Figure 6. miR-22 downregulates protein levels of MAPK14/p38 and tp53inp1.

A) Luciferase constructs expressing the Renilla luciferase gene under the control of the MAPK14/p38 or Tp53inp1 3′ UTRs were co-transfected in HEK293T cells with either an empty plasmid (pcDNA3.1), plasmid expressing a control miR-153 not predicted to target the 3′ UTR or a plasmid expressing miR-22. The intensity of Renilla luciferase luminescence was normalized to firefly luciferase luminescence, which was not under the control of a 3′ UTR. Graphs represent average values ± SEM, n = 6. a indicates p-value <0.01, by unpaired t test compared to the control sample transfected with the empty vector pcDNA3.1. b indicates p<0.01, by unpaired t test compared to the control sample transfected with a control microRNA, miR-146a or -153, not predicted to bind the 3′ UTR. B) Primary cortical neurons were infected with lentivirus expressing miR-22 and proteins were harvested 3 weeks post-infection. MAPK14/p38 protein levels in miR-22 overexpressing cells were compared to MAPK14/p38 protein levels in the non-infected control cells (CTRL). Protein levels were normalized to β-3 tubulin, which is not a predicted target of miR-22 and whose expression did not change with miR-22 treatment. Graphs represent quantification of the Western blot, error bars represent SEM, ** represents p-value <0.01, by unpaired t-test. CTRL – non-infected cells. C) and D) Primary striatal neurons were infected on DIV1 with lentiviral vectors encoding WT (Htt18Q) or mutant (Htt82Q) Htt171 fragments under the control of the TRE promoter and co-infected on DIV4 with vector encoding miR-22 and 2.5 weeks post-infection proteins were harvested. Protein levels were normalized to β-3 tubulin, which is not a predicted target of miR-22. Graphs represent quantification of the signals from the Western blot, error bars represent SEM. * represents p-value <0.05, ** represents p-value <0.01, by unpaired t-test.