Chung H-C, Nguyen V-G, Oh W-T, Huynh T-M-L, Moon H-J, Lee J-H, Kim H-K, Park S-J, Park B-K
Department of Veterinary Medicine Virology Laboratory, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul, Korea.
Department of Veterinary Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi, Vietnam.
Transplant Proc. 2017 Nov;49(9):2225-2232. doi: 10.1016/j.transproceed.2017.09.026.
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.
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.
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.
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.
异种移植可能有诸多益处，然而，跨物种屏障的感染风险依然存在，尤其是猪内源性逆转录病毒（PERV）。迄今为止，人们已进行了诸多尝试，通过靶向PERV不同基因的抑制性RNA（RNAi）和微小RNA（miRNA）来敲除活性PERV。有一些研究探讨了靶向PERV启动子区域是否能发挥抑制作用。
基于在线程序BLOCK-iT RNAi Designer自动选择miRNA。根据miRNA对PERV不同基因（长末端重复序列（LTR）、gag和pol）的抑制作用比较其抑制效率。同时进行相对定量实时聚合酶链反应（PCR）和C型逆转录酶活性检测。
结果表明，靶向LTR区域的miRNA降解了靶序列，同时抑制了PERV的gag和pol基因的mRNA表达。LTR1、LTR2以及双LTR1 + LTR2 miRNA分别抑制了76.2%、22%和76.8%的gag基因表达。同样，对于单靶向miRNA（LTR1和LTR2）和多靶向miRNA（LTR1 + LTR2），发现其分别敲低了69.8%、25.5%和77.7%的pol基因表达。一个稳定的PK15克隆持续表达双LTR1 + LTR2 miRNA，对gag和pol基因表达的抑制率分别高达82.8%和92.7%。此外，双LTR1 + LTR2 miRNA转染的PK15细胞与人细胞系共培养的结果抑制了PERV的LTR、gag和pol基因表达。
总之，本研究表明LTR可能是PERV基因沉默的一个替代靶点，且多靶向miRNA比单靶向miRNA具有更好的抑制效果。在体外模型中，miRNA的存在能够降低人细胞系中PERV的感染性。
