Figure 1.
miR-205 is down-regulated in breast cancer.
(A) Expression pattern of miR-205-5p in MCA10A, MCF7, BT549, MDA-MB-231, and MDA-MB-436 cells. The data in cancer cell lines are expressed as fold-change compared to MCA10A, which was assigned a value of “1”. (B) miR-205-5p levels in the normal adjacent breast tissues (N), non-metastatic tumor (T), and metastatic breast cancer specimens (M) were determined by qPCR. Data are presented relative to 18S rRNA. Mean values are indicated by horizontal bars.
Figure 2.
Over-expression of miR-205 reduces the proliferation and in vitro invasion of breast cancer cell lines.
(A) MDA-MB-231 and BT549 cells were transfected with 100 nM of pre-miR-205 or control oligonucleotides. Following a 96 h exposure, cell proliferation was measured using a WST-1 assay. (B) Representative photographs showing in vitro Matrigel invasion assay of MDA-MB-231 and BT549 cells after transfection with pre-miR-control or pre-miR-205-5p 48 hr prior to seed into the upper chamber of 24-well transwell units. Bar graphs represent the mean±SD values of the relative number of invasive cells (n=4). (C) Real time TaqMan analysis of miR-205 levels in stable cell lines over-expressing primary transcripts of miR-205. (D) Relative representation of the number of colonies in MDA-MB-231 and BT549 cells stably expressing miR-205-5p. (E) Overall proliferation of miR-205-5p stably expressing MDA-MB-231 and BT549 cells up to 96 hrs.
Figure 3.
HMGB3 is a target of miR-205 in breast cancer.
(A) Schematic diagram of miR-205 binding sites in the 3' UTR region of HMGB3 mRNA. (B) Luciferase reporter plasmids carrying the full length of HMGB3 3' UTR were transiently co-transfected with the negative control of pre-miR precursor (N) or the miR-205 precursor (205) at 50 and 100 nM concentrations. Luciferase activity was measured 24 h after transfection. The data are the mean ± SD of at least 3 independent transfections. (C) Lysates were immunoblotted for expression levels of HMGB3 in MDA-MB-231 and BT549 cells. Normalized expression to β-actin is included. (D) Expression of the selected predicted oncogenic target genes of miR-205 was evaluated in both MDA-MB-231 and BT549 cells 48 h after transfection of either control oligonucleotides or pre-miR-205-5p. Expression levels were calculated relative to 18S rRNA and the data are expressed as fold-increase compared to cells treated with control oligo (assigned a value of “1”).
Figure 4.
Knockdown of HMGB3 reduces the proliferation and in vitro invasion of breast cancer cells.
(A) Proliferation of MDA-MB-231 and BT549 cells transfected with HMGB3 siRNA or control siRNA. (B) in vitro Matrigel invasion assay of MDA-MB-231 and BT549 cells after transfection with HMGB siRNA or control siRNA 48 hr prior to seed into the upper chamber of 24-well transwell units. Bar graphs represent the mean ± SD values of the relative number of invasive cells.
Figure 5.
HMGB3 is over-expressed in primary breast cancer tissues.
(A) Expression pattern of HMGB3 mRNA level in normal adjacent (N), non-metastatic tumor (T) and metastatic (M) breast cancer tissues. mRNA expression levels were calculated relative to 18S rRNA. (B) HMGB3 and GAPDH protein levels in non-metastatic and metastatic breast cancer compared to paired normal tissues was determined by western blotting. HMGB3 expression was confirmed by immunohistochemistry staining in human breast tissues. Brown color indicates HMGB3 staining in adjacent benign tissue (C) and tumor (D). (E) Kaplan Meier plot of disease free survival based on HMGB3 expression in breast cancer samples. Blue, patients with gene down-regulated by more than 1.2 fold; red, patients with genes overexpressed by more than 1.2 fold; yellow patients with intermediate values of fold change. P-values of log-rank test for each pair of KM curves are provided at the bottom of the plot.
Figure 6.
Expression of miR-205 and HMGB3 are altered in 5-Aza and TSA treated cells.
(A) Increased miR-205 and (B) reduced HMGB3 expression in MDA-MB-231 cells treated with a combination of 5-Aza and TSA as described in Methods.
Figure 7.
Change in miR-205 and HMGB3 expression in REST-less cells.
(A) The amount of miR-205 in MCF7 cells stably expressing shRNA to REST (RESTless cells) was decreased compared to control but was unchanged in the RESTless MCF10A cell line. (B) HMGB3 mRNA levels increased only in the RESTless MCF7 cell line.