MicroRNA-203 suppresses cell proliferation and migration by targeting BIRC5 and LASP1 in human triple-negative breast cancer cells
Chen Wang
Xiangqian Zheng
Chunyan Shen
Yurong Shi
0
0
Tianjin Cancer Institute
,
Huanhuxi Ave, Tianjin 300060
,
China
Background: This study was performed to investigate the effect of microRNA-203 (miR-203) on cell proliferation and migration in triple-negative breast cancer (TNBC). Methods: Real-time PCR was performed to detect the expression of miR-203 in TNBC cell lines. miR-203 precursor and control microRNA (miRNA) were transfected into triple-negative breast cancer (TNBC) cell lines and the effects of miR-203 up-regulation on the proliferation and migration of cells were investigated. Meanwhile, the mRNA and protein levels of baculoviral IAP repeat-containing protein 5 (BIRC5) and Lim and SH3 domain protein 1 (LASP1) were measured. Luciferase assays were also performed to validate BIRC5 and LASP1 as miR-203 targets. Results: Both miR-203 and BIRC5 siRNA signicantly inhibited cell proliferation in TNBC cells. Both miR-203 and LASP1 siRNA signicantly inhibited cell migration in TNBC cells, also. Moreover, up-regulated of BIRC5 and LASP1 was able to abrogate the effects induced by transfection with the miR-203 precursor. Conclusions: These data suggest that miR-203 may function as a tumor suppressor in TNBC cells. Thus, miR-203 could be a potential therapeutic target for this disease.
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Background
Breast cancer is the most frequently diagnosed cancer
and the leading cause of cancer death in women
worldwide, accounting for 23% (1.38 million) of all new cancer
cases and 14% (458,400) of all cancer deaths in 2008.
Approximately half of all breast cancer cases and 60% of
breast cancer-related deaths are estimated to occur in
developing countries [1]. The large number of etiological
factors and the complexity of breast cancer present
challenge for prevention and treatment.
Triple-negative breast cancer (TNBC) is defined
histologically as invasive carcinoma of the breast that lacks
staining for estrogen receptor (ER), progesterone receptor
(PgR), and the human epidermal growth factor receptor-2
(HER2). TNBC is associated with high proliferative rates,
early recurrence, and poor survival rates. Much effort has
been spent on the study of the biological behavior of
TNBC cells to develop effective treatment strategies.
MicroRNAs (miRNAs) are small, non-coding RNAs of
1925 nucleotides in length that are endogenously
expressed in mammalian cells. miRNAs regulate gene
expression post-transcriptionally, by pairing with
complementary nucleotide sequences in the 3-UTRs of
specific target mRNAs [2,3]. This recently identified type of
gene regulators is involved in modulating multiple
cellular pathways, including cell proliferation, differentiation,
and migration. Thus, miRNAs may function as
oncogenic miRNAs or tumor suppressors [4-6]. Over 50% of
miRNA genes are located in cancer-associated genomic
regions [7]. The deletion or epigenetic silencing of a
miRNA that normally represses the expression of one or
more oncogenes might lead to carcinogenesis, tumor
growth and invasion, as has been demonstrated for
miR200, miR-122 and miR-203 [8-10].
miR-203 is significantly down regulated in several
cancers, including hepatocellular carcinoma [11], colon
cancer [12], prostate cancer [13], and laryngeal cancer [14].
Because the down-regulated of miR-203 is common to a
number of cancers, it has been hypothesized that
miR203 may play an important role in tumorigenesis and
tumor development. However, the function of miR-203
in breast cancer remains unclear, especially in TNBC.
In this paper, we showed that miR-203 was
downregulated in TNBC cell lines and that the ectopic
overexpression of miR-203 blocked tumor cell proliferation
and migration in vitro. Furthermore, BIRC5 and LASP1
were identified as two direct functional targets of
miR203 in TNBC cells. These data suggest that the reduced
expression of miR-203 facilitates the development and
metastasis of TNBC.
Materials and methods
Cell culture and treatment
Human triple-negative breast cancer cell lines
(MDAMB-468 and MDA-MB-231) and normal breast cell line
MCF-10A, were purchased from the American Type
Culture Collection. MDA-MB-468 and MDA-MB-231
cells were maintained in DMEM (Gibco) supplemented
with 10% FBS and 100 U/ml penicillin and 100 g/ml
streptomycin. MCF-10A cells were maintained in
DMEM/F-12 supplemented with 10% FBS, insulin
(10 g /ml), hydrocortisone (500 ng/ml) and EGF
(20 ng/ml). The cells were collected using 0.05% trypsin
EDTA following the specified incubation period.
Precursor miRNA/siRNA/plasmid transfection
Cells were seeded in 6-well plates at a concentration of
1 105 and cultured in medium without antibiotics for
approximately 24 h before transfection. Cells were
transiently transfected with miR-203 precursor (Applied
Biosystems) or negative control miRNA, BIRC5 siRNA
(Sigma), LASP1 siRNA (Sigma) or control siRNA at a
final concentration of 200nM. PcDNA-BIRC5 or
pcDNA-LASP1 plasmid was also transfected into
MDAMB-231 cells using Lipofectamine 20 (...truncated)