Long noncoding RNA ROR promotes breast cancer by regulating the TGF-β pathway

Abstract

Background: Breast cancer is the leading cause of oncological mortality among women. Efficient detection of cancer cells in an early stage and potent therapeutic agents targeting metastatic tumors are highly needed to improve survival rates. Emerging evidence indicates that lncRNAs (long noncoding RNAs) are critical regulators of fundamental cellular processes in a variety of tumors including breast cancer. The functional details of these regulatory elements, however, remain largely unexplored.

Methods: In this study, lncRNA ROR (linc-ROR) was examined by real-time PCR in different breast cancer cell lines and breast tumor tissues/non-tumor tissues were collected from both breast cancer patients and healthy controls. Linc-ROR was knockdown in breast cancer cell lines and the effects on cell proliferation, migration and invasion were tested both in vitro and in vivo tumor model. Effects of linc-ROR knockdown on TGF-β signaling pathway were investigated by Western blot.

Results: Our studies have suggested that linc-ROR, a critical factor for embryonic stem cell maintenance, probably acts as an oncogenic factor in breast cancer cells, causing poor prognostic outcomes. Overexpression of linc-ROR seems to be responsible for promoting proliferation and invasion of cancer cells as well as tumor growth in nude mice. The regulatory action of linc-ROR can affect the activity of the TGF-β signaling pathway, which has been proven critical for mammary development and breast cancer.

Conclusions: The results have highlighted the potential importance of linc-ROR in the progression of advanced breast cancer, and thus will stimulate efforts in the development of novel diagnostic and therapeutic strategies.

Keywords: Breast cancer; Long noncoding RNA; ROR; TGF-β.

Fig. 1

Upregulation of linc-ROR in breast cancer cell lines and tumor tissues. a Bar plot comparing the linc-ROR RNA levels in different cell lines. The MCF-7 and MDA-MB-231 cells are breast cancer cells, while the Hs578Bst cells are normal mammary fibroblast cells. The linc-ROR RNA levels were quantitated by qRT-PCR. GAPDH RNA was used as an internal control. Data were averaged from triplicate experiments. b Statistical comparison of linc-ROR expression levels in patient tissue samples. Linc-ROR RNA levels were quantitated from breast tumor tissues and the adjacent normal tissues using the same method described above. **p < 0.01, ***p < 0.001, vs the control

Fig. 2

Comparison of prognostic survival rates between Linc-ROR low- and high-expression groups. a Schematic presentation of two patient groups with low- and high-expression levels of linc-ROR. The expression levels of all 94 patients (black bars) were ordered from low to high. The control expression level (green bar) was normalized to 1. The demarcation was set to the patient (red bar) with 3.2-fold increase in the level of linc-ROR. b Kaplan–Meier overall survival curves of the low- and high-expression groups. Breast cancer patients with high expression of linc-ROR showed a poorer prognosis than those with low expression

Fig. 3

Linc-ROR knockdown reduced cell proliferation and invasion. a Comparison of the linc-ROR levels in the cultured MCF-7 breast cancer cells with or without linc-ROR knockdown. Linc-ROR was knocked down by the si-ROR interference RNA. Cells were also untransfected (control) or transfected with a scrambled negative-control siRNA (si-nc) for comparison. b Time courses of MCF-7 proliferation. Cell proliferation rates were compared between the cells transfected with si-ROR or si-nc. The proliferation rates were quantitated every 12 h over 3 days. Linc-ROR knockdown by si-ROR led to slower proliferation. c Comparison of MCF-7 cell invasion. The cells transfected with si-ROR or si-nc were assessed in transwell assays. The scale bars are 100 μm. The data points were averaged from triplicates. **p < 0.01 vs the control. d–f Comparison of proliferation and invasion for the MDA-MB-231 breast cancer cells transfected with si-ROR or si-nc. The panels are arranged as a–c

Fig. 4

Linc-ROR knockdown reduced tumor growth in nude mice. a Time-course comparison of tumor growth from the MCF-7 cells. The cells were transfected with si-ROR or si-nc and injected into nude mice. After 14 days, tumor sizes were measured twice a week over 3 weeks. b Image of tumors harvested at day 31. c Comparison of averaged tumor weights. The tumors grown from the MCF-7 cells transfected with si-ROR were smaller. d Relative ROR expression of tumors was confirmed. The expression of ROR of tumors from the MCF cells transfected with si-ROR was inhibited. e–h Comparison of tumors derived from the MDA-MB-231 cells, which were transfected with si-ROR or si-nc and injected into nude mice. The panels are arranged as a–c. **p < 0.01 vs the control

Fig. 5

Linc-ROR modulates the TGF-β signaling pathway. a Western blot probing TGF-β, Smad2 and α-SMA in the MCF-7 cells. The cells were transfected with si-ROR or si-nc. GAPDH was blotted as an internal reference. b Quantitative comparison of the expression levels of TGF-β, Smad2 and α-SMA. The protein expression levels in the MCF-7 cells transfected with si-nc were normalized to 1. The cells transfected with si-ROR showed reduced expression of all three proteins. c, d Comparison of the protein expression levels of TGF-β, Smad2 and α-SMA in the MDA-MB-231 cells. The panels were arranged as a, b. Data were Mean ± SD derived from three independent experiments. *p < 0.05 vs the control