In this study, we firstly detected overexpression of DHX15, a member of the DEAH-box RNA helicase family in BL patients. Then we explored the effect of DHX15 gene knockdown on BL both in vivo and in vitro. In the meantime, it is the first time to study the relationship between DHX15 and EBV. In accordance with our previous study, DHX15 gene knockdown significantly induced cell apoptosis and G2/M arrest, inhibited cell proliferation and growth of subcutaneous transplanted tumors in BL cells.
The transcription factor NF-κB is a key player in the inflammation, cancer development and progression (21–22). Aberrant NF-κB activation is a characteristic of various human malignances (21, 23). Activated NF-κB can stimulate cell proliferation, prevent apoptosis, and promote tumor angiogenesis, epithelial-to-mesenchymal transition, invasiveness, as well as metastasis (24–25). Previous studies have found that constitutive NF-κB activation is involved in the pathogenesis of BL and NF-κB seems to be required for the constitutive activation of c-myc and for of the upregulation of c-myc (26–28). In our experiments, we found that DHX15 gene knockdown inhibited the canonical NF-κB signaling transduction possibly via the following aspects: (1) inhibiting the synthesis and phosphorylation of p65/RelA protein, (2) inhibiting IκB kinase (IKK) to reduce the phosphorylation and proteasome-mediated degradation of IκBα, (3) inhibiting the activation of p105/NF-κB1 protein. Finally, DHX15 gene knockdown inhibited the homodimer or heterodimer formation of p65 with p50, leading to reduced translocation into the nucleus and subsequent inhibition of the transcription of target genes. We also found that there was no significant change in p100/NF-κB2 protein level, a member of the non-canonical NF-κB signaling pathway. However, whether DHX15 gene affects the non-canonical NF-κB signaling pathway is unclear and requires further study.
Several studies reported that DHX15 activates p38 MAPK and NF-κB signaling pathway during anti-virus infection (11–12). In our study, we found that the activity of NF-κB signaling pathway and downstream targets, including Bcl-2, Bcl-xl, survivin, are downregulated after DHX15 gene knockdown, indicating that DHX15 gene knockdown may affect the function of mitochondria via Bcl-2 family members. Subsequent studies confirmed the hypothesis that mitochondrial membrane potential was decreased and cytochrome C was released from mitochondria to cytoplasm, which activated the mitochondrial apoptotic pathway leading to Raji cell apoptosis. The above results suggested that mitochondria and Caspase cascade are involved in apoptosis after DHX15 gene knockdown in Raji cells. What’ more, we also found that the apoptosis rate of the Z-VAD-fmk pretreatment group is significantly higher than that of control group. The reasons we speculate are as follows: First, there may be other pathways that participate in cell apoptosis besides Caspase cascade, such as apoptosis inducing factor (AIF) signaling pathway (29–30), Bcl-2 inhibitor of transcription 1 (Bit1) signaling pathway (31). Second, the combination of the inhibitor and its substrate has a saturation effect, and Z-VAD-fmk cannot inhibit Caspase activity completely, which is also the cause of the higher cell apoptosis rate in the Z-VAD-fmk pretreatment group than that in control group. In addition, whether exogenous apoptotic pathways Caspase 8 or Caspase 10 participates in apoptosis needs to be further studied.
EBV, belonging to a family of human herpesviruses, contributes to life-long latent infection in B lymphocytes after primary infection (32). The virus is associated with various human malignancies, such as BL, nasopharyngeal carcinoma and Hodgkin lymphoma, which could be detected in almost all samples of endemic BL patients (33). In most BL patients, EBV shows type Ⅰ latent infection with expression of EBNA-1, EBER-1, EBER-2 and BART microRNAs (34). Previous studies had confirmed that EBV latent infection products EBNA-1, EBER-1 and EBER-2 were closely related to the occurrence and development of BL, and they could promote BL cell proliferation and inhibit BL cell apoptosis (6–9). In our study, we found that the expression of EBNA-1, EBER-1, EBER-2 and RNA polymerase Ⅲ transcripts 5S RNA, 7SL RNA and tRNAtyr are downregulated after DHX15 gene knockdown, which indicated that DHX15 may participate in the regulation of the expression of EBER-1 and EBER-2 via RNA polymerase Ⅲ. However, there are no direct approaches to detect the activity of RNA polymerase Ⅲ. In this experiment, we indirectly estimated the activity of RNA polymerase Ⅲ by the level of specific transcripts of RNA polymerase Ⅲ. Therefore, the methodology of direct detection of RNA polymerase Ⅲ activity needs to be further evaluated. In a word, DHX15 may participate in the occurrence and development of BL via regulation of the expression of the above EBV latent infection products.
Moreover, we found that DHX15 gene knockdown inhibits tumor growth and downregulates EBNA-1, EBER-1, EBER-2 in vivo. The tumor volume and weight of KD group are significantly smaller and lighter than those of the CON and NC group. Our results demonstrated that DHX15 could promoted tumor growth and upregulated EBV latent infection products.
In this study, we revealed that, compared with patients with low DHX15 expression, the overall survival time and progression-free survival time of patients with high DHX15 expression tended to shorten, but there was no significant difference. The reasons we speculate are as follows: First, the number of patients was relatively small because of low incidence rate. Second, the observation time was insufficient. Third, the patients in the group had a long-time span with different treatment options and compliance, for example, in the early years, patients with poor efficacy mostly used the CHOP chemotherapy regimen.
However, there are still some limitations in our study. For example, we did not determine whether DHX15 can also promote the expression of EBV latent infection products in other EBV-associated tumors or whether DHX15 can be used as a target for treatment of latent EBV infection. Further studies are required to explore the underlying mechanisms.