Journal: Molecular Therapy. Nucleic Acids

Article Title: microRNA-422a promotes HIV replication and innate immune evasion by targeting MECP2

doi: 10.1016/j.omtn.2026.102844

Figure Lengend Snippet: miR-422a directly targets MECP2 (A) Schematic diagram showing the predicted miR-422a target sites within the MECP2 3′UTR. The predicted target seed sites and their corresponding mutated sequences are highlighted in red. (B) HEK-293T cells were cotransfected with MECP2 3′UTR luciferase reporter vector (wild-type or mutant versions), pRL-TK, and miR-422a mimic or negative control (NC) for 24 h. Cells were then harvested for luciferase analysis. Relative luciferase activity is shown. (C) Unstimulated or stimulated primary CD4+ T cells were transfected with miR-422a mimic or NC for 72 h. Cells were then collected to detect MECP2 mRNA levels by RT-qPCR. (D) Stimulated primary CD4+ T cells were transfected with the indicated concentrations of miR-422a mimic or NC for 72 h. Cells were then collected to detect MECP2 protein levels by western blot. β-actin was used as the endogenous control. (E) Relative quantification of MECP2 protein from the western blot in (D). (F) MECP2 protein expression was analyzed by western blot following the establishment of stable cell lines. (G) MECP2 KO-Jurkat or scramble-Jurkat cells were infected with HIV-1 NL4-3 . The percentage of Gag-positive cells was detected by flow cytometry at the indicated time point. (H and I) Stimulated primary CD4+ T cells were nucleofected with Cas9-RNP targeting MECP2 for 24 h, then infected with HIV-1 NL4-3 for 6 days. Cells and supernatants were collected for Gag detection and p24 measurements by flow cytometry (H) and ELISA (I), respectively. (J) MECP2 KO-Jurkat or scramble-Jurkat cells were transfected with miR-422a mimic or its NC for 24 h, then infected with HIV-1 NL4-3 (or left uninfected). Six days after infection, cells were collected, fixed, and permeabilized, then stained with RD-fluorescent Gag antibody to detect Gag expression by flow cytometry. The percentage of Gag-positive cells was determined by flow cytometry. Each dot represents data from one donor. Data are representative of the results of three independent experiments ( n = 3 biologically independent samples, mean ± SEM). Statistical significance was analyzed by unpaired or paired student t tests. p ≤ 0.05 [∗], p ≤ 0.01 [∗∗], p ≤ 0.001 [∗∗∗], p ≤ 0.0001 [∗∗∗∗].

Article Snippet: HIV-1 NL4-3 viral stocks were generated by transfection of proviral DNA (BEI, #ARP-114) into HEK-293T cells via polyethylenimine (PEI, Polysciences, #26913-06-4) as described previously.

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Negative Control, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Control, Quantitative Proteomics, Expressing, Stable Transfection, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining