Department of Microbiology, Faculty of Medicine in Zabrze, University of Technology in Katowice, Katowice, Poland.
Department of Molecular Biology, Faculty of Pharmaceutical Sciences in Sosnowiec Medical University of Silesia, Katowice, Poland.
Xenotransplantation. 2021 Jan;28(1):e12650. doi: 10.1111/xen.12650. Epub 2020 Oct 9.
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).
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.
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.
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.
了解编码限制因子 (RF) 的基因的 miRNA 和 mRNA 之间的相互作用，可以开发新的抗逆转录病毒策略，旨在提高人类细胞对潜在 PERV 感染的抵抗力和降低易感性。在 RF 中，TRIM 家族在感染的各个阶段的免疫反应中发挥重要作用。本研究的目的是评估 NHDF 细胞中 TRIM 家族基因的转录谱，并在感染 PERV 并被革兰氏阴性脂多糖 (LPS) 诱导的 NHDF 细胞中鉴定互补 miRNA。
将人真皮成纤维细胞在 4 种不同条件下进行培养：2 种单核培养物：对照 (N) 和 LPS 处理 (NL) 以及 2 种与猪 PK15 细胞的共培养物：无 LPS (NP) 和 LPS 处理 (NLP)。本研究中使用细菌 LPS 作为 NHDF 细胞中炎症的诱导剂。从细胞中提取 DNA 和 RNA 后，通过 qPCR 和 RTqPCR 确认共培养物中的 PERV 感染。RNA 提取物用作 HGU 133A 2.0 和 miRNA 2.0 微阵列的基质，以充分评估所选 TRIM 家族基因和 miRNA 的表达谱。选择 TRIM 2、14、22 和 28 用于验证 HGU 133A 2.0 结果。使用 REST 2009 和 Genespring GX 13.0 进行统计分析。使用 Transcriptome Analysis Console 4.0 程序 (Affymetrix) 来识别所有条件下区分研究基因的 miRNA。
在每个测试组（NP 和 NLP）的 NHDF 细胞中均证实了猪内源性逆转录病毒在 DNA 和 RNA 水平上的感染。排除了 N 和 NL 组的污染。基于 HGU 133A 2.0 结果分析，选择了 93 个区分分析条件的 TRIM 家族基因的 mRNA ID，P<.05。使用 RTqPCR 验证了 HGU 133A 2.0 mRNA 荧光谱的 TRIM2、TRIM14、TRIM22 和 TRIM28，P<.05。TRIM14 的下调仅在 PERV 单感染（NP 组）中是特异性的。在 miRNA 2.0 微阵列中，鉴定出 346 个 miRNA 作为所有分析条件下区分 NHDF 细胞的差异，P<.05。根据 mirTAR 平台和 Microrna.org 数据库的分析，所选 miRNA 中没有一个具有调节所选 TRIM 家族基因表达的潜力。
猪内源性逆转录病毒感染 NHDF 细胞伴随着 TRIM14 的下调，表明 TRIM14 可能是逆转录病毒感染的一个可能标志物。在任何分析条件下，所选 miRNA 都没有统计学上显著调节所选 TRIM 表达的潜力。
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