Design of miRNA sponges for MDV-1 as a therapeutic strategy against lymphomas

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.

Keywords: MSB-1 cell; marek’s disease virus 1; meq miRNA cluster; miRNA sponge; tumorigenicity.

Figure 1. Stable expression of miRNA sponge for meq-cluster miRNAs by lentiviral vector

(A) Lentiviral vectors were used to stably express miRNA sponge targeting meq-cluster miRNAs, in which CMV promoter drives the expression of EGFP. (B) Schematic diagram of miRNA sponge. (C) Sequences of the targeted miRNA and the corresponding miRNA sponge.

Figure 2. MiRNA sponge degraded MDV-1 meq miRNA cluster in MSB-1 cells

(A) MSB-1 cells were infected with a miRNA sponge lentivirus or a miRNA sponge-NC expression virus (not bound to target miRNA). (B) The expression level of target miRNAs M2-3p, M3-5p, M5-3p, M9-5p, and M12-3p was measured in stable cell lines without infection, with miRNA sponge infection or miRNA sponge-NC infection. (C) mRNA levels of IL-18, smad2, and meq were measured in stable cell lines without infection, with miRNA sponge infection or miRNA sponge-NC infection. ^*P < 0.05.

Figure 3. Effect of expression of MDV-1 miRNA sponge on proliferation of MSB-1 cells

(A) miRNA sponge cells and control cells were incubated with EDU. After 24 h, EDU staining and DNA staining were used to observe the staining under fluorescence microscope. (B) The cells which were cultured in the incubator for 4 h were determined by OD value at 0 h. Then we determined OD at 24 h, 48 h, 72 h, respectively.

Figure 4. Effects of expression of MDV-1 miRNA sponge on apoptosis of MSB-1 cells

(A) DAPI staining under the microscope visual chart. (B) After the Annexin V-FITC / PI double staining, the scatter plot was analyzed. (C) On the basis of the analysis of the fourth quadrant of the ratio of apoptosis. Data are means of at least three independent experiments. ^**P < 0.01.

Figure 5. Effect of expression of MDV-1 miRNA sponge on invasion ability of MSB-1 cells

(A) The visual diagram under the microscope after crystal violet staining. (B) After decolorization with 33% acetic acid, the OD value of the decolorization solution at 570 nm was examined. ^**P < 0.01.

Figure 6. Effect of expression of MDV-1 miRNA sponge on tumorigenicity of MSB-1 cells in vitro

(A) The cells treated with different treatments were cultured in a 37° C cell incubator for 12 days in a low melting point agar with an upper layer of 0.33% in 8,000 wells per well. The resulting clones were stained with crystal violet. (B) The clones formed were observed and counted (under eye view and microscope of 40 magnifications). ^**P < 0.01.

Figure 7. Comparisons of the growth rates of birds inoculated with distinct treated cells in 60 days

^**P < 0.01.

Figure 8. Survival curves of the birds inoculated with MSB-1 cells or its miRNA sponge-treated cells over the 60 day experimental time period

Figure 9. Pathological lesions and tumorigenesis in the hearts, spleens and livers of birds inoculated with MSB-1 cells or its miRNA sponge-treated cells at 60 days p.i. Tumor foci are shown by black arrows