Oncogene GAEC1 regulates CAPN10 expression which predicts survival in esophageal squamous cell carcinoma

Abstract

Aim: To identify the downstream regulated genes of GAEC1 oncogene in esophageal squamous cell carcinoma and their clinicopathological significance.

Methods: The anti-proliferative effect of knocking down the expression of GAEC1 oncogene was studied by using the RNA interference (RNAi) approach through transfecting the GAEC1-overexpressed esophageal carcinoma cell line KYSE150 with the pSilencer vector cloned with a GAEC1-targeted sequence, followed by MTS cell proliferation assay and cell cycle analysis using flow cytometry. RNA was then extracted from the parental, pSilencer-GAEC1-targeted sequence transfected and pSilencer negative control vector transfected KYSE150 cells for further analysis of different patterns in gene expression. Genes differentially expressed with suppressed GAEC1 expression were then determined using Human Genome U133 Plus 2.0 cDNA microarray analysis by comparing with the parental cells and normalized with the pSilencer negative control vector transfected cells. The most prominently regulated genes were then studied by immunohistochemical staining using tissue microarrays to determine their clinicopathological correlations in esophageal squamous cell carcinoma by statistical analyses.

Results: The RNAi approach of knocking down gene expression showed the effective suppression of GAEC1 expression in esophageal squamous cell carcinoma cell line KYSE150 that resulted in the inhibition of cell proliferation and increase of apoptotic population. cDNA microarray analysis for identifying differentially expressed genes detected the greatest levels of downregulation of calpain 10 (CAPN10) and upregulation of trinucleotide repeat containing 6C (TNRC6C) transcripts when GAEC1 expression was suppressed. At the tissue level, the high level expression of calpain 10 protein was significantly associated with longer patient survival (month) of esophageal squamous cell carcinoma compared to the patients with low level of calpain 10 expression (37.73 ± 16.33 vs 12.62 ± 12.44, P = 0.032). No significant correction was observed among the TNRC6C protein expression level and the clinocopathologcial features of esophageal squamous cell carcinoma.

Conclusion: GAEC1 regulates the expression of CAPN10 and TNRC6C downstream. Calpain 10 expression is a potential prognostic marker in patients with esophageal squamous cell carcinoma.

Keywords: Calpain 10; Esophageal squamous cell carcinoma; Oncogene; RNA interference; Tissue microarray.

Figure 1

Expression level of GAEC1 in KYSE150 cells. A: Multiplex reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the overexpression of GAEC1 in KYSE150 compared with the non-tumor esophageal epithelial cell line NE1; B: Multiplex RT-PCR analysis demonstrated the down-regulation of GAEC1 expression in KYSE150 cells transfected with pSilencer P3-4 vector compared with the parental cells and those transfected with pSilencer control vector. The amount of RNA in each lane was normalized with the amplification of β-actin. M: 100 bp ladder marker; H₂O: Water control.

Figure 2

MTS cell proliferation assays for esophageal squamous cell carcinoma cell line KYSE150. Cells were transfected with pSilencer vector cloned with the P3-4 sequence (pSilencer P3-4) or control vector (pSilencer-ve). MTS assays were then performed every 24 h for 3 d on each type of transfected cells and the parental cells. The respective MTS activities on each day were compared with the corresponding activities of day 1. Representative data from 3 independent experiments are shown.

Figure 3

Flow cytometry analyses for KYSE150 cells. KYSE150 transfected with pSilencer cloned with P3-4 sequence demonstrated an increased apoptotic population by approximately 50% (C) compared with the parental cells (A) and cells transfected with pSilencer-ve control vector (B).

Figure 4

Immunohistochemical staining of CAPN10. A: In normal esophageal epithelial tissue showing weak CAPN10 staining; B: esophageal squamous cell carcinoma (ESCC) tissue showing strong CAPN10 staining; C: ESCC tissue showing weak CAPN10 staining in tumor. CAPN10 was mainly localized in the cytoplasm of the cancer cells (original magnification, ×100).

Figure 5

Overall 5-year survival rates as determined by the expression level of CAPN10 in esophageal squamous cell carcinoma patients. Low expression group of CAPN10 in ESCC patients showed a significantly lower 5-year survival rate than those of high expression group.