EC9706 Cells Research Articles

PRR11 and SKA2 promote the proliferation, migration and invasion of esophageal carcinoma cells.

Proline-rich protein 11 (PRR11) together with its upstream adjacent gene, spindle and kinetochore associated 2 (SKA2), represent a classic, head-to-head gene pair. The role of the PRR11 and SKA2 gene pair has been described in various types of cancer, including breast cancer, non-small cell lung cancer, hepatocellular carcinoma and ovarian carcinoma. However, its role in esophageal carcinoma (ESCC) remains unclear. The mRNA expression levels of PRR11 and SKA2 were examined in ESCC surgical specimens. In addition, the role of PRR11 and SKA2 in the proliferation and migratory and invasive capacities of EC9706 and EC109 cell lines was examined. The results from the present study demonstrated that PRR11 and SKA2 expression levels were upregulated in ESCC tissues compared with adjacent normal tissues. Furthermore, PRRl1 and SKA2 knockdown significantly inhibited the proliferation and migratory and invasive capacities of ESCC cells. Conversely, PRRl1 and SKA2 overexpression significantly promoted the proliferation and migratory and invasive capacities of ESCC cell lines via activation of the AKT signaling pathway and certain markers of epithelial-mesenchymal transition, including Snail and N-cadherin. The results from the present study suggested that the PRR11 and SKA2 gene pair may represent a potential target in the diagnosis and treatment of ESCC.

XPD inhibits cell growth and invasion and enhances chemosensitivity in esophageal squamous cell carcinoma by regulating the PI3K/AKT signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is a lethal disease due to its high aggressiveness. The aim of the present study was to investigate the role of xeroderma pigmentosum complementation group D (XPD) in the growth and invasion of ESCC and to elucidate the potential underlying molecular mechanisms. Western blot analysis and RT-qPCR were used to detect the expression level of XPD in ESCC tissue samples and adjacent normal esophageal tissue samples. The pEGFP-N2/XPD plasmid was transfected into human ESCC cell lines (EC9706 and EC109). The proliferation, apoptosis, migration and invasion of EC9706 or EC109 cells were assessed following transfection with the XPD overexpression plasmid. The chemosensitivity of EC9706 or EC109 cells to cisplatin or fluorouracil was evaluated by CCK-8 assay. The expression levels of phosphoinositide 3-kinase (PI3K)/AKT, nuclear factor (NF)-κB, Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) signaling pathway-related genes were detected by RT-qPCR and western blot analysis. The results demonstrated that the expression level of XPD was markedly lower in ESCC tissue samples than in adjacent normal esophageal tissue samples. The pEGFP-N2/XPD plasmid was successfully transfected into EC9706 or EC109 cells, inducing XPD overexpression. A High XPD expression markedly suppressed cell proliferation, migration and invasion, and increased the apoptotic rate of EC9706 and EC109 cells. Furthermore, the overexpression of XPD significantly increased the chemosensitivity of EC9706 and EC109 cells to cisplatin or fluorouracil. Following XPD overexpression, the expression levels of PI3K, p-AKT, c-Myc, Cyclin D1, Bcl-2, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 were markedly downregulated, while the expression level of p21 was markedly upregulated. On the whole, the findings of the present study demonstrate that XPD inhibits the growth and invasion of EC9706 and EC109 cells, whilst also enhancing the chemosensitivity of EC9706 and EC109 cells to cisplatin or fluorouracil by regulating the PI3K/AKT signaling pathway. XPD may thus be an underlying target for ESCC treatment and drug resistance.

Nicotinamide N-methyltransferase decreases 5-fluorouracil sensitivity in human esophageal squamous cell carcinoma through metabolic reprogramming and promoting the Warburg effect.

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor with poor prognosis. And different individuals respond to the same drug differently. Increasing evidence has confirmed that metabolism reprogramming was involved in the drug sensitivity of tumor cells. However, the potential molecular mechanism of 5-fluorouracil (5-FU) sensitivity remains to be elucidated in ESCC cells. In this study, we found that the 5-FU sensitivity of TE1 cells was lower than that of EC1 and Eca109 cells. Gas chromatography-mass spectrometry analysis results showed that nicotinate and nicotinamide metabolism and tricarboxylic acid cycle were significantly different in these three cell lines. Nicotinamide N-methyltransferase (NNMT), a key enzyme of nicotinate and nicotinamide metabolism, was significantly higher expressed in TE1 cells than that in EC1 and Eca109 cells. Therefore, the function of NNMT on 5-FU sensitivity was analyzed in vitro and in vivo. NNMT downregulation significantly increased 5-FU sensitivity in TE1 cells. Meanwhile, the glucose consumption and lactate production were decreased, and the expression of glycolysis-related enzymes hexokinase 2, lactate dehydrogenase A, and phosphoglycerate mutase 1 were downregulated in NNMT knockdown TE1 cells. Besides, overexpression of NNMT in EC1 and Eca109 cells caused the opposite effects. Moreover, when glycolysis was inhibited by 2-deoxyglucose, the roles of NNMT on 5-FU sensitivity was weakened. In vivo experiments showed that NNMT knockdown significantly increased the sensitivity of xenografts to 5-FU and suppressed the Warburg effect. Overall, these results demonstrated that NNMT decreases 5-FU sensitivity in human ESCC cells through promoting the Warburg effect, suggesting that NNMT may contribute to predict the treatment effects of the clinical chemotherapy in ESCC.

Expression of lncRNA AK058003 in esophageal carcinoma and analysis of its intervention effect.

The aim of this study was to investigate the expression of long non-coding ribonucleic acid (lncRNA) AK058003 in esophageal carcinoma (EC) tissues, and to analyze its intervention effect. The expression of lncRNA AK058003 in EC tissues and para-carcinoma tissues from 130 EC patients was detected via quantitative Polymerase Chain Reaction (qPCR). EC cell lines were selected for exogenous interference in lncRNA AK058003. Subsequently, the expression of lncRNA AK058003 in normal esophageal epithelial cell line (Het-1A) and EC cell lines (EC109, EC9706, KYSE-150, KYSE-30, and TE-1) was detected by qPCR. EC9706 cell lines with the highest expression of lncRNA AK058003 were selected and transfected with lncRNA AK058003 siRNA and lncRNA AK058003 control, respectively. After transfection, the expression of lncRNA AK058003 was determined using PCR. The changes in cell growth and proliferation were analyzed via cell growth curve and cell cycle assay. Meanwhile, the changes in cell migration and invasion were analyzed through wound healing assay. Protein expressions of matrix metalloproteinase-1 (MMP1) and MMP2 were determined by Western blot. Clinical data were collected from EC patients, and the association between lncRNA AK058003 expression and tumor-node-metastasis (TNM) stage was finally analyzed. LncRNA AK058003 was highly expressed EC tissues compared with para-carcinoma tissues (p<0.01). Compared with Het-1A cells, the expression of lncRNA AK058003 was significantly higher in EC109, EC9706, KYSE-150, KYSE-30, and TE-1 cells, with highest level in EC9706 cells (p<0.05). The expression of lncRNA AK058003 remarkably declined in lncRNA AK058003 siRNA group compared with lncRNA AK058003 control group (p<0.001). Compared with lncRNA AK058003 control group, the proliferation of EC cells was significantly weakened in lncRNA AK058003 siRNA group, with the greatest difference at 3 d. Flow cytometry results revealed that cell cycle was arrested in G0/G1 phase in lncRNA AK058003 siRNA group. Wound healing assay indicated that the intercellular distance became large, and cell migration ability was evidently enhanced in lncRNA AK058003 siRNA group with time (p<0.05). Besides, the protein expressions of MMP1 and MMP2 were remarkably lower in lncRNA AK058003 siRNA group than those in lncRNA AK058003 control group. This indicated remarkably declined invasion and metastasis ability. In addition, the postoperative prognosis was significantly worse in patients with higher expression of lncRNA AK058003 (p<0.05). All these findings suggested that lncRNA AK058003 could serve as a biomarker for EC prognosis. LncRNA AK058003 is highly expressed in EC patients, which promotes proliferation, migration, invasion, and metastasis of EC cells. In addition, the postoperative prognosis of EC patients with high expression of lncRNA AK058003 is relatively poor.

MS-275 combined with cisplatin exerts synergistic antitumor effects in human esophageal squamous cell carcinoma cells

MS-275 has been demonstrated to inhibit the growth of esophageal squamous cell carcinoma (ESCC) cells in our previous study, but its role in ESCC remains to be further explored. Cisplatin (cis-diamminedichloroplatinum II, DDP) is the first-line chemotherapeutic drug widely used in clinic for ESCC patients. However, the side effects of nephrotoxicity and drug resistance limit its clinical use. This study aimed to evaluate the anticancer effects of MS-275 combined with DDP on ESCC cell line EC9706 both in vitro and in vivo, and to investigate the possible mechanisms that mediate these effects. We found that MS-275 combined with DDP showed synergistic antitumor effects on EC9706 cells in vitro by decreasing cell proliferation, increasing apoptosis and oxidative damage, and inhibiting migration and stemness. The combination of MS-275 and DDP triggered pro-survival autophagy in EC9706. Moreover, MS-275 combined with DDP suppressed EC9706 xenografts growth and promoted apoptosis in vivo. Further study displayed that MS-275 combined with DDP suppressed Wnt/β-catenin signaling in EC9706 cells and xenografts. These results indicate that MS-275 combined with DDP exerts synergistic antitumor effects by enhancing the chemosensitivity of EC9706 cells to DDP, which may be a potential therapeutic strategy for the treatment of patients with ESCC.

Long noncoding RNA LINC01234 silencing exerts an anti-oncogenic effect in esophageal cancer cells through microRNA-193a-5p-mediated CCNE1 downregulation.

Long non-coding RNAs (lncRNAs) are transcribed pervasively in the genome and act to regulate chromatin remodeling and gene expression. Dysregulated lncRNA expression has been reported in many cancers, but the role of lncRNAs in esophageal cancer (EC) has so far remained poorly understood. In this study, we aimed to understand the effect of lncRNA LINC01234 on EC development through competitively binding to microRNA-193a-5p (miR-193a-5p). The Gene Expression Omnibus (GEO) database was used for microarray-based EC expression profiling. Gain- and loss-of-function analyses were carried out in human EC-derived Eca-109 and EC9706 cells. Expression analyses of miR-193a-5p, LINC01234, CCNE1, caspase-3, p21, Bax, cyclinD1 and Bcl-2 were performed using RT-qPCR and Western blotting. Cell proliferation, colony formation and apoptosis analyses were carried out using MTT, Hoechst 33258 and flow cytometry assays. A xenograft EC model in nude mice was used to evaluate in vivo tumor growth and CCNE1 expression. Microarray-based analyses revealed that LINC01234 expression was increased in primary EC samples, whereas that of miR-193a-5p was decreased. We found that CCNE1 was a target of miR-193a-5p and that LINC01234, in turn, sponges miR-193a-5p. After treatment with si-LINC01234 or miR-193a-5p mimic, EC cells (Eca-109 and EC9706) exhibited cyclinD1 and Bcl-2 downregulation, and caspase-3, p21, Bax and cleaved caspase-3 upregulation. LINC01234 silencing or miR-193a-5p upregulation resulted in decreased proliferation and colony formation, and increased apoptosis of EC cells. In addition, LINC01234 silencing or miR-193a-5p upregulation resulted in reduced in vivo EC tumor growth and CCNE1 expression in nude mice. We found that silencing of LINC01234 suppresses EC development by inhibiting CCNE1 through competitively binding to miR-193a-5p, which suggests that LINC01234 may represent a novel target for EC therapy.

MicroRNA-144 inhibits cell proliferation and invasion by directly targeting TIGAR in esophageal carcinoma.

microRNAs (miRNAs) have been identified to play vital roles in the development and progression of numerous different types of human malignancy, including esophageal squamous cell carcinoma (ESCC). In the present study, the biological function of microRNA-144 (miR-144) was investigated, as well as its underlying molecular mechanism in ESCC. The results revealed that miR-144 expression was significantly decreased, whereas the expression of TP53-inducible glycolysis and apoptosis regulator (TIGAR) was significantly increased in human ESCC tissues when compared with adjacent non-tumor tissues. An increase in TIGAR was significantly associated with tumor size and Tumor-Node-Metastasis staging in patients. Functional analysis revealed that the overexpression of miR-144 using lentivirus particles significantly inhibited cell proliferation and tumor colony formation, and induced cell apoptosis in EC9706 and EC109 cells. The autophagy activity was also enhanced by miR-144 activity. In addition, overexpression of miR-144 significantly inhibited tumor growth in vivo. In the present study, TIGAR was confirmed to be the downstream target of miR-144 in ESCC. siRNA-mediated downregulation of TIGAR inversely regulated the inhibition effect of miR-144 on ESCC cells. To conclude, the present study demonstrated that miR-144 inhibits proliferation and invasion in esophageal cancer by directly targeting TIGAR.

Expression of microRNA-17-5p in esophageal squamous cell carcinoma and its effects on cell proliferation and invasion

Objective: To explore the expression of microRNA-17-5p (miR-17-5p) in esophageal squamous cell carcinoma (ESCC) and its effects on cell proliferation and invasion ability. Methods: Real-time quantitative PCR (RT-qPCR) was used to detect the miR-17-5p level in ESCC tissues and cells. MiR-17-5p inhibitor and negative control (NC) were transfected into EC9706 and TE1 cells, and miR-17-5p expression was examined by using RT-qPCR. Cell counting kit-8 (CCK-8) and EdU were conducted to detect cell proliferation and Transwell chamber was used to investigate cell invasion ability. Dual-luciferase reporter assay was used to detect the direct interaction of miR-17-5p and retinoblastoma-like protein-2 (RBL2). Western blot and RT-qPCR were used to detect the expression of RBL2 in ESCC tissues, respectively. Finally, the correlation between RBL2 and miR-17-5p was analyzed. Results: The miR-17-5p level in ESCC tissues was 4.222±0.392, significantly higher than 1.081±0.046 in normal esophageal epithelial tissues (P<0.001). The expressions of miR-17-5p in ESCC cells, including EC9706, Eca109, TE1, KYSE450, KYSE70 and KYSE520, were 13.84±1.266, 6.453±0.293, 11.41±0.520, 2.613±0.548, 5.251±0.239 and 4.251±0.195, respectively, all obviously higher than (1.007±0.079) in normal esophageal epithelial cell Het-1A (P<0.05). The miR-17-5p level in patients with ESCC Ⅲ~Ⅳ was 5.094±0.562, markedly higher than 2.934±0.364 in patients with ESCCⅠ~Ⅱ(P<0.01). Moreover, the miR-17-5p level in ESCC patients with lymph node metastasis was 5.523±0.634, markedly higher than 3.533±0.461 in ESCC patients without lymph node metastasis (P<0.05). The survival ratio of ESCC patients with higher miR-17-5p level was evidently lower than that of ESCC patients with lower miR-17-5p level (P<0.05). MiR-17-5p inhibitor significantly downregulated the miR-17-5p expression in EC9706 and TE1, which suppressed cell proliferation and invasion ability. Dual-luciferase reporter assay revealed that co-transfection of 3' untranslated region-wild type (3'UTR-WT) of RBL2 and miR-17-5p mimic significantly reduced luciferase activity in EC9706 and TE1 cells (P<0.01), which implicated that RBL2 was the direct target of miR-17-5p. The result of western blot revealed that RBL2 protein expressions in miR-17-5p group of EC9706 and TE1 cells were 0.936±0.055 and 0.923±0.048, obviously higher than 0.087±0.019 and 0.102±0.010 in control group (P<0.001). The expression of RBL2 in ESCC tissues was 0.219±0.510, markedly lower than 0.983±0.324 in normal esophageal epithelial tissues (P<0.001). The miR-17-5p level exhibited a negative correlation with RBL2 level in ESCC tissues (r=-0.462, P<0.001). Downregulation of RBL2 reversed the miR-17-5p inhibitor induced suppression of cell proliferation and invasion ability. Conclusion: MiR-17-5p participates in the carcinogenesis and development of ESCC, thus may be a potential therapeutic target for ESCC patients.

RUNX3 inhibits the invasion and migration of esophageal squamous cell carcinoma by reversing the epithelial‑mesenchymal transition through TGF‑β/Smad signaling.

Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor, and its inactivation may play a crucial role in the carcinogenesis process of numerous cancer types, including esophageal squamous cell carcinoma (ESCC). We previously revealed that RUNX3 inactivation was correlated with lymph node metastasis (LNM) and ESCC recurrence. However, the exact mechanisms of this process are still under investigation. The aim of the present study was to examine the potential roles and underlying molecular mechanisms of RUNX3 in ESCC metastasis and the epithelial-mesenchymal transition (EMT). According to the results, RUNX3 expression in ESCC tissue was significantly reduced compared with that in adjacent normal tissue (0.50±0.20 vs. 0.83±0.16; P<0.001). In addition, statistical analysis revealed a close association between decreased RUNX3 expression and T status (P=0.027) and LNM (P=0.017) in ESCC patients. Pearson's correlation coefficient analysis was then used to evaluate correlations between RUNX3 and EMT-related marker expression. The results revealed that RUNX3 expression in ESCC tissues was negatively correlated with the expression of N-cadherin (r=−0.429; P<0.01) and Snail (r=−0.364; P<0.01) and positively correlated with the expression of E-cadherin (r=0.580; P<0.01). Moreover, Eca109 and EC9706 cell invasion, migration, MMP-9 expression and EMT were significantly inhibited by RUNX3 overexpression. Notably, further analysis revealed that RUNX3 overexpression markedly inhibited the phosphorylation of Smad2/3; RUNX3-overexpressing cells also displayed less sensitivity to TGF-β1-induced EMT than control cells. Thus, RUNX3 may inhibit the invasion and migration of ESCC cells by reversing EMT through TGF-β/Smad signaling and may be useful as a therapeutic target.

Nimotuzumab, an EGFR‑targeted antibody, promotes radiosensitivity of recurrent esophageal squamous cell carcinoma.

Local tumor recurrence is one of the main causes for the failure of esophageal cancer treatment following radiotherapy. Previous studies have demonstrated that epidermal growth factor receptor(EGFR)‑targeted therapy combined with radiotherapy is expected to become an effective means to control tumor recurrence. The aim of the present study was to investigate the effect and mechanism of nimotuzumab (an EGFR‑targeted antibody) in the treatment of recurrent esophageal carcinoma. The radiation responses of two esophageal squamous carcinoma cell lines, EC109 and TE‑1, with or without nimotuzumab, were first evaluated by CCK‑8 assay. Colony formation and apoptosis were used to measure anti‑proliferation effects. It was demonstrated that nimotuzumab arrested the cell cycle at the G2phase invitro. Western blotting and immunofluorescence analysis were used to determine signaling pathway changes. It was observed that nimotuzumab inhibited phosphorylation of EGFR in EC109 cells. Furthermore, recurrent tumor models were established and it was identified that the degree of tumor hypoxia was positively associated with EGFR overexpression. In EC109 cell xenografts, nimotuzumab combined with radiation led to a significant delay in recurrent tumor growth compared with that of radiation alone (P<0.001 for 0Gy pre‑irradiation, P=0.005 for 20Gy pre‑irradiation, P=0.005 for 10Gy pre‑irradiation). These results suggest that nimotuzumab combined with radiation may be an effective means to control recurrent esophageal squamous cell carcinoma with EGFR overexpression.

Activation of NLRP3 inflammasome promotes the proliferation and migration of esophageal squamous cell carcinoma.

Inflammasomes can identify endogenous danger signals as an inflammatory immune response. As the most common inflammasome, the NLR pyrin family domain containing 3 (NLRP3) inflammasome is associated with the pathogenesis of different tumors. However, the function of the NLRP3 inflammasome in esophageal cancer (EC) has rarely been reported. Herein, the expression levels of the components of NLRP3 inflammasome and Ki-67 were analyzed by immunohistochemistry. Furthermore, correlations between the NLRP3 inflammasome and Ki-67 along with the clinicopathological features of EC patients were evaluated. The components of the NLRP3 inflammasome were also assessed by western blot analysis and quantitative PCR. NLRP3 was silenced or overexpressed in different esophageal squamous cell carcinoma (ESCC) cell lines, and cell viability, migration and invasion were assessed by CCK-8 and Transwell assays. The present results showed that high NLRP3 expression in the tumor specimens was significantly associated with TNM stage and T category. Spearman's correlation analysis revealed a positive correlation between NLRP3 and the Ki-67 proliferation index. The mRNA and protein levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase-1, and interleukin (IL)-1β in tumor tissues were higher than those in non-cancerous tissues. The level of secreted IL-1β in tumor tissues was also increased, as compared to that in normal tissues. Silencing of NLRP3 in KYSE-70 and TE13 cells strongly attenuated cell viability, decreased cell mobility in wound-healing assays and greatly diminished the ability of cell migration and invasion in the Transwell system. Overexpression of NLRP3 in KYSE-510 and EC9706 cells markedly promoted the proliferation, migration and invasion. Collectively, these results revealed that the the NLRP3 inflammasome is upregulated in human ESCC tissues and promotes ESCC progression. Hence, NLRP3 could be a promising new candidate diagnostic and prognostic target.

RICTOR/mTORC2 affects tumorigenesis and therapeutic efficacy of mTOR inhibitors in esophageal squamous cell carcinoma

Dysregulation of mTORC1/mTORC2 pathway is observed in many cancers and mTORC1 inhibitors have been used clinically in many tumor types; however, the mechanism of mTORC2 in tumorigenesis is still obscure. Here, we mainly explored the potential role of mTORC2 in esophageal squamous cell carcinoma (ESCC) and its effects on the sensitivity of cells to mTOR inhibitors. We demonstrated that RICTOR, the key factor of mTORC2, and p-AKT (Ser473) were excessively activated in ESCC and their overexpression is related to lymph node metastasis and the tumor-node-metastasis (TNM) phase of ESCC patients. Furthermore, we found that mTORC1/ mTORC2 inhibitor PP242 exhibited more efficacious anti-proliferative effect on ESCC cells than mTORC1 inhibitor RAD001 due to RAD001-triggered feedback activation of AKT signal. Another, we demonstrated that down-regulating expression of RICTOR in ECa109 and EC9706 cells inhibited proliferation and migration as well as induced cell cycle arrest and apoptosis. Noteworthy, knocking-down stably RICTOR significantly suppresses RAD001-induced feedback activation of AKT/PRAS40 signaling, and enhances inhibition efficacy of PP242 on the phosphorylation of AKT and PRAS40, thus potentiates the antitumor effect of RAD001 and PP242 both in vitro and in vivo. Our findings highlight that selective targeting mTORC2 could be a promising therapeutic strategy for future treatment of ESCC.

Negative regulation of CDK6 expression by microRNA-126-5p and its influence on the proliferation and invasion of esophageal cancer cells.

The present study aimed to investigate the expression of cyclin-dependent kinase 6 (CDK6) and microRNA-126-5p (miR-126-5p) in esophageal cancer tissues and cells, and their effect on esophageal cancer cell proliferation and invasion, and to explore the potential molecular mechanisms. The relative expression levels of CDK6 and miR-126-5p in esophageal cancer tissue, paracancerous tissue, and HEEC and EC109 cells were determined and compared using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A miR-126-5p overexpression vector was constructed and a stable EC109 cell line expressing miR-126-5p was established. The EC109 cell line was transfected with a CDK6 small interfering RNA sequence. The rate of cell proliferation was determined using the WST-8 method, and cell invasion was determined using a Transwell assay. In addition, the relative expression levels of genes were determined using RT-qPCR; the relative expression levels of proteins were determined by western blot analysis; the binding sites of CDK6 and miR-126-5p were analyzed using TargetScan software; and the interaction of CDK6 and miR-126-5p was verified using dual-fluorescence reporter gene expression. Esophageal tissues and EC109 cells expressed higher levels of CDK6 but significantly lower levels of miR-126-5p compared with adjacent tissues and HEEC cells, and their correlation coefficient between esophageal tissues and matched adjacent tissues was -7.526. The overexpression of miR-126-5p and CDK6 knockdown in the EC109 cell line inhibited cell proliferation and invasion compared with the control and NC (negative control) groups. miR-126-5p overexpression reduced the relative expression level of CDK6, and CDK6 knockdown by siRNA increased the expression of miR-126-5p. miR-126-5p regulated CDK6 expression by binding to the 3'-untranslated region of its mRNA. Overexpression miR-126-5p inhibited the proliferation and migration of esophageal cancer cells by targeting CDK6 and negatively regulating its expression. These findings contribute to the understanding of the underlying molecular mechanism of esophageal cancer.

Gli1 interacts with YAP1 to promote tumorigenesis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the predominant esophageal cancer type in China. The aberrant activation of glioma-associated oncogene homolog1 (Gli1), a key factor in Hedgehog (Hh) signaling pathway, has been found in esophageal carcinoma. Moreover, Yes-associated protein 1 (YAP1), the major mediator of Hippo signaling pathway, has been linked to esophageal carcinoma progression. However, the precise roles and the underlying mechanism of both Gli1 and YAP1 in ESCC are unclear. Here, we found that Gli1 and YAP1 are overexpressed in ESCC and are associated with poor prognosis. In addition, we confirmed that knockdown of Gli1 or YAP1 suppresses ESCC cell growth, migration, and invasion in ESCC TE1 and EC109 cells. Significantly, Gli1 interacts with YAP1 in ESCC cells. Both Gli1 and YAP1 proteins are closely correlated with each other in human ESCC samples. Mechanistically, Gli1 upregulates YAP1 in a LATS1-independent manner. Conversely, YAP1 induces Gli1 by regulating phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Most importantly, we demonstrated that the interaction between Gli1 and YAP1 promotes ESCC tumor growth in vitro and in vivo. Our findings established a novel signaling mechanism by which the interaction between Gli1 and YAP1 promotes ESCC cell growth. This signaling regulation of the tumorigenesis provides a new therapeutic strategy for highly lethal ESCC.

Pigment Epithelium-Derived Factor Promotes the Growth and Migration of Human Esophageal Squamous Cell Carcinoma

Pigment epithelium-derived factor (PEDF) is an oncogene found in various types of cancers. However, how PEDF affects the development of human esophageal squamous cell carcinoma (ESCC) is unknown. This study investigates the role of PEDF in ESCC cell proliferation, migration, and cell cycle both in vitro and in vivo. The PEDF expression was examined in patient tumor samples and ESCC cell lines. Short hairpin RNA technology was used to inhibit the PEDF expression in ESCC EC9706 and KYSE150 cells. In vitro cell proliferation and migration assays were performed. The effects of PEDF on tumor growth and progression were examined in vivo in murine subcutaneous xenograft tumor models. It was found that PEDF was overexpressed in esophageal cancer cells and patient tumor tissues compared to normal control samples. PEDF enhanced cell cycle progression and inhibited cell apoptosis. Knock down of PEDF inhibited esophageal cell proliferation and migration in vitro. Moreover, Inhibition of PEDF significantly reduced tumor growth and tumor size in vivo. These results indicate that PEDF induce tumorigenesis in ESCC and can be a potential therapeutic target for cancer treatment.

Involvement of Glutathione Depletion in Selective Cytotoxicity of Oridonin to p53-Mutant Esophageal Squamous Carcinoma Cells.

Oridonin, a diterpenoid compound isolated from traditional Chinese medicine Rabdosia rubescens, has shown antitumor effects to esophageal cancer. However, its molecular mechanism is not fully understood, which limits its clinical application. In the present study, we used RNA-seq analysis to check the transcriptome changes after oridonin treatment and we found genes controlling the GSH-ROS system were up-regulated, namely SLC7A11, TXNRD1, TRIM16, SRXN1, GCLM, and GCLC. Furthermore, our data suggest that oridonin significantly increased the production of ROS in EC109 and TE1 cells, which can be inhibited by NAC. Interestingly, oridonin can dramatically reduce intracellular GSH levels in TE1 cells in a concentration and time-dependent manner. In addition, cell death caused by oridonin was strongly inhibited by GSH (1 mM), while GSSG (1 mM) had little effect. At the same time, we also found that oridonin showed selective cytotoxicity to esophageal squamous carcinoma cell with p53 mutation since mut-p53 cells had lower SLC7A11 expression, a component of the cystine/glutamate antiporter. We also found that γ-glutamyl cysteine synthetase inhibitor (BSO) synergizes with oridonin to strongly inhibit EC109 cells at a low dose. These results suggested that the antitumor effects of oridonin are based on its –SH reactivity and glutathione depletion. Esophageal squamous carcinoma cells with p53-mutation showed hypersensitivity to oridonin because of the suppression of SLC7A11 expression by p53 mutation.

Long Noncoding RNA LINC00634 Functions as an Oncogene in Esophageal Squamous Cell Carcinoma Through the miR-342-3p/Bcl2L1 Axis.

Many long noncoding RNAs reportedly have tumor suppressive roles or are oncogenic in esophageal cancer. We have previously performed a chip-based expression analysis of primary esophageal cancer tissues and found that the expression of LINC00634 in these tissues was higher than that in nontumor tissues. Quantitative real-time–polymerase chain reaction, cell counting kit-8, flow cytometry, caspase3/7 assay, dual-luciferase reporter assay, and restore assay were used to detect the proliferative and apoptotic effects of LINC00634 in esophageal cancer cells. The results showed that the expression of LINC00634 in these tissues was higher than that in nontumor tissues and associated with tumor–node–metastasis (TNM) stage of patients. Knockdown of LINC00634 decreased cell viability and increased cell apoptosis levels in EC9706 and EC1 cells. LINC00634 could target Bcl2L1 through miR-342-3p. In this study, we show that LINC00634 is upregulated in esophageal cancer. We also show that the knockdown of LINC00634 decreased cell viability and increased cell apoptosis levels in EC9706 and EC1 cells through the miR-342-3p/Bcl2L1 axis.

LncRNA SNHG1 Regulates the Progression of Esophageal Squamous Cell Cancer by the miR-204/HOXC8 Axis.

ObjectiveLong noncoding RNA small nucleolar RNA host gene 1 (SNHG1) has been reported to be aberrantly expressed and plays an important role in human cancers, including esophageal squamous cell cancer. However, the regulatory mechanism underlying SNHG1 in the progression of esophageal squamous cell cancer is poorly defined.Materials and MethodsFifty-three esophageal squamous cell cancer patients were recruited and overall survival was analyzed. EC9706 and KYSE150 cells were cultured for study in vitro. The expression levels of SNHG1, microRNA (miR)-204 and homeobox c8 (HOXC8) were detected by quantitative real-time polymerase chain reaction and Western blot. Cell cycle distribution, apoptosis, migration and invasion were determined by flow cytometry and transwell assays, respectively. The target interaction among SNHG1, miR-204 and HOXC8 was validated by luciferase reporter assay and RNA immunoprecipitation. Xenograft model was established to investigate the role of SNHG1 in vivo.ResultsHigh expression of SNHG1 was exhibited in esophageal squamous cell cancer and indicated poor outcomes of patients. SNHG1 silence led to cell cycle arrest at G0-G1 phase, inhibition of migration and invasion and increase of apoptosis. miR-204 was validated to sponge by SNHG1 and target HOXC8 in esophageal squamous cell cancer cells. miR-204 knockdown or HOXC8 restoration reversed the inhibitive role of SNHG1 silence in the progression of esophageal squamous cell cancer cells. Furthermore, inhibiting SNHG1 decreased xenograft tumor growth by regulating miR-204 and HOXC8.ConclusionSNHG1 knockdown suppresses migration and invasion but induces apoptosis of esophageal squamous cell cancer cells by increasing miR-204 and decreasing HOXC8.

Effects of HOXA1 Gene Antisense Oligonucleotides on Growth of Esophageal Cancer Cells

To investigate the effect of antisense oligodeoxynucleotides (ASODN) of Homeobox A1 gene ( HOXA1) on proliferation, apoptosis, invasion and migration of esophageal carcinoma cells. The expression of HOXA1 protein in normal esophageal epithelial cells Het-1A and esophageal cancer TE-1, EC9706 and Eca109 cells was detected by Western blot. Screening of highly expressed of HOXA1 protein esophageal squamous cell carcinoma cells for follow-up experiments. HOXA1 antisense oligonucleotide (ASODN) chains, sense oligodeoxynucleotides (SODN) chain, and nonsense oligodeoxy nucleotides (N-ODN) chain were designed. The screened esophageal squamous cell carcinoma cells with high expression were divided into HOXA1 ASODN group (5, 10, 15 μmol/L HOXA1 ASODN transfected Eca109 cells), control group (conventional culture medium, no cell transfection), SODN group (cells transfected with 15 μmol/L of SODN) and N-ODN group (cells transfected with 15 μmol/L N-ODN). Cell viability, apoptosis rate and invasion and migration ability were detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method, flow cytometry, transwell chamber respectively; The expression of HOXA1, phosphorylation serine/threonine kinase (p-AKT), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP-2) and B-cell lymphoma2 (Bcl-2) associated X protein (Bax) protein was detected by Western blot. Compared with normal esophageal epithelial cells Het-1A, the expression of HOXA1 protein in human esophageal squamous cell carcinoma cells TE-1, EC9706 and Eca109 was significantly higher ( P<0.05). The expression of HOXA1 protein was the highest in Eca109 cells, therefore, Eca109 cells were selected for follow-up experiments. The expression of HOXA1 protein in Eca109 cells transfected with HOXA1 ASODN was significantly decreased ( P<0.05). After transfection of Eca109 cells with HOXA1 ASODN, the viability of Eca109 cells decreased with the increase of concentration and time, the difference was significant compared with the control, SODN and N-ODN groups ( P<0.05). 15 μmol/L HOXA1 ASODN significantly inhibited cell viability. After 15 μmol/L HOXA1 ASODN was transfected into Eca109 cells, the invasion and migration abilities of cells were significantly decreased, the apoptosis rate was increased, the expressions of p-AKT, PCNA and MMP-2 were significantly decreased, and the expression of Bax was significantly increased ( P<0.05). Antisense oligodeoxynucleotides of HOXA1 gene can inhibit the proliferation, invasion and migration of esophageal cancer cells, and induce apoptosis. The mechanism is related to the inhibition of PI3K/AKT signaling pathway.

Nipped-B-like Protein Sensitizes Esophageal Squamous Cell Carcinoma Cells to Cisplatin via Upregulation of PUMA.

Nipped-B-like protein plays a pivotal role as a cohesin loading factor in the segregation of chromosomes when cells divide. Accumulating evidence indicates that alterations of this protein are involved in human carcinogenesis, especially in the regulation of chemotherapeutic drug response. However, the role of Nipped-B-like protein in esophageal squamous cell carcinoma remains unknown. In this study, we investigated the relevance of Nipped-B-like protein in the regulation of cisplatin sensitivity in esophageal squamous cell carcinoma. Ectopic expression of Nipped-B-like protein inhibited the growth of COLO-680N cells with low endogenous expression levels of Nipped-B-like protein, and increased sensitivity to cisplatin, a commonly used chemotherapy drug for patients with esophageal squamous cell carcinoma. In contrast, loss of Nipped-B-like protein stimulated the growth of EC9706 and Eca-109 cells with high levels of the protein, and resulted in resistance to cisplatin. P53-upregulated modulator of apoptosis, which is essential in the modulation of cisplatin sensitivity in a variety of cancers, acts as a downstream effector of Nipped-B-like protein. Restoration of this pro-apoptotic protein in Nipped-B-like protein-overexpressing esophageal squamous cell carcinoma cells effectively increased cisplatin sensitivity. Conversely, the silencing of P53-upregulated modulator of apoptosis in Nipped-B-like protein-depleted esophageal squamous cell carcinoma rendered cells resistant to cisplatin. Moreover, Nipped-B-like protein could bind directly to the promoter region of P53-upregulated modulator of apoptosis. In summary, our study addresses the involvement of Nipped-B-like protein in the development of esophageal squamous cell carcinoma, and the modulation of cisplatin sensitivity via regulation of P53-upregulated modulator of apoptosis.