The full recovery of mice (Mus Musculus C57BL/6 strain) from virus-induced sarcoma after treatment with a complex of DDMC delivery system and sncRNAs

Abstract

Background: Virus-induced cellular genetic modifications result in the development of many human cancers.

Methods: In our experiments, we used the RVP3 cell line, which produce primary mouse virus-induced sarcoma in 100% of cases. Inbreed 4-week-old female C57BL/6 mice were injected subcutaneously in the interscapular region with RVP3 cells. Three groups of mice were used. For treatment, one and/or two intravenous injections of a complex of small non-coding RNAs (sncRNAs) a-miR-155, piR-30074, and miR-125b with a 2-diethylaminoethyl-dextran methyl methacrylate copolymer (DDMC) delivery system were used. The first group consisted of untreated animals (control). The second group was treated with one injection of complex DDMC/sncRNAs (1st group). The third group was treated with two injections of complex DDMC/sncRNAs (2nd group). The tumors were removed aseptically, freed of necrotic material, and used with spleen and lungs for subsequent RT-PCR and immunofluorescence experiments, or stained with Leishman-Romanowski dye.

Results: As a result, the mice fully recovered from virus-induced sarcoma after two treatments with a complex including the DDMC vector and a-miR-155, piR-30074, and miR-125b. In vitro studies showed genetic and morphological transformations of murine cancer cells after the injections.

Conclusions: Treatment of virus-induced sarcoma of mice with a-miR-155, piR-30074, and miR-125b as active component of anti-cancer complex and DDMC vector as delivery system due to epigenetic-regulated transformation of cancer cells into cells with non-cancerous physiology and morphology and full recovery of disease.

Keywords: DDMC vector; Epigenetic therapy; Mice; Sarcoma; Small non-coding RNAs; Src tyrosine kinase.

Fig. 1

Kaplan-Meier plots of the lifespan of animals from the 1st (n = 10) (A.) and 2nd (n = 10) (B.) groups compared to that of the controls (n = 10) (p < .05).

Fig. 2

The dynamics of tumor growth in mice from all three groups (means ±SEMs (p < .05)).

Fig. 3

Photographs of mice from the control (A.) and 1st (B.) groups on the 20th day after tumor inoculation.

Fig. 4

Annexin A4 translocation to the nucleus in labeled tumor cells from animals in the 1st group. A. Tumor cells from the control group (24 days after beginning the experiment); B. tumor cells from mice in the 1st group (16 days after beginning the experiment); C. tumor cells from mice in the 1st group (24 days after beginning the experiment). Magnification 60x.

Fig. 5

Tumor cells from animals in the 1st group. Immunofluorescence labeling of cells with CD4⁺ (A.), CD117+ (B.), and Oct4 (C.). Magnification 40x. Cells from tumors from animals in the 1st group were stained with the Leishman-Romanowsky method (D.). Magnification 60x.

Fig. 6

Cells from animals in the 1st group were treated in vitro with the complex of sncRNAs with the DDMC delivery system 11 days after treatment. Cells were stained with the Leishman-Romanowsky method. Magnification 60x (A., B.). Cells labeled with anti-CD4⁺ antibodies and analyzed by immunofluorescence. Magnification 40x (C.).

Fig. 7

Heat map of the hierarchical clustering of gene expression profiles in different cells from mice in all investigated groups.

Fig. 8

Images of RT-PCR products obtained after electrophoresis on 2% agarose gels, stained with ethidium bromide.

Fig. 9

Summary figure of influence of complex a-miR-155, miR-125b, and piR-30074 with the DDMC delivery system on the processes of tumor suppression.