| Objective:The pathogenesis of systemic lupus erythematosus(SLE)is not fully understood so far,and T lymphocyte dysfunction plays an important role in the pathogenesis of SLE.In addition,some studies have found that abnormal expression of microRNA-181a(miR-181a)is closely related to the occurrence and development of SLE.Some scientific research and clinical experiments in recent years have shown that mesenchymal stem cells(MSCs)have a certain therapeutic effect on patients with SLE,but the mechanism is not fully understood.In this study,we will reveal the target genes of miR-181a,investigate the effects of MSCs on the expression of miR-181 and SLE-related inflammatory factors in peripheral blood T lymphocytes of SLE patients,to discuss the pathogenesis of SLE and the mechanism of MSCs in treating SLE.Methods:1.Analysis of target sites of microRNA-181a by dual luciferase reporter vector.To predict the target site of microRNA-181a(miR-181a)by useing bioinformatics software,and select the downstream target gene for the next verification test.After plasmid construction,HEK293T cells were cultured and transfected the plasmid;Then samples were collected for Luciferase detection.2.Effect of microRNA-181a on T lymphocytes and its mechanism.Construction of miR-181a and its target gene OPN(SPP1)overexpression and interference expression lentiviral vectors and corresponding lentiviral control vectors.The viral vectors were transduced to Jurkat cells and stable transfected cell lines were obtained.Then,cells from each strain were collected to extract total RNA and total protein.MRNA levels of OPN,NF-kB,TNF-?,?-catenin and miR181a were dected by Real-time PCR quantitatively(Rt-qPCR)and the OPN,NF-kB,TNF-? and ?-catenin protein expression were detected by using Western-blot method.3.Effects of mesenchymal stem cells on microRNA-181a in T lymphocytes of patients with systemic lupus erythematosus and its mechanism.(1).Jurkat cells cultured alone and Jurkat cells co-cultured with UC-MSCs in different time periods were collected to extract total RNA and total protein,then mRNA and protein expression levels of OPN,NF-kB,TNF-? and ?-catenin were detected by Rt-qPCR and Western-blotting,mRNA level of miR-181a was detected by Rt-qPCR;(2).Extraction whole blood from SLE patients and healthy people.After extracting PBMC,total T lymphocytes were sorted by magnetic beads.Each specimen was divided into four groups:co-culture of T lymphocytes from SLE patients and UC-MSCs(SLE+UC-MSCs),mono-culture T lymphocytes from SLE patients(SLE),co-culture of T lymphocytes from healthy control and UC-MSCs(HC+UC-MSCs)and mono-culture of T lymphocytes from healthy control(HC);(3).CCK8 method was used to detect the proliferation activity of cells in each group,and calculate cell viability and inhibition rate;The IFN-r,IL4,IL6 and IL10 levels in the supernatant of each group of culture cells were detected by ELISA;The mRNA and protein expression levels of OPN,NF-kB,TNF-? and P-catenin of T lymphocytes from each group were dectede by Rt-qPCR and Western-blot,the mRNA level of miR-181a was detected by Rt-qPCR.4.Statistical method.SPSS 19.0 was used for comparative analysis.Results:1.Analysis of target sites of microRNA-181a by dual luciferase reporter vector.The quantitative of fluorescence expression of miRNA mimic+H TNF WT+TK(91.88± 1)and miRNA mimic+H_SPP1 WT+TK(65.6±0.71)were significantly lower than other groups,and the differences were statistically significant(p<0.05).2.Effect of microRNA-181a on T lymphocytes and its mechanism.The protein and mRNA expressions of OPN,TNF-?,NF-kB and ?-catenin were significantly increased in SPP1(OPN)overexpression cell lines,while the mRNA expression of miR-181a was significantly decreased,the difference was statistically significant(P<0.05).On the contrary,the protein and mRNA expressions of OPN,TNF-?,NF-kB and ?-catenin were significantly decreased in OPN-shRNAl interference cell lines,while the mRNA expression of miR-181a was significantly increased,the difference was statistically significant(P<0.05).The mRNA and protein expressions of OPN,TNF-?,NF-kB and ?-catenin in miR-181a overexpression cell lines were significantly reduced,while the mRNA expression of miR-181a was significantly increased,the differences were statistically significant(P<0.05).In contrast,the expressions of mRNA and protein of OPN,TNF-?,NF-kB and?-catenin in miR-181a interference cell lines were significantly increased,while the mRNA expression of miR-181a was significantly decreased,the differences were statistically significant(P<0.05).3.Effects of mesenchymal stem cells on microRNA-181a in T lymphocytes of patients with systemic lupus erythematosus and its mechanism.(1)Compared with Jurkat cells cultured alone,the mRNA and protein expression of OPN,TNF-?,NF-kB and ?-catenin in Jurkat cells co-cultured with UC-MSCs were significantly reduced,while the mRNA expression of miR181a was significantly increased.The differences were statistically significant(P<0.05);(2)The results of CCK8 showed that the proliferation activity of the SLE group is significantly higher than that of HC group,and the SLE+UC-MSCs group is significantly lower than the SLE group(p<0.001).Meanwhile,compared with HC+UC-MSCs group,the SLE+UC-MSCs group had significantly lower cell viability and significantly higher cell inhibition rate(p<0.001);(3)The results of ELISA showed that the values of IFN-r,IL4,IL6 and IL10 in the T lymphocyte supernatant of the SLE group were significantly higher than those in the HC group,but the values in SLE+UC-MSCs decreased obviously than SLE group,the differences were significantly(P<0.01 or 0.001);(4)The results of RT-qPCR showed that compared with the HC group,OPN,TNF-?,NF-kB and ?-catenin in T lymphocytes of SLE group increased significantly,while miR-181a decreased significantly,the differences were significant(P<0.001).Compared with the SLE group,the expressions of OPN,TNF-?,NF-kB and ?-catenin in T lymphocytes of SLE+UC-MSCs group were significantly decreased,and the expression of miR-181a was significantly increased,the differences were significant(P<0.008 or 0.001);(5)Western-blot results showed that the protein expression levels of OPN,TNF-?,NF-kB and ?-catenin in T lymphocytes of SLE group were significantly higher than those in HC group(P<0.001),the protein expressions of OPN,TNF-?,NF-kB and ?-catenin in T lymphocytes of SLE+UC-MSCs group were significantly lower than those of SLE group(P<0.001).Conclusions:The overexpression of miR-181a has a significant inhibitory effect on SLE-related pathway proteins and inflammatory factors in T lymphocytes of SLE patients.Down-regulation of miR-181a expression may promote the occurrence and development of SLE.In our vitro experiments show that UC-MSCs can inhibit the expression of OPN,TNF-?,NF-kB and ?-catenin in T lymphocytes from the SLE patients,while up-regulates the expression of miR-181a,indicating that MSCs can significantly regulate T lymphocytes in patients with SLE.We speculate that MSCs in humans may play a therapeutic role in SLE by up-regulating miR-181a to suppress the expression of SLE-related inflammatory factors in T lymphocytes. |
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