Variation of Long Non-Coding RNA And mRNA Profiles in Breast Cancer Cells With Influences of Adipocytes

Abstract

Background: It is well known that obesity is one of the risks for incurrence and development in breast cancer patients. Long non-coding RNAs (lncRNAs) are reported to participate in the composition of tumor microenvironment and to regulate breast cancer cell metabolic activities. However, there was rare study focused on the lncRNAs in breast cancer with the influences of adipocytes. The study aimed to investigate lncRNAs expression profiles and discover potential biomarkers to predict the incidence and progression of adipocyte-associated-breast cancer.

Methods: We co-cultured adipocytes with breast cancer cells and profiled the expression of lncRNAs as well as mRNAs by using the RNA-sequencing method. Wound Healing, Migration assays and Invasion assays were applied to verify the invasion and metastasis of cancer cells.

Results: MDA-MB-231/Hpa-V and SK-BR-3/Hpa-V cells showed elevated migration and invasiveness compared to the control group. A sum of 371 mRNAs (181 upregulated and 190 downregulated) and 850 lncRNAs(414 upregulated and 436 downregulated) were differentially expressed in MDA-MB-231/Hpa-V comparing to MDA-MB-231(P < 0.05; |log2 (fold change)|>1.2). GO enrichment, KEGG pathway and interaction networks demonstrated that differentially expressed lncRNAs were involved in functional categories, such as material metabolism, which might lead to the progression of breast cancer.

Conclusion: Our study detected a lncRNA profile in breast cancer cells affecting by adipocytes and provided a better understanding of the tumor microenvironment. LncRNAs may be helpful to predict the therapeutic responses and prognosis of obese breast cancer patients.

Keywords: adipocytes; breast cancer; long-non-coding RNA; mRNA; tumor microenvironment.

Figure 1

Adipocytes promote invasion and migration of breast cancer cells. (A) wound healing experiment of the MDA-MB-231 or SkBr3 and Hpa-V co-cultured group and the control group (40 magnification); (B) a histogram shows the detailed healing area of the wound healing experiment; (C) transwell migration tests of co-cultured group and the control(200 magnification); (D) a histogram shows the specific vision cell number of transwell migration tests; (E) matrigel invasion tests of co-cultured group and the control(200 magnification); (F) a histogram shows the specific invaded cell number of matrigel invasion tests. MDA-MB-231/H: MDA-MB-231/Hpa-V; SkBr3/H: SkBr3/Hpa-V. **P < 0.01, *P < 0.05. The experiments were performed in triplicate.

Figure 2

The hierarchical clustering heatmaps of differential mRNAs (A), transcripts (B) and lncRNAs (C) after filtered by DEseq2. (P < 0.05, |log2 (fold change)|>1.2). The red color means upregulated, while the green color means downregulated. Exp: the experimental group, namely the MDA-MB-231/Hpa-V co-cultured group and the three samples were listed as A1, A2, A3. Con: the control group, namely the MDA-MB-231 group and the three samples were listed as B1, B2, B3.

Figure 3

The volcano plots of the differentially expressed mRNAs (A), transcripts (B) and lncRNAs (C). (P < 0.05; |log2 (fold change)|>1.2). The red dots mean upregulated, while the green dots mean downregulated, and the black dots mean no differential expressions.

Figure 4

Verification of 3 differentially expressed lncRNAs(ENST00000621974.1, ENST00000473798.1, ENST00000608741.1). The relative expressions of ENST00000621974.1, ENST00000473798.1 and ENST00000608741.1 were significantly increased in MDA-MB-231/Hpa-V in comparison with MDA-MB-231 cells. MDA-MB-231/H: MDA-MB-231/Hpa-V. *P<0.05. All experiment was performed in triplicate.

Figure 5

Distribution of differential lncRNAs (A: upregulated, B: downregulated) in breast cancer cells. (C) Distribution of differential lncRNAs abundance(shorter than 2000bp). (A) among the upregulated lncRNAs shorter than 2000bp, 45.90% were processed transcripts, 26.23% were lincRNAs and 21.31% were antisense RNAs; (B) among the downregulated lncRNAs shorter than 2000bp, 47.83% were processed transcripts, 23.19% were lincRNAs and 20.29% were antisense RNAs; (C) 39.9% of the lncRNAs FPKM expression were over 0.4, and 17.05% were over 1.

Figure 6

Significant GO enrichment terms correlated to differential mRNAs. (A) upregulated mRNAs were involved in interferon receptor activity, pancreatic polypeptide receptor activity, LBD domain binding(the first three biological processes), lipopolysaccharide receptor complex, ripoptosome, chromatoid body (the first three cellular components), glycerol-3-phosphate metabolic process, alditol phosphate metabolic process and positive regulation of protein insertion into mitochondrial(the first three molecular functions); (B) downregulated mRNAs were associated with hexokinase activity, glucokinase activity, N-acetylgalactosamine 4-O-sulfotransferase activity (the first three biological processes), dense body, pi-body, pyruvate dehydrogenase complex (the first three cellular components), maintenance of protein location in the mitochondrion, prostate epithelial cord arborization involved in prostate and prostate glandular acinus morphogenesis(the first three molecular functions).

Figure 7

KEGG pathway of differential genes. (A) upregulated gene pathway enrichments; (B) downregulated gene pathway enrichments; (C) metabolism associated pathway network. The size of dots represent the gene count; LgP means the statistical significance, the red color means a greater significance than the green color and the horizontal axis shows the concrete value.

Figure 8

Metabolism related coding-non-coding gene co-expression networks (Blue nodes represent downregulated lncRNAs and mRNAs; red nodes represent upregulated lncRNAs and mRNAs).