MicroRNA-181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2
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ABSTRACT:
SUBMITTER: Ms. Gisela Gordino
PROVIDER: S-SCDT-EMBOR-2020-52234V1 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:γδ T cells are a conserved population of lymphocytes that contributes to anti-tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL-2 or IL-15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA-181a as a key modulator of human γδ T cell differentiation. We observe that miR-181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR-181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR-181a overexpression restricts their levels of NKG2D and TNF-α. Upon in silico analysis, we identify two miR-181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR-181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next-generation immunotherapies.
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Project description:Activin, a member of the transforming growth factor-? superfamily, promotes the growth of preantral follicles and the proliferation of granulosa cells. However, little is known about the role of microRNAs in activin-mediated granulosa cell proliferation. Here, we reported a dose- and time-dependent suppression of microRNA-181a (miR-181a) expression by activin A in mouse granulosa cells (mGC). Overexpression of miR-181a in mGC suppressed activin receptor IIA (acvr2a) expression by binding to its 3'-untranslated region (3'-UTR), resulting in down-regulation of cyclin D2 and proliferating cell nuclear antigen expression, leading to inhibition of the cellular proliferation, while overexpression of acvr2a attenuated the suppressive effect of miR-181a on mGC proliferation. Consistent with the inhibition of acvr2a expression, miR-181a prevented the phosphorylation of the activin intracellular signal transducer, mothers against decapentaplegic homolog 2 (Smad2), leading to the inactivation of activin signaling pathway. Interestingly, we found that miR-181a expression decreased in ovaries of mice at age of 8, 12, and 21 days, as compared with that in ovaries of 3-day old mice, and its level was reduced in preantral and antral follicles of mice compared with that in primary ones. Moreover, the level of miR-181a in the blood of patients with premature ovarian failure was significantly increased compared with that in normal females. This study identifies an interplay between miR-181a and acvr2a, and reveals an important role of miR-181a in regulating granulosa cell proliferation and ovarian follicle development.
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Project description:PurposeHuman lens epithelial cell (HLEC) apoptosis is a common pathogenic mechanism in age-related cataracts (ARC). While the function of microRNAs (miRNAs) in the eye is beginning to be explored using miRNA expression array, the role of miR-34a in regulating HLEC apoptosis remains unknown and requires further investigation.MethodsQuantitative reverse-transcript polymerase chain reaction (RT-PCR) was used to determine the expression level of miR-34a in cataractous and control samples. MiR-34a mimics and small interfering RNAs were transfected into SRA01/04. Cell apoptosis and oxidative stress were assessed by flow cytometry. The Dual-Luciferase Reporter Assay System was used to confirm whether miR-34a bound to the 3'-UTR of the target gene and blocked its activity. The potential roles of the identified target genes in apoptosis and mitochondria dysfunction were also evaluated.ResultsThe expression of miR-34a increased in lens epithelial samples of ARC compared with the transparent group (cataract 2.41±0.81 vs. control 1.20±0.44, P=0.005). In cultured SRA01/04, miR-34a increased reactive oxygen species production and induced apoptosis (early apoptosis: 45.55%±5.96% vs. 15.85%±4.93%, P<0.01; late apoptosis: 6.10%±2.67% vs. 0.95%±0.42%, P<0.01). Overexpression of miR-34a promoted mitochondria-mediated apoptosis through activation of caspase-9, disruption of the mitochondrial membrane potential, blocking of mitochondrial energy metabolism and enhancement of cytochrome C release. Furthermore, Notch1 and Notch2 were confirmed as putative targets of miR-34a, but only Notch2 was verified as the effector that triggered mitochondria-mediated apoptosis.ConclusionMicroRNA-34a is increased in the cataractous lens and triggers mitochondria-mediated apoptosis and oxidative stress by suppressing Notch2.
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