Circ SMARCA5 Inhibited Tumor Metastasis by Interacting with SND1 and Downregulating the YWHAB Gene in Cervical Cancer

Abstract

Cervical cancer is one of the diseases that seriously endanger women's health. Circular RNA plays an important role in regulating the occurrence and development of cervical cancer. Here, we investigated the mechanisms of circ SMARCA5 in the development of cervical cancer. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) results showed that the expression of SMARCA5 was downregulated in cervical cancer tissues and cell lines. Then we found that overexpression of SMARCA5 inhibited proliferation and invasion, but promoted apoptosis in cervical cancer cells. These were detected by Cell Counting Kit-8, Transwell, and Annexin V-fluorescein isothiocyanate/propidium iodide detection kit, respectively, and the expression of the apoptosis-related proteins was determined by western blotting. Then we predicted that SMARCA5 combined with Staphylococcal nuclease domain-containing 1 (SND1) by starBase, and verified by RNA pull-down assay. To further reveal the molecular mechanisms of SMARCA5 in the progression of cervical cancer, the interaction protein of SND1 was predicted by STRING, and the interaction was verified by co-immunoprecipitation assay. Then, the effects of SND1 or YWHAB on the development of cervical cancer were detected by the gain and loss function test, and we found that knockdown of SND1 or YWHAB reversed the effects of SMARCA5 short interfering RNA on proliferation, invasion, and apoptosis of cervical cancer cells. Overexpression of SMARCA5 inhibited cervical cancer metastasis in vivo. Our results showed that overexpression of circ SMARCA5 inhibits the binding of SND1 to YWHAB, and inhibits the proliferation and invasion, but promotes apoptosis in cervical cancer cells, thus inhibiting the metastasis of cervical cancer.

Keywords: Circ SMARCA5; SND1; YWHAB; cervical cancer; tumor metastasis.

Figure 1.

The expression of SMARCA5 was downregulated in cervical cancer. (A) The expression of SMARCA5 in cervical cancer tissue and adjacent tissue samples collected from 20 patients (aged 44.52 ± 10.73 years) was detected by RT-qPCR. n = 20. **P < 0.01, unpaired t-test (B) The expression of SMARCA5 in normal cervical cell line Ect1/E6E7 and different cervical cancer cell lines (Hela, HT-3, C33A, and CaSki) was detected by RT-qPCR. n = 3. *P < 0.05, **P < 0.01, one-way analysis of variance. RT-qPCR: quantitative reverse transcriptase polymerase chain reaction.

Figure 2.

Overexpression of SMARCA5 inhibited proliferation and invasion but promoted apoptosis in cervical cancer cells. SMARCA5 was overexpressed or silenced in HeLa cells. (A) The transfection efficiency was detected by RT-qPCR. n = 3. **P < 0.01, one-way ANOVA. (B) The cell proliferation was evaluated by Cell Counting Kit-8 assay. n = 3. **P < 0.01, one-way ANOVA. (C) The invasion of HeLa cells was tested by Transwell assay. n = 3. *P < 0.05, one-way ANOVA. (D-E) The cell apoptosis was evaluated using Annexin V-FITC/PI detection kit. The expression of anti-apoptotic proteins was detected by western blotting, and the quantification was performed using Image J software. n = 3. *P < 0.05, **P < 0.01, one-way ANOVA. ANOVA: analysis of variance; FITC: fluorescein isothiocyanate; NC: negative control; PI: propidium iodide.

Figure 3.

SND1 functioned as an RBP for SMARCA5, and the expression of SND1 was upregulated in cervical cancer cells. (A) The binding site of SMARCA5 and SND1 was predicted by starBase. (B) The expression of SND1 in normal cervical cell line and different cervical cancer cell lines was detected by western blotting, and the quantification was performed using Image J software. n = 3. *P < 0.05, **P < 0.01, one-way ANOVA. (C) RNA pull-down assay was performed to further verify the combination of SMARCA5 and SND1. n = 3. **P < 0.01, one-way ANOVA. ANOVA: analysis of variance; NC: negative control.

Figure 4.

Overexpression of SND1 promoted proliferation and invasion, and inhibited apoptosis in cervical cancer cells. SND1 was overexpressed or silenced in HeLa cells. (A) The transfection efficiency was detected by western blotting, and the quantification was performed using Image J software. n = 3. *P < 0.05, **P < 0.01, one-way ANOVA. (B-D) The cell proliferation, invasion, and apoptosis were evaluated by Cell Counting Kit 8, Transwell, and Annexin V-FITC/PI assay, respectively. n = 3. *P < 0.05, **P < 0.01, one-way ANOVA. (E) The expression of anti-apoptotic proteins was detected by western blotting, and the quantification was performed using Image J software. n = 3. *P < 0.05, one-way ANOVA. ANOVA: analysis of variance; FITC: fluorescein isothiocyanate; NC: negative control; PI: propidium iodide.

Figure 5.

The expression of YWHAB was upregulated in cervical cancer cells, and SND1 upregulated the expression of YWHAB. (A) The interaction between SND1 and YWHAB was verified by co-immunoprecipitation assay. (B) The expression of YWHAB in normal cervical cell line and different cervical cancer cell lines was detected by western blotting, and the quantification was performed using Image J software. n = 3. *P < 0.05, **P < 0.01, one-way ANOVA. (C) SND1 was overexpressed or silenced in HeLa cells. The expression of YWHAB was detected by western blotting, and the quantification was performed using Image J software. n = 3. *P < 0.05, **P < 0.01, one-way ANOVA. ANOVA: analysis of variance; IgG: immunoglobulin G; NC: negative control.

Figure 6.

Overexpression of YWHAB reversed the effects of SND1 siRNA on the proliferation, invasion, and apoptosis of cervical cancer cells. The HeLa cells were transfected with SND1 siRNA alone or together with pcDNA-YWHAB. (A) The expression of YWHAB was detected by western blotting, and the quantification was performed using Image J software. n = 3. **P < 0.01, one-way ANOVA. (B-D) The changes in cell proliferation, invasion, and apoptosis were detected by Cell Counting Kit-8, Transwell, and Annexin V-FITC/PI detection kit, respectively. n = 3. **P < 0.01, one-way ANOVA. (E) The expression of apoptosis-related proteins was detected by western blotting, and the quantification was performed using Image J software. n = 3. *P < 0.05, one-way ANOVA. ANOVA: analysis of variance; FITC: fluorescein isothiocyanate; NC: negative control; OD: optical density; PI: propidium iodide.

Figure 7.

Knockdown of SND1 or YWHAB reversed the effects of SMARCA5 siRNA on the proliferation, invasion, and apoptosis of cervical cancer cells. The HeLa cells were transfected with SMARCA5 siRNA alone or together with SND1 siRNA or YWHAB siRNA. (A-B) The expression of SMARCA5 was detected by RT-qPCR. The expression of SND1 and YWHAB was detected by western blotting, and the quantification was performed using Image J software. n = 3. *P < 0.05, **P < 0.01, one-way ANOVA. (C-E) The changes in cell proliferation, invasion, and apoptosis were detected by Cell Counting Kit-8, Transwell, and Annexin V-FITC/PI detection kit, respectively. n = 3. **P < 0.01, one-way ANOVA. (F) The expression of apoptosis-related proteins was detected by western blotting, and the quantification was performed using Image J software. n = 3. *P < 0.05, one-way ANOVA. ANOVA: analysis of variance; FITC: fluorescein isothiocyanate; NC: negative control; OD: optical density; PI: propidium iodide.

Figure 8.

Overexpression of SMARCA5 inhibited cervical cancer metastasis in vivo. HeLa cells transfected with the pLO-ciR empty vector or pLO-ciR-SMARCA5 were inoculated into nude mice to establish subcutaneous xenograft tumor models. (A) The tumor volume was monitored every week. n = 3. **P < 0.01, one-way ANOVA. After the mice were sacrificed, (B) the tumor mass was measured, (C-E) the expression of SMARCA5 was detected by RT-qPCR, and the expressions of SND1, YWHAB, and anti-apoptotic proteins were detected by western blotting, the quantification was performed using Image J software. n = 3. *P < 0.05, **P < 0.01, one-way ANOVA. ANOVA: analysis of variance.