Fig 1.
The lncRNA UCA1 expression correlates with development of breast cancer.
(A) Analysis of the lncRNA UCA1 expression in normal and breast cancer tissues from healthy donor or patients using quantitative RT-PCR (qRT-PCR). Each sample was triplicated, GAPDH was used as the endogenous control, and the formula 2-ΔΔ Ct was used to calculate the relative expression. (B) Representative H&E staining for the breast cancer tissues with different pathological stage I-IV. Magnification, 200×. (C) Analysis of the lncRNA UCA1 expression using qRT-PCR in two groups of breast cancer with different pathological stages (stage I+II vs. stage III+IV). (D) Expression pattern of the lncRNA UCA1 in all 54 cases of breast cancer samples. (E) Relationship between the survival probability of breast cancer patients and the lncRNA UCA1 expression level. 54 patients were divided into the UCA1 high and low groups by the median value, and the follow-up was done up to 5 years.
Fig 2.
Manipulation of LncRNA UCA1 expression changes breast cancer cell drug resistance.
(A) qRT-PCR analysis shows the lncRNA UCA1 expression in the tamoxifen-resistant and their parental breast cancer cells. Normal, the parental MCF-7 or T47D cells; TAM-R, tamoxifen resistant MCF-7 or T47D cells. Overexpression of lncRNA UCA1 using lentivirus (Lv-empty vector or Lv-UCA1) in MCF-7 cells (B) or T47D cells (C) promotes the proliferation of breast cancer cells detected with MTT assay. Knockdown of lncRNA UCA1 using small interfering RNA targeting UCA1 (si-UCA1) decreases the proliferation of tamoxifen-resistant MCF-7 (D) or T47D (E) breast cancer cells, respectively. All data represents at least three independent experiments. *, P<0.05; **, P<0.01.
Fig 3.
Knockdown of lncRNA UCA1 enhances breast cancer chemo-sensitivity.
Knockdown of UCA1 decreased cell survival of MCF-7-R (A) and T47D-R (B) cells from tamoxifen treatment. (C) Representative flow cytometry showed the apoptosis ofMCF-7-R and T47D-R cells to tamoxifen treatment after transfected with scramble control or si-UCA1 using FITC-Annexin V and PI staining. (D) Summary of the calculated half maximal inhibitory concentration (IC50) of the wild type (parental), tamoxifen resistant (TAM-R), and lncRNA UCA1 knockdown (si-UCA1) breast cancer cells. All data represents at least three independent experiments. *, P<0.05; **, P<0.01.
Fig 4.
Knockdown of lncRNA UCA1 obstructs the migration of breast cancer cells.
Representative pictures shows numbers of migrated of MCF-7-R (A) or T47D-R (B) breast cancer cells Transfected with scramble control or si-UCA1. The following histogram summarized cell numbers of 12 pictures from 3 independent experiments. *, P<0.05.
Fig 5.
Inhibition of the tumorigenicity of tamoxifen-resistant breast cancer cells after knockdown of lncRNA UCA1.
Bar graphs shows the development of tumors generated by MCF-7-R (A) and T47D-R (B) cells transfected with scramble (blank bars) or si-UCA1 (black bars). (C) Representative pictures shows the tumors generated by MCF-7-R and T47D-R cells transfected with scramble or si-UCA1 24 days after injection. Each group contained 5 cases and 4 mice were housed in each case. (D) qRT-PCR data showed the expressions of downstream target genes of the Wnt/β-catenin signaling in tumor tissues generated from MCF-7-R or T47D-R cells transfected with scramble (blank bar) or si-UCA1 (black bar). All data represents at least three independent experiments. *, P<0.05; **, P<0.01.
Fig 6.
The lncRNAUCA1 promotes Wnt signaling activity by facilitating β-catenin cyto-nuclear translocation.
Shown are immunocytochemistry staining for the localization of β-catenin in MCF-7-R (A) and T47D-R (B) cells transfected with scramble control or si-UCA1. Meganification, 400×. (C) Western blot assay shows the decreased β-catenin protein level in the nuclear fraction from cells transfected with si-UCA1. Nuclear Lamin B serves as the loading control. (D) TOP/FOP assay showing the decreased activity of Wnt signaling in the tamoxifen-resistant breast cancer cells when UCA1 was knockdown. *, P<0.05.
Fig 7.
Correlation of the expression of lncRNA UCA1 and the nuclear β-catenin protein level in vivo.
(A) Immunohistochemistry staining of human breast cancer tissue shows the nuclear different β-catenin level in the different pathological stage I-IV. (B) Spearman's Rank-Order Correlation analysis for the nuclear β-catenin level and the expression of lncRNA UCA1 in human breast cancer tissues. (C) Western blot shows the nuclear protein level of β-catenin in the tumors generated from MCF-7-R (upper panel) and T47D-R (lower panel) cells with UCA1 knocked down. Lamin B served as the loading control. (D) Immunohistochemistry staining shows the nuclear β-catenin level in tumor tissues generated from MCF-7-R and T47D-R cells, respectively. Magnification, 200×. Scale bar, 100μm.