| Objective: We designed the study to explore the function of miR-20b-5p in Mtb-infected RAW 264.7 macrophages by analyzing the expression of miR-20b-5p in Mtb-infected macrophages,as well as in Mtb-infected macrophages-derived exosomes.Methods: The RAW 264.7 macrophages was infected with Mtb at the MOI of 10.We assessed the expression of miR-20b-5p at 6 h,16 h,24 h and 36 h post-infection by RT-PCR.We isolated the exosomes from cell culture supernatant by ultracentrifugation method.The characteristics of exosomes were identified by transmission electron microscopy(TEM).The CD63 protein as the marker of exosomes wad detected by Western blot.We measured the expression of miR-20b-5p derived from exosomes by RT-PCR.We regulated the levels of miR-20b-5p by transfecting with miR-20b-5p mimics and inhibitor into RAW 264.7macrophages followed infected with Mtb by the MOI of 10 for 24 h.On the one hand,we released Mtb by cracking the infected macrophages,vaccinated the released Mtb on Lowenstein-Jensen culture medium for 28 days for the CFU counts of Mtb;On the other hand,we detected the activity of macrophages with the CCK-8 kits at 6 h,16 h,24 h and 36 h post-infection,qualitatively and quantitatively detected the influence on cell apoptosis at 24 h post-infection by the AO/EB staining and TEM.We obtained the intersection of target genes of miR-20b-5p predicted by Miranda,TargetScan and PicTar software.We analyzed the molecular function,biological process,cell components of target genes by the BiNGO Cytoscape plugin.We applied the DAVID software for the KEGG signal pathway enrichment analysis.We selected the apoptosis related gene Mcl-1 as the target gene of miR-20b-5p.We analyzed the match between miR-20b-5p and Mcl-1 by TargetScan.We assessed theexpression of Mcl-1 by up and down regulation the expression of miR-20b-5p.Results:(1)The PCR results showed that compared with normal control group(0.977±0.006),the expression of miR-20b-5p in infection group at 6 h(0.870±0.026)(t=0.552,P<0.001),16 h(0.727±0.021)(t=1.294,P<0.001),24 h(0.643±0.031)(t=1.725,P<0.001)and 36 h(0.520±0.026)(t=2.364,P<0.001)were reduced,and were of the time dependence.(2)Exosomal RNA agarose gel electrophoresis results showed that only a small RNA electrophoresis band wad present,28 s and 18 s ribosomal RNA bands were absent.miR-20b-5p was not in the exosomes derived from normal RAW 264.7 macrophages,but in the exosomes derived from Mtb-infected RAW 264.7 macrophages.(3)Mtb was cultured on the Lowenstein-Jensen culture medium for 28 days,compared with the negative control-mimics group(2.33±0.58),the CFU counts of miR-20b-5p mimics group(0.67±0.01)was decreased(t=5.000,P<0.05).Compared with the negative control-inhibitor group(2.67±0.58),the CFU counts of miR-20b-5p inhibitor group(4.33±0.58)was increase(t=5.000,P<0.05).(4)According to the results of CCK-8detection,compared with the Negative control-mimics group(56.46±1.88%)at 6 h post-infection,the cell vitality of miR-20b-5p mimics group(44.39±1.31%)were decreased(t=6.672,P<0.05);compared with the Negative control-inhibitor group(54.87±0.733%),the cell vitality of miR-20b-5p inhibitor group(64.76±2.79%)were increased(t=5.082,P<0.05).Compared with the Negative control-mimics group(66.15±3.05%)at 16 h post-infection,the cell vitality of miR-20b-5p mimics group(39.30±0.79%)were decreased(t=19.263,P<0.01);compared with the Negative control-inhibitor group(66.96±2.48%),the cell vitality of miR-20b-5p inhibitor group(72.88±1.19%)were no significant difference(t=3.023,P>0.05).Compared with the Negative control-mimics group(75.62±3.25%)at 24 h post-infection,the cell vitality of miR-20b-5p mimics group(35.07±3.28%)were decreased(t=21.738,P<0.01);compared with the Negative control-inhibitor group(72.56±2.07%),the cell vitality of miR-20b-5p inhibitor group(97.14±1.63%)were increased(t=11.600,P<0.01).Compared with the Negative control-mimics group(74.73±4.41%)at 36 h post-infection,the cell vitality of miR-20b-5p mimics group(27.22±0.30%)were decreased(t=17.678,P<0.01);compared with the Negative control-inhibitor group(74.12±2.07%),the cell vitality of miR-20b-5p inhibitor group(88.55±0.98%)were increased(t=10.680,P<0.01).The AO/EB staining results under the fluorescence microscope showed that cells in the negative control-mimics group and negativecontrol-inhibitor group were dyed green fluorescence,the nucleus density was relatively uniform yellow green fluorescence.Cells in miR-20b-5p mimics group were yellow-green fluorescence,nuclear chromatin partial rendering intense orange fluorescent,contraction and round in shape.Cells in miR-20b-5p inhibitor group were green or dark green fluorescence,almost evenly distributed on the whole cell.Compared with the Negative control-mimics group(28.11±6.49%),the cell apoptosis index of miR-20b-5p mimics group(66.36±4.68%)was increased(t=10.643,P<0.01).Compared with the Negative control-inhibitor group(25.08±2.40%),the cell apoptosis index of miR-20b-5p inhibitor group(16.73±2.25%)decreased(t=5.362,P<0.05).According to the TEM results,the cellular structure of normal control group was complete,the nucleus and mitochondria were clear.In the infection control group,partial nucleus chromatin was edge gathered,mitochondrial cristae was vague and fracture.In the miR-20b-5p mimics group,the pseudopodiums of macrophages were disappear,the cell membrane was shrinking,and cell volume decreased.The nucleus fragmented,and membranous parcel nucleus fragments of apoptotic bodies was visible in the cytoplasm.The mitochondria swelled larger and cavitation.The rough endoplasmic reticulum was expansion within cytoplasm.In the negative control-mimics group the nucleus chromatin condensed and presented the“black hole”.The mitochondrial cristae was vague,fracture and cavitation.The autophagosome was visible in the cytoplasm.The morphological structure between negative control-inhibitor group and miR-20b-5p inhibitor group were nearly showed accordance that the cell membrane was intact,nucleus was neat and clear,and most mitochondria were clear but several mitochondria of cavitation.(5)miR-20b-5p was predicted 171 target genes.Molecular function analysis results showed that miR-20b-5p target genes mainly occurred in protein bining,activity of transcriptional regulation and DNA bining.According to the results of biological process analysis,miR-20b-5p target genes mainly enriched in the process of the metabolism of intracellular molecules,protein modification process,regulation of gene expression and cell apoptosis process.Cell component analysis results show that miR-20b-5p target genes mainly involved in cell components organelles and vesicles.KEGG pathway enrichment analysis revealed that significant enrichment in the MAPK signaling pathways,prostate,PI3K-Akt signaling pathways,mTOR signaling pathway,p53 signaling pathways,and T cell receptor signal pathway.The miR-20b-5p and Mcl-1 match analysis found that the type of match betweenapoptosis related gene Mcl-1 and miR-20b-5p seed region was 8 mer,and exist the complementary characteristics between Mcl-1 and miR-20b-5p nonseed 3'end.(6)According to the PCR results,compared with the normal control group(0.860±0.020)and negative control-mimics group(0.850±0.017),the expression of Mcl-1 mRNA in the miR-20b-5p mimics group(0.657±0.025)was decreased(t=21.922,P<0.01).Compared with the normal control group(0.860±0.020)and negative control-inhibitor group(0.853±0.029),the expression of Mcl-1 mRNA in the miR-20b-5p inhibitor group(1.103±0.075)was increased(t=8.183,P<0.05).Conclusions:(1)The expression of miR-20b-5p in Mtb-infected RAW 264.7macrophages was decreased,and was of time dependence.(2)miR-20b-5p was sorted and assembled into the exosomes derived from Mtb-infected RAW 264.7macrophages.(3)miR-20b-5p negatively regulation the survival of Mtb in RAW264.7 macrophages in part possibly through a mechanism by targeting the anti-apoptosis gene Mcl-1 to inhibitor apoptosis. |
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