Inhibition of Porcine Endogenous Retrovirus by Multi-Targeting Micro RNA Against Long Terminal Region.

ABSTRACT

BACKGROUND: There might be much benefit in xenotransplantation, however, the risk of infections across species barriers remains, especially porcine endogenous retrovirus (PERV). To date, many attempts have been made to knock down active PERVs by inhibitory RNA (RNAi) and micro RNA (miRNA), which target different genes of PERV. There are a few studies that have explored whether targeting promoter regions of PERV could exert an inhibition effect. METHODS: miRNAs were automatically selected based on an online program BLOCK-iT RNAi Designer. The inhibition efficiency between miRNAs was compared based on their inhibition of different PERV genes long terminal repeats (LTR), gag, and pol. Both relative quantitative real-time polymerase chain reaction (PCR) and C-type reverse transcriptase activity were performed. RESULTS: The results demonstrated that miRNA targeting the LTR region degraded the target sequence, and simultaneously inhibited the mRNA expression of both gag and pol genes of PERV. The LTR1, LTR2, and dual LTR1 + LTR2 miRNA inhibited 76.2%, 22%, and 76.8% of gag gene expression, respectively. Similarly, the miRNA was found to knock down the pol gene expression of 69.8%, 25.5%, and 77.7% for single targeting miRNA (LTR1 and LTR2) and multi-targeting miRNA (LTR1 + LTR2), respectively. A stable PK15 clone constitutively expressed dual LTR1 + LTR2 miRNA and exhibited higher inhibitory up to 82.8% and 92.7% of the expressions of the gag and pol genes, respectively. Also, the result of co-cultivation of dual LTR1 + LTR2 miRNA transfected PK15 cell with a human cell line inhibited expression of LTR, gag, and pol genes of PERV. CONCLUSIONS: In conclusion, this study suggested that the LTR might be an alternative target for gene silencing of PERV, and that multi-targeting miRNA had better inhibitory effect than single- targeting miRNA. In an in vitro model, the presence of miRNA was able to reduce PERV infectivity in a human cell line.