The present study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Urumqi, China). The ESCC tissue microarray, consisting of 86 cases of ESCC and 78 cases of paired adjacent normal control tissues, was obtained from Shanghai Outdo Biotech Company (Shanghai, China). The clinicopathological information accompanying the tissue array, including tumor-node-metastasis grading and staging, were also obtained. Staging and grading were assessed in accordance with the World Health Organization 2014 version classification and grading system (17). Prognosis and demographics were all available.

The human ESCC ECa109, EC9706, TE-1, KYSE-30, KYSE-150, KYSE-450 and KYSE-510 cell lines were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin in a 5% CO₂ humidified incubator at 37°C. For knockdown experiments, KIAA1377 short hairpin RNA (shRNA) vector and shRNA scramble control were purchased from Santa Cruz Biotechnology, Inc. (cat. no. sc-96891-SHH, Dallas, TX, USA). ESCC cells were grown to 80–90% confluence in 6-well plates and were transfected with 4.0 µg of shRNA-KIAA1377 vector or control vector together with 10 µl Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were grown for two days, and media was changed daily.

Immunohistochemical stains were performed using heat-induced epitope retrieval (18) and avidin-biotin complex (19) method. The rabbit anti-human KIAA1377 polyclonal antibody (clone, N-16; catalogue no. sc-168347; Santa Cruz Biotechnology, Inc.) was diluted at 1:200. The sections were evaluated by light microscopy, and cellular localization of the protein and immunostaining level in each section was assessed by 2 pathologists. The staining patterns were scored as follows: negative, defined as <15%; weak (≥15% but <30% of cells with positive staining), defined as +; moderate (≥30% but <60%) of cells with positive staining), defined as ++; and strong positive (≥60% of cells with positive staining), defined as +++, according to the immunostaining intensity area.

Total RNA was extracted from frozen tissue with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed to cDNA using the Prime Script RT-qPCR kit (Takara Biotechnology Co., Ltd., Dalian, China). The RT-qPCR assay was performed with the IQ5 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. All reactions were performed in triplicate. Subsequent to normalization with β-actin controls, relative gene expression was determined using a comparative standard curve. PCR was performed with the following primer sets: KIAA1377 forward, 5′-TCCTAACACAGCCCTAAATGC-3′ and reverse, 5′-TCCAGATTGTAAAGCGTCCAG-3′; and β-actin forward, 5′-TGGCACCCAGCACAATGAA-3′ and reverse, 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. The reaction mixtures for KIAA1377 and β-actin were incubated at the following thermal cycling conditions: 95°C for 3 min; 40 cycles at 95°C for 5 sec; and 58°C for 30 sec. the final relative expression of KIAA1377 was calculated after normalization to β-actin, as internal loading control, using standard curve RT-qPCR approach (20).

In total, 72 h after transfection, TE-1 and EC-9706 cells were harvested in radioimmunoprecipitation assay lysis buffer (BioTeke Corporation, Beijing, China) and 80 µg of cellular protein was subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation. Proteins were transferred to polyvinylidene fluoride microporous membrane (EMD Millipore, Billerica, MA, USA) and blots were probed with go at polyclonal antibody against human KIAA1377 (catalogue no. sc-168347; dilution at 1:800) and mouse monoclonal antibody against human glyceraldehyde 3-phosphate dehydrogenase (GAPDH; catalogue no. sc-25778; dilution at 1:500) were obtained from Santa Cruz Biotechnology Inc. GAPDH was chosen as an internal control and the blots were visualized with Western Breeze kit (catalogue no., WB7105; Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

MTT spectrophotometric dye assay (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was used to observe and compare cell proliferation ability. TE-1 and EC-9706 cells were plated in 96-well plates at a density of 5×10³ cells per well. Subsequent to transfection experiments, cell proliferation was assessed. Cells were incubated for 4 h in 20 µl MTT at 37°C. The color was developed by incubating the cells in 150 µl dimethyl sulfoxide; the absorbance was detected at 490 nm wavelength. The data were obtained from 3 independent experiments.

Cell migration ability was calculated by the wound-healing assay. TE-1 and EC-9706 cells were plated in 6-well plates at a concentration of 4×10⁵ cells/well and allowed to form a confluent monolayer for 24 h. Subsequent to the transfection experiment, the monolayer was scratched with a sterile pipette tip (10 µl), washed with serum free medium to remove floated and detached cells and images were captured (at 0 and 48 h) by an inversion fluorescence microscope (Olympus Corporation, Tokyo, Japan). Cell culture inserts (24-well, pore size 8 µm; BD Biosciences, Franklin Lakes, NJ, USA) were seeded with 5×10³ cells in 100 µl of medium with 0.1% FBS. Inserts pre-coated with Matrigel (40 µl, 1 mg/ml; BD Biosciences) were used for invasion assays. Medium with 10% FBS (400 µl) was added to the lower chamber and served as a chemotactic agent. Noninvasive cells were wiped from the upper side of the membrane and cells on the lower side were fixed in cold methanol (−20°C) and air-dried. Cell were stained with 0.1% crystal violet (dissolved in methanol) and counted using the inverted microscope. Each individual experiment had triplicate inserts, and 4 microscopic fields (magnification, ×100) were counted per insert.

Data were expressed as the mean ± standard deviation and were analyzed by Student's t-test, one-way analysis of variance and χ² test, as appropriate, using SPSS for Windows version 16.0 (SPSS, Inc., Chicago, IL, USA). Kaplan-Meier survival curves were plotted and log rank test was performed. The significance of various variables for survival was analyzed by Cox proportional hazards model in a multivariate analysis. P<0.05 was considered to indicate a statistically significant difference.

To understand the clinicopathological significance of KIAA1377 expression, a total of 78 distal normal tissues and 86 ESCC tissues were analyzed for KIAA1377 expression using IHC. Expression of KIAA1377 was heterogeneous in ESCC tissues, ranging from weak to moderate to strong positive staining. All paired normal control tissues had positive expression of KIAA1377 compared with ESCC tissues. It can be observed that KIAA1377 positive staining localized at the cytoplasm region of tumor and normal mucosal cells (Fig. 1). Representative results of KIAA1377 expression are shown in Fig. 1. Clinicopathological analyses of KIAA1377 expression are shown in Table I. The KIAA1377 expression level is significantly different in ESCC and normal tissues (P=0.001). There was a significant difference of KIAA1377 expression levels when the tumor samples were classified based on differentiation (P=0.030) and lymph node metastases (P=0.043). No significant association was found between KIAA1377 expression and other parameters (Table I).

To confirm whether or not there was an association between KIAA1377 expression and prognosis, the Kaplan-Meier survival curve for different expression statuses of KIAA1377 was used. Based on 86 patients with ESCC, no significant difference in survival was found comparing patients with weak, moderate or strong KIAA1377 staining in tumors (P=0.924) (Fig. 2). Similarly, multivariate analysis showed there were no significant differences in survival without lymph node metastasis (N₀) and with lymph node metastasis (N_1–3) or between stage I and II KIAA1377 immunostaining in tumors (data not shown).

To determine the basal expression of KIAA1377 in a panel of ESCC cell lines, RT-qPCR was performed on a panel of 7 different types of ESCC cell lines. It was found that in TE-1 and EC9706, the 2 different ESCC cell lines, endogenous mRNA level of KIAA1377 were higher than that in other cell lines (Fig. 3A). To understand the role of KIAA1377 in the metastasis of ESCC, specific short hairpin RNA (shRNA) interference vectors were transfected into TE-1 and EC9706 cells. The knockdown efficiency was evaluated using western blot analysis. It was shown that the shRNA vectors against KIAA1377 may effectively knockdown the KIAA1377 protein level (Fig. 3B). Based on which, the proliferation, migration and invasion were assessed by MTT, wound healing and Transwell assays, respectively. It was observed that knockdown of KIAA1377 hardly affected the proliferation (Fig. 4A and B), migration (Fig. 4C and D) and invasion (Fig. 4E and F) abilities subsequent to statistical analysis.