Chaojun Duan

Identificating Cathepsin D as a Biomarker for Differentiation and Prognosis of Nasopharyngeal Carcinoma by Laser Capture Microdissection and Proteomic Analysis

In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cathepsin D in the above two tissues as well as four NPC cell lines was determined by Western blotting. Next, siRNA was used to inhibit the expression of cathepsin D in highly metastatic NPC cell line 5-8F to examine whether it associates with NPC metastasis. Immunohistochemistry was also performed to detect the expression of cathepsin D in 72 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cathepsin D in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Down-regulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Significant cathepsin D down-regulation was observed in NPC versus NNET, whereas significant cathepsin D up-regulation was observed in lymph node metastasis versus primary NPC. In addition, cathepsin D down-regulation was significantly correlated with poor histological differentiation, whereas cathepsin D up-regulation was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. Furthermore, survival curves showed that patients with cathepsin D up-regulation had a poor prognosis. Multivariate analysis confirmed that cathepsin D expression was an independent prognostic indicator. The data suggest that cathepsin D is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.

Circular RNAs in cancer: an emerging key player

Circular RNAs (circRNAs) are a class of endogendous RNAs that form a covalently closed continuous loop and exist extensively in mammalian cells. Majority of circRNAs are conserved across species and often show tissue/developmental stage-specific expression. CircRNAs were first thought to be the result of splicing error; however, subsequent research shows that circRNAs can function as microRNA (miRNA) sponges and regulate splicing and transcription. Emerging evidence shows that circRNAs possess closely associated with human diseases, especially cancers, and may serve as better biomarkers. After miRNA and long noncoding RNA (lncRNA), circRNAs are becoming a new hotspot in the field of RNA of cancer. Here, we review biogenesis and metabolism of circRNAs, their functions, and potential roles in cancer.

Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

BackgroundmicroRNAs are small noncoding RNAs that modulate a variety of cellular processes by regulating multiple targets, which can promote or inhibit the development of malignant behaviors. Accumulating evidence suggests that miR-675-5p plays important roles in human carcinogenesis. However, its precise biological role remains largely elusive. This study examined the role of miR-675-5p in non- small cell lung cancer (NSCLC).MethodsThe expression of miR-675-5p was analyzed by real-time quantitative PCR (qRT-PCR). The effect of miR-675-5p on proliferation was evaluated through MTT and colony formation assays, and cell migration and invasion were evaluated through transwell assays. The expression of target proteins and downstream molecules was analyzed by western blotting and immunohistochemical staining. The luciferase reporter assay was used to assess the target genes of miR-675-5p in non-small cell lung cancer cells.ResultsThe expression levels of miR-675-5p in NSCLC tissues were significantly reduced compared to those in adjacent non-cancerous tissues (P < 0.001). The expression of miR-675-5p in patients with non-small cell lung cancer had a negative correlation with lymph node metastasis (P < 0.01) and TNM stage (P < 0.05). Down-regulation of miR-675-5p promoted cell growth, proliferation, colony formation, invasion and migration, and promoted the tumorigenicity graft growth of nude mice in vivo (P < 0.01); whereas up-regulation of miR-675-5p had the contrary effects. The luciferase reporter assay showed that GPR55 was a direct target gene of miR-675-5p. Overexpression of miR-675-5p can lead to the down-regulation of GPR55 and its signaling pathway, whereas the effect can be reversed by down-regulation of miR-675-5p expression.ConclusionsmiR-675-5p functions as a novel tumor suppressor in NSCLC and the anti-oncogenic activity may involve its inhibition of the target gene GPR55. These findings suggest the possibility for miR-675-5p as a therapeutic target in NSCLC.

Quantitative proteomic analysis of differential proteins in the stroma of nasopharyngeal carcinoma and normal nasopharyngeal epithelial tissue

The importance of stromal cells and the factors that they expressed during cancer initiation and progression have been highlighted by recent literature. To identify the stromal proteins involved in nasopharyngeal carcinoma (NPC) carcinogenesis, we assessed differences in protein expression of the stroma from NPC and normal nasopharyngeal epithelium tissues (NNET) using a quantitative proteomic approach combined with laser capture microdissection (LCM). LCM was performed to purify stromal cells from the NPC and NNET, respectively. The differential proteins between the pooled microdissected tumor and normal stroma were analyzed by two‐dimensional difference gel electrophoresis (2D‐DIGE) combined with mass spectrometry (MS). Twenty differential proteins were identified, and the expression and location of two differential proteins (L‐plastin and S100A9) were further confirmed by Western blotting and immunohistochemical analysis. Our results will be helpful to study the role of stroma in the NPC carcinogenesis, as well as discover the interaction between NPC cells and their surrounding microenvironment. J. Cell. Biochem. 106: 570–579, 2009.

MicroRNA-361-3p suppresses tumor cell proliferation and metastasis by directly targeting SH2B1 in NSCLC.

BackgroundLung cancer is the most common malignancies worldwide. However, the detailed molecular mechanisms underlying lung cancer progression are still not completely clear. MicroRNAs are small noncoding RNAs which occupy a crucial role of cancer metastasis. Accumulating evidence suggests that miR-361 plays important roles in human carcinogenesis. However, its precise biological role remains largely elusive, especially in lung cancer. This study examined the role of miR-361-3p in non-small cell lung cancer (NSCLC).MethodsReal-time quantitative PCR (qRT-PCR) was used to analyze the expression of miR-361-3p in NSCLC tissue and in compared adjacent non-cancerous tissues. The effect of miR-361-3p on proliferation was evaluated by CCK8 and colony formation assays. The effect of miR-361-3p on migration and invasion was evaluated by transwell assays. Western blotting and immunohistochemical staining were applied to analyze the expression of target proteins and downstream molecule, and the luciferase reporter assay to assess the target genes of miR-361-3p in non-small cell lung cancer cells.ResultsmiR-361-3p was significantly decreased in NSCLC tissue and cell lines, and its expression levels were highly correlated with lymph node metastasis (P < 0.01) and TNM stages (P < 0.05). Down-regulation of miR-361-3p promoted cell growth, proliferation, colony formation, invasion and migration in vitro, and promoted proliferation and metastasis in vivo (P < 0.01); whereas up-regulation of miR-361-3p had the contrary effects. The luciferase reporter assay showed that SH2B1 was a direct target gene of miR-361-3p. Enforced expression of miR-361-3p inhibited the expression of SH2B1 significantly and the restoration of SH2B1 expression reversed the inhibitory effects of miR-361-3p on NSCLC cell proliferation and metastasis.ConclusionsmiR-361-3p functions as a novel tumor suppressor in NSCLC and the anti-oncogenic activity may involve its inhibition of the target gene SH2B1. These findings suggest the possibility for miR-361-3p as a therapeutic target in NSCLC.

Identification of JAK2 as a mediator of FIP1L1-PDGFRA-induced eosinophil growth and function in CEL.

The Fip1-like1 (FIP1L1)-platelet-derived growth factor receptor alpha fusion gene (F/P) arising in the pluripotent hematopoietic stem cell (HSC),causes 14% to 60% of patients with hypereosinophilia syndrome (HES). These patients, classified as having F/P (+) chronic eosinophilic leukemia (CEL), present with clonal eosinophilia and display a more aggressive disease phenotype than patients with F/P (–) HES patients. The mechanisms underlying predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. Given that the Janus tyrosine kinase (JAK)/signal transducers and activators of transcription (Stat) signaling pathway is key to cytokine receptor-mediated eosinophil development and activated Stat3 and Stat5 regulate the expression of genes involved in F/P malignant transformation, we investigated whether and how JAK proteins were involved in the pathogenesis of F/P-induced CEL. F/P activation of JAK2, Stat3 and Stat5, were confirmed in all the 11 F/P (+) CEL patients examined. In vitro inhibition of JAK2 in EOL-1, primary F/P(+) CEL cells (PC) and T674I F/P Imatinib resistant cells(IR) by either JAK2-specific short interfering RNA (siRNA) or the tryphostin derivative AG490(AG490), significantly reduced cellular proliferation and induced cellular apoptosis. The F/P can enhance the IL-5-induced JAK2 activation, and further results indicated that JAK2 inhibition blocked IL-5-induced cellular migration and activation of the EOL-1 and PC cells in vitro. F/P-stimulation of the JAK2 suppressed cells led to a significantly reduction in Stat3 activation, but relatively normal induction of Stat5 activation. Interestingly, JAK2 inhibition also reduced PI3K, Akt and NF-κB activity in a dose-dependent manner, and suppressed expression levels of c-Myc and Survivin. These results strongly suggest that JAK2 is activated by F/P and is required for F/P stimulation of cellular proliferation and infiltration, possibly through induction of c-Myc and Survivin expression via activation of multiple signaling pathways, including NF-κB, Stat3, and PI3K/Akt.

Clinical controlled comparison between lobectomy and segmental resection for patients over 70 years of age with clinical stage I non-small cell lung cancer

AIMS The standard surgical procedure for elderly (≥ 70 years) patients with clinical stage I non-small cell lung cancer (NSCLC) was investigated. METHODS A non-randomized prospective controlled study was conducted to compare lobectomy with segmental resection for the treatment of elderly clinical stage I lung cancer patients under different pulmonary function. Perioperative indicators including time and volume of thoracic drainage, incidence of postoperative complications, locoregional recurrence rates, and 1, 3, and 5-year survival rates were analyzed. RESULTS A total of 184 patients were included in the study. Patients were classified into two groups according to pulmonary function: group 1 included 64 patients who had poor pulmonary function, with a forced expiratory volume in 1 s (FEV(1)) of less than 1.5 L, whereas group 2 consisted of 120 patients with an FEV(1) ≥ 1.5 L. The patients in group 1 had a longer postoperative mechanical ventilation time and a higher incidence rate of respiratory associated complications than those in group 2 (21.9 vs. 8.35%, p = 0.009). The local recurrence and long-term survival rates were not significantly different between lobectomy and segmental resection. Among the patients who underwent segmental resection, those who had regional lymph node dissection showed a higher 3-year and 5-year survival rate than those undergoing selected lymph node resection (77.8 vs. 51.7%, p = 0.042; 55.6 vs. 27.6%, p = 0.034), but this was not significant in lobectomy. CONCLUSIONS Segmental resection combined with regional lymph node resection could be the best choice for elderly clinical stage I NSCLC patients with FEV(1) < 1.5 L.

miR-675-5p enhances tumorigenesis and metastasis of esophageal squamous cell carcinoma by targeting REPS2

Recently H19 has been demonstrated to be up-regulated in esophageal squamous cell carcinoma (ESCC) and shown to be the precursor of miR-675 that encodes miR-675-5p conservatively. miR-675 is overexpressed in many human cancers; however, the function of miR-675-5p is largely unknown in ESCC. In this study, we found that miR-675-5p expression was significantly increased in ESCC tissues and cell lines and related with ESCC progression and poor prognosis. We also showed here that down-regulation of miR-675-5p in ESCC cells dramatically induced cell G1 arrest and reduced cell proliferation, colony formation, migration and invasion in vitro as well as tumorigenesis and tumor metastasis in vivo. We subsequently identified that REPS2 was a target gene of miR-675-5p. We found that inhibition of miR-675-5p up-regulated the expression of REPS2, inhibited RalBP1/RAC1/CDC42 signaling pathway. Inversely, interference of REPS2 abrogated the effect induced by miR-675-5p inhibition, which resembled the function of miR-675-5p up-regulation. Taken together, our findings suggested that miR-675-5p might play an oncogenic role in ESCC through RalBP1/RAC1/CDC42 signaling pathway by inhibiting REPS2 and might serve as a valuable prognostic biomarker and therapeutic target for ESCC patients.

Proteomic analysis of the stroma-related proteins in nasopharyngeal carcinoma and normal nasopharyngeal epithelial tissues

The stroma surrounding cancer cell population is increasingly recognized as playing an important role in cancer proliferation, invasion, and metastasis. To identify the stromal proteins involved in nasopharyngeal carcinoma (NPC) carcinogenesis, differences in protein expression of the stroma from NPC and normal nasopharyngeal epithelium tissues (NNET) were assessed using a comparative proteomic approach combined with laser capture microdissection (LCM). LCM was performed to purify stromal cells from NPC and NNET, respectively. Proteins between the pooled microdissected tumor and normal stroma were separated by two-dimensional electrophoresis (2-DE) and differential proteins were identified by mass spectrometry (MS). Sixty differential proteins between normal stroma (NS) and tumor stroma (TS) were identified, and the expression of CapG protein was further confirmed by western blotting and immunohistochemical analysis. Our results will be helpful to study the role of stroma in the NPC carcinogenesis and may provide helpful clues for pathogenesis, early diagnosis, and progression of NPC.

Proteomic analysis of differentially expressed proteins in normal human thyroid cells transfected with PPFP

The fusion gene encoding the thyroid-specific transcription factor PAX8 and peroxisome proliferator-activated receptor γ (PPARγ (PPARG)) (designated as the PPFP gene) is oncogenic and implicated in the development of follicular thyroid carcinoma (FTC). The effects of PPFP transfection on the biological characteristics of Nthy-ori 3-1 cells were studied by MTT assay, colony formation, soft-agar colony formation, and scratch wound-healing assays as well as by flow cytometry. Furthermore, the differentially expressed proteins were analyzed on 2-DE maps and identified by MALDI-TOF-MS. Validation of five identified proteins (prohibitin, galectin-1, cytokeratin 8 (CK8), CK19, and HSP27) was determined by western blot analysis. PPFP not only significantly increased the viability, proliferation, and mobility of the Nthy-ori 3-1 cells but also markedly inhibited cellular apoptosis. Twenty-eight differentially expressed proteins were identified, among which 19 proteins were upregulated and nine proteins were downregulated in Nthy-ori 3-1(PPFP) (Nthy-ori 3-1 cells transfected with PPFP). The western blot results, which were consistent with the proteome analysis results, showed that prohibitin was downregulated, whereas galectin-1, CK8, CK19, and HSP27 were upregulated in Nthy-ori 3-1(PPFP). Our results suggest that PPFP plays an important role in malignant thyroid transformation. Proteomic analysis of the differentially expressed proteins in PPFP-transfected cells provides important information for further study of the carcinogenic mechanism of PPFP in FTCs.