Hek293t cells

HEK293T cells were purchased from BeNa Culture Collection, China. Cells were cultured in a Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin solution (Hyclone Laboratories, Logan, UT, USA).
The NiVpv and HeVpv used in the study were kindly gifted from Dr. Youchun Wang (Division of HIV/AIDS and Sexually Transmitted Virus Vaccines, National Institutes for Food and Drug Control (NIFDC), Beijing, China).

FOXO3 wild-type and mutant (FOXO3-wt/mut) primers were designed at the predicted binding sites of the FOXO3 3′-UTR to miR-200a-3p and further synthesized by RT-qPCR. Subsequently, the above FOXO3 wild-type fragment of interest and mutant fragment were ligated separately with pMIR-REPORTTM vector using T4 ligase. The luciferase activity was assessed using a Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA). Using Lipofectamine 2000, HEK293T cells (BeNa Culture Collection, Shanghai, China) were transfected with miR-200a-3p mimics/NC and FOXO3-WT/MUT vector plasmids at room temperature. Then, the above mixture was detected luciferase activity.

The PCAT1 and TRL4 sequences containing the predicted potential miR‐145‐5p binding sites were amplified. The PCAT1‐WT and TRL4‐WT plasmids were constructed using a PCR method. PCAT1‐MUT and TLR4‐MUT were generated by site‐directed mutagenesis, replacing the first six ribonucleotides of the miR‐145‐5p complementary sequence. HEK293T cells (BeNa Culture Collection, China) were grown in 24‐well plates and cotransfected with miR‐145‐5p mimics and PCAT1‐WT, miR‐145‐5p mimics and PCAT1‐MUT, control mimics and PCAT1‐WT, control mimics and PCAT1‐MUT, miR‐145‐5p mimics and TLR4‐WT, miR‐145‐5p mimics and TLR4‐MUT, control mimics and TLR4‐WT, control mimics and TLR4‐MUT. The media was replaced after 6 hours; 24 hours after cotransfection, the Dual‐Luciferase Reporter Gene Test Kit (Promega, Fitchburg, WI, USA) was used to detect the luciferase activities in different groups. The firefly and the renal luciferase reagent were added to detect the luciferase activity of each group.