J Breast Cancer.  2016 Mar;19(1):26-33. 10.4048/jbc.2016.19.1.26.

Prolactin Inhibits BCL6 Expression in Breast Cancer Cells through a MicroRNA-339-5p-Dependent Pathway

• Yan H
• Zhao M
• Huang S
• Chen P
• Wu WY
• Huang J
• Wu ZS
• Wu Q

• KMID: 2176318
• DOI: http://doi.org/10.4048/jbc.2016.19.1.26

Abstract

PURPOSE
Prolactin (PRL) plays a critical role in breast cancer progression by activating its cognate receptor and promotes the growth and differentiation of breast cancer cells. Studies have shown that B-cell lymphoma 6 (BCL6) is the target gene of microRNA-339-5p (miR-339-5p) and that BCL6 expression contributes to breast cancer progression. Herein, we identified PRL as a potent suppressor of BCL6 expression in human breast cancer cells.
METHODS
Western blotting and quantitative reverse transcription-polymerase chain reaction were used to investigate molecular mechanisms underlying miR-339-5p expression and BCL6 manipulation in MCF-7, T47D, and SKBR3 breast cancer cells. Phenotypic changes in these breast cancer cell lines were assessed by performing cell viability (MTT), colony formation, migration, and invasion assays.
RESULTS
PRL suppressed BCL6 protein and mRNA expression and upregulated miR-339-5p expression in MCF-7 and T47D breast cancer cells. Selective downregulation of miR-339-5p expression significantly reversed PRL-induced suppression of BCL6 mRNA and protein expression. Exogenous PRL stimulation significantly decreased the proliferation, colony formation, migration, and invasion of breast cancer cells, and suppression of miR-339-5p expression reversed these processes in vitro.
CONCLUSION
These results indicated that PRL inhibited BCL6 expression and regulated breast cancer progression through a miR-339-5p-dependent pathway.

Keyword

B-cell lymphoma 6 proteins; Breast neoplasms; MicroRNA-339-5p; Prolactin

MeSH Terms

Blotting, Western
Breast Neoplasms*
Breast*
Cell Line
Cell Survival
Down-Regulation
Humans
Lymphoma, B-Cell
Prolactin*
RNA, Messenger
Prolactin
RNA, Messenger

Figure

• 

  Figure 1 PRL suppresses BCL6 protein and mRNA levels in breast cancer cells. (A) T47D cells were treated with or without the indicated doses of PRL for 24 hours. Detergent cell extracts were resolved by Western blot. (B) Western blot of representative over time showing protein levels of BCL6 and GAPDH in MCF-7 and T47D cells treated with or without PRL for up to 48 hours. (C) Corresponding densitometry data of BCL6 normalized to GAPDH loading controls. (D) Time course of BCL6 mRNA levels in MCF-7 and T47D cells in response to PRL treatment by qRT-PCR. The data are represented as mean±SD from three independent experiments.PRL=prolactin; BCL6=B-cell lymphoma 6; GAPDH=glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR=quantitative reverse transcription-polymerase chain reaction.*p<0.05; †p<0.01.

• 

  Figure 2 PRL inhibits BCL6 expression via miR-339-5p pathways. (A) qRT-PCR analysis of miR-339-5p mRNA in MCF-7 and T47D cells treated with or without PRL for up to 48 hours. (B) MCF-7 and T47D cells were grown and transiently transfected with miR-339-5p ASO or scrambled sequence oligonucleotides as negative control and subjected to western blot assays. Forty-eight hours later, cells were treated with or without PRL for 6 hours. (C) Corresponding densitometry data of BCL6 normalized to GAPDH loading controls. (D) qRT-PCR analysis of BCL6 was performed in MCF-7 and T47D cells, respectively.PRL=prolactin; BCL6=B-cell lymphoma 6; GAPDH=glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR=quantitative reverse transcription-polymerase chain reaction; ASO=antisense oligonucleotide.*p<0.05; †p<0.01.

• 

  Figure 3 Inhibition of miR-339-5p expression alters the effects of prolactin (PRL) in breast cancer cells in vitro. (A) T47D cells were grown and transiently transfected with miR-339-5p antisense oligonucleotide (ASO) or negative control. Forty-eight hours later, cells were treated with or without PRL for 6 hours, and cell proliferation was determined afterwards. The experiments were performed in triplicate and repeated thrice. (B) Colony formation of T47D cells transfected by miR-339-5p ASO or control and 48 hours later after transfection, cells were treated with or without PRL for 6 hours. Cells were shown 2 weeks after plating. Right panel showed the quantification of the relative colony formation. Values are the mean±SD of triplicate experiments. Representative photographs (right) and quantification (left) are shown. (C) Transwell migration and invasion assays. MCF-7 cells were grown and transiently transfected with miR-339-5p ASO or control for 2 days. Later, cells were treated with or without PRL for 6 hours and subjected to migration and invasion assays. Representative photographs (right) and quantification (left) are shown (Crystal violet stain, ×100).OD=optical density.*p<0.05; †p<0.01.

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