MiR-216a-5p targets TCTN1 to inhibit cell proliferation and induce apoptosis in esophageal squamous cell carcinoma

Abstract

Background: MiR-216a-5p has been reported to be associated with several tumors, including prostate cancer and melanoma. However, its expression level and potential role in esophageal squamous cell carcinoma (ESCC) remain uncertain.

Results: Here, we found that miR-216a-5p expression was significantly down-regulated in clinical ESCC tissues and cells. Functional assays were performed to evaluate the biological effects of miR-216a-5p on cell proliferation and cell apoptosis by CCK-8 assay and flow cytometry in ESCC cell lines, EC9706 and TE-9. The results showed that miR-216a-5p overexpression repressed cell proliferation and induced cell apoptosis. Through bioinformatics prediction and luciferase reporter assay, we revealed that miR-216a-5p could directly target tectonic family member 1 (TCTN1). Moreover, TCTN1 was obviously suppressed by miR-216a-5p overexpression. In addition, TCTN1 expression was significantly increased and inversely correlated with the levels of miR-216a-5p in ESCC tissues. More importantly, down-regulation of TCTN1 imitated, while restoration of TCTN reversed the effects of miR-216a-5p on cell proliferation and apoptosis. At the molecular level, we further found that TCTN1 overexpression reversed the effects of miR-216a-5p transfection on the expression of PCNA, Bcl-2 and Bad.

Conclusions: Our results demonstrate that miR-216a-5p might serve as a tumor suppressor in ESCC cells through negatively regulating TCTN1 expression, indicating the possibility that miR-216a-5p and TCTN1 might be attractive targets for ESCC therapeutic intervention.

Keywords: Apoptosis; ESCC; Proliferation; TCTN1; miR-216a-5p.

Fig. 1

MiR-216a-5p was significantly down-regulated in ESCC tissues and cell lines. a MiR-216a-5p expression levels in 25 pairs of ESCC tissues compared with matched adjacent normal tissues were determined using qRT-PCR. b MiR-216a-5p expression in four ESCC cell lines and normal esophageal epithelial HET-1A cells was measured using qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001 vs. normal group; ESCC, esophageal squamous cell carcinoma; qRT-PCR, quantitative real-time PCR

Fig. 2

MiR-216a-5p inhibited cell proliferation and induced apoptosis in ESCC cells. a qRT-PCR was used to determine miR-216a-5p in EC9706 and TE-9 cells following transfection with miR-216a-5p or miR-NC. b CCK-8 assay was used to analyze the effect of miR-216a-5p overexpression on ESCC cell proliferation. c Flow cytometry with double Annexin V/PI staining was used to detect the effect of miR-216a-5p overexpression on ESCC cell apoptosis. ***p < 0.001 vs. miR-NC

Fig. 3

TCTN1 was a direct target of miR-216a-5p. a Putative binding sites of 216a-5p within the 3′-UTR region of TCTN1 mRNA, and the sequences of wild-type and mutant-type vector. b The relative luciferase activities were inhibited in 293 T cells co-transfected with wild-type TCTN1 3′-UTR vector and miR-216a-5p, not with the mutant-type vector. Firefly luciferase activity was normalized to Renilla luciferase. Data are presented as the mean value ± SD from triplicate experiments. ***p < 0.001 vs. miR-NC; c mRNA and d protein levels of TCTN1 were detected by qRT-PCR and western blot in EC9706 and TE-9 cells transfected with miR-216a-5p or miR-NC. e The protein level of TCTN1 was determined by western blot in HET-1A cells transfected with miR-216a-5p inhibitor or miR-NC. f Relative expression levels of TCTN1 mRNA in ESCC tissues and adjacent tissues were detected by qRT-PCR. g Pearson’s correlation analysis for the relationship between miR-216a-5p levels and TCTN1 mRNA levels in ESCC tissues

Fig. 4

TCTN1 knockdown inhibited cell proliferation and induced apoptosis in ESCC. EC9706 and TE-9 cells were transfected with siTCTN1 or siNC, respectively. a qRT-PCR and b western blot analysis were performed to measure the TCTN1 expression at mRNA and protein levels, respectively. c CCK-8 assay was used to analyze cell proliferation in EC9706 and TE-9 cells. d Flow cytometry with double Annexin V/PI staining was used to detect the ESCC cell apoptosis. ***p < 0.001 vs. siNC

Fig. 5

Addition of TCTN1 reversed miR-216a-5p-mediated effects on cell proliferation and apoptosis. EC9706 cells were co-transfected with miR-216a-5p mimic/miR-NC and with TCTN1 overexpression plasmid/empty vector. a CCK-8 assay was used to analyze cell proliferation. b Flow cytometry with double Annexin V/PI staining was used to detect cell apoptosis. **p < 0.01, ***p < 0.001 vs. siNC+vector; #p < 0.05, ##p < 0.01 vs. miR-216a-5p + vector

Fig. 6

Addition of TCTN1 reversed the regulation of miR-216a-5p on the proliferation and apoptotic markers in ESCC. EC9706 cells were co-transfected with miR-216a-5p mimic/miR-NC and with TCTN1 overexpression plasmid/empty vector. Western blot analysis was performed to analyze the expression of PCNA, Bcl-2 and Bad in EC9706 cells