Downregulation of miR-138 sustains NF-κB activation and promotes lipid raft formation in esophageal squamous cell carcinoma.

Constitutive activation of NF-κB signaling plays vital roles in esophageal squamous cell carcinoma (ESCC) progression. The aim of this study was to evaluate the effect of miR-138 on NF-κB activation and ESCC progression. Expression of miR-138 in ESCC cell lines, ESCC tissues, and 205 archived ESSC specimens was determined using real-time PCR analysis. Anchorage-independent growth, chicken chorioallantoic membrane, Transwell matrix invasion and Annexin V-binding assays, and a xenograft tumor model were used to determine the role of miR-138 in ESCC progression. The effect of miR-138 on NF-κB activation was investigated using IKK in vitro kinase, electrophoretic mobility shift, lipid raft isolation, and luciferase reporter assays. miR-138 was downregulated and inversely correlated with tumor progression and patient survival in ESCCs. Downregulation of miR-138 enhanced, whereas upregulation of miR-138 reduced, the aggressive phenotype of ESCC cells both in vitro and in vivo. Silencing miR-138 promoted K63-linked polyubiquitination of the NF-κB signaling intermediaries TRAF2 and RIP1 and sustained NF-κB activation. Furthermore, downregulation of miR-138 induced lipid raft formation via upregulating multiple components of lipid rafts, including FLOT1, FLOT2, and caveolin-1. Importantly, the in vitro analysis was consistent with a significant inverse correlation between miR-138 expression and NF-κB hyperactivation in a cohort of human ESCC specimens. Our results show that miR-138 functions as a tumor-suppressive miRNA and that downregulation of miR-138 contributes to constitutive NF-κB activation and ESCC progression.

Gong H ，Song L ，Lin C ，Liu A ，Lin X ，Wu J ，Li M ，Li J ... -  《-》

Flotillin-1 promotes tumor necrosis factor-α receptor signaling and activation of NF-κB in esophageal squamous cell carcinoma cells.

The flotillin family of proteins, including flotillin-1 (FLOT1 or Reggie-2), are lipid raft proteins that initiate receptor kinase signaling and are up-regulated in several tumor types. We investigated the role of FLOT1 signaling and activation of the transcription factor nuclear factor (NF)-κB in esophageal squamous cell carcinoma (ESCC) cells. We used immunoblot and immunochemical analyses to determine levels of the lipid raft-associated protein FLOT1 in ESCC cell lines and 432 ESSC samples from patients; primary normal esophageal epithelial cells and matched adjacent nontumor tissues were used as controls. We determined the ability of FLOT1 to activate NF-κB using kinase, electrophoretic mobility shift, and luciferase reporter assays. We measured the effects of FLOT1 overexpression and knockdown with short hairpin RNAs in ESCC cell lines using colony formation, anchorage-independent growth, chicken chorioallantoic membrane, transwell matrix penetration, and Annexin V-binding assays. We analyzed growth of ESCC xenograft tumors in nude mice. Levels of FLOT1 were increased in ESCC cell lines and samples from patients, compared with controls; protein levels correlated with disease stage and survival time. Overexpression of FLOT1 in Kyse30 and Kyse510 ESCC cell lines increased proliferation, anchorage-independent growth, and invasive activity and protected them from apoptosis. FLOT1-transduced ESCC cells formed larger tumors in nude mice than control cells (transduced with only the vector). FLOT1 facilitated recruitment of the tumor necrosis factor-α receptor to lipid rafts; promoted K63-linked polyubiquitination of the signaling intermediaries tumor necrosis factor receptor associated factor 2, receptor interacting protein, and NEMO; and sustained the activation of NF-κB. Levels of FLOT1 correlated with activation of NF-κB in ESCC samples from patients. The lipid raft protein FLOT1 is up-regulated in ESCC cell lines and samples from patients and promotes ESCC cell proliferation and tumor growth in mice. FLOT1 activates tumor necrosis factor-α receptor signaling and sustains activation of NF-κB in ESCC cells.

Song L ，Gong H ，Lin C ，Wang C ，Liu L ，Wu J ，Li M ，Li J ... -  《-》

MiR-142-3p as a potential prognostic biomarker for esophageal squamous cell carcinoma.

microRNAs (miRNAs), small non-coding RNAs, are always aberrantly expressed in many diseases including human cancers. The aim of this study was to examine and determine the clinical significance of hsa-miR-31, hsa-miR-142-3p, hsa-miR-338-3p, and hsa-miR-1261 expression in esophageal squamous cell carcinoma (ESCC). Expression levels of four selected miRNAs, initially evaluated by microarray, were validated by qRT-PCR. Various statistical methods were used to analyze the relationship between miRNA expression and clinicopathologic features and prognosis in 91 patients with ESCC. MiR-31 and miR-142-3p expression were correlated to histological differentiation in ESCC (P < 0.05, Student's t-test); high miR-142-3p expression was associated with a poor prognosis in all 91 ESCC patients (P = 0.014, log-rank) and identified as an independent prognostic factor in ESCC (P = 0.017, univariate Cox; P = 0.022, multivariate Cox). More importantly, stratified analysis indicated that high miR-142-3p expression was correlated to a poor prognosis within good-prognosis groups comprised of ESCC patients with small tumor size, negative lymph node metastasis, or early stage (all P < 0.05). The main findings suggest that miR-142-3p is involved in the progression of ESCC and is a potential prognostic biomarker for ESCC.

Lin RJ ，Xiao DW ，Liao LD ，Chen T ，Xie ZF ，Huang WZ ，Wang WS ，Jiang TF ，Wu BL ，Li EM ，Xu LY ... -  《-》

miR-145, miR-133a and miR-133b: Tumor-suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma.

MicroRNAs (miRNAs), noncoding RNAs 21-25 nucleotides in length, regulate gene expression primarily at the posttranscriptional level. Growing evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. A search for miRNAs with a tumor-suppressive function in esophageal squamous cell carcinoma (ESCC) was performed using the miRNA expression signatures obtained from ESCC clinical specimens. A subset of 15 miRNAs was significantly downregulated in ESCC. A comparison of miRNA signatures from ESCC and our previous report identified 4 miRNAs that are downregulated in common (miR-145, miR-30a-3p, miR-133a and miR-133b), suggesting that these miRNAs are candidate tumor suppressors. Gain-of-function analysis revealed that 3 transfectants (miR-145, miR-133a and miR-133b) inhibit cell proliferation and cell invasion in ESCC cells. These miRNAs (miR-145, miR-133a and miR-133b), which have conserved sequences in the 3'UTR of FSCN1 (actin-binding protein, Fascin homolog 1), inhibited FSCN1 expression. The signal from a luciferase reporter assay was significantly decreased at 2 miR-145 target sites and 1 miR-133a/b site, suggesting both miRNAs directly regulate FSCN1. An FSCN1 loss-of-function assay found significant cell growth and invasion inhibition, implying an FSCN1 is associated with ESCC carcinogenesis. The identification of tumor-suppressive miRNAs, miR-145, miR-133a and miR-133b, directly control oncogenic FSCN1 gene. These signal pathways of ESCC could provide new insights into potential mechanisms of ESCC carcinogenesis.

Kano M ，Seki N ，Kikkawa N ，Fujimura L ，Hoshino I ，Akutsu Y ，Chiyomaru T ，Enokida H ，Nakagawa M ，Matsubara H ... -  《-》

miR-942 promotes cancer stem cell-like traits in esophageal squamous cell carcinoma through activation of Wnt/β-catenin signalling pathway.

Ge C ，Wu S ，Wang W ，Liu Z ，Zhang J ，Wang Z ，Li R ，Zhang Z ，Li Z ，Dong S ，Wang Y ，Xue Y ，Yang J ，Tan Q ，Wang Z ，Song X ... -  《Oncotarget》