LncRNA Expression Profiles in C6 Ceramide Treatment Reveal lnc_025370 as a Promoter in Canine Mammary Carcinoma CHMp Cells Progression

Abstract

Canine mammary carcinomas (CMCs) represent the most prevalent form of cancer in female dogs, characterized by a high incidence and mortality rate. C6 ceramide is recognized for its multifaceted anti-cancer properties, yet its specific influence on CMCs remains to be elucidated. Long noncoding RNAs (lncRNAs), now recognized as functional "dark matter" in precision oncology, are particularly intriguing, with 44% of canine lncRNAs exhibiting tissue-specific expression. In this study, we performed a thorough analysis of lncRNA expression profiles to uncover the mechanisms behind C6 ceramide's anti-cancer activity in CHMp cells. Our findings reveal that C6 ceramide notably inhibits the proliferation of CHMp cells. RNA sequencing identified 4522 lncRNAs with expression changes following C6 ceramide treatment, of which 2936 were upregulated and 1586 were downregulated. Further investigation into Lnc_025370 showed that it is predominantly nuclear-localized and is significantly downregulated by C6 ceramide treatment. Functional studies discovered that overexpression of Lnc_025370 enhances the growth and metastatic capabilities of CHMp cells, which is associated with an increase in NRG1, and concurrently diminishes the anti-cancer effectiveness of C6 ceramide in vitro. Mouse xenograft models also showed that Lnc_025370 overexpression promotes tumor growth and Ki67 expression. Together, our results suggest that Lnc_025370 acts as a pivotal target mediator of C6 ceramide's anti-cancer effects, facilitating the malignant progression of CHMp cells.

Keywords: C6 ceramide; Lnc_025370; NRG1; canine mammary carcinomas; expression profiles.

Figure 1

The growth inhibitory effect of C6 ceramide and its related lncRNAs in CHMp cells. (A) Cell viability was analyzed using CCK-8 in CHMp cells after exposure to C6 ceramide; (B,C) Colony formation of CHMp cells treated with C6 ceramide; (D) Heatmap plot of lncRNA. Red indicates high relative expression, blue indicates low relative expression; (E) Volcano plots of lncRNA expression; (F) Expression of lncRNAs by qRT-PCR; (G) Relative expression of Lnc_025370 after C6-ceramide treatment for 48 h; (H) Relative expression of Lnc_025370 in nuclear and cytoplasm of CHMp cells. The data are presented as the mean ± SD. ** p < 0.01, *** p < 0.001.

Figure 2

Overexpressing Lnc_025370 promoted CHMp cell proliferation. (A) The expression of Lnc_025370 and GAPDH was determined with qRT-PCR in cells transfected with the pcDNA3.1 vector (CHMp_NC) and pcDNA3.1-Lnc_025370 (CHMp_Lnc); (B) Growth curves of CHMp_NC and CHMp_Lnc cells; (C,D) Colony formation of CHMp_NC and CHMp_Lnc cells. The data are presented as the mean ± SD. ** p < 0.01, *** p < 0.001.

Figure 3

Overexpressing Lnc_025370 promoted CHMp cell metastasis migration and invasion. (A) The migratory ability of CHMp_NC and CHMp_Lnc cells. scale bar, 200 μm; (B) The migratory abilities of CHMp_NC and CHMp_Lnc were calculated by Image J. (C) Invading cells of CHMp_NC and CHMp_Lnc were stained with 0.1% (w/v) crystal violet. Scale bar, 100 μM; (D) The number of invading cells was calculated by Image J. The data are presented as the mean ± SD. * p < 0.05.

Figure 4

Overexpressing Lnc_025370 attenuated growth inhibition of C6 ceramide in CHMp cells (A) Cell viability was analyzed using CCK-8 in CHMp_NC and CHMp_Lnc cells after exposure to C6 ceramide for 48 h; (B) IC50 of C6-Ceramide on CHMp_NC and CHMp_Lnc cells; (C,D) Colony formation of CHMp_NC and CHMp_Lnc cells treated with C6 ceramide for 48 h. The data are presented as the mean ± SD. * p < 0.05, *** p < 0.001.

Figure 5

Overexpressing Lnc_025370 attenuated metastasis inhibition of C6 ceramide in CHMp cells. (A) The migratory ability of CHMp_NC and CHMp_Lnc cells after exposure to C6 ceramide for 24 h. scale bar, 200 μM; (B) The migratory abilities of CHMp_NC and CHMp_Lnc were calculated by Image J. (C) Invading cells of CHMp_NC and CHMp_Lnc were stained with 0.1% (w/v) crystal violet after exposure to C6 ceramide for 24 h. Scale bar, 100 μM; (D) The number of invading cells was calculated by Image J. The data are presented as the mean ± SD. * p < 0.05, ** p < 0.01.

Figure 6

Lnc_025370 promoted xenograft tumor growth. (A) Mice with subcutaneously transplanted tumors; (B) Representative photographs of tumors after the experiment; (C) Fluctuations in the size of subcutaneously transplanted tumors across the groups; (D) Weight measurements of the subcutaneously transplanted tumors; (E) The pathological histology of the tumor masses (Scale bar = 50 μM); (F) Illustrative images of immunohistochemistry (IHC) depicting Ki67 expression (Scale bar = 50 μM), with a quantitative analysis of Ki67 staining corresponding to the images in both groups. The data are presented as the mean ± SD. * p < 0.05, *** p < 0.001.

Figure 7

The promotion of Lnc_025370 in CHMp cells progression, with a possible association with NRG1 expression. (A) The expression of NRG1 and GAPDH was determined with qRT-PCR in CHMp cells after exposure to C6 ceramide for 48 h; (B,C) The expression of NRG1 was determined with western blotting in CHMp cells after exposure to C6 ceramide for 48 h; (D) The expression of NRG1 and GAPDH was determined with qRT-PCR in CHMp_NC and CHMp_Lnc cells; (E,F) The expression of NRG1 was determined with western blotting in CHMp_NC and CHMp_Lnc cells. The data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.