MicroRNA‑485‑5p suppresses the progression of esophageal squamous cell carcinoma by targeting flotillin‑1 and inhibits the epithelial‑mesenchymal transition

Abstract

As esophageal squamous cell carcinoma (ESCC) is one of the most frequently diagnosed cancers in Asia, it is crucial to uncover its underlying molecular mechanisms that support its development and progression. Several articles have reported that microRNA (miR)‑485‑5p inhibits the malignant phenotype in a number of cancer types, such as lung, gastric and breast cancer, but to the best of our knowledge, its function in ESCC has not been studied in depth until the present study. It is of great significance to probe the regulatory action and underlying mechanism of miR‑485‑5p in ESCC. In brief, this study identified that miR‑485‑5p expression in ESCC tissues was significantly lower than that in normal tissues. The decrease in miR‑485‑5p expression was associated with a larger tumour size and poor histology and stage. The expression of miR‑485‑5p was relatively high in Eca 109 and TE‑1 cells, but relatively low in KYSE 30. The overexpression of miR‑485‑5p inhibited cell proliferation, migration and invasion in vitro, whereas miR‑485‑5p knockdown did the opposite. Flotillin‑1 (FLOT‑1) can facilitate the malignant phenotype in various cancer types. The present study found that in ESCC tissue, the protein expression of FLOT‑1 was negatively correlated with miR‑485‑5p expression. Further experiments showed that miR‑485‑5p directly targeted the 3'‑untranslated region of FLOT‑1. The overexpression of miR‑485‑5p significantly suppressed the mRNA and protein expression levels of FLOT‑1, whereas knockdown had the reverse effects. Furthermore, overexpression of miR‑485‑5p restrained epithelial‑mesenchymal metastasis (EMT)‑related factors at both the mRNA and protein levels. At the same time, it also inhibited the growth of ESCC and restrained the EMT in vivo. In summary, miR‑485‑5p was found to be an inhibitor of ESCC and may have potential as a novel target candidate for ESCC treatment.

Keywords: esophageal squamous cell carcinoma; expression; miR‑485‑5p; FLOT‑1; clinicopathological features.

Figure 1.

miR-385-5p is downregulated in ESCC tissues. (A) The expression of miR-485-5p in 80 pairs of ESCC and paracarcinoma tissues was detected by RT-qPCR. (B) The expression of miR-485-5p in ESCC cell lines was assessed by PCR. (C) The miR-485-5p transfection efficacy was verified in Eca 109, KYSE 30 and TE-1 cells transfected with miR-485-5p mimics or miR-485-5p inhibitor via RT-qPCR. The data are expressed as the mean ± SD. *P<0.05 and **P<0.01 vs. control group. miR, microRNA; ESCC, esophageal squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control.

Figure 2.

miR-485-5p inhibits the proliferation, migration and invasion of ESCC cell lines. (A) The proliferation of Eca 109, KYSE 30 and TE-1 cells transfected with miR-485-5p mimics or miR-485-5p inhibitor was explored using an MTS assay. (B and D) Cell migration and Matrigel invasion assays (magnification, ×200) were performed to detect the effects of miR-485-5p on ESCC cell migration and invasion, and the migratory and invasive abilities were (C and E) quantified as cell numbers. (F) Wound healing assays were performed to detect the migratory ability of cells after transfection with miR-485-5p mimics (scale bar, 200 µm), and (G) the migratory ratio was determined by dividing the wound area by the total area. The data are expressed as the mean ± SD. *P<0.05 and **P<0.01 vs. control group. miR, microRNA; ESCC, esophageal squamous cell carcinoma; NC, negative control.

Figure 3.

FLOT-1 is a predicted target of miR-485-5p. (A) Bioinformatics analysis predicted a miR-485-5p-binding site in the 3′UTR of FLOT1 mRNA. (B) The FLOT-1 plasmid transfection efficacy was verified in Eca 109, KYSE 30 and TE-1 cells transfected with FLOT-1 plasmid or pcDNA3.1 via western blotting. (C) FLOT-1 mRNA levels in KYSE 30, Eca 109 and TE-1 cells transfected with miR-485-5p mimics, miR-485-5p inhibitor or their corresponding controls (miR-NC and inhibitor-NC). (D) FLOT-1 protein levels of Eca 109 and KYSE 30 cells transfected with miR-485-5p mimics, miR-485-5p inhibitor or their corresponding controls (miR-NC and inhibitor-NC). (E) Effects of miR-485-5p on the translation of the reporter gene inserted downstream of the 3′UTR of FLOT-1 mRNA or the mutated 3′UTR of FLOT-1 mRNA in 293T cells. 293T cells were co-transfected as shown in the figure, and the luciferase activities were measured using a Dual-luciferase reporter assay. The luciferase activity was normalized and expressed as the ratio of firefly/Renilla luciferase activities. (F) IHC staining of FLOT-1 expression in esophageal squamous cell carcinoma tissue sections (scale bar, 200 µm). (G) FLOT-1 protein levels in Eca 109 and KYSE 30 cells co-transfected with mimics-NC and pcDNA3.1, miR-485-5p mimics and pcDNA3.1, miR-485-5p mimics and FLOT-1, mimics-NC and FLOT-1. The data are expressed as the mean ± SD. *P<0.05 and **P<0.01 vs. control group. FLOT-1, flotillin-1; miR, microRNA; UTR, untranslated region; NC, negative control; IHC, immunohistochemistry; WT, wild-type; MUT, mutant.

Figure 4.

Overexpression of miR-485-5p inhibits the epithelial-mesenchymal transition. The mRNA levels of FLOT-1, E-cadherin, Vimentin, N-cadherin and ZEB1 in (A) Eca 109 and (B) KYSE 30 cells transfected with miR-485-5p or mimics-NC. (C) The protein levels of FLOT-1, E-cadherin, Vimentin, N-cadherin and ZEB1 in Eca 109 (left) and KYSE 30 (right) cells transfected with miR-485-5p or mimics-NC. The data are expressed as the mean ± SD. *P<0.05. FLOT-1, flotillin-1; miR, microRNA; NC, negative control; ZEB1, zinc finger E-box-binding homeobox 1.

Figure 5.

miR-485-5p suppresses the growth of esophageal squamous cell carcinoma in vivo. (A) Eca 109 cells transfected with lentivirus-miR-485-5p or lentivirus-NC (magnification, ×100). (B) Nude mice were injected into their flanks with Eca 109 cells transfected with lentivirus-miR-485-5p or lentivirus-NC. (C) Eca 109 cells stably expressing miR-485-5p or vector-implanted tumours from nude mice were dissected and imaged after euthanizing the mice. The (D) growth curve and (E) tumour weight of Eca 109 cells stably expressing miR-485-5p or vector implanted tumours from nude mice. n=6 mice per experimental group. **P<0.01 vs. vector group. miR, microRNA; NC, negative control.

Figure 6.

IHC staining of the tissue sections of implanted tumours. IHC staining of E-cadherin, N-cadherin, Vimentin, Ki-67 and FLOT-1 in Eca 109 cells stably expressing miR-485-5p or vector-implanted tumour sections from nude mice (magnification, ×40 or 200). n=6 mice per experimental group. IHC, immunohistochemistry; FLOT-1, flotillin-1; miR, microRNA.