LincRNA-ROR induces epithelial-to-mesenchymal transition and contributes to breast cancer tumorigenesis and metastasis

Abstract

LncRNAs have critical roles in various biological processes ranging from embryonic development to human diseases, including cancer progression, although their detailed mechanistic functions remain illusive. The lncRNA linc-ROR has been shown to contribute to the maintenance of induced pluripotent stem cells and embryonic stem cells. In this study, we discovered that linc-ROR was upregulated in breast tumor samples, and ectopic overexpression of linc-ROR in immortalized human mammary epithelial cells induced an epithelial-to-mesenchymal transition (EMT) program. Moreover, we showed that linc-ROR enhanced breast cancer cell migration and invasion, which was accompanied by generation of stem cell properties. Contrarily, silencing of linc-ROR repressed breast tumor growth and lung metastasis in vivo. Mechanistically, our data revealed that linc-ROR was associated with miRNPs and functioned as a competing endogenous RNA to mi-205. Specifically, linc-ROR prevented the degradation of mir-205 target genes, including the EMT inducer ZEB2. Thus our results indicate that linc-ROR functions as an important regulator of EMT and can promote breast cancer progression and metastasis through regulation of miRNAs. Potentially, the findings of this study implicate the relevance of linc-ROR as a possible therapeutic target for aggressive and metastatic breast cancers.

Figure 1

Linc-ROR expression in clinical breast cancer specimens and cancer cell lines. (a) The linc-ROR was significantly upregulated in 22 human breast cancerous tissues compared with the corresponding non-cancerous tissues. The relative linc-ROR mRNA level was normalized to β-actin. The statistical differences were analyzed using the Paired t-test. (b) The linc-ROR expression was upregulated in breast cancer cell lines compared with the immortalized human mammary epithelial cell (MCF10A). The linc-ROR mRNA level was normalized to β-actin. Error bars represent the mean±S.D. of triplicate experiments

Figure 2

Ectopic expression of linc-ROR in MCF10A cells induced an EMT program. (a) Ectopic expression of linc-ROR mRNA expression was confirmed by RT-PCR after retrovirus infection in MCF10A cells. (b) The morphology of MCF10A cells expressing the control vector or linc-ROR was examined by phase-contrast microscopy. Scale bar, 100 μm. (c) Immunoblotting analysis of the epithelial (E-cadherin and Occludin) and mesenchymal markers (Fibronectin, N-cadherin, Vimentin and α-SMA) in MCF10A cells expressing linc-ROR or the control vector. (d) Immunofluorescence staining for the EMT makers. Scale bar, 100 μm. (e) Expression of EMT marker mRNAs were assessed by real-time PCR. (f) The mRNA expression levels of EMT inducers (Snail, Slug, Twist, ZEB1 and ZEB2) were assessed by real-time PCR in linc-ROR or the control vector expressing MCF10A cells. (g) Immunoblotting analysis of EMT inducers (Snail, Slug, Twist, ZEB1 and ZEB2) in MCF10A cells expressing linc-ROR. The mRNA levels were normalized to β-actin. Error bars represent the mean±SD of triplicate experiments. Statistical differences were analyzed using the Paired t-test (***P<0.001, *P<0.05, non-significant (ns) P>0.05)

Figure 3

Effects of linc-ROR on invasion and migration in breast cancer cells. (a) Wound-healing assay to assess the effect of linc-ROR on cell mobility in MCF10A and MDA-MB-231 cells. (b) The effect of linc-ROR on migration ability was measured using transwell assays in MCF10A and MDA-MB-231 cells. (c) The effect of linc-ROR on invasion ability. The data represent the mean number of cells per field and are presented as the means±S.D. (**P<0.01, ***P<0.001). All the experiments were repeated three times

Figure 4

The linc-ROR-induced EMT generated stem cell-like phenotype in mammary epithelial cells. (a) Flow cytometric evaluation of CD44^high/CD24^low subpopulation in MCF-10A cells expressing vector or linc-ROR. The percentages of mean CD44^high/CD24^low population±S.D. of triplicate experiments are indicated. (b) Reprehensive images of mammospheres formed from MCF10A cells. Scale bar, 100 μm. (c) The quantification of mammosphere numbers formed from MCF10A cells in independent experiments (error bar, means±S.D., **P<0.01)

Figure 5

Knockdown of linc-ROR inhibited tumorigenesis and metastasis in nude mice. (a) Stable shCtrl or shROR-infected MDA-MD231 cells were subcutaneously injected into BALB/c female nude mice (n=6, for each experimental group), and 8 weeks later, the xenograft tumors were peeled off and weighted. The tumor weights are presented as the means±S.D., **P<0.01. (b) Linc-ROR knockdown MDA-MB-231 cells were injected via the lateral tail veins. Lung nodules were analyzed as percentage of mice that developed lung metastases after 9 weeks. Numbers over the bars indicate metastasis incidence. *P<0.05, χ² test. (c) Lung nodules at week 9 were analyzed as the numbers of nodules per mouse. **P<0.01, Student's t-test. (d) Representative lung images at week 9, corresponding hematoxylin–eosin-stained lung sections are shown

Figure 6

Linc-ROR was associated with miRNPs and acted as a molecular sponge for mir-205. (a) Prediction for miRNA-binding elements on linc-ROR by Miranda. (b) The indicated reporter constructs were transfected into 293T cells, together with control miRNA (NC), mir-205-5p or mir-200a-5p, at a final concentration of 48 nM, and additional detection of the indicated Rluc activity at a final concentration of 12, 24 and 48 nM. Numbers are mean±S.D. (n=3). (c) Detection of the indicated Rluc activity affected by mir-34a-5p or let-7-5p at a final concentration of 48 nM. (d) Co-expression of linc-ROR rescued the relative Rluc activity of reporters containing ZEB2 when co-transfected with mir-205-5p. (e) The binding ability of linc-ROR transcript to mir-205-5p that was precipitated by cDNA combined with MS2-binding sequences (MS2bs) and its binding protein Flag-MS2bp. Measured by real-time PCR. Numbers represent mean±S.D. (n=3) (f) Immunoblotting detection of AGO2 after Flag-MS2bp-MS2bs-based pull-down assay. ANTI-FLAG M2 Affinity Gel was used for pull-down assay. (g) RIP assay detection of the linc-ROR-binding efficiency to AGO2 in MCF10A-ROR cells. Error bars represent the mean±S.D. of triplicate experiments, **P<0.01