MicroRNA-218 inhibits EMT, migration and invasion by targeting SFMBT1 and DCUN1D1 in cervical cancer

Abstract

Repeated infection with high-risk HPV is a major cause for the development and metastasis of human cervical cancer, even though the mechanism of the metastasis is still not completely understood. Here, we reported that miR-218 (microRNA-218) was downregulated in cervical cancer tissues, especially in metastatic cancer tissues. We found that miR-218 expression was associated with clinicopathological characteristics of patients with cervical cancer. MiR-218 overexpression inhibited Epithelial-Mesenchymal Transition (EMT), migration and invasiveness of cervical cancer cells in vitro. Moreover, miR-218 repressed the expression of SFMFBT1 (Scm-like with four MBT domains 1) and DCUN1D1 (defective in cullin neddylation 1, domain containing 1) by direct binding to the 3'UTRs of the mRNAs. The overexpression of SFMBT1 induced EMT and increased the migration and invasiveness of cervical cancer cells, while the overexpression of DCUN1D1 increased the migration and invasiveness of these cells, but did not induce EMT. An inverse correlation was observed between the expression of miR-218 and DCUN1D1 protein in cervical cancer tissues. Importantly, HPV16 E6 downregulated the expression of miR-218 in cervical cancer, while miR-218 rescued the promotion effect of HPV16 E6 on the expression of SFMBT1 and DCUN1D1. Taken together, our results revealed that HPV16 E6 promoted EMT and invasion in cervical cancer via the repression of miR-218, while miR-218 inhibited EMT and invasion in cervical cancer by targeting SFMBT1 and DCUN1D1.

Keywords: DCUN1D1; EMT; SFMBT1; cervical cancer; miR-218.

Figure 1. MiR-218 expression is lower in cervical cancer tissues

In situ hybridization was used to detect miR-218 expression in a cervical cancer tissue microarray. A. Three representative cases of miR-218 expression are shown (scale bar, 100μm). Blue arrow demarcate normal cervical tissue; red arrows demarcate cervical cancer tissue. B. The percentage of specimens showing low or high miR-218 expression in relation to clinicopathologic parameters by Fisher's Exact Test. NAT, Cancer adjacent normal cervix tissue. (*P<0.05, **P<0.01, ***P<0.001.)

Figure 2. MiR-218 inhibits cervical cancer cell EMT in vitro

A. Phase contrast microscopy (up, scale bar, 50μm), F-actin staining (down, scale bar, 20μm) of SiHa cells transfected with the negative control (NC), miR-218 mimics, anti-NC, or miR-218 inhibitor for 48 hours. DAPI (4′,6-diamidino-2-phenylindole) staining was used to detect nuclei and is merged with F-actin. B. Immunoblotting of E-cadherin and N-cadherin in SiHa and HeLa cells transfected with NC, miR-218 mimics, anti-NC, or miR-218 inhibitor. β-actin was used as a loading control.

Figure 3. MiR-218 inhibits cervical cancer cell migration and invasiveness in vitro

A. SiHa cells were transfected with NC, miR-218 mimics, anti-NC, or miR-218 inhibitor subjected to transwell migration and invasion assays (scale bar, 100μm). B. HeLa cells were transfected with NC, miR-218 mimics, anti-NC, or miR-218 inhibitor subjected to transwell migration and invasion assays. C. SiHa and HeLa cells transfected with miR-218 inhibitors showed a significant increase in cell migration and invasion. The results are representative of three independent experiments. (**P<0.01, ***P<0.001.).

Figure 4. MiR-218 directly targets the SFMBT1 and DCUN1D1 3′UTR

A. Luciferase activity in SiHa cells transfected with miR-218 mimics or NC after transfection of the indicated 3′UTR-driven reporter constructs. B. Mutation in the putative binding sites of miR-218 in SFMBT1 3′UTRs abrogated the responsiveness to miR-218. Wt, wild-type; mut, mutation at 49–52 nt of the SFMBT1 3′UTR; C. Mutation in the putative binding sites of miR-218 in DCUN1D1 3′UTRs abrogated the responsiveness to miR-218. Wt, wild-type; mut, mutation at 349–352 nt of the DCUN1D1 3′UTR. D. The putative miR-218-binding site in the SFMBT1 and DCUN1D1 3′UTR. E. The protein levels of SFMBT1 and DCUN1D1 were determined by Western blot analyses after transfection with NC, miR-218 mimics, anti-NC, or anti-miR-218 in SiHa cells. β-actin was used as a loading control.

Figure 5. MiR-218 overexpression and SFMBT1 inhibition produce similar changes, which are restored by SFMBT1 ectopic expression in vitro

A. Transwell invasion assays of SiHa cells were performed after transfection with NC, siRNA against SFMBT1, and/or miR-218 as indicated (up); SFMBT1 protein were detected by Western blot analysis (down). B. Transwell invasion assays of SiHa cells were performed after transfection with NC, siRNA against DCUN1D1, and/or miR-218 as indicated (up); DCUN1D1 protein were detected by Western blot analysis (down). The results are representative of at least three independent experiments. C. F-actin staining of SiHa cells transfected as indicated (scale bar, 20μm). D. SiHa cells were transfected with SFMBT1 siRNAs, and then E-cadherin and N-cadherin protein levels were detected by Western blot analysis. β-actin was used as a loading control.

Figure 6. Clinical associations of miR-218 with DCUN1D1 expression

A. Immunostaining shown that DCUN1D1 expression is negatively correlated with miR-218 expression (scale bar, 100μm). The upper 2 photos are from a stage IV case, while the lower 2 photos are from a stage I case. B. Most specimens with high DCUN1D1 expression have low miR-218 expression, while more specimens with low DCUN1D1 expression have high miR-218 expression (^***, P<0.001).

Figure 7. The inhibition of miR-218 on EMT, migration and invasion of cervical cancer cells downregulated by HPV16 E6

A. qRT-PCR analysis of miR-218 expression in SiHa (HPV16⁺), HeLa (HPV18⁺) and C33A (HPV⁻) cells. B. qRT-PCR analysis of miR-218 expression in SiHa or HeLa cells transfected with NC or siRNAs as indicated. RT-PCR data were normalized to β-actin. Experiments were performed three times, and the data are presented as the mean±SEM. C. A graphical representation illustrates the role of the miR-218-mediated pathway in cervical cancer metastasis. D. and E. Transwell invasion assays showed SiHa cells transfected with HPV16 E6 siRNA had decreased invasiveness compared with NC (scale bar, 100μm). F. F-actin staining of SiHa cells transfected with HPV16E6 siRNA or negative control (scale bar, 20μm). G. and H. SiHa cells were transfected with NC or siRNAs, then E-cadherin, N-cadherin, SFMBT1 and DCUN1D1 proteins were detected by Western blot analysis. β-actin was used as a loading control.