PMC:7025468 / 17157-19952
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7025468","sourcedb":"PMC","sourceid":"7025468","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7025468","text":"Overexpression and Inhibition of miR-146a Confirms Immune Signaling Molecule Targets in CD4+ T-Cells\nTRAF6 and IRAK1 are known direct targets of miR-146a in several cell types, including murine macrophages and liver cells, primary human monocytes, and THP-1 monocytic cells (19, 27, 50, 51). We employed a miR-146a mimic to confirm that TRAF6 and IRAK1 are targets of the miRNA in primary CD4+ T-cells. The miR-146a mimic was transfected into CD4+ T-cells, resulting in an expected significant increase in miR-146a expression (Figure 3A). We observed no change in IL-1β mRNA expression (Figure 3B). RT-qPCR analysis showed that TRAF6 and IRAK1 mRNA levels were significantly decreased, indicating that miR-146a targeted these molecules and initiated mRNA degradation pathways (Figure 3B).\nFigure 3 Overexpression and inhibition of miR-146a confirms immune signaling molecule targets in CD4+ T-cells. CD4+ T-cells were un-transfected, transfected with a negative control mimic or miR-146a mimic. (A) Successful miR-146a overexpression was confirmed by RT-qPCR. Fold change was calculated by normalizing the transfected cells to non-transfected cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to non-transfected controls (***p \u003c 0.001). (B) Expression of IL-1β, TRAF6, and IRAK1 mRNA levels during miR-146a overexpression was determined by RT-qPCR. Fold change was calculated by normalizing miR-146a mimic-transfected cells to negative control-transfected cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to negative controls (*p \u003c 0.05). (C) CD4+ T-cells were un-transfected, transfected with a negative control inhibitor or a miR-146a inhibitor. Successful miR-146a inhibition was confirmed by using RT-qPCR. Fold change was calculated by normalizing the transfected cells to non-transfected cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to non-transfected controls (***p \u003c 0.001). (D) Expression of IL-1β, TRAF6, and IRAK1 mRNA levels during miR-146a inhibition was determined by RT-qPCR. Fold change was calculated by normalizing miR-146a inhibitor-transfected cells to negative control-transfected cells. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to negative controls (**p \u003c 0.01). We then used a miR-146a inhibitor and analyzed changes in TRAF6, IRAK1, and IL-1β mRNA expression. Transfection of the inhibitor significantly reduced miR-146a expression (Figure 3C). IL-1β mRNA expression was unchanged, but TRAF6 and IRAK1 mRNA levels were significantly increased (Figure 3D). Taken together, these data confirm IRAK1 and TRAF6 as targets of miR-146a in CD4+ T-cells.","divisions":[{"label":"title","span":{"begin":0,"end":100}},{"label":"p","span":{"begin":101,"end":788}},{"label":"figure","span":{"begin":789,"end":2409}},{"label":"label","span":{"begin":789,"end":797}},{"label":"caption","span":{"begin":799,"end":2409}},{"label":"p","span":{"begin":799,"end":2409}}],"tracks":[{"project":"TEST0","denotations":[{"id":"32117283-174-180-3683916","span":{"begin":275,"end":277},"obj":"[\"19596990\"]"},{"id":"32117283-178-184-3683917","span":{"begin":279,"end":281},"obj":"[\"23685241\"]"},{"id":"32117283-182-188-3683918","span":{"begin":283,"end":285},"obj":"[\"16885212\"]"},{"id":"32117283-186-192-3683919","span":{"begin":287,"end":289},"obj":"[\"24987958\"]"}],"attributes":[{"subj":"32117283-174-180-3683916","pred":"source","obj":"TEST0"},{"subj":"32117283-178-184-3683917","pred":"source","obj":"TEST0"},{"subj":"32117283-182-188-3683918","pred":"source","obj":"TEST0"},{"subj":"32117283-186-192-3683919","pred":"source","obj":"TEST0"}]},{"project":"2_test","denotations":[{"id":"32117283-19596990-35221722","span":{"begin":275,"end":277},"obj":"19596990"},{"id":"32117283-23685241-35221723","span":{"begin":279,"end":281},"obj":"23685241"},{"id":"32117283-16885212-35221724","span":{"begin":283,"end":285},"obj":"16885212"},{"id":"32117283-24987958-35221725","span":{"begin":287,"end":289},"obj":"24987958"}],"attributes":[{"subj":"32117283-19596990-35221722","pred":"source","obj":"2_test"},{"subj":"32117283-23685241-35221723","pred":"source","obj":"2_test"},{"subj":"32117283-16885212-35221724","pred":"source","obj":"2_test"},{"subj":"32117283-24987958-35221725","pred":"source","obj":"2_test"}]},{"project":"MyTest","denotations":[{"id":"32117283-19596990-35221722","span":{"begin":275,"end":277},"obj":"19596990"},{"id":"32117283-23685241-35221723","span":{"begin":279,"end":281},"obj":"23685241"},{"id":"32117283-16885212-35221724","span":{"begin":283,"end":285},"obj":"16885212"},{"id":"32117283-24987958-35221725","span":{"begin":287,"end":289},"obj":"24987958"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"attributes":[{"subj":"32117283-19596990-35221722","pred":"source","obj":"MyTest"},{"subj":"32117283-23685241-35221723","pred":"source","obj":"MyTest"},{"subj":"32117283-16885212-35221724","pred":"source","obj":"MyTest"},{"subj":"32117283-24987958-35221725","pred":"source","obj":"MyTest"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"TEST0","color":"#ec93e6","default":true},{"id":"2_test","color":"#93ecd8"},{"id":"MyTest","color":"#ecbe93"}]}]}}