Transduction with Lentiviral Vectors Altered the Expression Profile of Host MicroRNAs

Abstract

RNA interference (RNAi) is widely used in gene knockdown analysis and as a tool to screen host genes involved in viral infection. Owing to the limitations of transducing cells with synthetic small interfering RNAs (siRNAs), lentiviral short hairpin RNA (shRNA) vectors are more widely used. However, we found that stable transduction with lentiviral shRNA vectors inhibited hepatitis C virus (HCV) propagation in human hepatoma cells. We found by microRNA (miRNA) microarray analysis that this inhibition was induced by the alteration of host miRNA expression. In addition to one miRNA (miR-196b-5p) previously reported to be involved in HCV infection, other miRNAs (miR-216a-5p, -216b-5p, 217, and -30b-5p) were found to influence HCV infection in this study. Further studies suggested that this effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration site of the lentiviral vector might determine the change in miRNA expression. Moreover, the upregulation of JUN contributed to the dysregulation of miR-216a-5p, -216b-5p, and -217 in stably transduced cells. Although the changes in miRNA expression were beneficial for inhibiting HCV infection in our study, this off-target effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy. IMPORTANCE We found that stable transduction with lentiviral shRNA was able to nonspecifically inhibit HCV infection by the dysregulation of host miRNAs. Previous studies showed that the overexpression of shRNAs oversaturated the host miRNA pathways to inhibit HCV infection. In contrast, the miRNA machinery was not affected in our study. Knockout studies suggested that the nonspecific effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration sites in the host genome determined the changes in miRNAs. Stable transduction with lentiviral vectors was able to increase the expression of JUN, which in turn upregulated miR-216a-5p, miR-216b-5p, and miR-217. miR-216a-5p and miR-216b-5p might inhibit HCV by suppressing the host autophagic machinery. Our study suggested a novel nonspecific effect of lentiviral vectors, and this side effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.

Exosomes function as nanoparticles to transfer miR-199a-3p to reverse chemoresistance to cisplatin in hepatocellular carcinoma

“…Decrement of miR-196a strengthens the sensitivity of HCC cells to DDP treatment [ 47 ]. Viral vectors [ 48 ] and liposomal carriers [ 49 ] have been widely applied to transfer certain miRNAs and adjust the expression of them, but unfavorable biocompatibility, non-exact cytotoxicity and low transferring efficacy remain difficult to overcome for delivering miRNAs in the body [ 50 ]. In the present research, mice received intravenous injection of exosomes separated from cell culture medium, thus raising miR-199a-3p expression in tumors and exosomes, which may be a less cytotoxic treatment regimen with better biocompatibility.…”

Identification of key genes and long non-coding RNA associated ceRNA networks in hepatocellular carcinoma

“…miR-216b-5p is an anti-tumor miRNA found in several cancers (He et al, 2019; Ren et al, 2019; Sun et al, 2019b; You et al, 2017). It is believed that miR-216b-5p inhibits HCV by suppressing host autophagy (Huang et al, 2018). Paradoxically, miR-216b-5p represses the expression of UDP-glucuronosyltransferase 2B, a group of enzymes that detoxifies carcinogens by binding to their 3′UTRs (Dluzen et al, 2016).…”

Distinct Subsets of Noncoding RNAs Are Strongly Associated With BMD and Fracture, Studied in Weight-Bearing and Non–Weight-Bearing Human Bone

“…Still, we acknowledge that detection methodologies (microarrays with detection of biotin labeled ncRNAs versus TaqMan assays involving detection of PCR products after cleavage of quenching hybridizing probes) and quantification analyses (eg, different types of sample normalization, software, and thresholds) could have an influence on the results. However, several papers show that in general there is a very good concurrence in studies where miRNA levels was measured by both GeneChip miRNA 4.0 Array and TaqMan PCR analysis …”