Abstract
Lymphomas are solid-type tumors containing lymphoid cells. Some of latent herpesvirus infections established in B and/or T-lymphocytes could result in the formation of lymphomas. Marek's disease virus serotype 1 (MDV-1) is an avian herpes virus causing to lymphoproliferative tumors in birds, known as Marek’s disease (MD). MD has often been used as an ideal biological model for studying the pathogenesis of lymphoma diseases caused by viruses. Therefore, we used it as a research subject to study the effect of miRNA sponges on its tumorigenicity, and to develop the theoretical basis for a new anti-tumor small molecule. The miRNA sponges designed in this study specifically bind to and degrade the miRNAs of meq gene cluster of MDV-1, including miR-M2-3p, miR-M3-5p, miR-M5-3p, miR-M9-5p and miR-M12-3p.qPCR results showed that the knockdown efficiency was 85.03%, 74.97%, 47.06%, 75.33% and 62.55%, respectively. EDU staining and CCK-8 results showed that miRNA sponges inhibited the proliferation of MDV-1 transformed MSB-1 cells in vitro, and the proliferation rate of miRNA sponges-treated cells was about 50% of the control group. DAPI staining and Annxin V-FITC/PI double staining showed that miRNA sponges induced apoptosis in MSB-1 cells, and the apoptotic rate was increased by about 27.87% compared with the control group. The results of transwell showed that miRNA sponges could inhibit the invasion of MSB-1 cells in vitro, and the inhibitory rate was about 64.52%. The soft agar assay showed that miRNA sponges could inhibit the tumorigenic ability of MSB-1 cells in vitro, and the inhibitory rate was about 66.44%.The 60-days animal study showed that miRNA sponges could alleviate the growth inhibition of MSB-1 cells (about 14.78%) and reduce the mortality (about 16.00%). In addition, the tumor formation rate was 0 (8–12% in the control group).This study suggests that miRNA sponges can serve as an effective anti-tumor small molecule for the tumors caused by herpesvirus, with potential clinical implications.
Highlights
12% of global human cancers are reportedly caused by viral infections [1]
DAPI staining and Annxin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) double staining showed that miRNA sponges induced apoptosis in MSB-1 cells, and the apoptotic rate was increased by about 27.87% compared with the control group
We performed the transwell assay, and the results show that (Figure 5A, 5B) invasion of MSB-1 cells expressing miRNA sponges has been decreased compared to the control group, which reveals that miRNA sponges can inhibit the invasion of Marek’s disease virustransformed MSB-1 cells
Summary
12% of global human cancers are reportedly caused by viral infections [1]. Marek’s disease (MD) is an avian solid-type lymphoproliferative tumor mainly caused by Marek’s disease virus serotype 1 (MDV-1) This pathogen can rapidly develop lymphoma in a variety of visceral tissues and cause cell infiltration in peripheral nerves, leading to limb (wing) paralysis [5]. In the year 2007, Phillip Sharp and his colleagues have developed an efficient method for long-term suppression of miRNA function This technique is called “miRNA sponges”, which directly “adsorb” miRNAs so that miRNA molecules cannot further bind to their native mRNA targets [17]. The in vitro studies showed that the designed miRNA sponges bind to target miRNA molecules within host cells, and caused loss function of pathogenic MDV-1 miRNAs. Animal studies confirmed the effective roles of miRNA sponge in interfering with pathogenicity and tumorigenicity of MDV-1
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