Abstract: Objective To construct a recombinant lentiviral vector overexpressing leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), establish a stable LILRA2-overexpressing human myeloid leukemia mononuclear cell strain(THP-1), and preliminarily investigate the impact of LILRA2 on the expression of pro-inflammatory cytokines. Methods The pLV-SFFV-MCS-EF1-ZsGreen1-T2A-Puro backbone vector was double-digested with BamH Ⅰ/Xba Ⅰ to construct the LILRA2-overexpressing recombinant lentiviral vector. After lentiviral packaging, concentration, and titer determination, the optimal multiplicity of infection (MOI = 30) and puromycin screening concentration (2 μg/mL) for THP-1 cells were determined. Following lentiviral infection, puromycin selection was performed to obtain stable transfectants. LILRA2 expression was verified by RT-qPCR and Western blot. TNF-α and IL-1β expression was determined by RT-qPCR. Results Sequencing confirmed the successful construction of the LILRA2-overexpressing recombinant lentiviral vector. The fluorescence-positive rate in stably transfected THP-1 cells exceeded 90%. LILRA2 mRNA (P＜0.001) and protein (P＜0.05) expression levels in the THP-1-LILRA2 group were significantly higher than those in the THP-1-NC negative control group. TNF-α (P＜0.05) and IL-1β (P＜0.05) mRNA levels in the THP-1-LILRA2 group were significantly higher than those in the THP-1-NC negative control group. Conclusions The LILRA2-overexpressing lentiviral vector was successfully constructed and a stable LILRA2-overexpressing THP-1 cell strain was established. LILRA2 overexpression upregulates the expression of pro-inflammatory cytokines in monocytes.

Key words: leukocyte immunoglobulin like receptor subfamily A member 2(LILRA2), THP-1 cells, lentiviral vector, pro-inflammatory cytokines