RNA-binding protein QKI suppresses breast cancer via RASA1/MAPK signaling pathway

Abstract

Background: RNA-binding protein Quaking (QKI) has been linked with the pathogenesis and development of various human malignancies. Herein, we explored the particular role of QKI in breast cancer (BC) progression.

Methods: The methods employed in the study included public dataset analysis, western blot, quantitative real-time PCR (qRT-PCR), cell count kit-8 (CCK8) assay, colony formation assay, flow cytometric analysis, RNA immunoprecipitation (RIP), messenger RNA (mRNA) stability assay, QKI overexpression and knockdown, and Ras p21 protein activator 1 (RASA1) knockdown.

Results: Aberrant expression levels of QKI and RASA1 were detected in BC and compared with those in noncancerous tissues. A moderately positive correlation between QKI and RASA1 was verified within BC tissues. Low expression of QKI was associated with positive estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status, non-triple-negative breast cancer (TNBC), non-basal-like BC, and poor clinical outcomes in BC patients. QKI overexpression suppressed BC cell proliferation and colony formation, and arrested cell cycle at G1 phase. RIP assay and mRNA stability assay confirmed that QKI directly bound to RASA1 transcript and increased its stability, thus inactivating the MAPK pathway and inhibiting BC progression. RASA1 knockdown could partly attenuate the inhibitory effect of QKI on BC cell proliferation via activating the mitogen-activated protein kinase (MAPK) pathway.

Conclusions: QKI, which was frequently downregulated in BC, could significantly inhibit cell proliferation and arrest cell cycle at G1 phase by binding and enhancing RASA1 mRNA expression. Low expression of QKI was prominently associated with unfavorable clinical outcomes in BC patients, indicating the prognostic value of QKI in BC.

Keywords: Breast neoplasms; RNA-binding proteins; cell cycle; cell proliferation.

Figure 1

Downregulation of QKI in BC and its clinical significance. (A) Expression profile of QKI in BC and normal mammary tissues (data from GEPIA). (B) QKI mRNA expression in 96 paired primary BC samples and adjacent normal tissues (C) QKI protein level in 10 BC tissues comparing to paired noncancerous tissues. *, P<0.05; ***, P<0.001. GEPIA, Gene Expression Profiling Interactive Analysis; QKI, Quaking; N, normal; T, tumor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2.

Figure 2

Low expression of QKI indicated poor prognosis of BC patients (data from PrognoScan). HR, hazard ratio; DSS, disease-specific survival; DFS, disease-free survival; RFS, relapse-free survival; DMFS, distant metastasis-free survival; OS, overall survival.

Figure 3

The correlations between QKI expression and clinicopathological features in BC patients were evaluated using bc-GenExMiner v4.3. ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer.

Figure 4

QKI suppressed the proliferation capacity of BC cells. (A,B) Overexpression and knockdown efficiency of QKI in MCF-7 and T-47D cells was determined by western blot and qRT-PCR. (C,D) QKI significantly repressed cell growth and colony formation of MCF-7 and T-47D cells* (×40)*. (E) Quantification of colonies in colony formation assay. Colonies over 100 cells were scored. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; QKI, Quaking; BC, breast cancer; OD₄₅₀, optical density at 450 nm.

Figure 5

QKI blocked BC cell cycle progression at G1 phase. (A) Effect of QKI on cell cycle progression was detected by flow cytometry. (B) Cell cycle distribution showed that QKI overexpression induced cell cycle arrest at G1 phase. (C,D) Western blot and qRT-PCR analysis of cyclin-dependent kinases and cyclins in indicated BC cells. Data were plotted relative to expression levels in control groups. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; QKI, Quaking; CDK, cyclin-dependent kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6

QKI exerted inhibitory effects through RASA1-mediated inactivation of MAPK pathway. (A) QKI overexpression elevated RASA1 mRNA and protein levels in BC cells. (B,C) Expression of RASA1 and clinical relevance between QKI and RASA1 were examined in 96 paired tissue samples. (D,E) Cells with QKI overexpression were immunoprecipitated using anti-Flag antibody or negative control IgG. The presence of RASA1 mRNA in the immunoprecipitate was determined via ethidium bromide staining and qRT-PCR. (F) QKI-overexpressed cells and corresponding controls were treated with 5 µg/mL actinomycin D for the indicated times. RASA1 mRNA level was detected by qRT-PCR and the decay rates were calculated. **, P<0.01, ***, P<0.001. NC, negative control; QKI, Quaking; RASA1, Ras p21 protein activator 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitate.

Figure 7

Knockdown of RASA1 eliminated QKI-inhibited cell proliferation in BC. (A) Expression levels of phosphorylated Erk1/2, total Erk1/2, phosphorylated p38 MAPK and total p38 MAPK in QKI-overexpressed cells and control cells. (B) RASA1 knockdown restored the phosphorylation level of the MAPK pathway in QKI-overexpressed MCF-7 cells. C,D) Cell proliferation and colony formation ability were examined in QKI-overexpressed MCF-7 cells with RASA1 knockdown* (×40)*. (E) Quantification of colonies in colony formation assay. Colonies over 100 cells were scored. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; QKI, Quaking; MAPK, mitogen-activated protein kinase; RASA1, Ras p21 protein activator 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OD450, optical density at 450 nm.