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. 2018 Dec 7:13:55-63.
doi: 10.1016/j.omtn.2018.08.010. Epub 2018 Aug 22.

hsa_circ_0061140 Knockdown Reverses FOXM1-Mediated Cell Growth and Metastasis in Ovarian Cancer through miR-370 Sponge Activity

Qizhen Chen  1 , Jiarong Zhang  2 , Yinyan He  3 , Yanqiu Wang  4
Affiliations

hsa_circ_0061140 Knockdown Reverses FOXM1-Mediated Cell Growth and Metastasis in Ovarian Cancer through miR-370 Sponge Activity

"VSports手机版" Qizhen Chen et al. Mol Ther Nucleic Acids. .

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"VSports注册入口" Abstract

Circular RNAs (circRNAs) are a class of noncoding RNAs that regulate gene expression at the posttranscriptional level. The specific functions of circRNAs in ovarian cancer are yet to be established. Previous sequencing analyses have revealed an abnormal expression of hsa_circ_0061140 in ovarian cancer VSports手机版. The main aim of the present study is to establish the specific role of hsa_circ_0061140 in ovarian cancer. circRNA expression in ovarian cancer cells was detected via real-time qPCR. The effects on specific cellular characteristics (proliferation, migration, and the EMT) and subcellular localization of hsa_circ_0061140 were assessed via RNA fluorescence in situ hybridization, knockdown, and luciferase reporter assays in the SKOV3 and A2780 cell lines. Tumorigenesis was induced in nude mice to assess the effects of hsa_circ_0061140 on ovarian cancer growth in vivo. Our results showed that hsa_circ_0061140 was upregulated in ovarian cancer cell lines. Knockdown of hsa_circ_0061140 suppressed cell proliferation and migration, both in vivo and in vitro, by inhibiting FOXM1 expression through sponging miR-370. Overexpression of FOXM1 or suppression of miR-370 rescued hsa_circ_0061140 silencing-induced inhibition of cell proliferation, migration, and the EMT. The associations among hsa_circ_0061140, miR-370, and FOXM1 were confirmed via bioinformatic prediction and fluorescein reporter experiments. Thus, hsa_circ_0061140 appeared to function as a competing endogenous RNA of miR-370 that promoted cell growth and metastasis in ovarian cancer through regulation of the miR-370/FOXM1 pathway mediating EMT. .

Keywords: FOXM1; Ovarian cancer; cell proliferation and metastasis; hsa_circ_0061140; miR-370 V体育安卓版. .

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Figures

Figure 1
Figure 1
Expression and Subcellular Localization of hsa_circ_0061140 in Ovarian Cancer Cell Lines (A) Real-time qPCR showing expression of hsa_circ_0061140 in primary cultured normal ovarian epithelial and ovarian cancer cell lines. Data are presented as means ± SD (***p < 0.001 versus normal). (B) In situ hybridization was applied to determine the subcellular localization of hsa_circ_0061140.
Figure 2
Figure 2
Knockdown of hsa_circ_0061140 Suppresses Ovarian Cancer Cell Proliferation and Invasion (A and B) Expression of hsa_circ_0061140 in SKOV3 (A) and A2780 (B) cells detected via real-time qPCR after transfection with hsa_circ_0061140 interference vector (sicircRNA) or negative control (NC) for 48 hr. Data are presented as means ± SD (***p < 0.001 versus control). (C and D) Evaluation of cell proliferation with the CCK8 assay. (C) SKOV3 cells. (D) A2780 cells. Data are presented as means ± SD (***p < 0.001 versus NC). OD450 nm, optical density. (E–H) Wound-healing assay showing the effect of hsa_circ_0061140 on closure of scratch wounds in both SKOV3 (E and F) and A2780 (G and H) cells. Data are presented as means ± SD (***p < 0.001 versus NC). (I–L) Cell migration was assessed in SKOV3 (I and J) and A2780 (K and L) cells using the Transwell assay. Data are presented as means ± SD (***p < 0.001 versus NC). (M and N) Western blot of the expression of FOXM1 and EMT-related proteins Snail, E-cadherin, and vimentin in (M) SKOV3 and (N) A2780 cells. Data are presented as means ± SD (***p < 0.001 versus NC).
Figure 3
Figure 3
Downregulation of miR-370 Suppresses Ovarian Cancer Cell Proliferation and Invasion (A and B) Expression of miR-370 in SKOV3 (A) and A2780 (B) cells detected via real-time qPCR after transfection with miR-370 inhibitor or sicircRNA for 48 hr. Data are presented as means ± SD (***p < 0.001 versus control). (C and D) Evaluation of proliferation in both SKOV3 (C) and A2780 (D) cells with the CCK8 assay. Data are presented as means ± SD (***p < 0.001 versus control). (E–H) Wound-healing assays showing the effect of miR-370 on closure of scratch wounds in both SKOV3 (E and F) and A2780 (G and H) cells. Data are presented as mean values ± SD (***p < 0.001 versus control; ###p < 0.001 versus sicircRNA). (I–K) Transwell assay assessing cell migration in SKOV3 (I and J) and A2780 (I and K) cells. Data are presented as means ± SD (***p < 0.001 versus control; ###p < 0.001 versus sicircRNA). (L and M) Western blots of the expression of FOXM1 and the EMT-related proteins Snail, E-cadherin, and vimentin in SKOV3 (L) and A2780 (M) cells.
Figure 4
Figure 4
The miR-370 Is a Direct Target of hsa_circ_0061140 (A) Predicted binding sites of miR-370 and hsa_circ_0061140 and mutated hsa_circ_0061140. (B) Relative luciferase activity was determined 48 hr after transfection with miR-370 mimic/NC or hsa_circ_0061140 wild-type (WT)/Mutant (Mut) in 293T cells. Data are presented as means ± SD (***p < 0.001 versus control). (C) In situ hybridization showing that downregulation of hsa_circ_0061140 reverses the sponge effect on miR-370.
Figure 5
Figure 5
FOXM1 Is a Direct Target of miR-370 (A) Predicted binding sites of miR-370 in the 3′ UTR of FOXM1. The mutated FOXM1 3′ UTR sequence is presented. (B) Relative luciferase activity was determined 48 hr after transfection with miR-370 mimic/NC or the 3′ UTR of FOXM1 wild-type/Mut in 293T cells. Data are presented as means ± SD (***p < 0.001 versus control). (C and D) Real-time qPCR detection of miR-370 expression after transfection with miR-600 mimic or negative control (NC) in SKOV3 (C) and A2780 (D) cells. Data are presented as means ± SD (***p < 0.001 versus control). (E and F) Western blot of FOXM1 protein expression after transfection with miR-370 mimics in both SKOV3 (E) and A2780 (F) cells. Data are presented as means ± SD (***p < 0.001 versus control). (G and H) Assessment of cell proliferation in SKOV3 (G) and A2780 (H) cells with the CCK8 assay. Data are presented as means ± SD (***p < 0.001 versus control). (G) SKOV3 cells. (H) A2780 cells. (I–L) Wound-healing assays showing the effects of FOXM1 on SKOV3 (I and J) and A2780 (K and L) cells on scratch wound closure (***p < 0.001 versus control; ###p < 0.001 versus mimic). (M–O) Cell migration was assessed in SKOV3 (M and N) and A2780 (M and O) cells using the Transwell assay. Data are presented as means ± SD (***p < 0.001 versus control; ###p < 0.001 versus mimic). (P and Q) Western blot of the expression of EMT-related proteins Snail, E-cadherin, and vimentin in SKOV3 (P) and A2780 (Q) cells.
Figure 6
Figure 6
Silencing of hsa_circ_0061140 Suppresses Ovarian Cancer Growth Xenotransplantation studies with SKOV3 were performed in BALB/c nude mice. (A) Representative images of tumor formation in xenografts of nude mice. (B) Tumor volumes were measured every 5 days for 30 days. Data are presented as means ± SD (***p < 0.001 versus NC). (C and D) Real-time qPCR analysis of miR-370 (C) and FOXM1 (D) expression. Data are presented as means ± SD (***p < 0.001 versus control). (E) Western blot detection of FOXM1 and the EMT-related proteins Snail, E-cadherin, and vimentin is presented. Data are presented as means ± SD (***p < 0.001 versus control).
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