Long non-coding RNA NEAT1 confers oncogenic role in triple-negative breast cancer through modulating chemoresistance and cancer stemness

Abstract

Triple-negative breast cancer (TNBC) is a malignant subtype of breast cancer with the absence of targeted therapy, resulting in poor prognosis in patients. Chemotherapy remains the mainstay of treatment for TNBC; however, development of drug resistance is the main obstacle for successful treatments. In recent years, long non-coding RNA (lncRNA) has been implicated in multiple biological functions in various diseases, particularly cancers. Accumulating evidence suggested that lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) expression is dysregulated in many human cancers and thus is a useful prognostic marker for cancer patients. Nevertheless, the mechanism of how NEAT1 confers drug resistance in TNBC is still largely unknown. We performed lncRNA profiling by the LncRNA Profiler qPCR Array Kit in normal control (NC) and breast cancers (BC) blood samples and further validated in a larger cohort of samples by qRT-PCR. Gene expression level and localization were investigated by qRT-PCR, western blotting, and immunofluorescence staining. Flow cytometric analysis was carried out to detect cancer stem cells. Functional studies were performed both in vitro and in vivo xenograft model. Among 90 lncRNAs, NEAT1 was highly expressed in the blood samples of breast cancer patients than in NC. In particular, the expression of NEAT1 was higher in TNBC tissues than other subgroups. Functional studies revealed that NEAT1 conferred oncogenic role by regulating apoptosis and cell cycle progression in TNBC cells. We identified that knockdown of NEAT1 sensitized cells to chemotherapy, indicating the involvement in chemoresistance. Importantly, shNEAT1 reduced stem cell populations such as CD44+/CD24-, ALDH+, and SOX2+, implicating that NEAT1 was closely related to cancer stemness in TNBC. Our data highlighted the roles of NEAT1 chemoresistance and cancer stemness, suggesting that it could be used as a new clinical therapeutic target for treating TNBC patients especially those with drug resistance.

Fig. 1. Nuclear paraspeckle assembly transcript 1 (NEAT1) expression in breast cancer patients.

a The top 5 upregulated long non-coding RNAs in breast cancer patients; b NEAT1 was upregulated in peripheral blood of breast cancer (BC) patients when compared with normal controls (NC); c NEAT1 expression in tissues was higher in triple-negative BC than in other subtypes. ***P < 0.001 indicates statistically different from the control

Fig. 2. Nuclear paraspeckle assembly transcript 1 (NEAT1) is required for breast cancer cell growth.

a NEAT1 knockdown in triple-negative breast cancer cells led to significant decreased cell viability; b colony-formation ability was significantly impeded in NEAT1 stable knockdown cells; c NEAT1 silencing led to G1-phase cell cycle arrest and increased S-phase cell population; d Immunofluorescence staining and e western blotting showed that cyclin E1 and cyclin D1 were decreased upon NEAT1 knockdown; f shNEAT1 significantly enhanced early apoptosis and cleaved caspase-3 was increased in g immunofluorescence staining and h western blot analysis. *P < 0.05; **P < 0.01 indicates statistically different from the control

Fig. 3. Nuclear paraspeckle assembly transcript 1 (NEAT1) played important role in drug resistance.

a NEAT1 expression was upregulated in cisplatin (cisR) and taxol (taxR) resistant cells; b NEAT1 knockdown had synergistic effect with chemotherapeutic drugs by inhibiting cancer cells growth; c, d ATP7A/7B and SOX2 expression were decreased in shNEAT1 cells. *P < 0.05; **P < 0.01; ***P < 0.001 indicates statistically different from the control

Fig. 4. Nuclear paraspeckle assembly transcript 1 (NEAT1) was related to stemness in triple-negative breast cancer.

a NEAT1 knockdown significantly impeded single-cell clonogenic ability; NEAT1 silencing decreased b CD24−/CD44+; c ALDH+, and d SOX2+ cells populations by flow analysis; e western blot demonstrated that SOX2 protein level was decreased in shNEAT1 cells. *P < 0.05; **P < 0.01; ***P < 0.001 indicates statistically different from the control

Fig. 5. Nuclear paraspeckle assembly transcript 1 (NEAT1) knockdown inhibited tumor growth in vivo.

a Gross appearance of xenograft tumors in different groups of mice; b the tumor volume was smaller in shNEAT1-treated mice; c SOX2 mRNA expression was significantly downregulated in shNEAT1-treated xenograft tumors; d representative images of tumor sections on hematoxylin and eosin, aldehyde dehydrogenase, Ki-67, and SOX2 staining; e gross appearance of xenograft tumors; and f tumor volume in four different groups of mice; *P < 0.05, **P < 0.01, ***P < 0.001 indicates statistically different from the control