Expression Of MiR-27b In Spinal Tuberculosis And Its Effect On Amino Acid Cytochrome P450 Signaling Pathways

Objectives and background:Spinal tuberculosis is the highest incidence of extrapulmonary tuberculosis,morbidity is higher.The pathology of Mycobacterium tuberculosis in tuberculosis is not yet fully understood.Recent studies have shown that tuberculosis Bifidobacteria infection in macrophages in the process,through some way to control the expression of certain miRNAs,its cell activity and function regulation.And ultimately in the macrophages parasitic and long-term survival,under certain conditions,increased replication caused by tuberculosis.Aminoacetic acid metabolic pathway CYP450 metabolic pathway is a recent discovery of a new way in the body of biochemical and pathological significance has not yet known and clarified.The expression of CYP450 isoenzyme in monocytes and macrophages in human blood is mainly CYP450 lbl[15].Studies have shown that epoxidized eicosyltriacetic acid(EETs)produced by the arachidonic acid cytochrome P450 metabolic pathway can inhibit apoptosis by a variety of ways.Combination of bacilli to inhibit macrophage apoptosis may be related to this pathway.In our preliminary gene chip study,we found that the expression of miR-27b was significantly changed in patients with spinal tuberculosis.Further experiments showed that miR-27b also showed significant changes in Mycobacterium tuberculosis-induced macrophage apoptosis,indicating that miR-27b Which can play an important role in the process of Mycobacterium tuberculosis infection,which can affect the pathogenesis of tuberculosis by regulating the increase of macrophage apoptosis,but its specific pathogenesis is not clear.CYP450 1b1 is a target gene for miR-27b.We speculate that Mycobacterium tuberculosis may inhibit the apoptosis of mononuclear cells by regulating the expression of miR-27b and its target gene CYP450,regulating the arachidonic acid pathway and reducing EETs The purpose of immune escape.This study further validates this hypothesis.Materials and methods:1,miR-27b and predicted target gene CYP 450 lbl expression in clinical data of spinal tuberculosis:According to the clinical manifestations of patients,imaging data and related laboratory tests,combined with biopsy or surgical findings of histopathological examination results of the establishment of spinal tuberculosis(N = 30)and the control group(n = 12).5 mL of peripheral blood samples were taken before the implementation of anti-tuberculosis treatment.After adding Ficoll reagent,gradient centrifugation was performed to obtain mononuclear cells.The control group of people from our hospital health examination center health volunteers,with the law to obtain peripheral blood mononuclear cells peripheral blood mononuclear cell(PBMC).The mRNA and microRNAs were extracted and the mRNAs CYP450 lbl and miR-27b were detected by RT-PCR.The AACT values were analyzed statistically(p<0.05 was statistically significant)(ROC curve),and the area under the curve(AUC)was used toevaluate the diagnostic efficiency of spinal tuberculosis(TB).The specificity and sensitivity of spleen tuberculosis were measured.2,the validation of target gene relationship:the use of luciferase reporter gene to verify miR-27b and cytochrome P4501bl(CYP 4501b1)gene-target gene relationship.3,miR-27b on the metabolicpathway of arachidonic acid CYP450 macrophage apoptosis:a,miR-27b mimics transfection:Thp-1 induced differentiation into macrophages.The target genemiR-27b mimics was transfected with Lipofectamine2000 method.According to the transfection amount,the control group was divided into blank control group,2.5ul,5ul and lOul group.FAM control mimics were transfected and the negative control group)was established.The control group was also divided into blank control group according to the transfection concentration.B.The apoptotic level of miR-27b mimics transfected macrophages was detected by flow cytometry.C,miR-27b mimics transfection of CYP1b gene expression level:to take miR-27b mimics different concentrations of macrophages take total RNA,denatured agarose gel electrophoresis detection of RNA quality after reverse transcription.After transcription,RT-PCR was performed and the AACT of the CYP4501b1 gene in macrophages at different transfection concentrations was calculated and analyzed statistically(p<0.05 was statistically significant).The expression of CYP450 1b1 was detected by Western blot.E,ELSIA detected EETs expression.Result:1,the clinical sample test:the experimental group CYP450 lbl gene RT-PCR detection △△CT value compared with the healthy control group increased(1.76 ± 0.69/1.18 ± 0.27),miR-27b gene RT-PCR detection AACT value and healthy control group(0.92 ± 0.22/1.18 ± 0.32),both of which showed different expression(p<0.05).The sensitivity of miR-27b and CYP4501bl to detecting spinal tuberculosis were 0.67%and 0.585,respectively,83%and 91%,respectively,and AUC were 0.786 and 0.814.2,respectively.The luciferase reporter showed that when expression of miR(6.469 ± 0.886/8.901 ± 0.935),the expression of luciferase decreased with the control group(6.469 ± 0.886/8.901 ± 0.935),which was significantly higher than that of the control group(P<0.05)(P&It;0.05),and when overexpressed miR-27b can no longer specifically bind to the luciferase reporter gene vector after 11 base sequences,i.e.,the hypothetical binding site mutation,it is impossible to regulate fluorescein(12.305 ± 1.518/10.807 ± 2.287),there was no significant difference between the expression of luciferase and the control group(p>0.05).The above results indicate that miR-27b can down-regulate the expression of CYP4501bl gene in macrophages,and the CYP4501bl gene is the target gene of miR-27b.3,miR-27b function test:miR-27b mimics gene empty control group,2.5ul group,5ul group,lOul group macrophage apoptosis rates were 0%,9.4%,6.8%,8.4%,the results Indicating that increased expression of miR-27b can promote macrophage apoptosis.The miR-27b mimics gene was used to control the AACT value of the CYP450 lbl gene in 2.5ul,5ul,10ul macrophages.The values of △△CT were 1,0.36,0.15 and 0.02 respectively.The results showed that with the miR-27b transcription concentration High,CYP450 1b1 gene expression decreased.Western blot was used to detect the decrease of CYP4501bl level in macrophages after transfection.Elisa test results showed that the expression levels of EEt-14 and 15 in the empty control group,2.5ul group,5ul group and lOul group were 20.748pg/ml,11.106pg/ml,13.336pg/ml and 9.727pg/ml respectively.With the increase of miR-27b transcription concentration,the expression of EEt-14 and 15 decreased.Conclusions:1,the expression of miR-27b in peripheral blood mononuclear cells of patients with spinal tuberculosis was decreased,and the expression of CYP4501b1 gene was significantly different from that of healthy control group.MiR-27b and CYP4501b1 may be one of the early molecular markers of spinal tuberculosis diagnosis.2,The gene-target gene relationship between miR-27b and CYP4501b1 gene.3,miR-27b expression decreased,macrophage CYP4501b1 gene expression increased,the downstream product EETs expression increased,the apoptosis rate decreased.In view of the above,the target gene of miR-27b is CYP4501b1,and the expression of miR-27b is decreased in the course of Mycobacterium tuberculosis(MTB),and its effect is to increase the expression of target gene CYP4501bl and reduce the expression of EEts through the arachidonic acid pathway So that the apoptosis rate of PBMC decreased,so as to achieve immune escape.