Objective:To analyze the expression of LETM2 in KYSE150 and ECA109 cell lines and its effect on the proliferation, migra-tion, and invasion of esophageal squamous cell carcinoma (ESCC). Methods:The expression level of the LETM2 protein in 90 paired hu-man ESCC tissues and matched adjacent normal tissues was determined through immunohistochemistry. The expression level of LETM2 in ESCC cell lines was detected by real-time PCR and Western blot. The expression levels of LETM2 in KYSE150 and ECA109 cell lines were knocked down using lentivirus. MTT assays were performed to examine the effect of LETM2 on the proliferation of ESCC cells. Colony formation assay was used to detect the colony formation ability. Flow cytometry was performed to analyze the cell cycle. The effect of LETM2 depletion on the migration and invasion of ESCC cells was determined by Transwell assay. Results:LETM2 expres-sion was frequently upregulated in the ESCC tissues than in the adjacent normal tissues. The suppressed exogenous expression of LETM2 led to the inhibition of cell proliferation and colony formation. However, cell migration and invasion were not affected. The re-sults on the cell cycle distribution revealed that LETM2 knockdown acts as a negative regulator of the cell cycle at the G1 to S phase transition. Conclusion:LETM2 acts as a tumor-driven gene in the development and progression of ESCC. This finding suggests that LETM2 can be used as an efficient prognosis biomarker and a potential therapeutic target for ESCC.

OBJECTIVETo study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma.

METHODSMelanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression.

RESULTSIn melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector.

CONCLUSIONSBased on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.

Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Humans ; MAP Kinase Signaling System ; Melanoma ; genetics ; metabolism ; Mutation ; Phenotype ; Protein-Serine-Threonine Kinases ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Proto-Oncogene Proteins B-raf ; metabolism ; Signal Transduction ; Transfection

Objective To investigate the effects of methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC) on biological behavior of esophageal squamous carcinoma cell (ESCC) lines KYSE140 and KYSE150. Methods KYSE140 and KYSE150 cell lines were divided into the blank group, the control group and the experimental group. The cells in the blank group didn't do the treatment, and the cells in the control group were added to DMSO 2 μmol/L, while in the experimental group, cells were treated with different concentration (1, 2, 3 and 4 μmol/L) of 5-Aza-dC which affected respectively at different time (24, 48, 72 and 96 h). Cell proliferation was detected by using methyl thiazolyl tetrazolium (MTT) assay and the optimal drug concentration and time point were selected. Transwell assay was performed to detect the change of cell migration and invasion. Flow cytometry was used to observe the effects of drugs on cell apoptosis and cell cycle.The expression of PARP,Caspase-3,CCNB-1,and CCNE-1 were detected by Western blot. Results MTT result showed that the effective function time of 5-Aza-dC on KYSE140 and KYSE150 was 96 h at the concentration of 4 μmol/L. Under this condition, the cell ability of migration and invasion was decreased significantly. The migrated cell number of KYSE140 and KYSE150 respectively in the blank group, the control group and the experimental group was (193.3±8.6), (184.0±10.4), (61.7±7.1) and (112.0±6.4), (101.3± 7.9), (26.3±5.7). The invasive cell number was (47.3±7.3), (38.7±5.1), (8.0±3.9) and (83.3±6.8), (74.7±5.7), (21.0±2.7), respectively. The difference was statistically significant (P <0.05). Flow cytometry revealed that 5-Aza-dC increased the apoptosis of KYSE140 and KYSE150. The apoptosis rate of the blank group, the control group and the experimental group was (2.8±0.3) %, (11.2±0.7) %, (18.6±0.6) % for KYSE140 and (2.7±0.4)%,(9.8±0.4)%,(17.7±0.5)% for KYSE150.Compared with the other two groups,the cell number of G2/M phase in the experimental group was increased remarkably (P < 0.05). PARP and Caspase-3 were sheared evidently and the protein expression of CCNB-1 was up-regulated while the expression of CCNE-1 was down-regulated in the experimental group. Conclusion 5-Aza-dC can inhibit the proliferation and promote apoptosis of ESCC cells.

Objective: To explore the expression of FAT1 in esophageal squamous cell carcinoma (ESCC) tissues, and its effect on cell proliferation. Methods: The expression levels of FAT1 protein in human ESCC tissues and matched adjacent normal tissues were determined by immunohistochemistry (IHC). Lentivirus based knockdown of FAT1 was carried out in YSE2 and Colo680N cell lines and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assays was performed to examine the effect of FAT1 on the proliferation of these ESCC cells. Colony formation assay was used to detect the colony formation ability. Flow cytometry was performed to analyze the cell cycle and apoptosis. The expression levels of cell cycle markers in FAT1 knock out ESCC cell lines were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR) and Western blot. Results: The relative expression of FAT1 in ESCC tissues was 66.97±21.53, significantly lower than 78.13±16.76 of adjacent normal tissues(P<0.05). Knockdown of FAT1 promoted cell proliferation and colony formation. In YSE2 cell, the division time in negative control (NC) group was (1 570±51) min, significantly longer than (1 356±31) min in shFAT1 group. In Colo680N cell, division time in NC group was (1 532±53) min, significantly longer than (1 290±30) min in shFAT1 group (P<0.05). Knockdown of FAT1 promoted G1-to S-phase transition and resulted in the upregulation of CDK4/CDK6/CCND1. Conclusion FAT1 inhibits the proliferation and G1-to S-phase transition of ESCC cells through regulating the protein expression of CDK4/CDK6/CCND1 complex.

Objective: To investigate the function and mechanism of zinc finger protein 750 (ZNF750) in esophageal squamous cell carcinoma (ESCC). Methods: Xenograft in nude mice was applied to detect the tumorigenesis of ZNF750-depleted ESCC cells. Western blot was performed to observe the expression of downstream target protein of ZNF750 in ESCC cell lines and xenograft tumor tissues in which ZNF750 was knocked down. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay was used to determine the proliferation of ZNF750 stably depleted cells after restoration of its target protein. Results: The tumor weight of blank control, negative control and ZNF750 knockdown groups was 137±26 mg, 161±31 mg and 463±89 mg, respectively, with a statistically significant difference (P<0.01). The expressions of Kruppel-like factor 4 (KLF4) in ZNF750-depleted ESCC cells and its derived tumor tissue xenograft in nude mice were significantly down-regulated. Restoration of KLF4 in ZNF750 stably depleted cells significantly inhibited the cell proliferation (P<0.01). Conclusions ZNF750 may be a new tumor suppressor in the tumorigenesis of ESCC, and the inhibition of cell proliferation induced by ZNF750 may be partially through the regulation of KLF4 expression.