Circular RNA CDR1as-induced autophagy regulates the proliferation and migration of CD44⁺/CD24^- phenotype breast cancer stem cells in vitro

Figures & data

Figure 1. CDR1as expression in breast cancer. Correlation analysis between CDR1as and clinical prognosis in breast cancer (A), in luminal type (B) and in triple negative breast cancer (C) from TCGA database. D. The CD44⁺/CD24^-phenotype cells were assessed before and after sorting by flow cytometry. The expressions of CDR1as (E) and miR-7 (F) were determined by qRT-PCR in normal breast cells MCF10A, MCF7-Parent cells and CD44⁺/CD24^-MCF7 breast cancer stem cells. **P<0.01 vs. MCF10A or MCF7-Parent cells.

Figure 2. CDR1as enhanced the autophagy of breast CSCs. A. RNA-seq data of autophagy-associated genes in CD44⁺/CD24^- tumorigenic breast cancer cells obtained from GEO dataset of GSE6883. B. Schematic physical map of recombinant vectors of pLEX-EgCDR1as-CvG2L and pLEX-U6siCDR1as-CvG2L. C. The expressions of CDR1as (left) and miR-7 (right) in CD44⁺/CD24^- MCF7 breast cancer stem cells with knockdown or overexpression of CDR1as. D. The expression of LKB1-AMPK-mTOR pathway in CDR1as-knockdown and -overexpression breast cancer stem cells at 24hr after cultured in 1% FBS for autophagy-induction by Western blot. E. The activity of autophagy was assessed by Western blot after cultured in 1% FBS for autophagy-induction for 24hr.

Figure 3. The molecular mechanism of CDR1as in the activation of autophagy. A. Prediction of binding sites between CDR1as and miR-7 by TargetScan. B. The dual luciferase reporter vectors, pmirGLO-wCDR1as and pmirGLO-mCDR1as, were constructed. C. Identification of the interaction between CDR1as and miR-7 by the dual luciferase reporter gene assay. D-E. Expression of ATG4A, ATG7, and LKB1 in CD44⁺/CD24^-MCF7 breast cancer cells via RT-qPCR analysis (D) and western blotting (E).

Figure 4. CDR1as accelerates breast cancer stem cell growth and metastasis. A. Cell proliferation of breast cancer stem cells was assessed by CCK8 assay (n = 3/group). B. Flow cytometry was used to analyze the apoptosis in breast cancer stem cells when they were transfected with either siCDR1as, CDR1as, or CDR1as combined with miR-7 mimics after 48hr or with HCQ after 24hr. (**, P<0.01) C. Cell motilities of breast cancer stem cells transfected with siCDR1as, CDR1as, or CDR1as combined with miR-7 or HCQ were observed at 0 and 48hr following wounding by a pipette tip.

Data availability statement (DAS)

Because the main research has not been completed, participants of this study did not agree for their data to be shared publicly.