pLVX-IRES-mCherry

Case Study Human monocyte cell line U937 and human alveolar macrophages were used as in vitro models to explore the role of miR88 and miR99 in chronic abnormal activation of the body caused by human immunodeficiency virus (HIV). The functions and underlying mechanisms of miR88 and miR99 were investigated by real-time quantitative polymerase chain reaction, Transwell, and chromatin immunoprecipitation (ChIP) analyses. HIV-1-infected cells release miR88 and miR99 into the extracellular space through exosomes, and miR88 and miR99 promote the release of tumor necrosis factor α (TNFα), interleukin (IL)-6, and IL-12 by activating inflammatory factors. HIV-derived microRNAs miR88 and miR99 exert these functions by binding to TLR8 and stimulating macrophages to release pro-inflammatory factors such as TNFα, IL-6, and IL-12. These factors may be related to chronic abnormal immune activation caused by HIV infection.

In this study, the fragments of miR88, miR99 and control miRTAR were inserted into the pLVX-IRES-mCherry vector to construct pLVX-IRES-mCherry miR88, pLVX-IRES-mCherry miR99 and pLVX-IRES-mCherry miRRTAR vectors respectively. Lentiviruses obtained from these expression vectors were used to infect HEK293 cells. Fluorescence microscopy showed clear red fluorescence in HEK293, indicating that the lentivirus efficiently infected the cells (Figure 1a). The miRNA from lentivirus-infected HEK293 cells was extracted and reverse transcribed. RT-qPCR was used to determine the expression levels of miR88 and miR99. The results showed that the levels of miR88 and miR99 in lentivirus-infected HEK293 cells were significantly higher than those in the control, indicating that miR88 and miR99 were highly expressed in HEK293 cells (Figure 1b).

Figure 1. (a) Fluorescence microscopy showing pLVX-IRES-mCherry miR88, pLVX-IRES-mCherry miR99, and control pLVX-IRES-mCherry miRTAR expressed in HEK293 cells. (b) RT-qPCR assay showing lentiviral expression of miR88 and miR99 in HEK293 cells. (Zhao, Hui, et al. 2019)