Circ-ABCB10 knockdown inhibits the malignant progression of cervical cancer through microRNA-128-3p/ZEB1 axis

Abstract

Aims: We focused on the detailed functions of circ-ABCB10 in cervical cancer (CC) development and its mechanisms.

Background: The increasing findings have proposed the central roles of circular RNAs (circRNAs) in the tumorigenesis of various human cancers. Circ-ABCB10 displays promising oncogenic effect in several tumors.

Methods: Circ-ABCB10 and miR-128-3p production levels in CC tissues and cells were tested through RT-qPCR. The association of circ-ABCB10 expression with clinicopathologic parameters of CC patients was statistically analyzed. Cell proliferation, invasion, apoptosis, and epithelial-mesenchymal transition (EMT) were evaluated by MTT, transwell invasion assays, flow cytometry analyses, and western blot examination of EMT markers. The binding activity between miR-128-3p and circ-ABCB10 or zinc finger E-box binding homeobox 1 (ZEB1) was explored through pull-down assay or luciferase reporter assay. The influence of circ-ABCB10 on CC tumorigenesis was evaluated by in vivo xenograft experiments.

Results: The elevated circ-ABCB10 expression was determined in CC tissues and cells. Moreover, higher production level of circ-ABCB10 was close related to lymph-node metastasis, Federation of Gynecology and Obstetrics (FIGO) stage, and tumor size in CC patients. Loss of circ-ABCB10 weakened cell proliferative and invasive abilities, inhibited EMT, and induced apoptosis in CC. Loss of circ-ABCB10 inhibited ZEB1 expression by serving as a sponge of miR-128-3p in CC cells. Circ-ABCB10 sponged miR-128-3p to enhance cell proliferation, invasion, EMT and inhibit apoptosis in CC cells. Xenograft tumor assays confirmed that circ-ABCB10 knockdown inhibited CC tumor growth.

Conclusion: Our study suggests that circ-ABCB10 depletion inhibits proliferation, invasion and EMT and promotes apoptosis of cervical cancer cells through miR-128-3p/ZEB1 axis and represses CC tumor growth.

Keywords: Apoptosis; Cervical cancer; Circ-ABCB10; Invasion; Proliferation; ZEB1; miR-128-3p.

Fig. 1

Circ-ABCB10 expression analyses in CC tissues and cells. A-C Circ-ABCB10 expression levels were examined by RT-qPCR assays in CC tissues and adjacent normal tissues (A), CC tumors with different size (B), 5 CC cell lines and a normal cervical cell line (C). *P < 0.05

Fig. 2

Loss of circ-ABCB10 suppressed cell proliferation, invasion and EMT and induced apoptosis in CC cells. A-E Hela and C33A cells were infected with sh-Ctrl or sh-circ-ABCB10 lentiviruses. A Circ-ABCB10 expression levels were examined through qRT-PCR method. B Cell proliferative activity was estimated through MTT assays. C Cell invasive ability was assessed through transwell invasion assays. D E-cadherin, N-cadherin, Vimentin, and Snail production levels were measured through western blotting assays. E Apoptosis was assessed using flow cytometry analyses. *P < 0.05

Fig. 3

Circ-ABCB10 served as a molecular sponge of miR-128-3p in CC cells. A Putative complementary sites between circ-ABCB10 and miR-128-3p. B Luciferase activities were measured at 48 h post transfection. C The interaction between circ-ABCB10 and miR-128-3p in cells was further confirmed by pull-down assays. D The levels of miR-128-3p in CC tissues (n = 34) by RT-qPCR assay. E A negative correlation between miR-128-3p and circ-ABCB10 expressions in CC tissues. F MiR-128-3p level was determined by RT-qPCR assay in CC cells. G The effects of circ-ABCB10 loss or overexpression on miR-128-3p expression were tested through RT-qPCR assays. *P < 0.05

Fig. 4

miR-128-3p curbed cell proliferation, invasion, and EMT and promoted cell apoptosis and circ-ABCB10 reversed those effects in CC. A MiR-128-3p levels were examined through RT-qPCR assays in Hela and C33A cells infected with miR-128-3p or miR-Ctrl lentiviruses. B-E Hela and C33A cells were infected with miR-128-3p or miR-Ctrl along with control or circ-ABCB10 lentiviruses, followed by the detection of cell proliferative (B) and invasive (C) abilities, EMT marker expression levels (D) and cell apoptotic rate (E). *P < 0.05

Fig. 5

Circ-ABCB10 promoted ZEB1 expression by acting as a molecular sponge of miR-128-3p in CC cells. A Putative binding sites between miR-128-3p and ZEB1 3’UTR. B Luciferase activities were measured at 48 h after transfection. C ZEB1 protein levels were examined through western blotting assays at 48 h after transfection. *P < 0.05

Fig. 6

ABCB10 depletion inhibited the growth and EMT of CC xenograft tumors via miR-128-3p/ZEB1 axis. Mice were randomly divided into sh-ctrl or sh-circ-ABCB10 group with 6 mice per group. C33A cells stably transduced with sh-ctrl or sh-circ-ABCB10 lentiviruses were inoculated into the subcutaneous tissues of mice in sh-ctrl or sh-circ-ABCB10 group, respectively. A The tumor volume was monitored and recorded every 5 days. B At day 30, tumors were resected and weighed. C IHC analyses for Ki67 in xenograft tumors in sh-Ctrl and sh-circ-ABCB10 groups. D MiR-128-3p levels were tested through RT-qPCR assays in xenograft tumors. E ZEB1, E-cadherin, N-cadherin, Vimentin and Snail in xenograft tumors production levels were examined through western blotting assays. *P < 0.05