KIAA1377 is associated with lymph node metastasis in esophageal squamous cell carcinoma

Abstract

KIAA1377, of which there are few studies regarding cell biology and neurological diseases, has been found to be significantly amplified in esophageal squamous cell carcinoma (ESCC) with lymph node metastasis compared with ESCC without lymph node metastasis. This suggests that KIAA1377 may play a role in the lymph node metastasis of ESCC. There has, to the best of our knowledge, been no study performed to investigate the role of KIAA1377 in ESCC. In the present study, the expression of KIAA1377 was detected by immunohistochemistry, and its expression was statistically analyzed with clinicopathological parameters, using commercially obtained tissue arrays consisting of 86 cases of ESCC and 79 paired controls. KIAA1377 was knocked down ex vivo using transient transfection with specific small hairpin RNA (shRNA) vectors into ESCC TE-1 and EC9706 cell lines whose endogenous KIAA1377 level was highest. The variation of proliferation, migration and invasion were evaluated using methyl thiazolyl tetrazolium, wound healing and Transwell assay, respectively. It was found in vivo that KIAA1377 expression was significantly associated with lymph node metastasis and differentiation, and ex vivo that knockdown of KIAA1377 cannot significantly affect proliferation and mobility in the ESCC cell line TE-1. Overall, this is the first study suggesting that KIAA1377 may play a role in the lymph node micrometastasis of ESCC.

Keywords: ESCC; KIAA1377; lymph node metastasis.

Figure 1.

KIAA1377 was significantly upregulated in ESCC tissues compared with the normal control. Immunohistochemical analysis of expression of KIAA1377 in ESCC tissues is shown. ESCC, esophageal squamous cell carcinoma.

Figure 2.

KIAA1377 expression was not significantly associated with prognosis in ESCC. Immunostaining of KIAA1377 and overall survival in ESCC was analyzed by Kaplan-Meier survival curves. There was no significant difference between KIAA1377 expression and prognosis (P>0.05) subsequent to statistical analysis. ESCC, esophageal squamous cell carcinoma; -, negative; +, weak; ++, moderate; +++, strong; Cum., cumulative.

Figure 3.

Endogenous basal expression of KIAA1377 in a panel of 7 different ESCC cell lines and evaluation of knockdown efficiency in the TE-1 cell line. (A) Endogenous basal expression of KIAA1377 was detected by reverse transcription-polymerase chain reaction, and β-actin was used as the internal control. (B) Knockdown efficiency of shRNA-KIAA1377 and shRNA-scramble vector was determined using western blotting. The shRNA-KIAA1377 and shRNA-scramble vector were transfected into ESCC cell line TE-1. In total, 80 µg of protein was loaded per lane, which was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by visualization with Western Breeze kit. ESCC, esophageal squamous cell carcinoma; shRNA, small hairpin RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4.

KIAA1377 does not affect the proliferation, migration and invasion ability of cells subsequent to transient knockdown. (A) Methyl thiazolyl tetrazolium assay for the ESCC TE-1 and EC9706 cell lines, (B) subsequent to knockdown of KIAA1377 using transient transfection with a specific shRNA vector for 0, 24, 48, 72 and 96 h. (C) Qualitative wound-healing assay (left) and quantitative assay (right) for TE-1. (D) Similarly, qualitative wound-healing assay (left) and quantitative assay (right) for EC9706 subsequent to transient transfection for 48 h. (E) Quantitative Transwell assay for TE-1 and EC9706, (F) subsequent to knockdown for 48 h (P>0.05 compared with control group). Images of migratory cells from the scratched boundary were observed and acquired by light microscopy (magnification, 10×10). Similar results were obtained in 3 independent experiments, and representative images are shown. ESCC, esophageal squamous cell carcinoma.