Objective
To investigate the role of circular RNA (circRNA) in the occurrence of psoriasis.
Methods
Mesenchymal stem cells were isolated from skin lesions of 15 patients with psoriasis (psoriasis group) and skin tissues of 15 healthy human controls (control group) separately, and then subjected to cultivation. Flow cytometry and cell differentiation assay were performed to identify these mesenchymal stem cells. RNA sequencing was conducted to measure the expression of circRNA, and detailed bioinformatics analysis was performed. Then, 7 differentially expressed circRNAs were selected, and a circRNA-microRNA interaction network was established. In this network, 3 related microRNAs were selected and validated by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) . Statistical analysis was carried out by a two-sample t-test for comparison of qRT-PCR results between the psoriasis group and control group.
Results
As RNA sequencing showed, a total of 6 323 circRNAs were identified, and 3 227 out of these circRNAs were first reported in this study. Compared with the control group, 129 circRNAs were differentially expressed in the psoriasis group, including 123 up-regulated circRNAs and 6 down-regulated circRNAs. The predicted circRNA-microRNA interaction network showed that several psoriasis-related microRNAs were associated with the 7 circRNAs, such as miR-17-5p, miR-30e-5p, miR-142-3p/5p, miR-369-3p, miR-184, miR-4490, miR-654-3p and miR-423-5p. qRT-PCR also confirmed that the 7 differentially expressed circRNAs were up-regulated in the psoriasis group. Compared with the control group, the expression of the 3 selected microRNAs significantly decreased in the psoriasis group (t=3.993, 3.217, 2.918, respectively, all P < 0.05) .
Conclusion
There is aberrant expression of circular RNAs in the mesenchymal stem cells from psoriatic skin lesions, which may take part in the occurrence of psoriasis.
Key words:
Psoriasis; Mesenchymal stem cells; Circular RNA
Genome-wide association studies have identified over 120 risk loci for psoriasis. However, most of the variations are located in non-coding region with high frequency and small effect size. Pathogenetic variants are rarely reported except HLA-C*0602 with the odds ratio being approximately 4.0 in Chinese population. Although rare variations still account for a small proportion of phenotypic variances in complex diseases, their effect on phenotypes is large. Recently, more and more studies focus on the low-frequency functional variants and have achieved a certain amount of success.Whole genome sequencing and sanger sequencing was performed on 8 MZ twin pairs discordant for psoriasis to scan and verified the de novo mutations (DNMs). Additionally, 665 individuals with about 20 years' medical history versus 2054 healthy controls and two published large population studies which had about 8 years' medical history (including 10,727 cases versus 10,582 controls) were applied to validate the enrichment of rare damaging mutations in two DNMs genes. Besides, to verify the pathogenicity of candidate DNM in C3, RNA-sequencing for CD4+, CD8+ T cells of twins and lesion, non-lesion skin of psoriasis patients were carried out. Meanwhile, the enzyme-linked immunosorbent assay kit was used to detect the level of C3, C3b in the supernatant of peripheral blood.A total of 27 DNMs between co-twins were identified. We found six of eight twins carry HLA-C∗0602 allele which have large effects on psoriasis. And it is interesting that a missense mutation in SPRED1 and a splice region mutation in C3 are found in the psoriasis individuals in the other two MZ twin pairs without carrying HLA-C*0602 allele. In the replication stage, we found 2 loss-of-function (LOF) variants of C3 only in 665 cases with about 20 years' medical history and gene-wise analysis in 665 cases and 2054 controls showed that the rare missense mutations in C3 were enriched in cases (OR = 1.91, P = 0.0028). We further scanned the LOF mutations of C3 in two published studies (about 8 years' medical history), and found one LOF mutation in the case without carrying HLA-C*0602. In the individual with DNM in C3, RNA sequencing showed the expression level of C3 in skin was significant higher than healthy samples in public database (TPM fold change = 1.40, P = 0.000181) and ELISA showed protein C3 in peripheral blood was higher (~2.2-fold difference) than the other samples of twins without DNM in C3.To the best of our knowledge, this is the first report that DNM in C3 is the likely pathological mutations, and it provided a better understanding of the genetic etiology of psoriasis and additional treatments for this disease.
Abstract Psoriasis displays both increased angiogenesis and microvascular dilation in the skin, while human dermal microvascular endothelial cells (HDMECs) are involved in angiogenesis and microvascular dilation. Whether the functions of HDMECs are altered in psoriatic skin versus healthy skin remain unknown. Here, we isolated HDMECs from the skin of 10 patients with psoriasis and 10 healthy subjects and compared angiogenesis, proliferation, migration and cell metabolism between psoriatic HDMECs and normal HDMECs. We found that the morphology of primary HDMECs was comparable between psoriatic HDMECs and normal HDMECs. After passage, psoriatic HDMECs displayed larger cell size and wider intercellular space. In addition to DiI‐Ac‐LDL (DiI‐labelled acetylated low‐density lipoprotein) uptake, expression levels of CD31, vWF (von Willebrand factor) and LYVE‐1 were comparable in psoriatic HDMECs versus normal HDMECs. However, psoriatic HDMECs exhibited increased tube formation (numbers of nodes and meshes, p < 0.05) and migration (numbers of migrated cells, p < 0.001) and reductions in proliferation (growth rates, p < 0.05) and energy metabolism (oxygen consumption rate and extracellular acidification rate, p < 0.05) compared with normal HDMECs. Therefore, psoriatic HDMECs display an increased angiogenesis and migration and decreased proliferation and metabolic activity, suggesting a pathogenic role of HDMECs in psoriasis.
T cell-mediated inflammation plays an important role in the development of psoriasis. Mesenchymal stem cells (MSCs) are a population of multipotent cells that regulate the T cell-mediated immune response. To investigate the effects of psoriatic dermal mesenchymal stem cells (p-DMSCs) on proliferation, apoptosis and differentiation of T cells. p-DMSCs and normal DMSCs (n-DMSCs) were isolated from psoriatic skin and normal healthy controls, respectively, and co-cultured with activated T cells isolated from healthy volunteers using a Transwell system. Proliferation and apoptosis of T cells were assessed by cell count and flow cytometry, respectively. Expression levels of transcription factors associated with subtypes of T cells and cytokines were measured by qRT-PCR and western blot. Both p-DMSCs and n-DMSCs inhibited T cell proliferation and cytokine production. Similarly, the presence of p-DMSCs and n-DMSCs decreased the expression levels of both T-bet and ROR-γt in T cells. However, n-DMSCs exhibited a stronger inhibitory effect than p-DMSCs on T cell proliferation, cytokine production, and T-bet and ROR-γt expression. These results suggest that the effect of p-DMSCs on T cell function could contribute, at least in part, to the pathogenesis of psoriasis.
Thank you for your valuable comments on the paper.For the control problem, we made a blank control in the experiment, namely the control in the paper.Meanwhile, we also tested the indexes of the cells treated by the single autophagy inhibitor CQ and the p38 MAPK inhibitor SB203580.The results do not affect the final results and conclusions of the paper, but due to the space, we did not write the relevant results in the paper.In further studies we will add appropriate negative controls.
Abstract Background Cell proliferation, migration, and angiogenesis are aberrant in human dermal microvascular endothelial cells (HDMECs), resulting in abnormal endothelial function and microvascular dilation. Objective To explore the role of Integrin subunit alpha 9 (ITGA9) in proliferation and migration of dermal microvascular endothelial cells. Methods HDMECs were isolated from the skin of 6 psoriatic patients and 6 healthy controls. Expression levels of ITGA9 mRNA and protein were assessed with qRT-PCR and western blot, respectively, while miqRT-PCR was used to determine expression levels of miR-146a-3p. Cell proliferation and migration were assessed in human microvascular endothelial cell line (HMEC-1), following overexpression of either ITGA9 or miR-146a-3p, or co-transfection with miR-146a-3p-mimic and pLVX - ITGA9. Cell viability was detected by Cell Counting Kit-8 assay and 5ethynyl2′deoxyuridine (EdU) cell proliferation assay. Cell apoptosis was assessed, using annexin V-FITC/PI apoptosis detection kit, while cell migration was detected by wound healing and transwell assay. Results Expression levels of ITGA9 were significantly decreased in psoriatic HDMECs compared to normal controls. Moreover, expression levels of miR-146a-3p were higher in psoriatic HDMECs than in normal controls. Overexpression of miR-146a-3p lowered expression levels of ITGA9, accompanied by increased proliferation and migration of HMEC-1 in vitro . In contrast, overexpression of ITGA9 inhibited proliferation and migration of HMEC-1, while increasing expression levels of cdc42, ki67, focal adhesion kinase (FAK), c-Src tyrosine kinase (Src), RAC1 and RhoA. Conclusion ITGA9 can repress the proliferation and migration of HMEC-1, suggesting utility of ITGA9 as a potential therapeutic intervention for psoriasis.
Psoriasis is a chronic inflammatory skin disease, which the mechanisms behind its initiation and development are related to many factors. DMSCs (dermal mesenchymal stem cells) represent an important member of the skin microenvironment and play an important role in the surrounding environment and in neighbouring cells, but they are also affected by the microenvironment. We studied the glucose metabolism of DMSCs in psoriasis patients and a control group to reveal the relationship among glucose metabolism, cell proliferation activity，and VEC (vascular endothelial cell) differentiation in vitro, we demonstrated the biological activity and molecular mechanisms of DMSCs in psoriasis.We found that the OCR of DMSCs in psoriatic lesions was higher than that in the control group, and mRNA of GLUT1 and HK2 were up-regulated compared with the control group. The proliferative activity of DMSCs in psoriasis was reduced at an early stage, and mRNA involved in proliferation, JUNB and FOS were expressed at lower levels than those in the control group. The number of blood vessels in psoriatic lesions was significantly higher than that in the control group (p<0.05), which the mRNA of VEC differentiation, CXCL12, CXCR7, HEYL and RGS5 tended to be increased in psoriatic lesions compared to the control group, in addition to Notch3.We speculated that DMSCs affected local psoriatic blood vessels through glucose metabolism, and the differentiation of VECs, which resulted in the pathophysiological process of psoriasis.
Abstract Autophagy, an intracellular process of self-digestion, has been shown to modulate inflammatory responses. In the present study, we determined the effects of autophagy on inflammatory response induced by supernatant of psoriatic dermal mesenchymal stem cells (p-DMSCs). Human umbilical vein endothelial cells (HUVECs) were treated with supernatant of p-DMSCs cultures to induce inflammation and treated with rapamycin (RAPA) to induce autophagy. Expression levels of mRNA for inflammatory cytokines and BIRC2 were compared in HUVECs with vs. without induction of autophagy with rapamycin (RAPA) by PCR, while cell apoptosis was assessed by flow cytometry and caspase-3 activity assay kit. We found that induction of autophagy with RAPA decreased expression levels of IL6, IL8 and CCL20, in addition to reduction in inflammation-induced apoptosis in HUVECs; Expression levels of LC3, p62, p-p38 MAPK (Thr180/Tyr182), p-mTOR (Ser2445) and p-ULK1 (Ser555) proteins were measured by Western blotting. We found RAPA increased LC3Ⅱ, while decreasing p62 expression. Likewise, expression levels of p-p38 MAPK and p-mTOR proteins were markedly decreased by the treatment with RAPA; Finally, we evaluated thenitric oxide (NO) content, NO synthase (NOS) activity and cell angiogenesis. RAPA treatment increased the NO content and the NOS activity, and inhibited angiogenesis. Through the experimental results, we speculated that induced of autophagy can improve the function of endothelial cells in psoriasis, suggesting approaches to induce autophagy can be used to ameliorate psoriasis.
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