Study On The Mechanism Of Hsa＿circ＿0007694/hsa-miR-135b-5p/SMAD5 Axis In Systemic Lupus Erythematosus

Background Systemic Lupus Erythematosus（SLE）is an autoimmune disease associated with an increased risk of premature mortality.The mortality of this disease is high,and The incidence showed an increasing trend year by year.The limited efficacy,poor tolerance or ineffectiveness and large side effects in some patients with conventional therapies had been found.Therefore,it is urgent to explore the pathogenesis of SLE and seek new effective treatment methods.At present,the pathogenesis of SLE is not completely clear,and many factors such as heredity,environment,hormones and epigenetics are considered to be closely related to the pathogenesis of SLE.Circular RNA（circRNA）is a type of single-stranded RNA molecules which were characterized by covalently linked head-totail closed loop structures.It is lack of 5’ cap structure and 3’ poly-A tail structure.In recent years,with the development of high-throughput sequencing technology,a large number of circRNAs have been discovered,and more and more studies have shown that circRNA are related to the pathogenesis of autoimmune diseases.Previous studies have shown that circRNA is abnormally expressed in SLE and is closely related to the clinical features and disease activity of SLE.Therefore,its role in the pathogenesis of SLE has become increasingly prominent.The most frequently reported function of circRNA is their ability to act as miRNA sponges.This function depends on ceRNA regulatory network,and circRNA can adsorb miRNA competitively with mRNA,weakening miRNA-mediated gene suppression.At present,the research on the function and mechanism of circRNA in SLE is limited,so this study attempts to explore the potential function and mechanism of circRNA in SLE from the perspective of ceRNA.ObjectiveThe purpose of this study is to further comprehensively analyze and screen the differentially expressed circRNA in T cells from SLE patients On the basis of the previous sequencing results of circRNA in T cells of SLE patients and healthy controls,and expand the samples for verification in the population.To analyze the correlation between differentially expressed circRNA and clinical indexes of SLE,and then verify the cell function of differentially expressed circRNA,and explore its possible mechanism of action,so as to preliminarily explore the role of circRNA in the pathogenesis of SLE.MethodsStage I: Four candidate circRNA related to SLE were screened based on the results of our group’s previous high-throughput sequencing data of T cells in SLE patients and healthy controls（GSE84655）,combined with the literature.Phase II: The differentially expressed circRNA in T cells of SLE patients and healthy controls was examined by case-control study,and its structure,correlation with clinical markers of SLE and diagnostic value were explored.Phase III: Cell experiments were conducted to verify the effect of circRNA on T cell function in SLE patients from the perspective of ceRNA.hsa_circ₀007694 and SMAD5 lentivirus vectors were constructed to silence hsa_circ₀007694 and SMAD5,respectively.Reverse transcription-polymerase chain reaction（RT-PCR）was used to detect hsa_circ₀000175,hsa_circ₀007694,hsa_circ₀032959,hsa_circ₀072387,hsa-miR-135a-5p,hsa-miR-135b-5p,hsa-miR-136-5p and SMAD5.Flow cytometry was used to detect cell apoptosis and cycle.CCK8 assay was used to detect cell proliferation.Western blotting（WB）was used to detect thelevel of SMAD5 protein.Elisa was performed to detecting the secretion of inflammatory cytokines of IFN-α,IFN-γ,IL-10,IL-6,IL-17,and IL-21.The interaction between hsa_circ₀007694 and hsa-miR-135b-5p and hsa-miR-135b-5p and SMAD5 was determined using dual-luciferase reporter gene.Bioassay was used to predict downstream miRNA of circRNA and downstream target mRNA of miRNA.ResultIn this study,the results of high-throughput T cell sequencing data（GSE84655）from SLE patients and healthy controls in our research group were taken as the entry point.Four candidate circRNAs associated with SLE（hsa_circ₀000175,hsa_circ₀007694,hsa_circ₀072387 and Hsa_circ₀032959）were selected for RT-PCR verification.98 SLE patients and 100 healthy controls（HCs）were included in this study.The expression of hsa_circ₀000175,hsa_circ₀007694 and hsa_circ₀072387 in T cells of SLE group were lower than that in T cells of HCs（all P<0.05）.There was no significant difference in the expression of hsa_circ₀032959 in T cells between SLE group and the healthy control group（P>0.05）.Further studies on the correlation between the above circRNA levels and the demographic data,clinical symptoms and laboratory indicators of SLE patients showed that the above circRNA levels were correlated with multiple clinical features of SLE.The expression level of hsa_circ₀007694 was down-regulated in SLE patients with photosensitivity,hemolytic anemia,Anti-ANUA antibody positive,low complement,anticardiolipin antibody Ig M positive,anti-β2-glycoprotein 1 antibody Ig G positive,and anti-β2-glycoprotein 1 antibody Ig M positive（all P<0.05）and there is a correlation between hsa_circ₀007694 and uric acid（r=0.475,P=0.001）.However,neither SLEDAI nor clinical medication was associated with circRNAs.Given that hsa_circ₀007694 is associated with a variety of clinical indicators in SLE patients,it indicates that hsa_circ₀007694 may be involved in the pathogenesis of SLE and with simpler structure compared with the other three circRNAs,which is worthy of further study.Therefore,in order to further study the effect of circRNA on T cells,hsa_circ₀007694 was selected to continue the following functional experiments from the perspective of ceRNA.The results of in vitro cell function experiment showed that compared with negative control,the proportion of early apoptotic cells（P <0.001）,the proportion of late apoptotic cells（P <0.001）and the proportion of total apoptotic cells（P <0.001）in hsa_circ₀007694 silenced group were significantly decreased.Except for 0h,the cell proliferation activity of hsa_circ₀007694 silencing group was higher than that of negative control group at 24 h,48h and 72 h.Compared with negative control group,the proportion of cells in G1 phase（P <0.001）was significantly increased in Hsa_circ₀007694-sh RNA silencing group（P <0.01）,but the proportion of cells in G2 phase（P <0.001）was significantly decreased,and the difference in S phase between two groups was not statistically significant（P >0.05）.The levels of IFN-α,IFN-γ,and IL-10 in the Jurkat+Hsa_circ₀007694-sh RNA group were significantly lower than those in the Jurkat+ NC-sh RNA group,but the levels of IL-6,IL-17,and IL-21 were significantly higher.Through database Starbasehsa3.0（https://starbase.sysu.edu.cn/）and circ Bank（http://www.circbank.cn/index.html）to predict Hsa_circ₀007694 downstream of micro RNAs.Three miRNAs were obtained by intersection,namely,hsa-miR-135a-5p,hsa-miR-135b-5p and hsa-miR-136-5p.Among them,the expression of hsa-miR-135b-5p was significantly up-regulated both in T cells of SLE patients and Jurkat（clone E6-1）cell line.In addition,the dual luciferase reporter assay confirmed the presence of targeted binding between Hsa_circ₀007694 and hsa-miR-135b-5p.Is the effect of Hsa_circ₀007694_sh RNA lentivirus on cell cycle,apoptosis,proliferation and inflammatory factor secretion affected by hsa-miR-135b-5p inhibitor? Hsa-miR-135b-5p inhibitor could partially reverse the inhibitory effect of Hsa_circ₀007964-sh RNA on apoptosis,the promotion effect of cell proliferation,the effect of cell cycle and the secretion of inflammatory factors.Through the downstream mRNAs of hsa_miR-135b-5p predicted from the database Starbase 3.0（https://starbase.sysu.edu.cn/）,miRDB（http://mirdb.org/）and Target Scan 8.0（http://www.targetscan.org/vert₈0/）intercrossing with the down-regulated mRNAs in SLE patients’ T cell high-throughput sequencing data,four mRNAs were got,namely BCAT1,ATP8A1,RAB33 B and SMAD5.The expression level of SMAD5 in T cells of SLE patients was significantly lower than that of healthy controls.Compared with Jurkat+ NC-sh RNA group,the expression of SMAD5 was decreased in Jurkat+Hsa_circ₀007694-sh RNA group at mRNA（P <0.001）and protein（P <0.01）.Compared with Jurkat+Hsa_circ₀007694_sh RNA+miR-NC,SMAD5 was significantly up-regulated at the mRNA level in Jurkat+Hsa_circ₀007694_sh RNA+hsa_miR-135b-5p-inhibitor group（P <0.001）.Although there was a trend of up-regulation at the protein level,the difference was not statistically significant（P <0.05）.Dual luciferase reporter gene assay confirmed the targeted binding between SMAD5 and hsa-miR-135b-5p.These results suggested that SMAD5 might be a downstream mRNA of hsa-miR-135b-5p.Thus,We further explore its function using SMAD5 lentivirus.Compared with the Jurkat+SMAD5-sh RNA-NC group,the percentage of late apoptotic cells and the percentage of total apoptotic cells decreased in the Jurkat+ SMAD5-sh RNA group,while the percentage of early apoptotic cells decreased,there was no statistical difference.Compared with the Jurkat+SMAD5-sh RNA-NC group,the cell proportion in G1 phase was significantly increased in Jurkat+ SMAD5-sh RNA group,while G2 phase was downregulated,and the difference was statistically significant.However,there was no significant difference in the cell proportion in S phase between the two groups.Compared with Jurkat+SMAD5-SHRNA-NC group,the proliferation activity of Jurkat+ SMAD5-sh RNA group was significantly up-regulated at 24 h,48h,and 72 h,except at 0h time point.Compared with the Jurkat+ SMAD5-sh RNA-NC group,IFN-α,IFN-γ and IL-10 were significantly down-regulated in the Jurkat+ SMad5-shr NA knockdown group,while IL-6,IL-17 and IL-21 were significantly up-regulated.The results showed that hsa-miR-135b-5p inhibitors could reverse the inhibitory effect of SMAD5-sh RNA on apoptosis,promote cell proliferation,influence on cell cycle,and secretion of inflammatory cytokines.Conclusion（1）The down-regulated expression of hsa_circ₀000175,hsa_circ₀007694 and hsa_circ₀072387 were found in T cells of SLE patients,while the expression of hsa_circ₀032959 showed no significant change.（2）Silencing of hsa_circ₀007694 inhibited cell apoptosis,promoted cell proliferation,affected cell cycle and inhibited the secretion of IFN-α,IFN-γ and IL-10,as well as promoted IL-6,IL-17 and IL-21.（3）Hsa_circ₀007694 acted as a sponge of hsa_miR₁35b-5p to regulate the expression of SMAD5,thereby involving in cell apoptosis,proliferation,cycle and secretion of inflammatory factors.