TNF-α mRNA(a), and protein expression (b) in human primary keratinocytes transfect with hsa_circ_0105040 or control siRNAs upon 2×10\u003csup\u003e8\u003c/sup\u003eCFU/ml heat-inactivated\u003cem\u003e C. acnes \u003c/em\u003ebiofilm. (c) Relative p-IκBα, IκBα, p-P38, P38 and p-ERK1/2 protein expression in human primary keratinocytes transfect with hsa_circ_0105040 or control siRNAs upon 2×10\u003csup\u003e8\u003c/sup\u003eCFU/ml heat-inactivated\u003cem\u003e C. acnes \u003c/em\u003ebiofilm. (d) the staining of immunofluorescence for NF-κB P65(red) and DAPI (blue) in human primary keratinocytes transfect with hsa_circ_0105040 or control siRNAs upon 2×10\u003csup\u003e8\u003c/sup\u003eCFU/ml heat-inactivated\u003cem\u003e C. acnes \u003c/em\u003ebiofilm. Scale bars:5μm. Bio-C. acnes, \u003cem\u003eC. acnes\u003c/em\u003e biofilm; si-NC, scrambled control; si-circ_105040, hsa_circ_0105040 siRNA. Student’s\u003cem\u003e t\u003c/em\u003e-test: *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/f250511d399327137b5f9829.png"},{"id":36005949,"identity":"2dd97c7a-e716-423b-9edc-21adef735f52","added_by":"auto","created_at":"2023-04-19 14:20:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2793818,"visible":true,"origin":"","legend":"\u003cp\u003eHsa_circ_0105040 acts as a ceRNA to competitively sponge miR-146a in human primary keratinocytes\u003c/p\u003e\n\u003cp\u003e(a) miR-146a binding sequence prediction for hsa_circ_0105040. Red nucleotides are visible in the mutationsequence. (b) Luciferase assay analysis of hsa_circ_0105040 or mutant luciferase activity in human primary keratinocytes transfect with miR-146a or control mimics. (c) RIP analysis of hsa_circ_0105040 relative fold enrichment in human primary keratinocytes. RIP efficiency is discovered using WB analysis with the Ago2 protein. IgG is utilized as a negative control. (d) Enrichment of hsa_circ_0105040 in human primary keratinocytes transfect with biotin-miR-146a or miR-NC using miR-146a pull-down assay. (e) qRT-PCR analysis of miR-146a expression in human primary keratinocytes transfect with hsa_circ_0105040 or control siRNAs. (f) qRT-PCR analysis of miR-146a expression in human primary keratinocytes transfect with hsa_circ_0105040 plasmid or empty control plasmid (g) qRT-PCR analysis of hsa_circ_0105040 in human primary keratinocytes transfect with miR-146a or miR-NC mimics. (h) qRT-PCR analysis of hsa_circ_0102678 in human primary keratinocytes transfect with anti-miR-NC or anti-miR-146a. Student’s \u003cem\u003et\u003c/em\u003e-test: **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/e389762003e65e3bfcd735c7.png"},{"id":36004511,"identity":"b7449510-bca6-429b-b749-0a41140b47ac","added_by":"auto","created_at":"2023-04-19 14:12:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4549643,"visible":true,"origin":"","legend":"\u003cp\u003eHsa_circ_0105040 sponge miR-146a to alleviate targeted IRAK1 and TRAF6\u003c/p\u003e\n\u003cp\u003eRelative TRAF6 and IRAK1 mRNA (a), and protein expression (b) in human primary keratinocytes transfect with hsa_circ_0102678 or control siRNAs. (c) WB analysis of TRAF6 and IRAK1 expression in human primary keratinocytes under the prescribed conditions. Relative IL6, IL8 and TNF-α mRNA (d), and protein expression (e) in human primary keratinocytes under the specified circumstances. Student’s \u003cem\u003et\u003c/em\u003e-test: **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/43bfca51a2db094b5c389985.png"},{"id":36004517,"identity":"b17351fd-5458-441a-8ffa-aa30cb37ba5d","added_by":"auto","created_at":"2023-04-19 14:12:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3311009,"visible":true,"origin":"","legend":"\u003cp\u003eA model posits on the relationship of the hsa_circ_0105040/miR-146a/TRAF6/IRAK1 axis in regulating inflammatory response in human primary keratinocytes.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/5b506cfc6146e85b9c5cf938.png"},{"id":36431586,"identity":"f9e5b1d5-04c9-45f2-919c-1d80faec7546","added_by":"auto","created_at":"2023-04-28 14:29:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4158456,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/8379a0c3-2016-41d4-980d-8ececa2caaf1.pdf"},{"id":36006282,"identity":"7fad55bf-fed9-4dfb-a5e9-8655d2afe6f7","added_by":"auto","created_at":"2023-04-19 14:28:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":300872,"visible":true,"origin":"","legend":"","description":"","filename":"supplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-2825777/v1/c0ed37b7343bbd74958f4edb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hsa_circ_0105040 promotes Cutbacterium acnes biofilm induced inflammatory via sponge miR-146a in human keratinocyte","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcne is a pervasive, persistent, relapsing inflammatory skin disease that affects 90% teenagers and likely to be negative physical and psychological repercussions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The complicated skin disease known as acne is brought on by various causes, such as hyperkeratosis of the sebaceous units, infection with \u003cem\u003eCutibacterium acnes\u003c/em\u003e (\u003cem\u003eC. acnes\u003c/em\u003e), increased sebum production, and inflammation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, studies on the molecular processes by which \u003cem\u003eC. acnes\u003c/em\u003e produce acne are scarce [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, a better comprehension of the potential functions of circRNAs underlying the occurrence and development of acne will may shed fresh light on the potential pathogenic mechanism causing acne.\u003c/p\u003e \u003cp\u003eEukaryotic genomes can present different transcriptional features, which generate diverse RNA transcripts and most of them are non-coding RNAs (ncRNAs). However, the primary reports on the pathogenesis of acne are confined to protein-coding genes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Only a few studies have suggested that ncRNAs including long non-coding RNAs (lncRNA) and microRNAs are strongly related to acne [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Apparently, little research has been focused on the functions of ncRNAs in acne.\u003c/p\u003e \u003cp\u003eCircRNAs is a novel type of non-coding RNA that is created when linear RNA is back-spliced in eukaryotes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It is significant to note that circRNAs may function as competitive endogenous RNAs (ceRNAs) that regulate and sponge miRNAs on their target genes. According to reports, circRNAs has significant regulatory functions in several illnesses and are connected to the pathophysiology and development of inflammatory skin conditions through diverse molecular pathways [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. For example, Lower-level expression of hsa_circ_0045272 in T-cells of patients with systemic lupus erythematosus, which contains miR-6127 binding sites and regulates downwards DTX4 and PAX8 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, 538 differently expressed circRNAs are linked to the emergence and development of severe acne in severe acne tissues and adjacent normal skin tissues [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, circRNAs in acne has not yet been fully comprehended in terms of their underlying biochemical roles and molecular processes.\u003c/p\u003e \u003cp\u003eIn our study, we employ in circRNA microarray analysis to identify the differential expression of circRNAs, miRNAs and mRNAs in acne and normal tissues from six pairs of volunteers. Furthermore, we find that hsa_circ_0105040, which is associated with miR-146a and positively controls IRAK1 and TRAF6, leads to elevated levels of inflammation in acne, is dramatically downregulated in acne. Our research suggests that hsa_circ_0105040 can be a critical indicator for acne patients.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical specimen acquisition\u003c/h2\u003e \u003cp\u003eThe clinical and demographic properties are performed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This experiment follows the Declaration of Helsinki's criteria. This study is approved by the Medical Ethics Council of the Skin Diseases Hospital of the Chinese Academy of Medical Sciences (CAMS) (approval number:2018-KY-012), and all patients provide in forms consent.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic characteristics and clinical sampling information of the acne patients and nomal group.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"14\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNomal1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNomal2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNomal3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNomal4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNomal5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNomal6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAcne1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eAcne2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAcne3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eAcne4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eAcne5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eAcne6\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge(year)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eskin region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCheek\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForehead\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCheek\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCheek\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCheek\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eChin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eForehead\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCheek\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCheek\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eCheek\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eForehead\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eCheek\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicroarray Analysis Of Circrna\u003c/h3\u003e\n\u003cp\u003eThe samples (six pairs of normal and acne-prone skin tissues) are hybridized and pretreated with Aksomics (Shanghai, China). The amply circular RNAs are then converted into fluorescent circRNA using the Arraystar Super RNA Labeling Kit after digestion with RNase R (Epicentre Technologies, Madison, WI, USA) to remove linear RNAs (Arraystar). NanoDrop ND-1000 is used to detect the concentration and specific activity of labeled cRNA (Pmol Cy3/g cRNA), follow by fragmentation in 5\u0026micro;l 10 \u0026times; block and 1\u0026micro;l 25 fragment buffer. Samples are then heat at 60\u0026deg;C for 30 min, and dilute with 25\u0026micro;l 2 \u0026times; hybridization buffer. After filling the gasket slides with the 50\u0026micro;l of hybridization solution. The Slides are cultivated in an Agilent hybridization chamber at 65\u0026deg;C for 17 h. The hybrid array is detected by Agilent Scanner G2505C.\u003c/p\u003e\n\u003ch3\u003eMicroarray Analysis Of Mirna\u003c/h3\u003e\n\u003cp\u003eThe total RNAs are extracted by TRIzol\u0026reg; Reagent (Invitrogen life technologies) and then labeled using miRCURYTM Array Power Labeling Kit (Cat #208032-A, Exiqon). Briefly, total RNAs are incubated at 65\u0026deg;C for 15 min, followed by hybridization at 95\u0026deg;C for 2 min. Carefully placed the miRCURYTM Array on top of the coverslip and spacer, where the printed side of the array should face the coverslip to form the hybrid assembly. After hybridization, the slides were washed with washing buffer at 56\u0026deg;C for 2min. Finally, scans were performed using an Axon GenePix 4000B microarray scanner.\u003c/p\u003e\n\u003ch3\u003eMicroarray Analysis Of Mrna\u003c/h3\u003e\n\u003cp\u003eRNA concentration is detected by Agilent ND-1000. Hybridization is carried out by Arraystar RNA Flash Labeling Kit. Briefly, the slides are washed and scanned using an Agilent DNA Microarray Scanner. The probe signal is collected by Agilent Feature Extraction software (V11.0.1.1). Agilent GeneSpring GX V12.1 software is used to measure differentially expressed mRNA in acne and normal skin tissues.\u003c/p\u003e\n\u003ch3\u003eCell Cultures And Stimulation\u003c/h3\u003e\n\u003cp\u003eInfant foreskin tissue served as the source of the human primary keratinocyte, and the detail experimental approach was detailed in [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. At 80\u0026deg;C for 30 min, the \u003cem\u003eC. acne\u003c/em\u003e biofilm was rendered inactive. \u003cem\u003eC.acne\u003c/em\u003e biofilm is introduced and cultured for six or twenty-four hours when the cell density was above 90%. Our earlier research has followed an explicit protocol.\u003c/p\u003e\n\u003ch3\u003eRna Extraction, Rnase R Treatment And Quantitative Reverse Transcriptase-polymerase Chain Reaction (Qrt-pcr)\u003c/h3\u003e\n\u003cp\u003eTRIzol reagent was used to isolate total RNA (Invitrogen, USA). Additionally, 3 U/mg of RNase R (Geneseed, China) is added to 2g of total RNA for 15 min at 37\u0026deg;C. After that, cDNA was produced using First Strand cDNA Synthesis Kit (Thermo Scientific in the United States), and RT-PCR is performed using SYBR Green SuperMix (YEASEN, China). As an internal reference, U6 or GAPDH are applied. The primers are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003ePlasmids, Short Interfering Rnas (Sirnas) And Cell Transfection\u003c/h3\u003e\n\u003cp\u003eThe circRNA overexpression plasmid (pcDNA3.1 CircRNA Mini) is provided by MiaolingBio (Wuhan, China). The hsa_circ_0105040 sequence is cloned into pcDNA3.1 circRNA vector (Miaoling, China) by EcoRI and NotI cleavage sites, and then obtained hsa_circ_0105040 overexpression plasmid. Human primary keratinocyte is seeded into 6-well plates and transfection with hsa_circ_0105040 or control siRNA (50nM, General Biosystems, China) using jetPRIME (Polyplus, France) following the manufacturer\u0026rsquo;s instructions. The siRNA sequences are shown in Table S2.\u003c/p\u003e\n\u003ch3\u003eWestern Blotting (Wb)\u003c/h3\u003e\n\u003cp\u003eRIPA lysis buffer (Beyotime, China) with 1\u0026times; protease inhibitors and phosphorylase inhibitors are used to obtain cell lysates (Thermo Fisher, USA). The WB is carried out as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. On Bio-Rad gel imaging system, immunoreactive findings are anticipated using the immobilon upgraded chemiluminescence kit (YEASEN, Shanghai, China). The antibodies used are listed in Table S3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA fluorescence\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein situ\u003c/span\u003e \u003cb\u003ehybridization (FISH)\u003c/b\u003e \u003c/p\u003e \u003cp\u003e5\u0026rsquo;-FAM-labeled hsa_circ_0102678 and control probe sequence are created by Gene Pharma (Shanghai, China). The FISH kit (Gene Pharma) is utilized for hybridization research. Briefly, 4% paraformaldehyde (PFA) is used to fix human primary keratinocytes or tissues. Slices are hybridized at 37\u0026deg;C in a moist, dark environment with specific probes. And then covered in DAPI-containing paraffin to seal slices. The pictures are taken using Olympus FV1000 confocal laser scanning microscope.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eHuman primary keratinocytes are grown using confocal dish (Corning, United States) and then fixed with 4% PFA for 20min at 37\u0026deg;C. Following permeabilization in 0.1% TritonX-100, the cell is washed three times with PBS. After blocking for 1 hour at 37\u0026deg;C with 5% normal goat serum, the primary antibody is incubated for overnight at 4\u0026deg;C. The next day, cells are incubated with secondary antibodies for 1 hour at 37\u0026deg;C after been rinsed three times with PBS. Nuclear staining is carried out using DAPI. A confocal laser scanning microscope is used to take fluorescent micrographs (Olympus FV1000).\u003c/p\u003e\n\u003ch3\u003eLuciferase Reporter Assay\u003c/h3\u003e\n\u003cp\u003epGL3 Basic luciferase reporter vector is provided by Tsingke (Nanjing, China), which contain miR-146a binding from hsa_circ_0105040 wild-type (WT) or mutant sequences. These recombinant luciferase reporter plasmids are co-transfected with miR-146a mimics, inhibitors, or controls into human primary keratinocytes. A Dual-luciferase assay equipment (Promega, E2920, USA) is used to measure the level of luciferase activity inside transfected cells.\u003c/p\u003e\n\u003ch3\u003eRna Immunoprecipitation Assay (Rip)\u003c/h3\u003e\n\u003cp\u003eRNA immunoprecipitation (RIP) analysis is performed using the EZ Magna RNA immunoprecipitation Kit (Millipore, USA). In brief, human primary keratinocytes are lysed in RIP lysis buffer 24 hours after transfection. After incubation with anti-Ago2 antibody or IgG in magnetic beads. The beads are then washed and incubated with cell lysates for 12 hours at 4\u0026deg;C. After being purified, the coprecipitated RNAs are discovered using qRT-PCR. The RNA sample preparation variations in each group are normalized using the input fraction, and the background fraction is using the negative controls (IgG).\u003c/p\u003e\n\u003ch3\u003eBiotin-labeled Mirna Pull-down Assay\u003c/h3\u003e\n\u003cp\u003eFirstly, the 3\u0026prime;-end biotinylated miR-146a mimics or the miRNA control (50nM, General Biosystems, Anhui, China) are transfected into human primary keratinocytes, and then cell lysates are harvested 48 hours post transfection. After centrifugation at 12000 rpm for 10 min at 4\u0026deg;C, 30\u0026micro;l of cell lysates are used as an input group. Followed by enrichment of the biotin-linked RNA complex with the Dynabeads MyOne Streptavidin C1 kit (Invitrogen, United States). qRT-PCR is applied to evaluate efficacy. All sequences are listed in Table S4.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe results of each experiment are provided as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard error of the mean after being replicated at least three times using biological. One-way ANOVA or Student's t-tests statistical analysis is used to compare the difference. Statistical significance is defined as a value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDifferent expression profiles of circRNAs-miRNAs-mRNAs between acne patients and normal controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the significance expression of genes involved in acne, we first show by circRNA\u0026nbsp;microarray\u0026nbsp;analyses that differentially expressed circRNAs exist in acne compared to normal skin tissues. The circRNA expression profile is statistically assessed using hierarchical clustering (Figure\u0026nbsp;1a). Subsequent analysis of scatter plots and volcano plots shows a total of 1,594 significant circRNAs expressions, of which 605 are upregulated and 989 circRNAs are downregulated in acne (Figure\u0026nbsp;1b-c).\u003c/p\u003e\n\u003cp\u003eWe then use an unsupervised hierarchical classification to evaluate differential miRNA expression patterns in acne and normal skin tissues (Figure 2a). As indicated in (Figure 2b), differential miRNA profiles are exposed in acne versus normal skin tissues. A total of 582 mRNAs are expressed differentially significantly (fold change \u0026gt;2.0, P \u0026lt; 0.05), of which 384 mRNAs are elevated and 198 mRNAs are downregulated (Figure 2c).\u003c/p\u003e\n\u003cp\u003eIt is well acknowledged that miRNA regulates mRNA expression by suppressing its activity in biological processes [9, 15, 16]. To explain whether mRNA is significantly expressed in acne in comparison with normal skin tissue, we show by hierarchical clustering and volcano plot analysis that a total of 13075 mRNA significantly differentiated expressed (fold-change \u0026gt;2.0, P \u0026lt; 0.05), among these 4955 upregulated and 8120 are downregulated (Figure 3a-b). These results indicate that circRNAs-miRNAs-mRNAs may be involved in the occurrence and development of acne.\u003c/p\u003e\n\u003ch3\u003eFunctional Analysis Of Differentially Expressed Mrna Between Acne Patients And Normal Controls\u003c/h3\u003e\n\u003cp\u003eFurther investigate the differential function of mRNA in acne and normal skin tissues. KEGG signaling pathway enrichment demonstrates that these associated genes are strongly involved in herpes simplex virus 1 infection, Fc gamma R-mediated phagocytosis, endocytosis, apoptosis, pyrimidine metabolism, Notch signaling pathway, Fc epsilon RI signaling pathway, sphingolipid signaling pathway, and AGE-RAGE signaling pathway in diabetic complications (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). GO enrichment examination of the relationship mRNAs reveal that they are predominantly enriched in biological processes, including the regulation of cellular (GO: 0050794), biological (GO: 0050789), cellular metabolic (GO: 0031323), primary metabolic (GO: 0080090), cellular macromolecule metabolic (GO: 0044260), and nitrogen compound metabolic processes (GO: 0051171), in addition to biological regulation (GO: 0065007) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eHsa_circ_0105040 Is Downregulated In Acne Patients, And Primarily Located In The Cytoplasm\u003c/h3\u003e\n\u003cp\u003eOur previous studies have shown that miR-146a is overexpressed and regulates inflammatory reactions in acne [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, we use Cytoscape software (version 3.4.0) to assess possible circRNAs to find out whether circRNAs regulate the expression of miR-146a and three circRNAs (hsa_circ_0105040, hsa_circ_0102678, hsa_circ_0102680) are predicted. To assess potential circRNAs in acne, we first choose hsa_circ_0105040 to further study. We show by histological analysis that expression of hsa_circ_0105040 expression shapely decreases in acne tissues compared to corresponding adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHsa_circ_0105040 is produced from the head-to-tail splicing of the exon17 (170bp) within the filamin A (FLNA) locus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). To clarify the molecular characteristics of hsa_circ_0105040, we first use divergent and convergent primers to generate the FLNA mRNA and hsa_circ_0105040 by qRT-PCR analyses. Electrophoresis on an Agarose gel assay shows that hsa_circ_0105040 is only detected in cDNA and not in gDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Next, the RNase R degradation analysis demonstrates that hsa_circ_0105040 is not degraded, while the quantity of FLNA mRNA decreases significantly. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eAdditionally, nuclear and cytoplasmic fractionation and FISH assay indicate that the majority of hsa_circ_0105040 is found in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-f). These results show that hsa_circ_0105040 may be involved in acne progression and is a stable circRNA expressed in the cytoplasm of keratinocytes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHsa_circ_0105040 promotes inflammatory response induced by\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eC. acnes\u003c/span\u003e \u003cb\u003ebiofilm in human primary keratinocytes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt is generally accepted that the development of acne is correlated with inflammation caused by \u003cem\u003eC. acnes\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To determine whether hsa_circ_0105040 affects acne by modulating the inflammatory response, we establish an accessible biofilm model of \u003cem\u003eC. acnes\u003c/em\u003e in vitro. Our previous study found that the 8th day can be the most effective stimulation period to construct a model of acne [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We then transfect with hsa_circ_0105040 siRNA (si-circ_0105040) and the control siRNA (si-NC), or overexpression hsa_circ_0105040 plasmid (pcDNA-circ_0105040) and the empty control plasmid (pcDNA-circ-NC) in human primary keratinocytes (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-b). To assess whether hsa_circ_0105040 is involved in acne inflammatory reaction, we suggest by qRT-PCR and enzyme-linked immunosorbent assay (ELISA) analysis that knockdown of hsa_circ_0105040 significantly reduces the expression of TNF-α, IL-6 and IL-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b), while hsa_circ_0105040 overexpression elevates the expression of these inflammatory genes (Figure S2a-b). Meanwhile, si-circ_105040 transfection reduces the positive effects of \u003cem\u003eC. acnes\u003c/em\u003e biofilm on the expression of p-P38, p-IκBα, and p-ERK1/2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Consistently, the level of these inflammatory genes is remarkably elevated with the transfection of pcDNA-circ_105040 (Figure S2c). Furthermore, immunofluorescence examination revealed that hsa_circ_0105040 positively affects the ability of \u003cem\u003eC. acnes\u003c/em\u003e biofilm to induce NF-κB-p65 nuclear translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed; S2d). Together, these results show that hsa_circ_0105040 promotes inflammation of human primary keratinocytes via MAPK and NF-κB pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eHsa_circ_0105040 Acts As A Cerna To Competitively Sponge Mir-146a In Human Primary Keratinocytes\u003c/h3\u003e\n\u003cp\u003eThis is clearly recognized that cytoplasmic circRNAs function as ceRNAs to sponges for microRNAs which have precisely reduced the synthesis of target mRNAs [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We then investigate whether hsa_circ_0105040 has similar effects in keratinocytes. Our previous investigation reveals that miR-146a suppressed the expression of IL-6, IL-8 and TNF-α in acne [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Hence, we further explore the interaction between hsa_circ_0105040 and miR-146a. Indeed, bioinformatics instruments predicts that hsa_circ_0105040 may bind to miR-146a (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Meanwhile, luciferase experiment shows that hsa_circ_0105040 can bind directly to miR-146a (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Furthermore, RNA immunoprecipitation (RIP) assay indicates that hsa_circ_0105040 exists in Ago2-containing miRNA complexes with ribonucleoprotein via binding to miR-146a (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Moreover, biotin-labelled miRNA pulldown assays detect that hsa_circ_0105040 expression improves transfect with biotin-labelled miR-146a as opposed to Bio-NC control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). These results indicate that hsa_circ_0105040 is acting as a ceRNA for miR-146a in human primary keratinocytes cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate these findings in vivo, we transfect human primary keratinocytes with si-circ_0105040 and si-NC or pcDNA-circ_105040 and pcDNA-circ-NC. We discover that knockdown of hsa_circ_0105040 dramatically increased the expression of miR-146a, while overexpression of hsa_circ_0105040 decreased the expression of miR-146a (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee-f). In addition, hsa_circ_0105040 expression is reduced with miR-146a transfection, but elevated with anti-miR-146a transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-h), suggesting that hsa_circ_0105040 may be directly regulating to miR-146a.\u003c/p\u003e\n\u003ch3\u003eHsa_circ_0105040 Sponge Mir-146a To Alleviate Targeted Irak1 And Traf6\u003c/h3\u003e\n\u003cp\u003eIt is essential for miR-146a to bind directly to IRAK1 and TRAF6 and inhibit expression of IRAK1 and TRAF6 in human primary keratinocytes cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. To test whether hsa_circ_0105040 is upstream of miR-146a to regulate IRAK1 and TRAF6, we find by qRT-PCR and WB analyses that the expression of IRAK1 and TRAF6 was significantly suppressed upon hsa_circ_0105040 siRNA compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCoincidentally, we observe that both IRAK1 and TRAF6 expressions are increased upon transfection with anti-miR-146a, while silence hsa_circ_0105040 can significantly reduce the positive impact of anti-miR-146a on IRAK1 and TRAF6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). Furthermore, TNF-, IL-8 and IL-6 expression induced by \u003cem\u003eC. acnes\u003c/em\u003e biofilm is elevated with anti-miR-146a, but reduced with si-circ_105040 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed-e). Taken together, these data indicate that hsa_circ_0105040 decoy miR-146a to promote the expression of \u003cem\u003eIRAK1\u003c/em\u003e and \u003cem\u003eTRAF6\u003c/em\u003e, and an increase inflammation in human primary keratinocytes trigger by \u003cem\u003eC. acnes\u003c/em\u003e biofilm.\u003c/p\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt is widely accepted that the development and occurrence of acne are caused by cellular inflammation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. During this time, circRNAs are emerging as an important moderator controlling cell inflammation in many human diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, circRNAs has not been investigated in the pathophysiology of acne\u003c/p\u003e \u003cp\u003eHere, we unexpectedly identify numerous differential expression circRNAs/mRNAs/mRNAs in acne and normal skin tissues. In addition, we suggest that hsa_circ_0105040 can increase inflammation levels upon \u003cem\u003eC. acnes\u003c/em\u003e biofilm in human primary keratinocytes. We first show mechanically that, during the pathology of acne, hsa_circ_0105040 inhibits miR-146 expression; Given that TRAF6 and IRAK1 are co-targets for miR-146, hsa_circ_0105040 attenuates the negative effect of TRAF6 and IRAK1 on miR-146a, which increase cellular inflammation by \u003cem\u003eC. acnes\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). These results demonstrate that hsa_circ_0105040 may be an indicator gene for controlling the development of acne inflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCircRNAs are single stranded covalently RNA molecules, which connect with occurrence and development of different diseases [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Moreover, it is supposed that dysregulation of circRNAs may be a factor in inflammatory skin illnesses [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Clinically, the \u003cem\u003eC. acnes\u003c/em\u003e infection, which causes skin inflammation, is one of the causes of acne. It is not clear, however, if circRNAs has physiologic effects on acne. The only relevant information comes from RNA sequencing study of the circRNA expression profile in severe acne, with circRNAs being associated with inflammatory and immunological responses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Here, circRNAs microarray analysis demonstrates that hsa_circ_0105040 affects acne inflammation, which may provide additional evidence for this study and enhance the comprehending of the novel functional relationships between circRNA and the pathophysiology of acne.\u003c/p\u003e \u003cp\u003eAs previously mentioned, circRNAs may act as ceRNA to bind miRNAs, thereby inhibiting their activity. For instance, hsa_circRNA_0088036 can interact with miR-140-3p and function as a miRNA sponge to regulate the expression of FOXQ1, which promotes bladder cancer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similar to this, circRAB3B sponge miR-1228-3p and negatively regulated miR-1228-3p, resulting in reduced psoriasis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Parallel to these observations, our research reveals that hsa_circ_0105040 expression within acne is negatively connected with miR-146a expression, but positively correlated with IRAK1 and TRAF6, suggesting that hsa_circ_0105040 may sponge miR-146a to control inflammation in acne. Our results here offer a fresh perspective on how the circRNA/miRNA/mRNA axis regulates inflammation in acne.\u003c/p\u003e \u003cp\u003eIn summary, we first identify the regulating mechanism by which hsa_circ_0105040, exerts its inhibition of miR-146a on TRAF6 and IRAK1, leading to increased levels of inflammation. Our study, which reveals the clinical significance of the hsa_circ_0105040/miR-146a/TRAF6/IRAK1 axis diagnosis of acne, perhaps gives novel perspectives on the pathological mechanism of acne.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCircular RNAs (circRNAs)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDifferentially expressed genes (DEGs)\u003c/p\u003e\n\u003cp\u003eGene Ontology (GO)\u003c/p\u003e\n\u003cp\u003eKyoto Encyclopedia of Genes and Genomes (KEGG)\u003c/p\u003e\n\u003cp\u003eCompetitive endogenous RNA (ceRNA)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFluorescence in situ hybridization (FISH)\u003c/p\u003e\n\u003cp\u003eQuantitative real-time polymerase chain reaction (qRT-PCR)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWestern blotting (WB)\u003c/p\u003e\n\u003cp\u003eParaformaldehyde (PFA)\u003c/p\u003e\n\u003cp\u003eRNA immunoprecipitation assay (RIP)\u003c/p\u003e\n\u003cp\u003eGenomic DNA (gDNA)\u003c/p\u003e\n\u003cp\u003eShort interfering RNAs (siRNAs)\u003c/p\u003e\n\u003cp\u003eInterleukin (IL)\u003c/p\u003e\n\u003cp\u003eTumor necrosis factor (TNF)\u003c/p\u003e\n\u003cp\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/p\u003e\n\u003cp\u003eInterleukin 1 receptor-associated kinase 1 (IRAK1)\u003c/p\u003e\n\u003cp\u003etumor necrosis factor receptor-associated factor 6 (TRAF6)\u003c/p\u003e\n\u003cp\u003eNuclear Factor Kappa B (NF-\u0026kappa;B)\u003c/p\u003e\n\u003cp\u003eMitogen-Activated Protein Kinase (MAPK)\u003c/p\u003e\n\u003cp\u003eFilamin A (FLNA)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is supported by National Natural Science Foundation of China (81773338, 82103749); CAMS Innovation Fund for Medical Sciences (2017-I2M-1-017, CIFMS-2021-I2M-1-001); Science and Technology Program of Nanjing, China (2019060001); Jiangsu Provincial \u0026ldquo;Double Innovation Doctors\u0026rdquo; Program (grant number JSSCBS20211610); Science and Technology development foundation of Nanjing Medical University (NMUB2020154).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are very grateful to patients for their participation and cooperation in providing us with important clinical evidence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eML, YZL and MZ conceived the study. YZL, MZ, NNZ, XC, HXX performed the study. MZ, HXX and RZ analyzed RNA sequencing data. ML, YZL and ZMD provided fundings. MZ, RZ and TL wrote the manuscript.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTan JK, Bhate K. A global perspective on the epidemiology of acne. Br J Dermatol 2015; 172 Suppl 1:3-12.\u003c/li\u003e\n\u003cli\u003eGollnick HP. From new findings in acne pathogenesis to new approaches in treatment. J Eur Acad Dermatol Venereol 2015; 29 Suppl 5:1-7.\u003c/li\u003e\n\u003cli\u003eTilles G. Acne pathogenesis: history of concepts. Dermatology 2014; 229:1-46.\u003c/li\u003e\n\u003cli\u003eGu H, An HJ, Gwon MG, Bae S, Leem J, Lee SJ, et al. Bee Venom and Its Major Component Melittin Attenuated Cutibacterium acnes- and IGF-1-Induced Acne Vulgaris via Inactivation of Akt/mTOR/SREBP Signaling Pathway. Int J Mol Sci 2022; 23.\u003c/li\u003e\n\u003cli\u003eAn integrated encyclopedia of DNA elements in the human genome. Nature 2012; 489:57-74.\u003c/li\u003e\n\u003cli\u003eXia X, Li Z, Liu K, Wu Y, Jiang D, Lai Y. Staphylococcal LTA-Induced miR-143 Inhibits Propionibacterium acnes-Mediated Inflammatory Response in Skin. J Invest Dermatol 2016; 136:621-630.\u003c/li\u003e\n\u003cli\u003eYang S, Fang F, Yu X, Yang C, Zhang X, Wang L, et al. Knockdown of H19 Inhibits the Pathogenesis of Acne Vulgaris by Targeting the miR-196a/TLR2/NF-\u0026kappa;B Axis. Inflammation 2020; 43:1936-1947.\u003c/li\u003e\n\u003cli\u003eZeng R, Xu H, Liu Y, Du L, Duan Z, Tong J, et al. miR-146a Inhibits Biofilm-Derived Cutibacterium acnes-Induced Inflammatory Reactions in Human Keratinocytes. J Invest Dermatol 2019; 139:2488-2496 e4.\u003c/li\u003e\n\u003cli\u003eChen LL, Yang L. Regulation of circRNA biogenesis. RNA Biol 2015; 12:381-8.\u003c/li\u003e\n\u003cli\u003eHao JQ. Targeting interleukin-22 in psoriasis. Inflammation 2014; 37:94-9.\u003c/li\u003e\n\u003cli\u003eLiu X, Frost J, Bowcock A, Zhang W. Canonical and Interior Circular RNAs Function as Competing Endogenous RNAs in Psoriatic Skin. Int J Mol Sci 2021; 22.\u003c/li\u003e\n\u003cli\u003eQiao M, Ding J, Yan J, Li R, Jiao J, Sun Q. Circular RNA Expression Profile and Analysis of Their Potential Function in Psoriasis. Cell Physiol Biochem 2018; 50:15-27.\u003c/li\u003e\n\u003cli\u003eLi LJ, Zhu ZW, Zhao W, Tao SS, Li BZ, Xu SZ, et al. Circular RNA expression profile and potential function of hsa_circ_0045272 in systemic lupus erythematosus. Immunology 2018; 155:137-149.\u003c/li\u003e\n\u003cli\u003eLiang J, Wu X, Sun S, Chen P, Liang X, Wang J, et al. Circular RNA expression profile analysis of severe acne by RNA-Seq and bioinformatics. J Eur Acad Dermatol Venereol 2018; 32:1986-1992.\u003c/li\u003e\n\u003cli\u003eMuniategui A, Nogales-Cadenas R, V\u0026aacute;zquez M, Aranguren XL, Agirre X, Luttun A, et al. Quantification of miRNA-mRNA interactions. PLoS One 2012; 7:e30766.\u003c/li\u003e\n\u003cli\u003eSalim U, Kumar A, Kulshreshtha R, Vivekanandan P. Biogenesis, characterization, and functions of mirtrons. Wiley Interdiscip Rev RNA 2022; 13:e1680.\u003c/li\u003e\n\u003cli\u003eDr\u0026eacute;no B, Dagnelie MA, Khammari A, Corvec S. The Skin Microbiome: A New Actor in Inflammatory Acne. Am J Clin Dermatol 2020; 21:18-24.\u003c/li\u003e\n\u003cli\u003eDr\u0026eacute;no B, P\u0026eacute;castaings S, Corvec S, Veraldi S, Khammari A, Roques C. Cutibacterium acnes (Propionibacterium acnes) and acne vulgaris: a brief look at the latest updates. J Eur Acad Dermatol Venereol 2018; 32 Suppl 2:5-14.\u003c/li\u003e\n\u003cli\u003eZheng N, Zhou M, He Y, Xu H, Chen X, Duan Z, et al. Low curcumin concentrations combined with blue light inhibits cutibacterium acnes biofilm-induced inflammatory response through suppressing MAPK and NF-\u0026kappa;B in keratinocytes. Photodiagnosis Photodyn Ther 2022; 40:103204.\u003c/li\u003e\n\u003cli\u003eZhang S, Liao K, Miao Z, Wang Q, Miao Y, Guo Z, et al. CircFOXO3 promotes glioblastoma progression by acting as a competing endogenous RNA for NFAT5. Neuro Oncol 2019; 21:1284-1296.\u003c/li\u003e\n\u003cli\u003eZhong Y, Du Y, Yang X, Mo Y, Fan C, Xiong F, et al. Circular RNAs function as ceRNAs to regulate and control human cancer progression. Mol Cancer 2018; 17:79.\u003c/li\u003e\n\u003cli\u003eKircik LH. Advances in the Understanding of the Pathogenesis of Inflammatory Acne. J Drugs Dermatol 2016; 15:s7-10.\u003c/li\u003e\n\u003cli\u003eWilliams HC, Dellavalle RP, Garner S. Acne vulgaris. Lancet 2012; 379:361-72.\u003c/li\u003e\n\u003cli\u003eJin X, Gao J, Zheng R, Yu M, Ren Y, Yan T, et al. Antagonizing circRNA_002581-miR-122-CPEB1 axis alleviates NASH through restoring PTEN-AMPK-mTOR pathway regulated autophagy. Cell Death Dis 2020; 11:123.\u003c/li\u003e\n\u003cli\u003eLiu R, Zhang L, Zhao X, Liu J, Chang W, Zhou L, et al. circRNA: Regulatory factors and potential therapeutic targets in inflammatory dermatoses. J Cell Mol Med 2022; 26:4389-4400.\u003c/li\u003e\n\u003cli\u003eCardamone G, Paraboschi EM, Rimoldi V, Duga S, Sold\u0026agrave; G, Asselta R. The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis. Int J Mol Sci 2017; 18.\u003c/li\u003e\n\u003cli\u003eDong W, Bi J, Liu H, Yan D, He Q, Zhou Q, et al. Circular RNA ACVR2A suppresses bladder cancer cells proliferation and metastasis through miR-626/EYA4 axis. Mol Cancer 2019; 18:95.\u003c/li\u003e\n\u003cli\u003eDube U, Del-Aguila JL, Li Z, Budde JP, Jiang S, Hsu S, et al. An atlas of cortical circular RNA expression in Alzheimer disease brains demonstrates clinical and pathological associations. Nat Neurosci 2019; 22:1903-1912.\u003c/li\u003e\n\u003cli\u003eYang J, Qi M, Fei X, Wang X, Wang K. Hsa_circRNA_0088036 acts as a ceRNA to promote bladder cancer progression by sponging miR-140-3p. Cell Death Dis 2022; 13:322.\u003c/li\u003e\n\u003cli\u003eLu J, Xu X, Li Y, Yu N, Ding Y, Shi Y. CircRAB3B suppresses proliferation, motility, cell cycle progression and promotes the apoptosis of IL-22-induced keratinocytes depending on the regulation of miR-1228-3p/PTEN axis in psoriasis. Autoimmunity 2021; 54:303-312.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"acne vulgaris, circRNA, ceRNA, miR-146a, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-2825777/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2825777/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCircular RNAs (circRNAs) are thought to play a crucial function in controlling gene expression, according to expanding findings. However, the importance of circRNAs in the regulation of acne inflammation is unclear.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMicroarray analysis has been carried out to investigate circRNAs/miRNAs/mRNAs that express abnormally in acne. RNase R digestion assay is used for confirmation of the hsa_circ_0105040 characteristic. The functional roles of hsa_circ_0105040 on inflammatory response induced by \u003cem\u003eCutibacterium acnes\u003c/em\u003e (\u003cem\u003eC. acnes\u003c/em\u003e) biofilm in human primary keratinocytes were revealed by Fluorescence in situ hybridization (FISH), Reverse transcription quantitative (PCR), Western blotting analysis, Immunoprecipitation, Luciferase reporter assay, Biotin-labeled miRNA pull-down assay, RNA immunoprecipitation (RIP).\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe first evaluate the human circRNA expression patterns in acne tissues and find that hsa_circ_0105040 expression is considerably reduced in acne tissues. Moreover, we discover that the majority of hsa_circ_0105040 is found to be localized in the cytoplasm of primary human keratinocytes. Hsa_circ_0105040 overexpression significantly enhances the production of proinflammatory factors (interleukin-8, interleukin-6, and tumor necrosis factor-α). Mechanistic research reveals that the microRNA miR-146a binds to hsa_circ_0105040, which then actively sponges miR-146a to prevent the level of IRAK1 and TRAF6.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThese findings point to hsa_circ_0105040 as a critical circRNA that function as \"microRNA sponges\" for the controlled inflammatory response in the development of acne. Our findings may provide valuable insights into the progression of acne.\u003c/p\u003e","manuscriptTitle":"Hsa_circ_0105040 promotes Cutbacterium acnes biofilm induced inflammatory via sponge miR-146a in human keratinocyte","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-19 14:12:44","doi":"10.21203/rs.3.rs-2825777/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2f234dfb-172b-4922-9486-019e5044a01c","owner":[],"postedDate":"April 19th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-04-28T14:29:26+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-19 14:12:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2825777","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2825777","identity":"rs-2825777","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}