Study On The Role Of Circular RNAs In Systemic Lupus Erythematosus

Background Systemic lupus erythematosus?SLE?is a typical autoimmune disease characterized by production of various autoantibodies,complement consumption,and chronic inflammation.The abnormal behavior of T cells in SLE patients is crucial for the initiation and progression of this disorder.The pathogenesis of SLE is multifactorial,with genetic,environmental,hormonal,and epigenetic factors contributing to its development.Studies from home and abroad have shown that a subset of noncoding RNAs,including micro RNAs?miRNAs?and long non-coding RNAs?lncRNAs?,play a key role in SLE.Owing to the development of high-throughput sequencing and computational approaches,studies in the past few years have revealed the pervasive expression of a new type of noncoding RNAs,namely circular RNAs?circRNAs?,in eukaryote.Circ RNAs are characterized by covalently closed loop structures without 5' to 3' polarity or polyadenylated tail.Circ RNAs are more stable than linear transcripts.Furthermore,researches have shown that circRNAs can sponge miRNAs and then sequester miRNAs from their targets to inihibit the function of miRNAs.They can also regulate gene expression through promoting the gene transcription of their parent genes or competing with pre-mRNA splicing.Accumulating evidence supports the implication of circRNAs in the diagnosis,initiation,progresseion,and prognosis of various human diseases including cancers.Yet,study exploring the role of circRNAs in SLE is relatively lacking.Objective This study was undertaken to learn the expression profile of circRNAs in SLE T cells by virtue of circ RNA microarrays and to screen and validate the differentially expressed circRNAs in SLE T cells;Then,this study will investigate the functions of the differentially expressed circ RNA and explore its mechanisms to tentatively reveal the potential role of circ RNA and its functional mechanism in the initiation and progression of SLE.Methods This study screened the T cells obtained from 6 SLE patients and 3 healthy controls with Human circ RNA Array v2.0 to identify differentially expressed circRNAs between the two groups.The differential expression of two circRNAs,hsa_circ₀045272 and hsa_circ₀000694,was validated in T cells from 24 SLE patients and 12 healthy controls using real-time quantitative PCR?q RT-PCR?.The correlations between the expression level of differentially expressed circRNAs and clinical paramaters of SLE patients were also analyzed.To reveal the function of differentially expressed circRNAs,this study simultaneously screened the aforementioned T cells to identify differentially expressed message RNAs?mRNAs?and lncRNAs between the two groups using Human Lnc RNA Array v3.0.The differential expression of four lncRNAs?ENST00000448942,ENST00000440578,uc001 ykl.1,and TCONS₀0026993?was also validated in T cells from 24 SLE patients and 12 healthy controls with q RT-PCR to test the validity of microarray.To explore the function of hsa_circ₀045272,two specific short hairpin RNA?sh RNAs?targeting hsa_circ₀045272 were designed,and lentiviral hsa_circ₀045272-sh RNA was used to knockdown hsa_circ₀045272.The silencing efficiency of hsa_circ₀045272-sh RNA was assessed with q RT-PCR.The hsa_circ₀045272-sh RNA showing higer silencing efficiency was used to generate Jurkat cells with stable hsa_circ₀045272 knockdown.Following staining with Annexin V-PE and 7-Amino-Actinomycin?7-AAD?,the influence on apoptosis of hsa_circ₀045272 knockdown was examined with flow cytometric analysis.Enzyme-linked immunosorbent assay?ELISA?was performed to evaluate the effect on interleukin-2?IL-2?and interferon gamma?IFN-??secretion of activated jurkat cells with hsa_circ₀045272 knockdown.The miRNAs binding to hsa_circ₀045272 and competitive endogenous RNAs?ce RNAs?of hsa_circ₀045272 were predicted through bioinformatic analyses.The predicted results were combined with the expression information of these ce RNAs obtained from lncRNA microarrays to explore its mechanism.The expression of two potential ce RNAs,NM₀03466?PAX8?and NM₀15177?DTX4?,in cells with stable hsa_circ₀045272 knockdown was assessed with q RT-PCR,and the levels of their corresponding proteins were examined with western blot?WB?.Dual luciferase reporter assay was performed to test the binding of hsa_circ₀045272 with hsa-mi R-6127 and explore whether NM₀03466?PAX8?and NM₀15177?DTX4?are targets of hsa-mi R-6127.To further investigate the function of those differentially expressed circRNAs,this study constructed the coexpression networks of the differentially expressed circRNAs with differentially expressed mRNAs in T cells.Results A total of 127 circRNAs were found to be differentially expressed?fold change ? 2;P < 0.05?in T cells from SLE patients compared with healthy controls.Among them,55 were upregulated and 72 were downregulated in SLE T cells.q RT-PCR validated the significantly lower expression levels of hsa_circ₀045272 in T cells from SLE patients?P = 0.0076?,whereas no significant difference in expressions of hsa_circ₀000694 between these two groups was noticed?P = 0.2149?.No associations between hsa_circ₀045272 level and clinical parameters including SLEDAI score of SLE patients were found?P > 0.05 for each?.A total of 1977 mRNA were found to be differentially expressed?fold change ? 1.5;P < 0.05?in T cells of SLE patients compared with healthy controls.Among them,1119 mRNA were upregulated and 858 mRNA were downregulated in SLE T cells.Additionally,869 upregulated lncRNAs and 1066 downregulated lncRNAs were identified in SLE T cells.q RT-PCR validated significantly lower expression levels of ENST00000448942 and uc001 ykl.1 in SLE T cells?P < 0.05 for each?,which confirmed the validity of lncRNA microarray.hsa_circ₀045272-sh RNA2# showed a silencing efficiency of 98.4% as suggested by q RT-PCR,and its was used for generation of Jurkat cells with stable hsa_circ₀045272 knockdown.The percentage of cells of early apoptosis was significantly increased in hsa_circ₀045272-sh RNA group compared with NC-sh RNA group?P = 0.0268?,whereas the proportion of cells in late stage apoptosis or already dead was not significantly different between hsa_circ₀045272-sh RNA group and NC-sh RNA group?P = 0.1084?.After activation with anti-CD3 and anti-CD28 for 24 h,significantly elevated IL-2 level in hsa_circ₀045272-sh RNA group compared with NC-sh RNA was noticed?P < 0.0001?.Similarly,after activation for 48 h,IL-2 level in hsa_circ₀045272-sh RNA group was also significantly higher than NC-sh RNA group?P = 0.001?.744 mRNAs which could competitively bind to 73 distinct miRNAs were predicted as ce RNAs for hsa_circ₀045272.Combinging with their expression information,22 of them were considered promising hsa_circ₀045272 ce RNAs.As compared to NC-sh RNA group,the mRNA lvels of NM₀03466?PAX8??P < 0.001?and DNM₀15177?DTX4??P < 0.0001?were significantly decreased in these cells with hsa_circ₀045272 knockdown,whereas their corresponding protein levels in these cells are not significantly changed?P > 0.05 for both?.Dual-luciferase assays showed hsa_circ₀045272 acted as a sponge of hsa-mi R-6127 and suggested that NM₀03466?PAX48?or NM₀15177?DTX4?were not targets of hsa-mi R-6127.Circ RNA-mRNA coexpression networks showed that circRNAs were highly correlated with mRNAs.Conclusions This present study found a total of 127 differentially expressed circ RNA,1935 lncRNA,and 1977 mRNA in T cells from SLE patients and validated the significantly decreased expression of hsa_circ₀045272.Moreover,the study domenstrated that hsa_circ₀045272 could bind with hsa-mi R-6127 and negatively regulate apoptosis and IL-2 production.Yet,the mechanisms by which hsa_circ₀045272 modulated apoptosis and IL-2 production remain unclear.The roles of hsa_circ₀045272 and other dysregulated circRNAs and the relevant mechanisms in the initiation and progression of SLE await further investigation.