MicroR-142-3p Down-regulates IRAK-1in Response To BCG Infection In Macrophages

Objective In order to better understand the role of MicroRNAs（miRNAs） inthe immunological regulation of macrophages against Mycobacterium tuberculosis(MTB) infection, and it’s potential mechanism.Method1.Small RNA was extracted at different times(4h,8h,12h,24h)afterstimulated with BCG in cultured mouse RAW264.7cells.After using stem-loopreverse transcription primers to reverse transcribed into cDNA, the expression ofmiR-203, miR-142-3p and miR-21、miR-142-3p、miR-155、miR-132、miR-200c wasdetected by Real-time PCR.At the same time, building the T vector of the six maturemiRNA to make the standard curve.2. PCR primers were designed according to miR-142-3p sequences, miR-142-3pshRNA by chemical synthesis was inserted into pSicoR vector,and then identified bydouble digestion and nucleotide sequencing.The positive recombinant vector wasdesignated as pSicoR-miR-142-3p. Moreover, we synthesized miR-142-3p inhibitorswhich is23nt2’-methoxy-modified RNA oligonucleotide and these can effectivelyinhibit the expression of mature miR-142-3p.3. Using miRanda, Target Scan and the PicTar target gene prediction software tofind the potential target genes of miR-142-3p.Construction of vector containingIRAK-13’UTR luciferase reporter and validation of miR-142-3p target using Dualluciferase reporter gene system and Western blot analysis.4. RAW264.7cells was transfected by pSicoR/miR-142-3p or miR-142-3pinhibitors for24h,then using BCG stimulate cells.Cells were harvested12h, total RNA was isolated.Real Time PCR was used to detect the cytokines NF-κB,TNF-α and IL-6expression.Results1.The expression profile of six miRNAs known to be related to innateimmunity in RAW264.7macrophage cells following a BCG infection was determinedby qRT-PCR. The expressions of five miRNAs, miR-132, miR-142-3p, miR-155,miR-200c and miR-203were significantly decreased at various time points followingBCG infection, except that an increased expression of miR-203was found at24h afterthe infection (p<0.05),However, miR-21expression was significantly elevated inresponse to BCG infection at all tested time point in this study (p<0.05). This resultimplied that miRNAs might play an important regulatory role in macrophages againstBCG infection.2. RAW264.7cells transfected with pSicoR/miR-142-3p and miR-142-3pinhibitor showed ectopic and inhibitory expression of miR-142-3p by twenty-one foldand four fold in comparison with the control transfected with pSicoR/nc respectively.3. Luciferase reporter assays were performed to verify whether is a direct targetof miR-142-3p using293T cells, The data suggest that miR-142-3p directlysuppresses IRAK-1expression by targeting the3’UTR of IRAK-1mRNA.miR-142-3p showed an inability to alter IRAK-1expression at mRNA level in bothnon-infected and BCG infected cells; however, it demonstrated an ability todown-regulate IRAK-1protein expression by5/6in cells transfected withpSicoR/miR-142-3p as compared with the pSicoR/nc control; and cells transfectedwith miR-142-3p inhibitor exhibited an1.8-fold increase in IRAK-1proteinexpression. These data indicated that miR-142-3p was able to regulate the immuneresponse in macrophages against MTB infection through a mechanism in part bypost-transcriptionally down-regulating IRAK-1expression.4. To test the hypothesis of whether the miR-142-3p induced down-regulation of IRAK-1may alter the expressions of inflammatory cytokine genes in the signalingpathway initiated by IRAK-1, the function of miR-142-3p was ascertained byevaluation of NF-κB, TNF-α and IL-6expression in RAW264.7cells. As expected,the expression of NF-κB, TNF-α and IL-6were down-regulated in cells transfectedwith pSicoR/miR-142-3p in comparison with the pSicoR/nc control; on the other hand,cells transfected with miR-142-3p inhibitor showed an increased expressions ofNF-κB, TNF-α and IL-6as compared with the control. Thus an overall increasedexpression of these inflammatory factors weas observed in RAW264.7cells inresponse to BCG infection..Conclusions Collectively, the results reported in this study demonstrated analteration of immunity-related miRNA profile in macrophage RAW264.7cells inresponse to BCG infection. Moreover, miR-142-3p demonstrated a potential tonegatively regulate the production of pro-inflammatory mediators NF-κB, TNF-α andIL-6in the macrophages in part through a mechanism of targeting IRAK-1gene andpost-transcriptionally down-regulating IRAK-1protein expression.