Identification And Functional Study On MiRNA Associated With Spontaneous Apoptosis Of Neutrophils

Objective: Neutrophils (PMN) are important inflame-matory cells found in the human body. Neutrophils participate in hostdefense against pathogenic microorganisms and serve an importantfunction in the non-specific immune response of the human body. The lifespan of neutrophils is only24~48h. Once they enter the circulation,neutrophils immediately start the spontaneous apoptosis program tomaintain the balance between the quantity and stability of internal theenvironment, thus serving an important function in the occurrence,development, and prognosis of inflammation. MiRNA refers to a class ofsmall non-protein coding RNA length of approximately18~24nucleotides. MiRNA regulates gene expression by targeting of the3’endnon-protien coding regions of target mRNAs. MiRNA serves an importantfunction in negative regulation by promoting the degradation orpreventing the translation of mRNA. This study screens differentiallyexpressed miRNAs between normal and spontaneous apoptosis PMN anddetermines the progress of the spontaneous apoptosis of PMN. The studyalso provides new insights into the mechanism underlying PMNspontaneous apoptosis and offers a unique basis for the diagnosis andtreatment of inflammatory diseases. Methods:(1) PMNs were isolatedfrom healthy volunteers and cultured according to different groups:0h as control and12and24h as the experimental groups.(2) Cells werecollected for total RNA extraction.(3) Different levels of mRNAexpression were determined through the application of digital geneexpression profiling technology (DGE) at0and24h in neutrophils, andapoptosis-related genes were detected.(4) Affymetrix miRNA microarraychips were used to detect the differential miRNA expressions of0,12, and24h PMN.(5) Four important candidates, namely, miR-4419a, miR-487b,miR-4417, and miR-432were selected for verification through Northernblot analysis.(6) The target genes of differential expression wereevaluated based on three databases: www.mirbase.org, www.targetscan.org,and mirdb.org. Results of the databases were analyzed to predict thepossibility that miRNA target genes will take the intersection. The resultswere then compared with the genes detected by DGE, which againexhibited intersection.(7) Two closely related apoptosis genes wereselected according to the target genes and the candidates from DGE,PKCβ and MCL1. These candidates were verified by RT-PCR and Westernblot analysis to identify the relationship between these genes and thecorresponding miRNA target genes. Results:(1) A total of1,317differentgenes, including many of those associated with apoptosis were detected byDGE.(2) Twenty-one miRNAs were up-regulated, whereas21weredown-regulated at12h, whereas41were up-regulated, and22weredown-regulated in24h PMN compared with those in the control group. Among these miRNAs,13were up-regulated, and11were down-regulatedin both12and24h PMN.(3) Northern blot analysis results wereconsistent with the miRNA microarrays.(4) Predicted target genes werePKCβ and MCL1, and many other mRNAs were associated with apoptosis.Furthermore, PKCβ may be the target gene of miR-432and miR-199a,whereas MCL1may be the target gene of miR-4539.(5) RT-PCR showedthat PKCβ and MCL1were both down-regulated. The result wasconsistent with those of DGE.(6) Western blot analysis results wereconsistent with those of RT-PCR. Conclusion:(1) The mRNA profiles ofthe spontaneous apoptosis of PMN were obtained.(2) The miRNA profilesof the spontaneous apoptosis of PMN were obtained.(3) PKCβ and MCL1may be involved in the regulation of the spontaneous apoptosis of PMN.(4) MiR-432and miR-199a may be involved in the regulation of thespontaneous apoptosis of PMN by down-regulating PKCβ.(5) MiR-4539may be involved in the regulation of spontaneous apoptosis of PMN bydown-regulating MCL1.