Expression profile of Tripartite motif family (TRIM) in human fibroblasts (NHDF) infected with porcine endogenous retrovirus (PERV)

Abstract

Background: Understanding the interactions between the microRNA (miRNA) and mRNA of genes encoding restriction factors (RFs) can lead to the development of new antiretroviral strategies aimed at providing the resistance and reducing susceptibility of human cells to potential PERV infection. Among RFs TRIM family play an important role in shaping the immune response during various stages of infection. The aim of the study was to evaluate in vitro the transcriptional profile of TRIM family genes and identify complementary miRNAs in NHDF cells infected with PERVs and induced by gram negative lipopolysacharide (LPS).

Methods: Human dermal fibroblasts cells were cultured in four separate conditions- 2 monocultures: control (N), treated with LPS (NL) and 2 co-cultures with porcine PK15 cells: without LPS (NP) and treated with LPS (NLP). Bacterial LPS was used in this study as an inducer of inflammation in NHDF cells. After extraction of DNA and RNA from cells PERV infection was confirmed in co-cultures by qPCR and RTqPCR. RNA extracts served as a matrix for HGU 133A 2.0 and miRNA 2.0 microarrays to evaluate the expression profile of the selected TRIM family genes and miRNAs adequately. TRIM 2, 14, 22, and 28 were selected for the validation of HGU 133A 2.0 results. Statistical analyses were performed with the use of REST^© 2009 and Genespring GX 13.0. Transcriptome Analysis Console 4.0 program (Affymetrix) was used to identify miRNAs that differentiate the studied genes in all conditions.

Results: Porcine endogenous retrovirus infection at DNA and RNA level was confirmed in NHDF cells in each of the tested groups (NP and NLP). Contamination was excluded in N and NL groups. Based on the analysis of HGU 133A 2.0 results 93 mRNA IDs of TRIM family genes differentiating analyzed conditions were selected P < .05. HGU 133A 2.0 mRNA fluorescence profile was confirmed with RTqPCR of TRIM2, TRIM14, TRIM22 and TRIM28 P < .05. TRIM14 down regulation was specific only in PERV monoinfection (group NP). In miRNA 2.0 microarray 346 miRNAs were identified as differentiating NHDF cells in all analyzed conditions, P < .05. According to the analysis with mirTAR platform and Microrna.org datatbase none of the selected miRNAs had the potential to regulate the selected genes of the TRIM family.

Conclusion: Porcine endogenous retrovirus infection of NHDF cells is accompanied by TRIM14 down regulation suggesting TRIM14 as a possible marker of retroviral infection. None of the selected miRNAs had statistically significant potential to regulate the expression of the selected TRIMs in any of the analyzed conditions.

Keywords: PERVs; TRIM; miRNA; restriction factors.