| Objective: In order to screen the differentially expressed circ RNA between SLE patients and healthy people,and to explore the molecular genetic mechanism of the differentially expressed circ RNA in SLE patients.Method:1.In the screening stage,the PBMCs of 3 newly-emerged SLE patients and 4 healthy controls were taken as the research objects.High-throughput sequencing was used to screen the differentially expressed circ RNAs between SLE patients and healthy controls,and the differentially expressed circ RNAs were genetically identified.Ontology(GO)and Kyoto Gene and Genome Encyclopedia(KEGG)pathway analysis.2.In the verification stage,31 patients with SLE were selected as the case group(SLE group),and 35 healthy volunteers were selected as the healthy control group(HC group).The expression levels of hsa＿circ＿0007683,hsa＿circ＿0042519,hsa＿circ＿0008647,hsa＿circ＿0006689,hsa＿circ＿0070562,and hsa＿circ＿0006117 in PBMC were detected by RT-q PCR,and compared with clinical parameters(including SLEDAI score,WBC,RBC,PLT,C3,C4,24 h urinary protein,ANA,ds DNA antibody,Sm antibody and Anu A)correlation analysis.Receiver operating characteristic curve(ROC)was used to evaluate the clinical value of circ RNA as a diagnostic indicator.3.At the stage of preliminary exploration of the molecular genetic mechanism of circ RNA formation,174 SLE patients and 176 healthy controls were included,and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS)typing technology was used to analyze the introns on both sides of hsa＿circ＿0006689 The Alu element performs SNP detection,performs allele and genotype frequency distribution,linkage disequilibrium,and haplotype analysis.Results:1.Screened 362 differentially expressed circ RNAs between SLE patients and healthy controls,of which 167 were significantly up-regulated and195 were significantly down-regulated;GO analysis suggested that these differentially expressed circ RNA-derived genes are mainly distributed in the cytoplasm,by affecting proteins and RNA Participate in the metabolism and modification of proteins and other macromolecules;KEGG analysis suggests that the differentially expressed circ RNA-derived genes are mainly enriched in lysine degradation,tumor erroneous transcription,and B cell receptor signaling pathways.2.(1)Compared with healthy controls,hsa＿circ＿0007683,hsa＿circ＿0042519,hsa＿circ＿0008647 were all increased in PBMC of SLE patients(P<0.05),and hsa＿circ＿0006689 was decreased in PBMC of SLE patients(P<0.05).There was no significant difference in the expression of hsa＿circ＿0070562 and hsa＿circ＿0006117 in PBMC of SLE patients and healthy controls(both P >0.05).(2)Correlation analysis suggests that hsa＿circ＿0006689in PBMC is correlated with SLEDAI score(P <0.05),and is correlated with WBC,RBC,PLT,C3,C4,24 h urine protein,ANA,anti-ds DNA antibody,Sm antibody,Anu A and other indicators No correlation(P>0.05).hsa＿circ＿0007683,hsa＿circ＿0042519,hsa＿circ＿0008647 had no correlation with the above clinical parameters(P>0.05).(3)ROC analysis showed that the AUC area of hsa＿circ＿0008647 and hsa＿circ＿0006689 when distinguishing HC group and SLE group were 0.911(sensitivity 93.55%,specificity 74.29%,P<0.001)and0.736(sensitivity 51.61%,specific The sex was 88.57%,P<0.001).Combined with hsa＿circ＿0008647 detection,the sensitivity of ANA,anti-ds DNA antibody,anti-Sm antibody and Anu A to distinguish HC group and SLE group from96.77%,29.03%,32.26%,32.26% to 100.00%,83.87%,80.65%,80.65,respectively %(All P<0.001),compared with the detection of HC group and SLE group using anti-ds DNA antibody,anti-Sm antibody and Anu A alone,the combined detection can improve the sensitivity of these three indicators to distinguish HC group and SLE group(all P<0.001),compared with using ANA alone to detect HC group and SLE group,ANA combined with hsa＿circ＿0008647 can improve the sensitivity of ANA to distinguish HC group and SLE group,but there is no statistical significance(P>0.05).Combined with hsa＿circ＿0006689 detection,the sensitivity of anti-ds DNA antibody,anti-Sm antibody and Anu A to distinguish HC group and SLE group can be increased to61.30%,71.00%,67.70%,the difference before and after the combination is statistically significant(P<0.05);when hsa＿circ＿0006689 and The combined detection of ANA and HC group and SLE group can slightly increase the sensitivity of ANA from 96.77% to 96.80%,but it is not statistically significant compared with that before ANA combination(P> 0.05).3.(1)The 7 sites rs10847698,rs11610968,rs12813317,rs144234620,rs186471269,rs4760536 and rs4760537 were accurately measured.(2)The genotype T/C and allele C at rs10847698 are enriched in the SLE group(P<0.05),and the C/A genotype at rs4760537 is enriched in the SLE group(P<0.05),possibly It is a risk factor for SLE.(3)The results of linkage disequilibrium analysis show that there is a high linkage disequilibrium relationship between rs10847698,rs11610968,rs4760536 and rs4760537;(4)Haplotype analysis suggests that rs10847698,rs11610968,rs12813317,rs144234620,rs186471269,rs4760536 and rs4760537 alleles The haploid CGGCCCC haplotype genotype was enriched in the SLE group(P<0.05).Conclusions:1.There are a large number of differentially expressed circ RNAs between SLE patients and healthy people.These differentially expressed circ RNAs are related to the metabolism and modification of proteins and other large molecules.2.The expression of hsa＿circ＿0008647 in PBMC of SLE is significantly increased,ROC analysis shows that hsa＿circ＿0008647 has a higher sensitivity in distinguishing SLE patients from healthy people;the expression of hsa＿circ＿0006689 in PBMC of SLE patients is significantly reduced,which is related to the SLEDAI score of SLE patients.It is speculated that hsa＿circ＿0008647 and hsa＿circ＿0006689 have the potential as molecular markers in the diagnosis and evaluation of SLE,respectively.3.The difference between rs10847698 and rs4760537 on the Alu elements on both sides of hsa＿circ＿0006689 in SLE patients and healthy people may be related to the difference in hsa＿circ＿0006689 cyclization efficiency,and it is worth further exploration. |
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