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. 2019 Oct;18(4):4203-4209.
doi: 10.3892/ol.2019.10749. Epub 2019 Aug 16.

MicroRNA-544 Inhibits Esophageal Squamous Cell Carcinoma Cell Proliferation and Enhances Sensitivity to Cisplatin by Repressing E2F Transcription Factor 5

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Free PMC article

MicroRNA-544 Inhibits Esophageal Squamous Cell Carcinoma Cell Proliferation and Enhances Sensitivity to Cisplatin by Repressing E2F Transcription Factor 5

Fengrong Sun et al. Oncol Lett. .
Free PMC article

Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. MicroRNA (miRNA)-544 is an important cancer-associated RNA that is downregulated in multiple types of cancer. However, the role of miR-544 in ESCC progression remains unknown. In the present study, miR-544 expression level was determined via RT-qPCR in 30 pairs of ESCC and adjacent normal tissues and in a panel of ESCC cell lines. Cell proliferation and cell apoptosis were assessed by MTT and flow cytometry assays. Luciferase reporter assay and western blot analysis were conducted to verify E2F transcription factor 5 (E2F5), an oncogene in ESCC, as a novel target gene of miR-544. The results illustrated that miR-544 is frequently downregulated in ESCC tissues and cell lines. Overexpression of miR-544 in ESCC cells resulted in decreased cell proliferation and increased cell apoptosis. Thus, E2F5 was identified as a target of miR-544, and its expression was negatively correlated with miR-544 expression in clinical ESCC tissues. More importantly, overexpression of miR-544 led to increased sensitivity of ESCC cells to cisplatin, an anticancer drug. Overall, these findings indicate that miR-544 serves as a tumor suppressor by targeting E2F5; thus, miR-544 may be a therapeutic target for the treatment of ESCC.

Keywords: E2F transcription factor 5; apoptosis; cisplatin; esophageal squamous cell carcinoma; microRNA-544; proliferation.

Figures

Figure 1.
Figure 1.
Expression of miR-544 and E2F5 in ESCC tumors and cell lines. (A) The expression of miR-544 in ESCC tissues and normal tissues was detected by RT-qPCR. (B) The expression of miR-544 in ESCC cells lines and normal esophageal epithelial HET-1A cells was detected by RT-qPCR. (C) The mRNA expression of E2F5 in ESCC tissues and normal tissues was detected by RT-qPCR. (D) Western blot analysis was performed to determine the expression of E2F5 in human ESCC and adjacent normal tissues. (E) The protein expression of E2F5 in ESCC cell lines and normal HET-1A cell line was detected by western blot analysis. (F) The mRNA expression of E2F5 in ESCC cells lines and HET-1A cell line was detected by RT-qPCR. (G) The correlation between miR-544 and E2F5 expression in ESCC tumor tissues. *P<0.05. miR, microRNA; E2F5, E2F transcription factor 5; ESCC, esophageal squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative PCR.
Figure 2.
Figure 2.
Overexpression of miR-544 inhibited cell proliferation of esophageal squamous cell carcinoma cells. (A and B) The expression level of miR-544 in KYSE450 and TE-1 cells transfected with miR-544 mimic or miR-NC was detected by RT-qPCR. U6 acted as the internal control. (C) MMT assay was used to detect the proliferation of KYSE450 and TE-1 cells. (D) Colony-formation assay was used to analyze the proliferative capability of KYSE450 and TE-1 cells. *P<0.05. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control.
Figure 3.
Figure 3.
E2F5 is a direct target gene of miR-544. (A) Sequence alignment of miR-544 with 3′-UTR of wt and mut E2F5 predicted by TargetScan. (B) The luciferase activity of cells was examined via the dual-luciferase reporter assay. (C) The mRNA expression level of E2F5 in KYSE450 and TE-1 cells was analyzed by RT-qPCR. (D) The protein expression level of E2F5 in KYSE450 and TE-1 cells was analyzed by western blot analysis. β-actin acted as the internal control. *P<0.05. miR, microRNA; E2F5, E2F transcription factor 5; RT-qPCR, reverse transcription-quantitative PCR; wt, wild-type; mut, mutant; NC, negative control; UTR, untranslated region.
Figure 4.
Figure 4.
E2F5 is a functional target of miR-544 in esophageal squamous cell carcinoma cells. The KYSE450 and TE-1 cells were co-transfected with miR-544 mimic and E2F5 plasmid or empty vector. The protein expression of E2F5 in (A) KYSE450 and (B) TE-1 cells was detected by western blot analysis. β-actin acted as the internal control. Cell proliferation was detected by MTT assay with (C) KYSE450 and (D) TE-1 cells, and (E) colony-formation assays. *P<0.05. miR, microRNA; E2F5, E2F transcription factor 5; NC, negative control.
Figure 5.
Figure 5.
miR-544 increases the chemosensitivity of KYSE450 and TE-1 cells to cisplatin. Apoptosis rate was detected in (A) KYSE450 and (B) TE-1 cells transfected with miR-544 mimics and E2F5 plasmid, and treated with or without 15-µM cisplatin for 24 h. MMT assay was used to detect the proliferation of (C) KYSE450 and (D) TE-1 cells treated with cisplatin following no transfection or transfection with miR-544 mimics. β-actin acted as the internal control. (E) The protein expression of (E) cleaved caspase-3 and cleaved PARP and (F) E2F5 was detected by western blot analysis. β-actin acted as the internal control. *P<0.05. miR, microRNA; E2F5, E2F transcription factor 5; NC, negative control; PI, propidium iodide; OD, optical density; PARP, poly(ADP-ribose) polymerase.

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