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. 2019 Dec 6:18:88-98.
doi: 10.1016/j.omtn.2019.07.023. Epub 2019 Aug 14.

"V体育2025版" circFBXW7 Inhibits Malignant Progression by Sponging miR-197-3p and Encoding a 185-aa Protein in Triple-Negative Breast Cancer

Feng Ye  1 , Guanfeng Gao  1 , Yutian Zou  1 , Shaoquan Zheng  1 , Lijuan Zhang  1 , Xueqi Ou  1 , Xiaoming Xie  2 , Hailin Tang  3
Affiliations

"VSports注册入口" circFBXW7 Inhibits Malignant Progression by Sponging miR-197-3p and Encoding a 185-aa Protein in Triple-Negative Breast Cancer

"V体育官网入口" Feng Ye et al. Mol Ther Nucleic Acids. .

Abstract

Accumulating evidence indicates that circular RNAs (circRNAs) are vital regulators of various biological functions involved in the progression of multiple cancers. Circular F-box and WD repeat domain containing 7 (circFBXW7) (hsa_circ_0001451) has been reported to act as a tumor suppressor by encoding a novel protein in glioma; however, its functions and mechanisms in triple-negative breast cancer (TNBC) remain elusive. In the current study, we validated by qRT-PCR that circFBXW7 was downregulated in TNBC cell lines and found that low expression of circFBXW7 was associated with poorer clinical outcomes. circFBXW7 expression was negatively correlated with tumor size and lymph node metastasis, and it was an independent prognostic factor for TNBC patients. We performed cell proliferation, colony formation, transwell, wound-healing, and mouse xenograft assays to confirm the functions of circFBXW7. Overexpression of circFBXW7 obviously inhibited cell proliferation, migration, and tumor growth in both in vitro and in vivo assays. Luciferase reporter assays and RNA immunoprecipitation assays revealed that circFBXW7 serves as a sponge of miR-197-3p and suppresses TNBC growth and metastasis by upregulating FBXW7 expression VSports手机版. In addition, the FBXW7-185aa protein encoded by circFBXW7 inhibited the proliferation and migration abilities of TNBC cells by increasing the abundance of FBXW7 and inducing c-Myc degradation. In summary, our research demonstrated that circFBXW7 sponges miR-197-3p and encodes the FBXW7-185aa protein to suppress TNBC progression through upregulating FBXW7 expression. Thus, circFBXW7 may act as a therapeutic target and prognostic biomarker for TNBC. .

Keywords: FBXW7; circFBXW7; circular RNAs; competitive endogenous RNAs; triple negative breast cancer V体育安卓版. .

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Figures

Figure 1
Figure 1
circFBXW7 Is Downregulated in TNBC and Correlated with Better Clinical Outcomes (A) The relative expression level of circFBXW7 in breast cancer cell lines. (B and C) Kaplan-Meier analysis of the (B) overall survival and (C) disease-free survival of 473 TNBC patients with high (green) or low (blue) circFBXW7 expression levels. SD is shown as error bars.
Figure 2
Figure 2
Overexpression of circFBXW7 Inhibits the Proliferation and Metastasis of TNBC Cells (A) Overexpression of circFBXW7, as assessed by qRT-PCR. (B) circFBXW7 inhibits the colony-forming ability of BT549 and 4T1 cells. (C) Statistical graph of the colony-forming assays. (D) CCK-8 assays were used to assess cell proliferation. (E) Transwell assays were used to evaluate the cell migration capability. (F) Statistical graph of the transwell assays. (G) Wound-healing assays were used to assess the impact of circFBXW7 on the cell migration ability. (H) Statistical graph of the wound-healing assays. (I) Xenograft models were established. (J) Tumor weights were measured and are shown. (K) H&E-stained sections of lung metastases. (L) The number of metastases was counted and recorded. *p < 0.05, **p < 0.01. SD is shown as error bars.
Figure 3
Figure 3
circFBXW7 Acts as a Sponge for miR-197-3p (A) U6 (nuclear control transcript), GAPDH (cytoplasmic control transcript), and circFBXW7 transcription in the nuclear and cytoplasmic fractions, as analyzed by qRT-PCR. (B) Predicted binding sites of miR-197-3p in circFBXW7. (C) Kaplan-Meier analysis of the association between miR-197-3p and overall survival in patients with breast cancer from TCGA database. (D) The relative expression level of miR-197-3p in breast cancer cell lines. (E and F) Luciferase reporter assay of BT549 (E) and 4T1 (F) cells cotransfected with miR-197-3p mimics and the circFBXW7 wild-type or mutant luciferase reporter. (G) MS2-based RIP assay of cells transfected with MS2bs-circFBXW7, MS2bs-circFBXW7-mt, or control. *p < 0.05, **p < 0.01. SD is shown as error bars.
Figure 4
Figure 4
miR-197-3p Decreases the Expression of the Tumor Suppressor Gene FBXW7 (A) Predicted binding sites of miR-197-3p in the 3′ UTR of FBXW7 mRNA according to TargetScan. (B) The relative expression level of FBXW7 in breast cancer cell lines. (C and D) Luciferase reporter assay of BT549 (C) and 4T1 (D) cells cotransfected with miR-197-3p mimics and the FBXW7 3′ UTR wild-type or mutant luciferase reporter. (E) The expression of FBXW7 was decreased after transfection with miR-197-3p mimics. (F) The expression of FBXW7 was increased after transfection with miR-197-3p inhibitors. (G and H) Kaplan-Meier analysis of the (G) overall survival and (H) disease-free survival of 473 TNBC patients with high (green) or low (blue) FBXW7 expression levels. *p < 0.05, **p < 0.01. SD is shown as error bars.
Figure 5
Figure 5
circFBXW7 and FBXW7 Act as ceRNAs in TNBC through the Regulation of miR-197-3p (A) Enrichment of circFBXW7, FBXW7, and miR-197-3p on Ago2, as assessed by a RIP assay. (B) The enrichment of Ago2 on circFBXW7 was decreased, while FBXW7 expression was increased after knockdown of circFBXW7. (C) Silencing FBXW7 reduced the relative enrichment of Ago2 on FBXW7 and increased the abundance of circFBXW7. (D) Knockdown of circFBXW7 resulted in a reduction in FBXW7 expression, which was reversed by miR-197-3p inhibitors. (E) Spearman correlation analysis showed that circFBXW7 expression was positively correlated with FBXW7 expression in 473 TNBC samples (r = 0.568, p < 0.001). *p < 0.05, **p < 0.01. SD is shown as error bars.
Figure 6
Figure 6
FBXW7-185aa, Encoded by circFBXW7, Inhibited the Proliferation and Metastasis of TNBC Cells through Downregulating c-Myc Expression (A) Schematic diagram of plasmid construction. The linearized FBXW7-185aa ORF and a FLAG tag were cloned downstream of a cytomegalovirus (CMV) promoter (FBXW7-185aa-FLAG). (B) The expression level of circFBXW7 was measured in BT549 cells after transfection with the FBXW7-185aa-FLAG vector. (C–E) FBXW7-185aa inhibits (C) cell proliferation, as assessed by CCK-8 assays; (D) the colony-forming ability and (E) migration ability were assessed via transwell assays. (F and G) The prooncogenic effect enhanced by sh-circFBXW7 was reversed after cotransfection with miR-197-3p inhibitors and the FBXW7-185aa protein expression plasmid, as evaluated by (F) colony formation assays and (G) transwell assays. (H) FBXW7-185aa-FLAG-overexpressing BT549 cells were cotransfected with sh-FBXW7 or USP28 vectors. The FBXW7-185aa-FLAG, FBXW7, USP28, and c-Myc expression levels were quantified by western blot analysis. (I) The schematic illustrates the biological mechanism of circFBXW7, which sponges miR-197-3p and encodes FBXW7-185aa to suppress TNBC progression through upregulating FBXW7 expression. SD is shown as error bars.
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