Circular RNA 0001823 aggravates the growth and metastasis of the cervical cancer cells through modulating the microRNA-613/RAB8A axis

Abstract

Cervical cancer (CC) is a gynecological cancer, which has become the second malignant tumor with mortality in developing countries. The purpose of current study was to explore the influence of Circular RNA 0001823 (circ_0001823) in the CC development. Thirty CC tissues and paracancerous tissues were obtained, and Hela and CaSki CC cells were purchased for this study. The cell growth was analyzed by CCK-8 and colony formation assays. The cell metastasis was determined with Transwell assay. The circ_0001823, miR-613, and RAB8A expression were analyzed with qRT-PCR analysis. The specific mechanisms of circRNA_0001823 were analyzed by Dual luciferase reporter and RNA pull-down assays. The circ_0001823 and RAB8A expressions were increased, and miR-613 were decreased in the CC cells and tissues. Knockdown of circ_0001823 inhibited the malignant behavior of the CC cells, which was antagonized by miR-613 inhibitor. Over-expressed RAB8A reversed the miR-613 effects in the CC cells. Knockdown of circ_0001823 inhibited the malignant behaviors of the CC cells via regulating the miR-613/RAB8A axis.

Keywords: Cervical cancer; RAB8A; metastasis; miR-613; proliferation.

Graphical abstract

Figure 1.

Circ_0001823 was over-expressed in CC tissues as well as cells.A Volcano map showed significant differentially expressed circRNAs via log2-fold change and log10 p-values. The circ_0001823 expression in the CC patients (b) and CC cells (c) were measured by qRT-PCR asssay. D-E Verification of circ_0001823 stability. *P < 0.05. **P < 0.01.

Figure 2.

Circ_0001823 knockdown inhibited the proliferation of the CC cells. A Validation of sh-circ_0001823 transfection efficiency. B-C After sh-circ_0001823 transfection, CCK-8 and colony formation assays were performed to measure the cell viability and cloned cells numbers. **P < 0.01.

Figure 3.

Circ_0001823-silenced inhibited the malignant behavior of the CC cells. A-B After sh-circ_0001823 transfection, transwell assay was conducted to analyze the migration and invasion of the CC cells . **P < 0.01.

Figure 4.

Circ_0001823 overexpression promoted the proliferation of the CC cells. A Validation of oe-circ_0001823 transfection efficiency. B-C After oe-circ_0001823 transfection, CCK-8 and colony formation assays were performed to measure the cell viability and cloned cells numbers. *P < 0.05. **P < 0.01.

Figure 5.

Circ_0001823-overexpressed promoted the malignant behavior of the CC cells. A-B After oe-circ_0001823 transfection, transwell assay was conducted to analyze the migration and invasion of the CC cells . *P < 0.05.

Figure 6.

Circ_0001823 acted as a miR-613 sponge in CC cells. A The predicted circ_0001823 binding site in the miR-613 3’-UTR. B-C Double Luciferase Report and RNA pull-down assays were carried out to confirmed circ_0001823 could bind to miR-613. D The miR-613 expression in the CC cells was measured with qRT-PCR assay after sh-circ_0001823 transfection. E-F The miR-613 expression in the CC tissues as well as cells was detected with qRT-PCR assay. **P < 0.01.

Figure 7.

miR-613 inhibitor treatment reversed the effects of sh-circ_0001823 on the cell viability as well as cloned cells numbers in the CC cells.A Validation of miR-613 inhibitor or mimic transfection efficiency. B-C After sh-circ_0001823 and miR-613 inhibitor transfection, the cell viability and cloned cells numbers were measured by CCK-8 and colony formation assays. **P < 0.01 VS control group. #P < 0.05, ##P < 0.01 VS sh-circ_0001823+ miR-613 inhibitor nc group.

Figure 8.

miR-613 inhibitor treatment inverted the effects of sh-circ_0001823 on the metastasis of the CC cells. A-B After sh-circ_0001823 as well as miR-613 inhibitor transfection, transwell assay was performed to detect the migration and invasion of the CC cells. **P < 0.01 VS control group. ##P < 0.01 VS sh-circ_0001823+ miR-613 inhibitor nc group.

Figure 9.

RAB8A targeted to miR-613 in CC cells. A The predicted RAB8A binding site in the miR-613 3’-UTR. B the 3D structure of RAB8A protein. C-D Double Luciferase Report and RNA pull-down assays were carried out to confirmed RAB8A could bind to miR-613. E The RAB8A expression in the CC cells was measured with qRT-PCR assay after sh-circ_0001823 and miR-613 inhibitor transfection. F-G The RAB8A expression in the CC tissues as well as cells was detected with qRT-PCR assay.

Figure 10.

RAB8A-silenced inhibited the malignant behavior of the CC cells. A Validation of sh-RAB8A transfection efficiency. B-C After sh-RAB8A transfection, CCK-8 and colony formation assays were performed to measure the cell viability and cloned cells numbers. D-E After sh-RAB8A transfection, transwell assay was conducted to analyze the migration and invasion of the CC cells . **P < 0.01.

Figure 11.

Over-expressed RAB8A inverted the effects of miR-613 mimic on the cell viability and cloned cells numbers of the CC cells. A Validation of RAB8A transfection efficiency. B-C After RAB8A and miR-613 mimic transfection, the cell viability and cloned cells numbers were measured by CCK-8 and colony formation assays. **P < 0.01 VS control group. #P < 0.05, ##P < 0.01 VS miR-613 mimic+vector group.

Figure 12.

Over-expressed RAB8A inverted the effects of miR-613 mimic on the metastasis of the CC cells. A-B After RAB8A and miR-613 mimic transfection, the migration and invasion of the CC cells were determined with transwell assay. **P < 0.01 VS control group. ##P < 0.01 VS miR-613 mimic+vector group.