Circ_0051428 targeting miR-885-3p/MMP2 axis enhances the malignancy of cervical cancer

Abstract

Circular RNAs (circRNAs) are key regulators of cervical cancer (CC) progression. This study aimed to elucidate the role and mechanism of circ_0051428, a novel circRNA, in CC tumorigenesis. Quantitative real-time polymerase chain reaction and western blotting analyses confirmed that circ_0051428 and matrix metalloprotein-2 (MMP2) were overexpressed in CC, whereas the microRNA miR-885-3p was poorly expressed. After performing a series of in vitro and in vivo experiments, circ_0051428 knockdown was shown to repress CC cell invasion and proliferation in vitro, and hamper tumor formation in vivo. Dual-luciferase reporter and RNA-binding protein immunoprecipitation experiments verified that circ_0051428 interacts with miR-885-3p to regulate the target gene MMP2 of miR-885-3p in CC. In addition, miR-885-3p knockdown offset the anticancer effects of circ_0051428 or MMP2 knockdown on CC cell malignancy. Overall, this study revealed that circ_0051428 executes a tumor-promoting function in CC pathogenesis by modulating the miR-885-3p/MMP2 axis. Our findings provide a novel approach for CC treatment.

Keywords: MMP2; cervical cancer; circ_0051428; invasion; miR-885-3p; proliferation.

Figure 1

Circ_0051428 is overexpressed in cervical cancer. (a) The circ_0051428 expressions in normal and CC tissues were estimated via qRT-PCR. P < 0.0001 vs Normal. (b) The circ_0051428 levels in the CC cell lines (CaSki, C33A, HeLa, and SiHa) as well as in the HcerEpic cells were quantified via qRT-PCR. ^** P < 0.01 vs HcerEpic. (c) Circ_0051428 levels within the HeLa and CaSki nuclei and cytoplasm. (d) The circ_0051428 and linear RELB expressions in total cellular RNA incubated with RNase R. ^** P < 0.01 vs Control.

Figure 2

Silencing circ_0051428 represses the invasion and proliferation of cervical cancer cells in vitro as well as the growth of tumor xenograft in vivo. (a) The circ_0051428 expression in CaSki and HeLa cells, following si-circ-1, si-circ-2, or si-NC transfection, was estimated via qRT-PCR. ^** P < 0.01 vs si-NC. (b) HeLa and CaSki cells carrying si-circ-1, si-circ-2, or si-NC were evaluated for viability by means of the CCK-8 experiment. ^** P < 0.01 vs si-NC. (c) The number of invasive HeLa and CaSki cells following their transfection with si-circ-1, si-circ-2, or si-NC was assessed in the Transwell invasion experiment. ^** P < 0.01 vs si-NC. (d) The number of HeLa and CaSki cell colonies following their transfection with si-circ-1, si-circ-2, or si-NC was tallied in the colony formation experiment. ^** P < 0.01 vs si-NC. (e) Pictures, weights, and volumes of xenograft that resulted from injecting each mouse with HeLa cells carrying sh-circ-1, sh-circ-2, or sh-NC. The tumor volume was documented weekly. The tumor weight was measured after 5 weeks. ^** P < 0.01 vs sh-NC.

Figure 3

Circ_0051428 targets miR-885-3p. (a) CircInteractome predicted the binding site between circ_0051428 and miR-885-3p. (b) Luciferase activities in HeLa and CaSki that have a combination of pGL3-circ_0051428 WT/MUT and miR-885-3p mimic/NC had been assessed via the dual luciferase experiment. ^** P < 0.01 vs miR-NC. (c) Circ_0051428’s interaction with miR-885-3p was verified using the results of the RIP experiment. ^** P < 0.01 vs anti-IgG. (d) The miR-885-3p levels in normal and CC tissues were estimated via qRT-PCR. P < 0.0001 vs Normal. (e) The miR-885-3p levels among the CC cell lines (HeLa and CaSki) as well as in HcerEpic cells were gauged by conducting qRT-PCR. **P < 0.01 vs HcerEpic. (f) In CC tissues, the correlation of circ_0051428 expression with that of miR-885-3p had been ascertained using Pearson’s correlation coefficient.

Figure 4

mir-885-3p inhibition partly abates the anti-tumor effect of circ_0051428 knockdown on cervical cancer cell invasion and proliferation. Si-circ, miR-885-3p inhibitor (inhibitor), inhibitor-NC, si-NC, and si-circ + inhibitor were introduced into HeLa and CaSki cells. (a) The miR-885-3p levels in transfected HeLa and CaSki cells were estimated via qRT-PCR. (b) The transfected CaSki and HeLa cells were tested for viability via the CCK-8 experiment. (c) The number of transfected invasive cells was determined in the Transwell invasion experiment. (d) The number of transfected HeLa and CaSki cell colonies was determined by executing the colony formation experiment. ^* P < 0.05, ^** P < 0.01 vs si-NC. ^# P < 0.05, ^## P < 0.01 vs inhibitor-NC. ^& P < 0.05, ^&& P < 0.01 vs si-circ + inhibitor.

Figure 5

mir-885-3p targets MMP2. (a) TargetScan predicted the putative sites between miR-885-3p and MMP2. (b) The target relationship of MMP2 with miR-885-3p was substantiated by the outcomes of the dual luciferase experiment. ^** P < 0.01 vs miR-NC. (c) The amount of MMP2 in CC and normal tissues was estimated via qRT-PCR. P < 0.0001 vs Normal. (d) The MMP2 expression in HcerEpic and CC (HeLa and CaSki) cells was quantified via qRT-PCR. ^** P < 0.01 vs HcerEpic. (e) The association of miR-885-3p expression in CC tissues with that of MMP2 was ascertained using Pearson’s correlation coefficient.

Figure 6

mir-885-3p interacts with MMP2 to influence cervical cancer progression in vitro. si-MMP2-1, si-MMP2-2, inhibitor, inhibitor-NC, si-NC, si-MMP2-1 + inhibitor, and si-MMP2-2 + inhibitor were transfected into CaSki and HeLa cells. (a) The levels of MMP2 protein in the transfected HeLa and CaSki were estimated via western blotting. (b) The viabilities of the transfected CaSki and HeLa were evaluated via the CCK-8 experiment. (c) The number of transfected invasive cells was assessed via the Transwell invasion experiment. (d) The number of transfected CaSki and HeLa cell colonies was assessed via the colony formation experiment. ^** P < 0.01 vs si-NC. ^## P < 0.01 vs inhibitor-NC. ^&& P < 0.01 vs si-MMP2-1 (si-M-1) + inhibitor. ^$$ P < 0.01 vs si-MMP2-2 (si-M-2) + inhibitor.