The Role Of MiR-301b-3p In Regulation Of Monocyte Autophagy Induced By M-CSF

BACKGROUNDInflammation has been proven to play an important role in the pathophysiology of atherosclerosis. Inflammation is also related with the recruitment and activation of monocyte-derived macrophages and the differentiation of monocytes. However, because of the short half-life of monocytes, the monocytes patrolling in the circulation would naturally apoptosis in case of absence of autophagy. It has been reported that autophagy can be induced by GM-CSF or M-CSF in monocytes.Autophagy is a ubiquitous phenomenon in eukaryotes,which is a general mechanism for degradation and recycling of unnecessary or dysfunctional cellular components that is characterized by the formation of double-membrane vesicles in order to sustaining cellular homeostasis, ageing, differentiation, and development. During autophagy, LC3 and p62/SQSTM1,a ubiquitin like binding protein,are important marker proteins. Therefore,they are often used to detect the levels of autophagy acitivities.In short chain noncoding RNAs, microRNAs (miRNAs, miR-) can regulate a wide variety of cellular biological activities at post-transcriptional level. In this study, we induced autophagy of THP-1 human monocytes by M-CSF and investigated the changes of the expression levels of miRNAs to select the miRNA of which the change is the most significant. Furthermore,we explored the potential target of the selected miRNA in regulating the autophagy in monocytes.METHODS1. Cell cultureTHP-1 Human monocytes were incubated with modified RPMI 1640 medium(90%RPMI-1640+10% fetal bovine serum +1% Penicillin-Streptomycin solution).Monocytes were cultured under condition in modified RPMI 1640 medium and 100 ng/ml M-CSF to induce autophagy.2. Flow cytometry analysisTHP-1 Human monocytes were maintained via standard culture practice mentioned above, and were treated by CYTO-ID Green stain solution. Analyze the samples in green(FL1) channel of a flow cytometer.3. Western blot analysisProteins were collected from monocytes after M-CSF treatment by HelixGen RIPA Lysis Buffer. Western blot analysis was used to detect the expressions of LC3-Ⅰ, LC3-Ⅱ,p62/SQSTM1 and GAPDH. The concentrations of primary antibodies were as follows: anti-LC3 (1:1000), anti-p62 (1:1000), and anti-GAPDH (1:1000). The concentrations of second antibodies were as follows: anti-rabbit (1:1000) and anti-mouse (1:1000). Protein bands were detected by ImageQuant LAS 4000 imaging system.4. Transmission electron microscopeMonocytes were treated by M-CSF for 6 h, and then the cells were solidified by paraformaldehyde fixative over 6 h. Under the electron microscope, autophagosomes as the vesicles with double membrane structure were observed as the standard of the qualitative detection of autophagy.5. Agilent miRNA microarrayTotal RNA extracted from monocytes by mirVana PARIS kit (ABI Ambion am1556).After hybridization, microarray slides were scanned by Aglient G2505C Scanner.6. qRT-PCR analysisMiRNAs were extracted from monocytes by miRcute miRNA isolation kit. Reverse transcription reactions were carried out by miRcute Plus miRNA First-Strand cDNA Synthesis Kit. Real-time PCR was performed by miRcute miRNA qPCR Detection Kit(SYBR Green). U6 expression served as control. △Ct values were normalized to the expression levels of U6. The relative quantitation of gene expression was determined by the 2-△△Ct method.7. Construction of adenoviruses and cell transfectionMiR-301b-3p was delivered to the monocytes via adenovirus vectors after the construction and amplification of recombinant adenoviruses. The efficiency of miRNAs transfection was determined by qRT-PCR.8. Dual luciferase reporter assayWe fused luciferase reporter plasmids with 3’ UTR of the mRNAs of MYB and CYLD respectively, which were then co-transfected into 293T cells with miR-301b-3p mimic. The cells were lysed after 24 h. Dual luciferase reporter assay kit was used to determin the luciferase activities on a luminometer and make the quantitative analysis of it.9. Statistical analysisMeasurement data are expressed as mean ± standard deviation. The experimental data of each group were repeated at least 3 times. The two groups were compared by independent-samples t test, and the differences between multi-groups were analyzed by single factor ANOVA analysis. The standard of significant difference was supposed as P <0.05.RESULTS1. M-CSF induced autophagy in monocytesUnder treatment by M-CSF for 6 h, autophagy of THP-1 human monocytes was activated significantly. Flow cytometry analysis showed that the level of autophagy in monocyte which treated by M-CSF for 6 h was higher than that of the control group. Western blot analyses showed that the densitometry ratio of LC3- Ⅱ/Ⅰ was increased at 6 h compared with the control group and the expression of p62/SQSTM1 decreased. After using bafilomycin Al to inhibit lysosomal activity, the densitometry ratio of LC3-Ⅱ/Ⅰ incresed further under the same treatment condition, and the expression of p62/SQSTM1 was higher than that of the control group. Under the transmission electron microscope, it was observed that autophagosomes were as the vesicles with double membrane structure, which was not shown in the control group. The above results imply that autophagy in monocytes was activated significantly after induced by M-CSF for 6 h.2. The expression of miRNAs during autophagy in monocytesAgilent miRNA microarray analysis show that the expression of miR-1249-3p, miR-301b-3p, miR-4653-3p, miR-513a-5p, and miR-6734-5p in the monocytes which previously treated by M-CSF for 6 h differed from that in the control group significantly. And qRT-PCR analysis indicated that the expression of miR-301b-3p increased significantly in the experiment group compared with the control group.3. Overexpression of miR-301b-3p enhanced autophagy in monocytes induced by M-CSFWhen monocytes were infected by the adenovirus expressing miR-301b-3p (Ad-miR-301b-3p) for 48 h, the expression level of miR-301b-3p was detected by qRT-PCR analysis.The result showed that the expression of miR-301b-3p increased markedly in the monocytes infected by Ad-miR-301b-3p compared with the control group in which the cells were infected by adenovirus vector (Ad-vector). After being infected for 48 h, the cells were stimulated by M-CSF for 6h as previous. The results of western blot analysis showed that the densitometry ratio of LC3-Ⅱ/Ⅰ in Ad-miR-301b-3p group increased significantly compared with the control group while the expression of p62/SQSTM1 decreased. The results indicated that overexpression of miR-301b-3p enhanced autophagy in monocytes induced by M-CSF.4. Target gene prediction and verificationThe potential target genes of miR-301b-3p were screened and predicted in the databases of TargetScan and microRNAorg. The results showed that the potential target genes may be MYB or CYLD. Luciferase reporter vectors containing 3’UTR of MYB and CYLD were constructred. The reporter assay showed that luciferase expression of pSicheck2-MYB-3’-UTR was suppressed by miR-301b-3p overexpression markedly.CONCLUSION1. M-CSF could induce autophagy in THP-1 human monocytes, which was showed by the further analysis to be activated markedly after treatment by M-CSF for 6 h.2. During autophagy induced by M-CSF in monocytes, miR-301b-3p was upregulated significantly.3. Overexpression of miR-301b-3p in monocytes could enhance the activity of autophagy.4. MYB and CYLD were the targets of miR-301b-3p and may play a role in the regulation of autophagy induced by M-CSF in monocytes.