Prediction And Identification Of MicroRNA Effect On Expression Of Granulysin

Objective:To predict microRNA (miRNA) related to Granulysin (GLS) usingbioinformatics analysis, construct miRNA eukaryotic expression vector anda GLS3’-UTR fused to firefly luciferase reporter vector, and then screenout the related miRNA with GLS3’-UTR expression by dual-luciferase test.To obtain stable expression cell lines THP-218and THP-NC by infectingTHP-1cells with lentivirus containing the selected miRNA, and stimulatethe two cell lines with phorbol12-myristate13-acetatefor (PMA). The GLSexpression of two cell lines was detected to further confirm mir-218interference effect on endogenous GLS protein expression.Methods:1. The miRNA (mir-218、mir-514、 mir-185、mir-611) targeting GLS3’-UTR was predicted by bioinformatics analysis and cloned into miRNAeukaryotic expression vector, and a pair of scrambled sequences wasdesigned for control. The synthetic3’-UTR fragment of GLS was clonedinto pGL3-control to construct firefly luciferase reporter vector pGLS-3UTR. Five different miRNA eukaryotic expression vectors,pGLS-3UTR and pRL-TK which express renilla luciferase wereco-transferred into293T cells. At48hours post transfection, fireflyluciferase activity and renilla luciferase activity of each group weredetected. To prevent the off-target effect, anti-mir-inhibitor was transfectedat the same time in order to select the miRNA related to GLS3’-UTR.2. THP-1cells were induced with PMA (at a final concentration of160nM) for12h,24h and48h respectively, and then total RNA and proteinwere extracted.12h,24h and48h post stimulation, expression of GLSmRNA was detected by RT-PCR and sequence of GLS mRNA wasidentified by DNA sequencing.24h and48h post stimulation, expression ofGLS protein was detected by Western blot, and expression of GLS protein48h after stimulation was detected by immunocytochemistry3. THP-1cells were infected with lentivirus LV3-mir-218andLV3-mir-control respectively, stable expression cells THP-218andTHP-NC were obtained after selected by puromycin. This two cell lineswere stimulated by PMA, total RNA and protein were isolated48h afterstimulation to detect the differential expression of GLS mRNA and protein.Results:1. Bioinformatics analysis showed that mir-218, mir-514, mir-185andmir-611have the complementary binding sites with GLS3’-UTR. Results of restriction analysis and DNA sequencing revealed that miRNA eukaryoticexpression vector and pGLS-3UTR were constructed correctly. After293Tcells were co-transferred with five different miRNA eukaryotic expressionvectors, pGLS-3UTR and pRL-TK, the firefly luciferase activity ofpGLS-3UTR treated with mir-218was decreased observably about75%(P<0.01), while the other miRNAs could not inhibit the expression offirefly luciferase. Moreover, firefly luciferase expression gradually returnedto normal levels after transfected with anti-mir-218.2. Transcription of GLS mRNA in THP-1cells was observed24h aftertreated with PMA, and Western blot and immunocytochemistry showedexpression of GLS48h after treated with PMA.3. THP-218and THP-NC cell lines were screened out by puromycinsuccessfully. After the two cell lines were treated with PMA, expression ofendogenous GLS protein in THP-218cells compare to THP-NC cells wassignificantly inhibited by55%(P<0.01) as detected by Western blotanalysis, while expression of GLS mRNA remains unchanged.Conclusions:1. Four miRNAs related to GLS3’-UTR was predicted by in silicoanalysis and miRNA eukaryotic expression vector and GLS3’-UTR-fireflyluciferase reporter vector were constructed successfully. Besides,miRNA-218which is closely related to GLS3’-UTR expression was selected.2. PMA activated the expression of endogenous GLS in humanmonocytic cell line THP-1.3. mir-218negatively regulates endogenous GLS expression at apost-transcriptional level.Our research confirmed that miRNA could inhibit expression of GLSin THP-1cells, which lay a foundation for further exploring the mechanismof miRNA regulates GLS expression and provide a reasonable therapeuticstrategy for the treatment of tuberculosis in the future.