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. 2015 Sep 25;5:56.
doi: 10.1186/s13578-015-0047-5. eCollection 2015.

MicroRNA-720 Promotes in Vitro Cell Migration by Targeting Rab35 Expression in Cervical Cancer Cells

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Free PMC article

MicroRNA-720 Promotes in Vitro Cell Migration by Targeting Rab35 Expression in Cervical Cancer Cells

Yunlan Tang et al. Cell Biosci. .
Free PMC article

Abstract

Background: MicroRNA-720 (miR-720), a nonclassical miRNA, is involved in the initiation and progression of several tumors. In our previous studies, miR-720 was shown to be significantly upregulated in cervical cancer tissues compared with normal cervical tissues. However, the precise biological functions of miR-720, and its molecular mechanisms of action, are still unknown.

Results: Microarray expression profiles, luciferase reporter assays, and western blot assays were used to validate Rab35 as a target gene of miR-720 in HEK293T and HeLa cells. The regulation of Rab35 expression by miR-720 was assessed using qRT-PCR and western blot assays, and the effects of exogenous miR-720 and Rab35 on cell migration were evaluated in vitro using Transwell(®) assay, wound healing assay, and real-time analyses in HeLa cells. The influences of exogenous miR-720 on cell proliferation were evaluated in vitro by the MTT assay in HeLa cells. In addition, expression of E-cadherin and vimentin associated with epithelial-mesenchymal transition were also assessed using western blot analyses after transfection of miR-720 mimics and Rab35 expression vectors. The results showed that the small GTPase, Rab35, is a direct functional target of miR-720 in cervical cancer HeLa cells. By targeting Rab35, overexpression of miR-720 resulted in a decrease in E-cadherin expression and an increase in vimentin expression and finally led to promotion of HeLa cell migration. Furthermore, reintroduction of Rab35 3'-UTR(-) markedly reversed the induction of cell migration in miR-720-expressing HeLa cells.

Conclusions: The miR-720 promotes cell migration of HeLa cells by downregulating Rab35. The results show that miR-720 is a novel cell migration-associated gene in cervical cancer cells.

Keywords: Cell migration; Cervical cancer cells; Rab35; miR-720.

Figures

Fig. 1
Fig. 1
MiR-720 promotes cell migration but does not affect cell proliferation in HeLa cells. a The qRT-PCR analysis of miR-720 expression indicated that HeLa cells transfected with miR-720 mimics (M-720) showed a significant increase in miR-720 expression compared with cells transfected with the microRNA mimic negative control (M-NC). b Representative photographs of wound healing assays (100×) show that HeLa cells transfected with miR-720 mimics result in a significant improvement in wound healing ability. The statistical results of cell migration area measured by Image J software demonstrate a significant difference in cell migration ability caused by overexpression of miR-720. **p < 0.01. c Transwell® migration assay shows that the upregulation of miR-720 dramatically enhances cell migration ability. The top panel shows representative photographs of the Transwell® migration assay and the bottom panel shows the statistical results. **p < 0.01. d Twenty-four hours after transfection, real-time cell analysis (RTCA) experiments were performed to contrast cell migration indices between cells transfected with miR-720 mimics and the microRNA mimic negative control. The results show that after 8 h, the migration indices of cells transfected with miR-720 mimics were greater than the controls. e The results of qRT-PCR analysis of miR-720 expression show that the miR-720 inhibitors can effectively knockdown miR-720 in HeLa cells. **p < 0.01. The (3-4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays show upregulation (f) or downregulation (g) of miR-720, does not show an effect on cell proliferation viability in HeLa cells
Fig. 2
Fig. 2
Identification of miR-720 targets. a Microarray assays were performed on HEK293T cells transfected with pre-miR-720 and the pre-miR-control. The red dots represent genes upregulated with a ≥2-fold change, the green dots represent genes downregulated with a >0.5-fold change, and the gray dots indicate genes with expression levels ranging from −0.5-fold change to +2-fold change in the scatterplot. b The Venn diagram shows the relationships between the mRNA microarray and two miRNA-target prediction algorithms on the quantity of miR-720 targets. The TargetScan program and the miRanda program predicted that 827 candidate genes and 1328 candidate genes, respectively, were possible targets of miR-720. Expressions of 192 genes in HEK293T cells were changed ≥2-fold with a p value cut-off of 0.05 by ectopic expression of pre-miR-720. Among these 192 genes, 14 and 20 were predicted by TargetScan 5.1 and miRanda, respectively. Among these 20 genes, 10 were classified as the intersection targets. c The heat map shows the change in expression levels of 10 genes with overexpression of miR-720
Fig. 3
Fig. 3
Dual luciferase reporter assays verify that miR-720 regulates its targets by binding directly to their 3′-UTR. a The diagram shows that 3′-UTR of Rab35 was cloned downstream into the open reading frame of the pcDNA3.0-Vector, and also displays the detailed binding sites or the mutated binding sites of the 3′-UTR for miR-720 targeting; b The results of analysis indicate that the relative luciferase activities of the pMIR-REPORT vector containing wide-type 3′-UTR of INSIG1, KCTD15, METTL2B, NRXN3, Rab35, RASAL2, or TET2 could be decreased by upregulating miR-720; Firefly luciferase activity was normalized to Renilla luciferase activity. The relative luciferase expression level is expressed as the mean ± standard deviation (SD). Three independent experiments were performed, and representative data are shown. *p < 0.05 shows the significant difference. c Normalized luciferase activity in cells transfected with reporter vector containing mutant type 3′-UTR of KCTD15, INSIG1, or RAB35 could not be affected by overexpression of miR-720 compared with cells transfected with wild type 3′-UTR. The relative luciferase expression level is expressed as the mean ± standard deviation (SD). Three independent experiments were performed, and representative data are shown. *p < 0.05 shows the significant difference
Fig. 4
Fig. 4
Rab35 is negatively regulated by miR-720 at the translational level in HeLa cells. Transfection of miR-720 mimics (a) or miR-720 inhibitors (b) in HeLa cells did not significantly affect Rab35 mRNA expression levels, as assessed by qRT-PCR analysis. c Representative photographs of Rab35 protein expression detected by western blotting, and the results of gray intensity analysis using Image J software showed that Rab35 protein levels were negatively regulated by miR-720
Fig. 5
Fig. 5
Rab35 has the opposite effect on the migration of HeLa cells, relative to miR-720. a Representative western blot assays show that transfection with the constructed vector pcDNA3.0-Rab35 results in a high expression level of Rab35. b Representative photographs of wound healing assays show that the forced expression of Rab35 decreased cell wound healing ability. The statistical results of cell migration area measured by Image J software demonstrate a significant reduction in cell migration caused by overexpression of Rab35. **p < 0.01. c Representative photographs of Transwell® migration assays and the statistical results show that the forced expression of Rab35 led to a dramatic decrease in cell migration. *p < 0.05. d Real-time cell analysis shows that overexpression of Rab35 decreased the cell migration index when compared with the corresponding negative control
Fig. 6
Fig. 6
Restoration of Rab35 expression reverses the promoting effect of miR-720 on cell migration. Representative photographs (a) and statistical results (b) of Transwell® assays show that while transfecting miR-720 mimics could lead to a significant increase in cell migration ability compared with cells transfected with the microRNA mimic negative control, cotransfecting pcDNA3.0-Rab35 and miR-720 mimics resulted in a dramatic decrease in cell migration ability compared with the negative group cotransfected with miR-720 mimics and the pcDNA3.0-Vector. *p < 0.05
Fig. 7
Fig. 7
Detection of E-cadherin and vimentin expression by western blotting. a HeLa cells transfected with miR-720 mimics show a significant increase in vimentin protein expression and a dramatic decrease in E-cadherin protein expression. Transfection of miR-720 inhibitors (b) or pcDNA3.0-Rab35 (c) had a negative effect (E-cadherin was increased and vimentin was decreased), in contrast to transfection of miR-720 mimics in HeLa cells

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