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  • br Introduction Human endogenous retroviruses HERVs are gene

    2019-05-23


    Introduction Human endogenous retroviruses (HERVs) are genetic remnants of ancient retroviral infections of the germ line produced during primate evolution which are now transmitted vertically. To date, approximately 8% of the human genome is composed of such retroviral sequences [1]. Most HERVs are dysfunctional due to numerous mutations and deletions. However, those belonging to HERV-K family, contain sequences which are likely to be transcribed. Many of these HERVs are transcribed and translated under normal physiological conditions. Nevertheless, reactivation of HERVs has frequently been observed in a variety of human tumors suggesting their potential to contribute to malignant progression [2]. Specifically during hemato-oncological processes, several studies have reported the presence of rxr receptor against HERV-K, overexpression of HERV genes and also the presence of retroviral particles in primary leukemia cells [2]. In addition, np9, a small regulatory gene encoded by HERV-K, has a role as a potent viral oncogene and as a critical molecular switch of multiple signaling pathways regulating the growth of certain human myeloid and lymphoblastic leukemia cells [3]. CLL is the most common form of leukemia in Western countries and mainly affects elderly individuals. It follows an extremely variable course, with survival ranging from months to decades. Available treatments often induce disease remission, but almost all patients will relapse and there is a consensus that CLL remains incurable. To date, both an unmutated (UM) profile of immunoglobulin (Ig) VH genes as well as the presence of genetic lesions at chromosome 17p13, or at 11q23 constitute poor prognosis indicators [4]. Several studies have begun to shed light on the nature of genetic predisposition of CLL but the basis of this disorder remains unknown [4,5]. Studies that screened for the presence of a virus expressed at the RNA level in human CLL, by using massive sequencing technology, gave no evidence of a putative exogenous viral candidate as a cause for this disease [6]. In order to gain insight into the role of endogenous retrovirus in leukemogenesis, we studied np9 and gag gene expression of HERV-K in Chronic Lymphocytic Leukemia (CLL) patients compared to healthy donors. The results revealed that 70% of CLL patients express np9 gene at least 5 times higher than healthy donors, and 33% express gag gene twice as high as normal donors. These findings suggest a significant relationship between CLL disease and HERV-K np9 (and possibly gag) expression.
    Materials and methods The peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll-Hypaque (GE Healthcare). RNA was isolated from 1 to 5×106 cells using the Trizol RNA Isolation Protocol. To minimize genomic DNA contamination each RNA sample was treated with DNA-free™ Kit (Ambion, USA). cDNA synthesis was performed as described previously [7]. Negative controls in which reverse transcriptase was omitted were performed to confirm the exclusion of residual genomic DNA contamination. For gene expression analyses of np9 and gag genes, we used the KAPA SYBR FAST qPCR Kit Master Mix (2×) Universal (Kapa Biosystems) and a Corbett Rotor-Gene 6000. HERVs-K primer sequences of np9 and gag were previously described [2,8]. The cellular c-myc expression level in CLL patients was also assessed with respect to healthy donors. The results of HERV-K and c-myc expression were normalized to the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcription using the following primers: forward: 5′- GGTGCTGAGTATGTCGTGGA-3′ and reverse: 5′-ATGCCAGTGAGCTTCCCGTT-3′. PCR cycling conditions for np9, c-myc and GAPDH amplification were: 3min at 95°C and 40 cycles at 95°C for 3s, 58°C for 20s and 72°C for 20s. For HERV-K gag specific amplification, cycling conditions were: 95°C for 10min, 40 cycles at 95°C for 15s and 60°C for 1min. The threshold cycle value (Ct), computed for each of these genes as the average of 2 determinations was used to measure the amount of PCR product. The mean ΔCt was calculated by subtracting the mean Ct of duplicated np9 and gag measurements from the mean Ct of duplicated GAPDH measurements. The ΔΔCt values were calculated with the mean ΔCt of the 6 healthy donors. The expression factor difference was calculated by the formula 2−ΔΔCt. Thus, the calculated relative expression provides data on how much np9 and gag gene expression differs from that found in healthy donors.