Radioresistance has been an important factor in restricting efficacy of radiotherapy for non-small cell lung cancer (NSCLC) patients and new approaches to inhibit cancer growth and sensitize irradiation were warranted. Despite the important role of ubiquitin/proteasome system (UPS) during cancer progression and treatment, the expression and biological role of ubiquitin (Ub) in human NSCLC has not been characterized. In this study, we found that ubiquitin was significantly overexpressed in 75 NSCLC tissues, compared to their respective benign tissues by immunohistochemistry (P < 0.0001). Knock-down of ubiquitin by mixed shRNAs targeting its coding genes ubiquitin B (UBB) and ubiquitin C (UBC) suppressed the growth and increased the radiosensitivity in NSCLC H1299 cells. Apoptosis and γ H2AX foci induced by X-ray irradiation were enhanced by knock-down of ubiquitin. Western blot and immunostaining showed that knock-down of ubiquitin decreased the expression and translocation of NF-κB to the nucleus by reduced phospho-IκBα after irradiation. Suppression of ubiquitin decreased the proliferation and radioresistance of H1299 transplanted xenografts in nude mice by promoting apoptosis. Taken together, our results demonstrate the critical role of ubiquitin in NSCLC proliferation and radiosensitivity. Targeting ubiquitin may serve as a potentially important and novel approach for NSCLC prevention and therapy.

BackgroundA critical mechanism in atrial fibrillation (AF) is cardiac autonomic nerve remodeling (ANR). MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Numerous miRNAs are involved in diseases of the nervous and cardiovascular systems.ObjectiveWe aimed to assess the underlying role of miRNAs in regulating cardiac ANR in AF by right atrial tachypacing (A-TP) in canines.Methods and ResultsFollowing 4-week A-TP, the superior left ganglionated plexuses (SLGPs), which are embedded in the fat pads of the left atrium, were subjected to miRNA expression profiling to screen preferentially expressed miRNAs. Sixteen miRNAs showed significantly differential expression between the control and A-TP groups, including miR-206, miR-203, miR-224 and miR-137. In particular, we focused on miR-206, which was elevated ~10-fold in A-TP dogs. Forced expression of miR-206 through lentiviral infection based on A-TP in vivo significantly shortened the atrial effective refractory period (AERP) (81 ± 7 vs. 98 ± 7 ms, P < 0.05). Immunohistochemical analysis showed that the regeneration of nerves increased more than 2-fold by miR-206 overexpression (P < 0.01). The expression of superoxide dismutase 1 (SOD1) was repressed by miR-206 overexpression by Western blot and luciferase assay, indicative of SOD1 as a direct target of miR-206. Overexpression of miR-206 increased reactive oxygen species (ROS) levels in vitro and in vivo, whereas miR-206 silencing attenuated irradiation- or A-TP-induced ROS. Knockdown of SOD1 effectively abolished ROS reduction caused by miR-206 silencing.ConclusionsOur results found the differential expression of miRNAs in response to ANR in AF and elucidated the important role of miR-206 by targeting SOD1. The study illustrated the novel molecular mechanism of ANR and indicated a potential therapeutic target for AF.

Radiotherapy is an important procedure for the treatment of inoperable non-small cell lung cancer (NSCLC). However, recent evidence has shown that irradiation can promote the invasion and metastasis of several types of cancer, and the underlying mechanisms are not fully understood. This study aimed to investigate the molecular mechanism by which radiation enhances the invasiveness of NSCLC cells. We found that after irradiation, hypoxia-inducible factor 1α (HIF-1α) was increased and translocated into the nucleus, where it bound to the hypoxia response element (HRE) in the CXCR4 promoter and promoted the transcription of CXCR4. Furthermore, reactive oxygen species (ROS) also plays a role in the radiation-induced expression of CXCR4. Our results revealed that 2 Gy X-ray irradiation promoted the metastasis and invasiveness of H1299, A549 and H460 cells, which were significantly enhanced by SDF-1α treatment. Blocking the SDF-1α/CXCR4 interaction could suppress the radiation-induced invasiveness of NSCLC cells. The PI3K/pAkt and MAPK/pERK1/2 pathways were found to be involved in radiation-induced matrix metalloproteinase (MMP) expression. In vivo, irradiation promoted the colonization of H1299 cells in the liver and lung, which was mediated by CXCR4. Altogether, our findings have elucidated the underlying mechanisms of the irradiation-enhanced invasiveness of NSCLC cells.

Esophageal squamous cell carcinoma (ESCC) has one of the highest mortality rates worldwide. AU-rich element RNA-binding factor 1 (AUF1) is an established RNA-binding protein. AUF1 influences the process of development, apoptosis and tumorigenesis via interacting with adenylate-uridylate rich elements (AREs) bearing mRNAs. However, the clinical relevance of AUF1 and its biological function in ESCC progression have not been reported. In the present study, we first investigated the expression of AUF1 in the ESCC tissue samles and normal samples. We found a significantly higher expression of AUF1 in ESCC tissues than that in normal tissues and tumor adjacent tissues. The expression of AUF1 correlated with ESCC stage (P=0.011) and marginally correlated with lymph node metastasis (P=0.055) of ESCC patients. Silencing of AUF1 by an siRNA inhibited the proliferation and enhanced the apoptosis of ESCC cells. mRNA profiling by microarray analysis revealed that AUF1 knockdown affected 285 genes (fold change ≥2) that function in multiple pathways. GTP cyclohydrolase I (GCH1), the rate limiting enzyme for BH4 synthesis, was found to be downregulated. One of the AU-rich elements in the 3'UTR of GCH1 was found to be responsive to AUF1 expression by luciferase assay. Knockdown of GCH1 suppressed cell proliferation and colony formation of ESCC cells. The expression of AUF1 significantly correlated with that of GCH1 in ESCC tissues. Taken together, we demonstrated the overexpression of AUF1 in esophageal carcinoma and identified GCH1 as AUF1's effector for the proliferation of ESCC cells.

Esophageal squamous cell carcinoma (ESCC), one of the most common gastrointestinal tumors, is known for its high mortality rate. microRNAs (miRNAs) have been reported to play important regulatory roles in cancer metastasis and progression. miR-34a has been demonstrated to be associated with the development of and metastasis in certain types of cancer via various target genes, but its function and targets in ESCC are unknown. The aim of this study was to examine whether the expression of miR-34a was significantly decreased in ESCC tissues, compared with normal esophageal tissues using RT-PCR and western blot analysis. The results showed that miR-34a overexpression increased apoptosis and decreased clonogenic formation, but inhibited invasion and migration in ESCC cells by suppressing MMP-2 and -9 expression. Yin Yang-1 (YY1), a widely distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins, was found to be a direct target of miR-34a in ESCC cell lines. Rescue experiments indicated that the suppressive effect of miR-34a on invasion and migration was mediated by activating YY1 expression. Results of the present study showed that miR-34a is associated with ESCC migration and provides a potential therapeutic and diagnostic target for ESCC.

Superoxide dismutase 1 (SOD1) is the ubiquitously expressed and predominant dismutase in the cytoplasm. While transcriptional regulation of the SOD1 gene has been well characterized, post-transcriptional regulation of the gene remains largely unknown in eukaryotes. In this study, a full-length 3’UTR of the SOD1 transcript was cloned and characterized for its ability to regulate SOD1 gene expression in human cancer cells. Inclusion of the SOD1 3’UTR in the pGL3 reporter construct dramatically enhanced the reporter activity by 10–220-fold in various cell lines. RT-PCR analysis, however, indicated that the reporter gene mRNA levels were only modestly altered by the SOD1 3’UTR, suggesting that SOD1 3’UTR enhances the reporter gene activity not simply by stabilizing the mRNA but primarily through promoting translation of the protein. Bioinformatics analysis showed multiple stem and loop structures of the SOD1 3’UTR and alterations of this secondary structure led to remarkably reduced reporter gene activity. Importantly, introducing the SOD1 3’UTR to cancer cells attenuated endogenous SOD1 expression in a concentration-dependent manner, indicating the involvement of RNA trans-acting factors in this process. Using siRNA and RNA immunoprecipitation techniques, we identified AUF-1, a RNA binding protein, as a positive regulator of SOD1 expression through its 3’UTR. Consequently, AUF-1 was found to regulate redox balance in our cell model systems. Furthermore, in human ovarian, esophageal, and pancreatic cancer tissues, the expression of SOD1 was significantly correlated with that of AUF-1, further supporting the importance of AUF-1 in regulating SOD1 gene expression.

Lugol's iodine staining (LIS) and narrow-band imaging (NBI) are currently the most common methods applied in demarcating early esophageal squamous cell carcinoma (EESCCs) during endoscopic submucosal dissection. The purpose of the present study was to investigate the effects on clinical outcomes in comparison between LIS and NBI for the demarcation of EESCCs during endoscopic submucosal dissection. This was a single-center, retrospective, cohort study. A total of 172 patients were involved. 109 patients received demarcation of the lesion by LIS and 63 patients by NBI. Data on baseline characteristics, clinical outcomes and follow-up information were collected for analyses. The mean diameter of the lesions was 3.9 ± 1.5 cm. R0 resection rate was 89.5%. The rate of total and in-hospital adverse events was 25.6% and 9.3%. The cumulative recurrence rate was 2.9% and 3-year disease-specific survival rate was 98.3%. Compared to patients of the LIS group, patients of the NBI group showed significantly shorter procedure time (44.8 ± 32.2 v.s. 57.0 ± 40.6, P = .044), lower rate of using of scopolamine butylbromide (19.0% vs 35.8%, P = 0.021), reduced number of clips used (1.3 ± 1.2  vs 1.8 ± 1.5, P = .017) and alleviated discomfort evaluated by visual analog system score after operation (4.7 ± 0.8 vs 5.5 ± 1.0, P < .001). There was no significant difference of R0 resection rate, margin status, adverse events, cumulative recurrence rate and 3-year disease-specific survival rate between the two groups. Demarcation of EESCCs by NBI could achieve comparable accuracy and clinical outcomes with more convenience and safety compared with demarcation by LIS.