Expression of Transcription Factor 21 (TCF21) and Upregulation Its Level Inhibits Invasion and Metastasis in Esophageal Squamous Cell Carcinoma

Abstract

BACKGROUND Transcription factor 21 (TCF21), a member of the class A of basic helix-loop-helix family, has been widely identified as a tumor suppressor. Growing evidence has demonstrated the downregulation of TCF21 in distinct cancers. The aim of this study was to explore the expression and biological functions of TCF21 in esophageal squamous cell carcinoma (ESCC). MATERIAL AND METHODS TCF21 expression in esophageal cancer cell lines and carcinomas tissues were detected, and its associations with clinical characteristics were analyzed. We carried out this study of biological functions and underlying mechanisms using TE10 and KYSE510 cell lines. RESULTS TCF21 mRNA and protein expression were both downregulated in esophageal cancer tissues compared with adjacent normal tissues. Low expression of TCF21 was closely correlated with N stage. In Kaplan-Meier survival analysis, patients with lower TCF21 expression had poorer prognosis. Overexpression of TCF21 greatly inhibited the proliferation, migration, and invasion in both TE10 and KYSE510 cell lines. Furthermore, mechanistic studies showed that with TCF21 gene overexpressed, the expression of tumor suppressor Kiss-1 was upregulated and epithelial-mesenchymal transition (EMT) related proteins (E-cadherin, N-cadherin, Snail, Twist, and Vimentin) which participate in cancer cell invasion and metastasis, were reversed. CONCLUSIONS TCF21 is downregulated in ESCC, and its low expression is closely correlated with N stage and predicts a poor prognosis. TCF21 functions as a tumor suppressor in ESCC progression, and enhancement of its expression levels may be partly through promoting Kiss-1 expression to reverse EMT by modulating EMT-related gene expression. Thus, TCF21 can potentially be used as a treatment target for ESCC.

Figure 1

Expression of TCF21 in ESCC samples and in transfected cells. (A) Relative expression of TCF21 mRNA was examined in 16 ESCC clinical fresh specimens. The data are presented as average ± standard deviation (SD); * P<0.05, ** P<0.01. (B) Immunohistochemical assay revealed TCF21 was downregulated in most cancer tissues compared with that in the distant tissues (magnification, 40×). (C) Expression of TCF21 in esophageal cancer cell lines ECA109, ECA9706, KYSE150, KYSE510, and TE10, and compared to its expression in HET-1A cells; * P<0.05, ** P<0.01. (D) Overall survival rate, using Kaplan-Meier survival analysis, was associated with TCF21 expression in 80 ESCC patients; P=0.0005. (E) Fluorescence images show high transfection efficiency of Lentivirus in 2 esophageal cancer cell lines (TE10 and KYSE510) (magnification, 40×). (F) RT-PCR was used to confirm the overexpression of TCF21.

Figure 2

Expression of proteins in 2 groups of cells with overexpression of TCF21 and the empty vector. Western blot assay verified the efficiency of target gene TCF21 and proteins regulated by TCF21.Overexpression of TCF21 promoted the expression of Kiss-1 and E-cadherin while downregulated the expression of N-cadherin, Snail, Twist and Vimentin. Each assay was performed in triplicate. Data were presented as mean ±SD; * P<0.05, ** P<0.01.

Figure 3

Overexpression of TCF21 decreased cell colony formation, proliferation, migration and invasion. (A) Colony formation was inhibited in the LV-TCF21 group compared with the LV-NC group. Data are presented as mean ±SD; ** P<0.01. (B) The CCK-8 proliferation assay showed that upregulating of TCF21 inhibited the cell proliferation of the TE10 and KYSE510 cells; * P<0.05, ** P<0.01. (C) The Transwell assay (without Matrigel) showed the migration was inhibited in the LV-TCF21 group; ** P<0.01. (D) The same results occurred in the invasion assay; ** P<0.01. The experiments were performed in triplicate, and the data were presented as mean ±SD (magnifications, 10×). (E, F) By using the scratch assay, the migration ability of the LV-TCF21 groups were also inhibited compared with the control (LV-NC) groups. The quantitative results of migration rates are shown; ** P<0.01 (magnifications, 40×).