MiR-17-5p Inhibits The Autophagy Of RAW264.7 Cells By Targeting ULK1 And Regulates The Infection Of BCG

Objective In order to study the role of MicroRNAs(miRNAs)in the regulation of autophagy participated with Mycobacterium tuberculosis,and it's potential mechanism.Method 1.Small RNA was extracted fome the mouse RAW264.7 cells after treated with Rapamycin for 2hours,4hours,6hours and 8hours.The expression of MiR-30 b ?MiR-106?MiR-214?MiR-183 ?MiR-376c?MiR-200a?MiR-17-5p?MiR-142-3p?MiR-30c?MiR-377 was detected in RAW264.7 cells by Real-time PCR.2.The target genes of miR-17-5p was predicted with bioinformatics software of miRanda.We want to study the gene is ULK1.We constructed double Luciferase expression vector to verify the relation between miR-17-5p and ULK1 3'UTR of ULK1 and we got the ULK1 3'UTR use PCR amplified and inserted into pMIR-Report Luciferase vector.By calculating the relative luciferase activity(the ratio of firefly and Renilla luciferase activity)and with the internal control PRL-TK,detect the effect on luciferase enzyme activity of miR-17-5p.To detecting the influence of MiRNA on ULK1 by real-time PCR and Western Blot.3.Cells were transfected with miR-17-5p mimic or inhibitors for 24 hours.After using rapamycin and BCG were treated cells for 2 hours.Total RNA and total protein were extracted from Cells to detect the target gene in the mRNA level of ULK1,and the protein levels of LC3 B.4.Using the same cell model,fixed cells,embedded sections,and using TEM to observe autophagy situation.Corresponding fixed cells,after immunofluorescence test,using confocal microscopy to observe FITC-labeled protein LC3 B.Results 1.Using Rapamycin treated RAW264.7 macrophages for 2 hours,4 hours,6 hours,and 8 hours,Real-Time PCR test results show: miR-17-5p,miR-214,miR-142,miR-106 a,miR-376 c,miR-30 c were upregulated(greater than 2.3 times the P <0.05)at 2,6,8 hours;miR30b,miR214,miR142-3p,miR376 c,miR200a were downregulated in 4 hours(P <0.05).Downregulation of miR-183(P <0.05)at 2,6,8 hours.Mir-200 a,mir-30 b,mir-377,mir-30 c,mir-376 c,mir-17-5p changes significantly and miR-17-5p in four periods are stablely upregulated,showed that these miRNAs may affect certain genes by regulating autophagy.2.By dual luciferase experiments confirmed the miR-17-5p target ULK1 binding its 3'UTR of conserved sites and inhibits its expression;Real-Time PCR results showed that: The role of miR-17-5p mimic and inhibitors on ULK1 had meaningless about mRNA expression;Western blot analysis: miR-17-5p mimic inhibit ULK1 protein expression(p <0.05),ULK1 protein expression was increased(p <0.05)when suppressed the expression of miR-17-p.The influence of miR-17-5p to ULK1 was mainly in post-transcriptional level,inhibiting the level of ULK1.3.In order to observe the effects on the regulation of autophagy about miR-17-5p,we detected LC3 B protein and ULK1,Meanwhile,the detection about ULK1 can also verify that the target genes of miR-17.The results showed that LC3 B protein reduced significantly(p <0.05)when transfected with miR-17-5p mimic,and LC3 B protein expression increased significantly(p <0.05)when transfected with miR-17-5p after inhibitors.The results of TEM and microscopy showed that,miR-17-5p mimic significantly inhibited rapamycin and BCG-induced autophagy.These datas suggest that miR-17-5p inhibits the expression of LC3 B protein by targeting ULK1,and plays a negative role for autophagy.Conclusion 1.High expression of miR-17-5p when rapamycin and BCG were infected RAW264.7 macrophages cells,indicating that it may regulates autophagy caused by rapamycin or BCG.2.miR-17-5p inhibits LC3 B protein(autophagy marker protein)or inhibits cell autophagy through targeting ULK1,plays a negative role in autophagy pathway and in RAW264.7 cells.