Figure 2.

KIAA0101 is a downstream target of miR-216a-5p, and its expression is inversely correlated with miR-216a-5p expression level in ESCC. (A) miR-216a-5p expression level in the human normal esophageal epithelial HET1A cells and six human ESCC cell lines (EC109, TE1, TE10, KYSE150, KYSE450, and EC9706) were quantified by qPCR. Values were calculated using the 2^−ΔΔCq method, normalized to U6. (B) Expression of KIAA0101 protein (15 kDa) and β-actin (42 kDa) in the indicated cell lines was quantified by western blot analysis. Representative western blot images are shown (upper panel), and the band intensities were summarized (lower panel; n=3 for each group). *P<0.05, HET1A cells compared with the ESCC cell lines. (C) The mRNA level of KIAA0101 in EC9706 and TE1 cells transfected with miR-216a-5p mimic, or inhibitor (Anti-miR-216a-5p), or the corresponding controls (Mock and Anti-NC) was determined by qPCR. n=3 for each group. Values were calculated using the 2^−ΔΔCq method, normalized to β-actin. ns not significant. (D) The protein levels of KIAA0101 in EC9706 and TE1 cells with the indicated transfection were determined by western blot analysis. Representative western blot images are shown (upper panel), and the band intensities were summarized (lower panel; n=3 for each group). *P<0.05 and **P<0.01. (E) Compared with the controls, miR-216a-5p inhibited the reporter activities of the vector with the wild-type (WT), but not the mutant-type (MUT) of 3′ UTR of KIAA0101 in EC9706 and TE1 cells in the dual-luciferase assays. **P<0.01 and ***P<0.001; ns, not significant. ESCC, esophageal squamous cell carcinoma.