CircRNA PDE3B regulates tumorigenicity via the miR-136-5p/MAP3K2 axis of esophageal squamous cell carcinoma.

BACKGROUND: CircRNA has a covalently closed circular conformation and a stable structure. However, the exact role of circRNA in esophageal squamous cell carcinoma (ESCC) remains uncertain. The purpose of this study was to explore the role of hsa_circ_0000277 (circ_PDE3B) in ESCC. METHODS: The expression levels of circ_PDE3B, miR-136-5p and mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in ESCC tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. The proliferation ability of EC9706 and KYSE30 cells was detected by clonal formation, 5-ethynyl-2'-deoxyuridine (EdU) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays. Flow cytometry was used to detect the apoptosis rate of cells. Transwell assay was used to detect the invasion ability of EC9706 and KYSE3 cells. The relationship between miR-136-5p and circ_PDE3B or MAP3K2 was verified by dual-luciferase reporter assay and RNA pull-down, and the effect of circ_PDE3B on tumor growth in vivo was explored through tumor transplantation experiment. Immunohistochemistry (IHC) assay was used to detect MAP3K2 and Ki67 expression in mice tumor tissues. RESULTS: The results showed that circ_PDE3B was highly expressed in ESCC tissues and cells. Downregulated circ_PDE3B expression in ESCC cells significantly reduced cell proliferation, migration and invasion. Circ_PDE3B served as a sponge for miR-136-5p, and miR-136-5p inhibition reversed the roles of circ_PDE3B knockdown in ESCC cells. MAP3K2 was a direct target of miR-136-5p, and miR-136-5p targeted MAP3K2 to inhibit the malignant behaviors of ESCC cells. Furthermore, circ_PDE3B regulated MAP3K2 expression by sponging miR-136-5p. Importantly, circ_PDE3B knockdown inhibited tumor growth in vivo. CONCLUSIONS: In conclusion, circ_PDE3B acted as oncogenic circRNA in ESCC and accelerated ESCC progression by adsorption of miR-136-5p and activation of MAP3K2, supporting circ_PDE3B as a potential therapeutic target for ESCC.

Circular RNA _0015278 inhibits the progression of non-small cell lung cancer through regulating the microRNA 1278/SOCS6 gene axis.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most lethal malignancies worldwide. Deepening understanding of the pathogenesis of NSCLC is quite important for its treatment. Circular (circ) RNA_0015278 has been found to be downregulated in NSCLC, but its role in NSCLC and the underlying regulatory mechanism is unknown. METHODS: Circ_0015278, microRNA (miR)-1278 and SOCS6 were analyzed with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) or western blot. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining were used to evaluate cell proliferation. The colony forming capacity and invasion of NSCLC cells were assessed with colony formation and transwell assays, respectively. The interaction among circ_0015278, miR-1278, and SOCS6 was evaluated using luciferase, receptor interacting protein (RIP), and RNA-pull down assays. Cell apoptosis was analyzed using flow cytometry. A subcutaneous NSCLC xenograft mouse model was established for evaluating circ_0015278-mediated effects on the growth of NSCLC in vivo. RESULTS: Circ_0015278 was downregulated in NSCLC tissues and cells, and its reduced expression indicated poor prognosis. Overexpression of circ_0015278 restrained the proliferation, colony formation, invasion, and epithelial-mesenchymal transition (EMT) of NSCLC cells and induced NSCLC cell apoptosis. Moreover, overexpression of circ_0015278 inhibited the growth of NSCLC in vivo. Mechanically, circ_0015278 acted as an miR-1278 sponge to reduce its quantity, and miR-1278 targeted SOCS6 to inhibit its expression in NSCLC cells. Circ_0015278 promoted SOCS6 expression by sponging miR-1,278 in NSCLC cells. Overexpression of circ_0015278 attenuated the malignant phenotypes of NSCLC through sponging miR-1278 and consequently promoting SOCS6 expression. CONCLUSIONS: We demonstrated for the first time that circ_0015278 attenuated the progression of NSCLC via targeting the miR-1278/SOCS6 axis, which provides potential diagnostic biomarkers and therapeutic targets.

Postoperative transient elevation of serum cancer antigen 125 in non-small cell lung cancer patients.

OBJECTIVE: The aim of this retrospective study was to investigate the correlation of transiently elevated postoperative serum cancer antigen 125 levels and prognosis in patients with non-small cell lung cancer. METHODS: A total of 181 non-small cell lung cancer patients with normal levels of preoperative serum cancer antigen 125 were statistically summarized in this study. RESULTS: Out of the analyzed patients, 22 (12.2%) showed elevation of serum cancer antigen 125 within one month after surgery. Serum cancer antigen 125 level decreased to normal at three months postoperation. Serum cancer antigen 125 was positively correlated with pro-brain natriuretic peptide in non-small cell lung cancer postoperative patients (p=0.00035). Univariate analysis did not find significant difference in disease progression survival between those who experienced cancer antigen 125 elevation in the early postoperation and those who did not (p=0.646). CONCLUSIONS: In conclusion, transient elevation of cancer antigen 125 is associated to pro-brain natriuretic peptide increase after pulmonary surgery in non-small cell lung cancer patients.

Postoperative transient elevation of serum cancer antigen 125 in non-small cell lung cancer patients

SUMMARY OBJECTIVE: The aim of this retrospective study was to investigate the correlation of transiently elevated postoperative serum cancer antigen 125 levels and prognosis in patients with non-small cell lung cancer. METHODS: A total of 181 non-small cell lung cancer patients with normal levels of preoperative serum cancer antigen 125 were statistically summarized in this study. RESULTS: Out of the analyzed patients, 22 (12.2%) showed elevation of serum cancer antigen 125 within one month after surgery. Serum cancer antigen 125 level decreased to normal at three months postoperation. Serum cancer antigen 125 was positively correlated with pro-brain natriuretic peptide in non-small cell lung cancer postoperative patients (p=0.00035). Univariate analysis did not find significant difference in disease progression survival between those who experienced cancer antigen 125 elevation in the early postoperation and those who did not (p=0.646). CONCLUSIONS: In conclusion, transient elevation of cancer antigen 125 is associated to pro-brain natriuretic peptide increase after pulmonary surgery in non-small cell lung cancer patients.

Thoracoscopic resection of a huge esophageal dedifferentiated liposarcoma: A case report.

BACKGROUND: Esophageal liposarcoma is a rare malignant tumor and an esophageal dedifferentiated liposarcoma (DDL) is extremely rare. There are no reports on the treatment of DDL by thoracoscopic surgery. CASE SUMMARY: A 38-year-old woman presented with dysphagia and dyspnea. Imaging examination showed a large mass in the posterior mediastinum. The patient also developed respiratory failure and it was unclear whether this was caused by a mass from inside or outside the esophagus. We decided to perform thoracoscopic exploration to relieve the obstruction caused by tracheal compression. The upper segment of the esophagus was split longitudinally, and most of the mass could be removed from the esophageal lumen to the thoracic cavity. The pedicle was excised by linear cutting closers under mirrors. Little residual mass was visualized by gastroscopy. The mucous and muscular layers were closed by interrupted sutures. Pathological examination showed that the mass was a DDL. The patient did not have any dysphagia or dyspnea 2 wk postoperatively and refused any further treatment. Computed tomography and esophagoscopy did not find any recurrence at up to 20 mo postoperatively. CONCLUSION: Thoracoscopy can be used to treat large esophageal masses.

TW37 enhances the pro-apoptosis and anti-migration ability of gefitinib in Non-Small Cell Lung Cancer.

B cell leukemia-2 (Bcl-2) plays important roles in the development of tumor and drug resistance. The growth of tumor cells can be inhibited by downregulating the abnormal expression of Bcl-2 protein. TW37, an effective inhibitor of Bcl-2 protein, has now been widely studied in many tumors. In our study, it was found that TW37 exerted a significant effect on the proliferation, apoptosis and migration of Non-Small Cell Lung Cancer cells. Bcl-2 is also a key downstream factor of many signaling pathways such as Epidermal Growth Factor Receptor (EGFR). TW37 enhanced the inhibition of tumorigenesis by gefitinib, an EGFR-Tyrosine Kinase Inhibitor drug. Moreover, TW37 can promote apoptosis ability by inhibiting the phosphorylation level of EGFR protein in H1975 cells. Overall, TW37 enhances the pro-apoptosis and anti-migration ability of gefitinib in Non-Small Cell Lung Cancer.

Exosomes from Irradiated Nonsmall Cell Lung Cancer Cells Reduced Sensitivity of Recipient Cells to Anaplastic Lymphoma Kinase Inhibitors.

Exosomes, released from various cell types, serve as vehicles of intercellular communication. Rearranged anaplastic lymphoma kinase (ALK) has been detected in exosomes released from cancer cells in ALK-positive nonsmall cell lung cancer (NSCLC), however, the functional consequence of ALK in exosomes has not been studied. This study aims to address whether exosomal ALK release is affected by stress, and whether exosomal ALK can modulate survival of recipient cells in vitro and in vivo. Exosomes, isolated from ALK-containing H3122 cells with (Exo-Apo) or without (Exo-Ctrl) irradiation treatment, were transferred to recipient H3122 cells in vitro or mouse xenograft in vivo. Western blot, flow cytometry, MTT, and xenograft were employed to respectively assess activation of the ALK pathway, apoptosis, cell viability, and tumor growth. Exo-Apo contained much higher levels of phosphorylated ALK (p-ALK) than that of Exo-Ctrl, and it activated AKT, STAT3, and the ERK pathway in recipient H3122 cells. ALK-specific inhibitors, including Crizotinib, Ceritinib, and TAE684, exhibited less effects on H3122 cells preincubated with Exo-Apo than on those treated with Exo-Ctrl in either inhibition of cell viability or promotion of apoptosis. Moreover, in an H3122 xenograft model, the Exo-Apo treatment resulted in a greater tumor growth and less sensitivity to Ceritinib than the Exo-Ctrl treatment. The ALK protein cargo in exosomes could be a key element to drive tumor growth and compromise therapeutic efficacy of ALK inhibitors for ALK-positive NSCLC.

Effect of TW37 on the growth of H1975 EGFR­TKI­resistant lung cancer cells and its underlying mechanisms.

Previous studies have suggested that the B­cell lymphoma 2 (Bcl­2) inhibitor, TW37, may induce apoptosis of the non­small cell lung cancer cell line, H1975/epidermal growth factor receptor­tyrosine kinase inhibitor (EGFR­TKI), which exhibits secondary resistance to EGFR­TKI. However, the effects of TW37 on H1975/EGFR­TKI cells remain unclear. The aim of the present study was to investigate the effects of TW37 on the growth of H1975/EGFR­TKI cells and explore the underlying mechanisms. An in vitro study was performed, whereby H1975/EGFR­TKI cells were treated with serially increasing concentrations of TW37. MTT, flow cytometry, migration and transwell invasion assays were preformed to investigate the proliferation, apoptosis, migration and invasion of H1975/EGFR­TKI cells, respectively. In addition, reverse transcription­polymerase chain reaction and western blot analyses were performed to detect the mRNA and protein expression levels of apoptosis­associated factors, respectively. An enzyme­linked immunosorbent assay was performed to detect phosphatidylinositol [3,4,5] tris­phosphate (PIP3) expression. The results suggested that the mRNA and protein expression levels of Bcl­2 were significantly decreased in TW37­treated cells when compared with the untreated control group. Following treatment with TW37, the proliferation, migration and invasion ability of H1975/EGFR­TKI cells decreased in a dose­dependent manner, while the percentage of apoptotic cells increased. In addition, the results demonstrated that TW37 reduced the expression of PIP3 and the phosphorylation of AKT serine/threonine kinase 1 (AKT) in H1975/EGFR­TKI cells in a dose­dependent manner. In conclusion, TW37 inhibited H1975/EGFR­TKI cell growth and induced cell apoptosis potentially via suppression of AKT signaling pathway activation.

Promoter methylation of TCF21 may repress autophagy in the progression of lung cancer.

Lung cancer is a leading cause of cancer mortality worldwide. Promoter methylation of transcription factor 21 (TCF21) was frequently observed in the early stage of non-small cell lung cancer (NSCLC). However, clinical relevance and molecular functions of TCF21 in NSCLC progression remain unclear. In this study, we analyzed the associations between TCF21 expression and clinicopathological features in 100 patients with NSCLC and revealed the underlying molecular mechanisms of TCF21 methylation on cell viability, apoptosis and invasion of H1299 cells. We found that the expression of TCF21 was significantly regulated by its methylation level in patients with NSCLC and was associated with tumor stage, metastasis and invasion. Demethylation of H1299 cells by 5-aza-2'-deoxycytine (5-Aza) demonstrated that a higher level of TCF21 expression led to remarkable decreases of cell viability and invasion ability but an increase of cell apoptosis. Accordingly, TCF21 knockdown showed converse results to high expression of TCF21. TCF21 knockdown cells exhibited significantly upregulated ATG-9, BECLIN-1, and LC3-I/II expressions but decreased p62 expression compared to wildtype cells. Inhibition of autophagy by 3-methyladenine (3-MA) elevated TCF21 expression and increased cell apoptosis. TCF21 expression is clinically related to the progress of lung cancer and may inhibit autophagy by suppressing ATG-9 and BECLIN-1. In turn, autophagy may also play an important role in regulation TCF21 expression.

Circulating exosomal microRNA-96 promotes cell proliferation, migration and drug resistance by targeting LMO7.

Detection and treatment of lung cancer still remain a clinical challenge. This study aims to validate exosomal microRNA-96 (miR-96) as a serum biomarker for lung cancer and understand the underlying mechanism in lung cancer progression. MiR-96 expressions in normal and lung cancer patients were characterized by qPCR analysis. Changes in cell viability, migration and cisplatin resistance were monitored after incubation with isolated miR-96-containing exosomes, anti-miR-96 and anti-miR negative control (anti-miR-NC) transfections. Dual-luciferase reporter assay was used to study interaction between miR-96 and LIM-domain only protein 7 (LMO7). Changes induced by miR-96 transfection and LMO7 overexpression were also evaluated. MiR-96 expression was positively correlated with high-grade and metastatic lung cancers. While anti-miR-96 transfection exhibited a tumour-suppressing function, exosomes isolated from H1299 enhanced cell viability, migration and cisplatin resistance. Potential miR-96 binding sites were found within the 3'-UTR of wild-type LMO7 gene, but not of mutant LMO7 gene. LMO7 expression was inversely correlated with lung cancer grades, and LMO7 overexpression reversed promoting effect of miR-96. We have identified exosomal miR-96 as a serum biomarker of malignant lung cancer. MiR-96 promotes lung cancer progression by targeting LMO7. The miR-96-LMO7 axis may be a therapeutic target for lung cancer patients, and new diagnostic or therapeutic strategies could be developed by targeting the miR-96-LMO7 axis.

Curcumin increases exosomal TCF21 thus suppressing exosome-induced lung cancer.

Curcumin is a novel drug for lung cancer treatment. However, the mechanism underlying the anti-tumor effect of curcumin remains elusive. Previous evidences indicated that, the methylating transferase DNMT1 is downregulated by curcumin, and the transcription factor 21 (TCF21) is suppressed by DNMT1. We hereby attempt to elucidate the correlation between curcumin treatment and TCF21 expression. Exosomes derived from curcumin-pretreated H1299 cells were used to treat BEAS-2B cells, which induced proliferation, colony formation and migration of BEAS-2B cells. An increase in TCF21 expression in response to curcumin was also seen, as revealed by real-time PCR (RT-PCR) and western blot. Analysis using the GEO database (access #GSE21210) indicated that a positive correlation existed between TCF21 levels and lung cancer patient survival. TCF21 overexpression and knockdown was introduced to H1299 cells through lentiviral system, which led to suppression and promotion of tumor growth, respectively. We also demonstrated that DNMT1 expression was downregulated by curcumin. Therefore, curcumin exerts its anti-cancer function by downregulating DNMT1, thereby upregulating TCF21.

Long-term survival after enucleation of a giant esophageal gastrointestinal stromal tumor.

Gastrointestinal stromal tumors (GISTs) are rare mesenchymal neoplasms of the gastrointestinal tract. Less than 1% occurs in the esophagus. Surgery is the primary treatment for patients with GISTs. We report a 29-year-old male was admitted after the detection of a posterior mediastinal mass during work-up with routine examination. He did not have any disease-related symptoms. The physical examination was unremarkable. Chest computed tomographic scan, the barium esophagogram and endoscopic esophageal ultrasound showed benign neoplasm. The patient was performed an enucleation surgery through the right posterolateral thoracotomy. The pathology revealed a 13.0 cm × 12.0 cm × 5.0 cm mass. The tumor was CD117 (C-kit), PDGFRA and DOG1 positive. These findings were consistent with a GIST of the esophagus. So the diagnosis of GIST of esophagus was confirmed. The pathological diagnosis of low grade of GIST of esophagus was confirmed. The patient has no evidence of recurrence and is in good clinical conditions up-to date, five years after surgery.

Carcinoid tumorlet in pulmonary sequestration with bronchiectasis after breast cancer: A case report.

Pulmonary sequestration (PS) is an uncommon lung disease. Carcinoid tumorlets in pulmonary sequestration are extremely rare. This case report presents a rare clinical case of carcinoid tumorlet in pulmonary sequestration with bronchiectasis after breast cancer. A 64-year-old female was diagnosed with infiltrating ductal carcinoma of the left breast in February 2009. Chest computer tomography (CT) revealed a cystic low-density mass of ∼2.5×4.7 cm in the right lower lung field, as well as cystic bronchiectasis in the right lower lobe. A right lower lobectomy was performed. In the surgery, abnormal vessel growth from the mass was found. Therefore, intralobar PS was diagnosed and pathological examination supported the diagnosis. Subsequently, pathological examination identified a carcinoid tumorlet in the PS. This report presents a rare clinical case of PS and bronchiectasis as well as carcinoid tumorlet in PS following diagnosis of breast cancer three years earlier. When a mass is found in the lung of patents with bronchiectasis with a history of breast cancer, aggressive therapy should be considered, since the mass may be a tumor or precancerous lesion.

RALY RNA binding protein-like reduced expression is associated with poor prognosis in clear cell renal cell carcinoma.

The molecular mechanisms involved in the progression of clear cell renal cell carcinomas (ccRCCs) are still unclear. The aim of this study was to analyse the relationships between expression of RALYL and clinical characteristics. In 41 paired samples of ccRCCs and adjacent normal tissues, we used real-time qPCR to evaluate the expression of RALYL mRNA. RALYL protein levels were determined in 146 samples of ccRCC and 37 adjacent normal tissues by immunohistochemistry. Statistical analysis was used to explore the relationships between expression of RALYL and the clinical characteristics (gender, age, tumor size, T stage, N stage, M stage, survival times and survival outcome) in ccRCC. In addition, these patients were follow-up period 64 months (range: 4~116 months) to investigate the influence on prognosis. We found significantly differences between ccRCC tissues and normal tissues (p<0.001, paired-sample t test) in mRNA levels of RALYL. Immunohistochemistry analyses in 146 ccRCC samples and 37 adjacent normal tissues showed significantly lower RALYL protein levels in ccRCC samples (χ2-test, p<0.001), inversely correlating with tumour size (p=0.024), T stage (0.005), N stage (p<0.001) as well as M stage (p=0.019), but not age (p=0.357) and gender (p=0.348). Kaplan-Meier survival analysis demonstrated that people with lower level of RALYL expression had a poorer survival rate than those with a higher level of RALYL expression, significantly different by the log-rank test (p=0.011). Cox regression analysis indicated that RALYL expression (p=0.039), N stage (p=0.008) and distant metastasis (p<0.001) were independent prognosis factors for the overall survival of ccRCC patients. We demonstrated that the expression of RALYL was significantly low in ccRCC and correlated with a poor prognosis in a large number of clinical samples. Our findings showed that RALYL may be a potential therapeutic target as well as a poor prognostic factor.

Whole-genome synthesis and characterization of viable S13-like bacteriophages.

BACKGROUND: Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template. METHODOLOGY/PRINCIPAL FINDINGS: The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl(2), the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition. CONCLUSIONS/SIGNIFICANCE: The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.

Chemical synthesis of bacteriophage G4.

BACKGROUND: Due to recent leaps forward in DNA synthesis and sequencing technology, DNA manipulation has been extended to the level of whole-genome synthesis. Bacteriophages occupy a special niche in the micro-organic ecosystem and have potential as a tool for therapeutic agent. The purpose of this study was to carry out chemical synthesis of the bacteriophage G4 and the study of its infectivity. METHODOLOGY/PRINCIPAL FINDINGS: Full-sized genomes of bacteriophage G4 molecules were completed from short overlapping synthetic oligonucleotides by direct assembly polymerase chain reaction and ligase chain reaction followed by fusion polymerase chain reaction with flanking primers. Three novel restriction endonuclease sites were introduced to distinguish the synthetic G4 from the wild type. G4 particles were recovered after electroporation into Escherichia coli and were efficient enough to infect another strain. The phage was validated by electron microscope. Specific polymerase chain reaction assay and restriction analyses of the plaques verified the accuracy of the chemical synthetic genomes. CONCLUSIONS: Our results showed that the bacteriophage G4 obtained is synthetic rather than a wild type. Our study demonstrated that a phage can be synthesized and manipulated genetically according to the sequences, and can be efficient enough to infect the Escherichia coli, showing the potential use of synthetic biology in medical application.