circHtra1/miR-3960/GRB10 Axis Promotes Neuronal Loss and Immune Deficiency in Traumatic Brain Injury

Figure 3

circHtra1 binds to miR-3960 and GRB10 in neurons. (a) Based on the interactome prediction, four miRNAs were predicted to bind to circHtra1. (b) The complementary site between circHtra1 and miR-3960 is shown. (c) Primary cultured neurons were cotransfected with each miRNA mimic and the wild-type circHtra1 reporter or mutant circHtra1 reporter. The -axis shows the luciferase intensity. The scrambled mimic was used as a control. (d) RNA FISH for circHtra1 detection in primary cultured neurons. Nuclei were stained with DAPI (blue), and circHtra1 appears green. circHtra1 was dominantly located in the cytoplasm. Scale bars represent 20 mm. (e, f) circHtra1 is predicted to interact with AGO2 (a miRNA binding protein), as supported by a RIP assay. (g, h) miR-3960 could bind to Htra1 in three positions, as predicted by miRanda. (i) After primary cultured neurons were transfected with NC, miR-3960, miR mutant, circHtra1, circHtra1+miR-3960, and circHtra1+miR mutant, mRNA levels of htra1 were assessed in primary cultured neurons , ; , ; , ; and , , respectively. (j) After transfection with si-NC, si-circHtra1, or si-circHtra1+miR-3960 antagonist, si-circHtra1+Htra1, miR-3960 antagonist and Htra1, CCK-8 levels were assessed. ^{, #, &}.

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