LncRNA Expression Profiles in Canine Mammary Tumors Identify lnc34977 as a Promoter of Proliferation, Migration and Invasion of Canine Mammary Tumor Cells

Abstract

Canine mammary tumor (CMT) is the most common tumor in canines after skin tumors. Long noncoding RNAs (lncRNAs) have crucial roles in human breast tumor initiation and progression, but the role of lncRNAs in canine mammary tumors is unclear. We analyzed the expression profiles of canine mammary tumors and their adjacent non-neoplastic tissue to explore abnormally expressed lncRNAs. LncRNA expression was detected by qRT-PCR. After overexpression of lnc40589 and knockdown of lnc34977 in CMT cells, CCK-8, colony formation, wound healing and Transwell assays were used to assess the proliferation, migration and invasive ability of canine mammary tumor cells. We also established a mammary tumor-bearing nude mouse model. GO analysis and KEGG pathway analysis demonstrated that the differentially expressed lncRNAs were closely related to the mammary tumor. lnc40589 was significantly upregulated and lnc34977 was significantly downregulated in CMTs. In addition, lnc40589 inhibits cell proliferation, migration and invasion, while lnc34977 promotes cell proliferation, migration and invasion. In addition, lnc34977 promotes the development of mammary tumors in animals. Taken together, our study results reveal the lncRNA expression profiles in CMTs and indicate that lnc34977 promotes the development of CMT both in cell culture and in vivo.

Keywords: canine mammary tumor; expression profiles; lncRNA; lncRNA34977.

Figure 1

Differentially expressed gene analysis from RNA-seq (fold change > 1.5, p < 0.05). (A,B) Heatmap plot of mRNA and lncRNA. Red indicates high relative expression; blue indicates low relative expression. (C,D) Volcano plots of mRNA and lncRNA expression.

Figure 2

Differentially expressed gene analysis from a public database. (fold change > 1.5, p < 0.05). (A) Volcano plots of gene expression. (B) Venn diagram of common differentially expressed genes. Green indicates data generated in this study and yellow indicates publicly available data.

Figure 3

GO and KEGG pathway analysis of common differentially expressed genes. (A) GO analysis of common differentially expressed genes. (B) KEGG pathway analysis of common differentially expressed genes.

Figure 4

lncRNA–miRNA–mRNA network. Triangles indicate miRNAs, V-shapes indicate lncRNAs and round shapes indicate mRNAs. Red indicates upregulated genes; green indicates downregulated genes.

Figure 5

The expression levels of lncRNAs in CMT and adjacent non-neoplastic tissue measured by qRT–PCR. Samples 1–10. (A) The expression levels of lncRNA40589 in CMT and adjacent non-neoplastic tissue. (B) The expression levels of lncRNA34977 in CMT and adjacent non-neoplastic tissue. (C) The expression levels of lncRNA32949 in CMT and adjacent non-neoplastic tissue. (D) The expression levels of lncRNA39112 in CMT and adjacent non-neoplastic tissue. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001).

Figure 6

The expression level of lncRNAs in CMT and adjacent non-neoplastic tissue measured by qRT–PCR. Samples 11–20. (A) The expression levels of lncRNA40589 in CMT and adjacent non-neoplastic tissue. (B) The expression levels of lncRNA34977 in CMT and adjacent non-neoplastic tissue. (C) The expression levels of lncRNA32949 in CMT and adjacent non-neoplastic tissue. (D) The expression levels of lncRNA39112 in CMT and adjacent non-neoplastic tissue. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001).

Figure 7

The expression level of lncRNAs in CMT and adjacent non-neoplastic tissue measured by qRT–PCR. Samples 21–30. (A) The expression levels of lncRNA40589 in CMT and adjacent non-neoplastic tissue. (B) The expression levels of lncRNA34977 in CMT and adjacent non-neoplastic tissue. (C) The expression levels of lncRNA32949 in CMT and adjacent non-neoplastic tissue. (D) The expression levels of lncRNA39112 in CMT and adjacent non-neoplastic tissue. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 8

Overexpression of lnc40589 and knockdown of lnc34977 inhibited the proliferation of CMT cells. (A,B) Relative expression of lnc40589 and lnc34977 after transfection was detected by qRT–PCR. (C,D) Growth curve of CHMp and CHMm after overexpressing lnc40589. (E) Survival rate of CHMp and CHMm after overexpression of lnc40589. (F,G) Growth curve of CHMp and CHMm after knocking down lnc34977. (H) Survival rate of CHMp and CHMm after knockdown of lnc34977. (I) Clone formation assay. (J,K) Clone efficiency of CHMp and CHMm. Three intra-group replicates were performed in each experimental subgroup. (** p < 0.01, *** p < 0.001 and **** p < 0.0001).

Figure 9

Overexpression of lnc40589 and knockdown of lnc34977 inhibited the migration of CMT cells. (A) CHMp migration observed under a microscope before and after overexpressing lnc40589 and knocking down lnc34977. (B) Comparison of cell migration areas of CHMp. (C) CHMm migration observed under the microscope before and after overexpressing lnc40589 and knocking down lnc34977. (D) Comparison of cell migration areas of CHMp. Three intra-group replicates were performed in each experimental subgroup. (*** p < 0.001 and **** p < 0.0001).

Figure 10

Overexpression of lnc40589 and knockdown of lnc34977 inhibited the invasion of CMT cells. (A,C) CHMp and CHMm invasion detected by Transwell assay. (B,D) Numbers of invading CHMp and CHMm cells. Three intra-group replicates were performed in each experimental subgroup. (* p < 0.05 and **** p < 0.0001).

Figure 11

Knockdown of lnc34977 inhibited mammary tumors in a nude mouse model. (A) Representative images of tumor-bearing mice. (B) The tumor volume growth curves after injections. (C) Representative images of hematoxylin and eosin (HE) staining of tumor samples, in which fusiform pleomorphic tumor cell proliferation infiltration is observed in all three images. (* p < 0.05).