MiR-34a reverses radiation resistance on ECA-109 cells by inhibiting PI3K/AKT/mTOR signal pathway through downregulating the expression of SIRT1.

BACKGROUND: Radiotherapy is an effective treatment for esophageal squamous cell carcinoma (ESCC). However, many ESCC patients relapsed after receiving radiotherapy due to the inherent resistance. The function of miR-34a and SIRT1, as well as the correlation between miR-34a and SIRT1 has been widely claimed in multiple types of malignant tumors. This study aimed to investigate the effects of miR-34a on radiation resistance against ESCC and the underlying mechanism. METHODS: In this study, CCK8, flow cytometry, wounding healing assays, and cell clone formation assay were used to determine the in vitro anti-tumor effects of radiation on radiation-resistant ESCC cell line (rECA-109). The luciferase activity and Western Blot assays were used to investigate the relationship among miR-34a, SIRT1, and the anti-radiation resistant effects. The xenograft experiments were used to verify the important function of miR-34a and SIRT1 in radiation resistance against ESCC. The apoptosis state of tumor tissues was evaluated by TUNEL assay. RESULTS: The introduction of miR-34a significantly induced the cell death and apoptosis of rECA-109 and inhibit the migration of rECA-109 treated by radiation. The anti-tumor effect was accompanied by the downregulation of SIRT1 and the inhibition of PI3K/AKT/mTOR signal pathway. The radiation resistance on rECA-109 cells was reversed by silencing SIRT1, accompanied by the PI3K/AKT/mTOR signal pathway inhibited. In vivo experiments revealed that the radiation resistance on ESCC was reversed by the introduction of miR-34a, the effect of which was promoted by the activation of SIRT1. CONCLUSION: Our results showed that miR-34a could reverse the radiation resistance on rECA-109 cells by downregulating the expression of SIRT1through inhibiting the PI3K-AKT-mTOR signal pathway.

Identification of candidate genes of nasopharyngeal carcinoma by bioinformatical analysis.

OBJECTIVE: This study aimed to identify candidate genes as potential biomarkers in nasopharyngeal carcinoma (NPC) by bioinformatical analysis. METHODS: Three microarray datasets: GSE32906, GSE15170, GSE53819 were download from public database and analyzed to identify the differentially expressed genes (DEGs) between NPC and normal samples. Functional and pathway enrichment analysis of the DEGs were performed. Protein-protein interaction network and gene-transcription factor regulatory network of DEGs were constructed. And the expression of hub genes in NPC was also validated based on the public database. RESULTS: A total of 16 up-regulated and 27 down-regulated genes were screened out from the microarray datasets. Functional and pathway enrichment analysis showed that DEGs were mostly enriched in positive regulation of angiogenesis, mesenchymal cell proliferation, cell surface and DNA binding, ECM-receptor interaction pathway, PI3K-Akt signaling pathway, and pathways in cancer. Five hub genes JUN, VEGFA, FOXM1, MYB, and WNT5A were identified from the protein-protein interaction network. Subsequently, the hub gene-transcription factor regulatory network revealed that STAT3, MYC, SOX2, RUNX2 present key relations with hub genes. The expression of these five hub genes were also validated to be differentially expressed among NPC and normal samples. CONCLUSIONS: The current study indicated that the hub DEGs JUN, VEGFA, FOXM1, MYB, and WNT5A we identified might be potential therapeutic biomarkers of NPC.

Bioinformatic identification of candidate biomarkers and related transcription factors in nasopharyngeal carcinoma.

BACKGROUND: The incidence of nasopharyngeal carcinoma (NPC) is rare, but a certain amount of mortality remains in NPC patients. Our study aimed to identify candidate genes as biomarkers for NPC screening, diagnosis, and therapy. METHODS: We investigated two microarray profile datasets GSE64634 and GSE12452 to screen the potential differentially expressed genes (DEGs) in NPC. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs were also performed. A protein-protein interaction (PPI) network of DEGs was constructed by STRING and visualized by Cytoscape software. The associated transcriptional factor regulatory network of the DEGs was also constructed. RESULTS: A total of 152 DEGs were identified from the GSE64634 and GSE12452 datasets, including 10 upregulated and 142 downregulated genes. Gene functional enrichment analysis indicated that these DEGs were enriched in the cilium movement, antimicrobial humoral response, O-glycan processing, mucosal immune response, carbohydrate transmembrane transporter activity, hormone biosynthetic process, neurotransmitter biosynthetic process, and drug metabolism-cytochrome P450 pathway. Five hub genes (DNALI1, RSPH4A, RSPH9, DNAI2, and ALDH3A1) and one significant module (score = 5.6) were obtained from the PPI network. Key transcriptional factors, such as SPI1, SIN3B, and GATA2, were identified with close interactions with these five hub DEGs from the gene-transcriptional factor network. CONCLUSIONS: With the integrated bioinformatic analysis, numerous DEGs related to NPC were screened, and the hub DEGs we identified may be potential biomarkers for NPC.

Clinical and prognostic analyses of 110 patients with N3 nasopharyngeal carcinoma.

OBJECTIVE: To analyze the clinical outcome and prognostic factors of N3 nasopharyngeal carcinomas (NPCs), provide a basis for rational treatment and improve the cure rate. METHODS: A total of 110 patients with a pathologically confirmed diagnosis of N3 (NPC 2008 stage in China) NPC from our hospital were retrospectively included in the study conducted from April 2007 to July 2011. All patients received intensity-modulated radiation therapy. Some of these patients received various chemotherapies. The doses of the planning gross primary tumor and retropharyngeal lymph node volume, high-risk planning tumor volume, low-risk planning tumor volume, and gross tumor volume of neck lymph nodes were 6000 to 7600, 5400 to 6600, 5000 to 6000, and 6000 to 6996 cGy, respectively. The Kaplan-Meier analysis and logrank test were carried out to calculate and compare the survival rates of the patients, and the Statistical Package for the Social Sciences software version 17.0 was used for all analyses. Meanwhile, the Cox model was used to analyze the prognostic factors. RESULTS: In this study, the 1-, 3-, and 5-year overall survival rates of the patients were 92.63%, 83.16%, and 70.53%, respectively. Based on the univariate analysis, T stage (P = .043) and chemotherapy (P = .003) were significant factors for survival. In the multivariate analysis, only chemotherapy influenced survival (). Recent toxicity included radioactive oral mucosa inflammation and skin injury, and difficulty opening the mouth and hearing loss were considered late adverse reactions. None of the patients died during treatment.(Table is included in full-text article.) CONCLUSIONS:: Patients with N3 NPC are at high risk of distant metastasis, and their 5-year survival rate is poor. The more important prognostic factors were T stage and chemotherapy. Patients with N3 NPC should be treated with combined chemotherapy and radiotherapy.

A comparison of neoadjuvant chemotherapy with gemcitabine versus docetaxel plus cisplatin in locoregionally advanced nasopharyngeal carcinoma: a propensity score matching analysis.

PURPOSE: To compare the efficacy and safety of neoadjuvant chemotherapy (NACT) with gemcitabine (GEM) vs docetaxel plus cisplatin (CDDP) in locoregionally advanced nasopharyngeal carcinoma (NPC). METHODS: A total of 222 patients with locoregionally advanced NPC between February 2012 and May 2014 in our hospital who received NACT with GEM or docetaxel plus CDDP combined with concurrent chemoradiotherapy (CCRT) were retrospectively analyzed. Fifty-two patients treated with GEM plus CDDP (GP) combined with CCRT were matched with 52 patients who received docetaxel plus CDDP (TP) combined with CCRT. RESULTS: With a median follow-up time of 60 months (range, 14-72 months), the 5-year overall survival, progression-free survival (PFS), local relapse-free survival and distant metastasis-free survival (DMFS) rates were 78.8%, 66.0%, 81.0% and 75.9%, respectively, in the GP group and 79.4%, 60.5%, 79.6% and 73.6%, respectively, in the TP group. No statistically significant survival differences were found between the two groups. In multivariate analysis, T3-4 and N2-3 were prognostic factors for poor 5-year PFS and DMFS (all P-values <0.05). Patients in the TP group experienced less grade 3-4 thrombocytopenia but more grade 3-4 leucopenia and neutropenia than those in the GP group (all P-values <0.05). There were no significant differences between the two groups in other toxicities (all P-values >0.05). CONCLUSION: NACT with GP or TP regimen achieved comparable clinical outcome with acceptable toxicities. Both regimens might be a treatment option for patients with locoregionally advanced NPC.

Neoadjuvant Chemotherapy with Fluorouracil plus Nedaplatin or Cisplatin for Locally Advanced Nasopharyngeal Carcinoma: a Retrospective Study.

The present study aimed to evaluate the efficacy, toxicity and long-term outcome of nedaplatin or cisplatin combined with 5-fluorouracil neoadjuvant chemotherapy (NF or PF regimen) followed by concurrent chemoradiotherapy (CCRT) for treatment of locally advanced nasopharyngeal carcinoma (NPC). In this study, a total of 186 patients with locally advanced NPC between January 2009 and November 2011 in our center were retrospectively analyzed. 103 cases were received NF neoadjuvant chemotherapy followed by nedaplatin concurrent intensity-modulated radiotherapy (IMRT), and 83 cases were received PF neoadjuvant chemotherapy followed by cisplatin concurrent IMRT. Overall survival (OS), progression-free survival (PFS), local relapse-free survival (LRFS), regional relapse-free survival (RRFS) and distant metastasis-free survival (DMFS), as well as acute toxicities were monitored. Results showed that there were no significant differences in 5-year OS, PFS, LRFS, RRFS and DMFS between NF and PF groups. NF group had a higher incidence of grade 3-4 neutropenia (46.6% vs. 31.3%, P=0.035) and thrombocytopenia (17.5% vs. 7.3%, P=0.042) compared with PF group. However, NF group was less common to suffer from grade 3-4 nausea (1.9% vs. 24.1%, P<0.001), vomiting (0% vs. 13.3%, P<0.001) and weight loss (0% vs. 4.8%, P=0.025). In multivariate analysis, N stage was an independent factor for OS, PFS, RRFS and DMFS. In conclusion, neoadjuvant chemotherapy with fluorouracil plus nedaplatin followed by nedaplatin concurrent with IMRT exhibited similar efficacy but more tolerable toxicity than cisplatin setting, which might be an effective and safe choice for treatment of locally advanced NPC.

Downregulation of miR­429 and inhibition of cell migration and invasion in nasopharyngeal carcinoma.

Viral, dietary and genetic factors have been implicated in nasopharyngeal carcinoma (NPC), however, the molecular mechanism underlying its pathogenesis remains to be fully elucidated. MicroRNAs (miRNAs) have been reported to be important in NPC tumorigenesis, with a previous miRNA microarray study showing the downregulation of miRNA (miR)­429 in NPC cells. However, the possible mechanisms of action of miR­429 have not been examined. In the present study, the expression profiles of miR­429 were detected using reverse transcription­quantitative polymerase chain reaction analysis in CNE­1 and CNE­2 cells, which are two generally used NPC cells with different degrees of differentiation. Subsequently, cell proliferation, invasion and migration were analyzed in miR­429­overexpressing CNE­2 cells, and the modulatory function of miR­429 was also investigated using two target genes, zinc finger E­Box­binding homeobox 1 (ZEB1) and CRK­like (CRKL), by transfection with miR­429 mimic or anti­miR­429. Significant changes in the expression of miR­429 were detected, particularly in low­differentiated CNE­2 cells, with higher levels of epidemicity and malignancy. Additional results revealed that miR­429 inhibited the invasion and migration of the CNE­2 cells, whereas no significant effect on cell growth was observed. In addition, the mRNA and protein expression levels of the two target genes, ZEB1 and CRKL, were negatively regulated by miR­429, demonstrated through gain­of­function and loss­of­function investigations, indicating that these two functional downstream targets may be involved in the inhibitory effects of miR­429 on NPC migration and invasion. miR­429 may act as a negative regulatory factor of NPC tumorigenesis, involving the functions of its downstream targets, ZEB1 and CRKL. The results suggested miR­429 as a potential candidate for miRNA­based prognosis or therapy against NPC.

miR-195 is a key regulator of Raf1 in thyroid cancer.

Proto-oncogene Raf1 serves as a part of the mitogen-activated protein kinases/extracellular signal-regulated kinase signal transduction pathway and regulates cell migration, apoptosis, and differentiation. Although a large number of studies have shown that Raf1 is overexpressed in various kinds of cancer, little is known about the association between Raf1 and miRNAs in thyroid carcinoma. This study proves that Raf1 is overexpressed in thyroid cancer, which has been confirmed by many other studies. Besides, we identify that Raf1 is a direct target of miR-15a/b, miR-16, and miR-195 by dual luciferase reporter assay. We also find that the expression of miR-195 is downregulated in 50 pairs of thyroid tumor tissues compared to the adjacent nontumor tissues, while there is no difference in the expression of miR-15a/b and miR-16 between the groups. Furthermore, exogenous overexpression of miR-195 significantly inhibits the protein expression of Raf1 and blocks the thyroid cancer cell proliferation. Our findings delineate a novel mechanism for the regulation of Raf1 in thyroid cancer, which may help to provide a new direction for the treatment of thyroid cancer.

Clinical and prognostic analysis in 32 pediatric nasopharyngeal carcinoma.

OBJECTIVES: To analyze the clinical outcome and prognostic factors of pediatric nasopharyngeal carcinomas, provide the basis for the rational treatment, and improve the cure rate. MATERIALS AND METHODS: Thirty-two pediatric nasopharyngeal carcinoma patients with pathologically confirmed diagnosis and aged from 11 to 18 years old were retrospectively analyzed. All patients received intensity-modulated radiation therapy, dose of GTVnx 6400-7425 cGy, PGTVnx 6400-7050 cGy, PTV 15,400-6000 cGy, PTV 25,000-5490 cGy, and GTVnd 6000-6996 cGy were given. Among these patients, 29 received various chemotherapies. The Kaplan-Meier test and log-rank test of SPSS 17.0 statistic software package were used to calculate survival rate, compare and analyze the survival rates of each group. Cox model was used to analyzing the prognosis factors. RESULTS: In this study, the median survival time was 44.5 months, and the median follow-up time is 62.5 months. The 1-, 3-, and 5-year overall survival rates were 100.0%, 93.2%, 85.7%, respectively, in stage III, and 94.8%, 88.3%, and 64.7% in stage IV. All patients 1-, 3-, and 5-year overall survival rates were 97.2%, 90.5%, and 74.2%. Univariate analysis resulted that N stage (P = 0.043), chemotherapy (P = 0.003) and the radiotherapy dose (P = 0.028) were significant factors for survival. On multivariate analysis, only the N stage influence survival. CONCLUSIONS: In pediatric nasopharyngeal carcinoma, the more important prognostic factors are N stage, chemotherapy, radiotherapy dose. Patients with N2-3 should be treated with combined chemotherapy and radiotherapy.

Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway (PPP) and plays an essential role in the oxidative stress response by producing NADPH, the main intracellular reductant. G6PD deficiency is the most common human enzyme defect, affecting more than 400 million people worldwide. Here, we show that G6PD is negatively regulated by acetylation on lysine 403 (K403), an evolutionarily conserved residue. The K403 acetylated G6PD is incapable of forming active dimers and displays a complete loss of activity. Knockdown of G6PD sensitizes cells to oxidative stress, and re-expression of wild-type G6PD, but not the K403 acetylation mimetic mutant, rescues cells from oxidative injury. Moreover, we show that cells sense extracellular oxidative stimuli to decrease G6PD acetylation in a SIRT2-dependent manner. The SIRT2-mediated deacetylation and activation of G6PD stimulates PPP to supply cytosolic NADPH to counteract oxidative damage and protect mouse erythrocytes. We also identified KAT9/ELP3 as a potential acetyltransferase of G6PD. Our study uncovers a previously unknown mechanism by which acetylation negatively regulates G6PD activity to maintain cellular NADPH homeostasis during oxidative stress.

Arterial interventional chemotherapy and IMRT with concurrent chemotherapy for nasopharyngeal carcinoma with intracranial involvement.

The aim of this study was to ensure a high dose of intensity modulated radiation therapy (IMRT) was delivered to tumor tissue with a low dose to normal organs. Seldinger interventional techniques were used to inject chemotherapy drugs for nasopharyngeal carcinoma (NPC). IMRT was conducted 3 weeks after intervention. Primary tumor volume was reduced by 42.76% after 2 doses of interventional chemotherapy and intracranial tumor volume was reduced by 55.63%. All patients presented grade II and above nasopharyngeal mucositis. In the 2 years following radiotherapy, overall survival (OS) was 83.3% and progression-free survival (PFS) was 75%. In conclusion, T4 NPC patients with intracranial extension received induction chemotherapy followed by IMRT and concurrent chemotherapy, which proved to be efficacious and well tolerated.

Unfavorable clinical implications of circulating CD44+ lymphocytes in patients with nasopharyngeal carcinoma undergoing radiochemotherapy.

BACKGROUND: To evaluate the use of cellular immunity parameters as predictors of therapy response. METHODS: Circulating lymphocytes were measued by flow cytometry in 94 nasopharyngeal carcinoma (NPC) patients following radiochemotherapy. RESULTS: Significantly decreased percentage of CD3(+), CD4(+), and CD8(+) lymphocytes, significantly increased proportion of CD44(+), CD25(+), NK lymphocytes, and an increased CD4(+)/CD8(+) ratio were indicated in NPC patients as compared with healthy controls. Circulating CD44(+) lymphocytes in both the N2/N3 and III/IV groups were significantly increased as compared to the N0/N1 and I/II groups, respectively (P<0.05). A significant decrease in CD19(+) lymphocytes was observed in the III/IV group as compared with the I/II group (P<0.05). After radiochemotherapy, NPC patients had significantly (P<0.05) decreased percentages of CD4(+), CD44(+), and CD19(+) lymphocytes and a decreased CD4(+)/CD8(+) ratio, whereas the mean percentages of CD8(+) and NK lymphocytes were significantly (P<0.05) increased. However, compared with the pre-radiochemotherapy values, no significant (P>0.05) changes in CD3(+) or CD25(+) lymphocytes were observed in the NPC-treated group. Follow-up analysis indicated significantly lower DFS for patients with high CD44(+) lymphocytes compared to those with low CD44(+) lymphocytes after radiochemotherapy. CONCLUSION: Circulating CD44(+) lymphocytes seems to be a promising criterion to predict survival in NPC patients undergoing radiochemotherapy.

Hypermethylation-modulated downregulation of RASSF1A expression is associated with the progression of esophageal cancer.

BACKGROUND AND AIMS: Chromosome 3p21 is an important locus harboring critical tumor suppressor genes (TSGs) implicated in the pathogenesis of multiple tumors including esophageal carcinoma (EC). Aberrant promoter methylation is a fundamental mechanism of inactivation of TSGs in cancer. RASSF1A, a candidate tumor suppressor gene, recently cloned from the lung tumor locus at 3p21.3, is frequently inactivated by hypermethylation of its promoter region in a number of malignancies. We undertook this study to investigate the methylation status of RASSF1A and its significance in esophageal squamous cell carcinoma (ESCC). METHODS: Real-time RT-PCR and real-time methylation-specific PCR (real-time MSP) were used to detect RASSF1A expression and the methylation status of the RASSF1A promoter, respectively, in 124 primary ESCC tissues. RESULTS: Hypermethylation, partial methylation and unmethylation of the promoter region of RASSF1A were detected in 56 (45.2%), 23 (18.6%) and 45 (36.2%) of 124 ESCC samples, respectively. Unmethylation of the promoter region of RASSF1A was detected in 119 (96%) of the 124 corresponding noncancerous tissues. Five (4.0%) of 124 noncancerous tissues showed partial methylation. The presence of hypermethylation was statistically associated with loss of RASSF1A mRNA expression in primary ESCC (p <0.05). There were statistically significant correlations between the presence of hypermethylation and regional lymph node involvement (p=0.000), histological differentiation (p=0.009) and tumor stage (p=0.000). CONCLUSIONS: Our results suggest that RASSF1A may be one of the ESCC-related TSGs located at 3p21, and hypermethylation of the CpG island promoter of the RASSF1A is associated with the progression of ESCC.

Aberrant methylation of different DNA repair genes demonstrates distinct prognostic value for esophageal cancer.

BACKGROUND: DNA mismatch repair (MMR) deficiency results in a strong mutator phenotype and high-frequency microsatellite instability (MSI-H), which are the hallmarks of many tumors. AIM: The objective of this study is to investigate the promoter CpG island methylation status of mismatch repair genes human mutL homolog 1 (hMLH1), human mutS homolog 2 (hMSH2), and O(6)-methylguanine-DNA methyltransferase (MGMT) in esophageal squamous cell carcinoma (ESCC) and its roles in alkylating agents chemotherapy. METHODS: Real-time methylation-specific polymerase chain reaction (PCR) (real-time MSP) was employed to detect promoter CpG island methylation of the hMLH1, hMSH2, as well as MGMT genes in 235 surgical tumor tissue samples from ESCC patients and their corresponding normal tissue samples. RESULTS: Promoter CpG island methylation of hMLH1, hMSH2, and MGMT were detectable in 43.4, 28.9, and 40.4% of ESCC tumor DNA, respectively, and the loss rates of hMLH1, hMSH2, and MGMT protein expression were 48.6, 34.5, and 40.9% in tumor tissues, respectively. For the entire population of 235 ESCC patients who were enrolled in operating treatment combined with radiotherapy and chemotherapy with alkylating agents, there was a significant difference in the overall survival between patients with methylated MGMT promoter and those with an unmethylated MGMT promoter (P < 0.05). CONCLUSION: Promoter CpG island methylation may be a frequent event in ESCC carcinogenesis. Detection of the methylated sequences of hMLH1, hMSH2, and MGMT appears to be promising as a predictive factor in primary ESCC.

Hypermethylation-modulated down-regulation of CDH1 expression contributes to the progression of esophageal cancer.

CDH1, a cell adhesion molecule, which plays a key role in maintaining the epithelial phenotype, is regarded as an invasion-suppressor gene in light of accumulating evidence from in vitro experiments and clinical observations. In an attempt to clarify the mechanism responsible for inactivation of this gene in carcinomas, we investigated the methylation status of the CDH1 gene 5'-CpG islands and its regulatory mechanism in the progression of esophageal squamous cell carcinoma. Real-time methylation-specific polymerase chain reaction (qMSP) and treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) were conducted to analyze the methylation status at the CDH1 promoter region in the human esophageal carcinoma cell lines, EC1 and EC9706. A total of 235 invasive esophageal squamous cell carcinomas (ESCC) at stages I-IV and their corresponding normal tissue samples, were included in an immunohistochemistry study and methylation analysis of CDH1. The results demonstrate that in EC1 and EC9706 cells, the CDH1 promoter is methylated and treatment with 5-Aza-CdR restored CDH1 expression. Enhanced CDH1 expression decreased cell migration, invasion ability and increased adhesion ability. Decreased CDH1 expression was detected in 59.6% of ESCC tissues, compared with their adjacent non-neoplastic epithelia, which had a close correlation with the primary tumor status, lymph node status, distant metatasis and clinicopathologic stage. Hypermethylation at the CDH1 promoter was detected in 97.9% of 140 cases of ESCC with low CDH1 expression. The methylation of CDH1 promoters (P=0.929) was closely correlated with the lack of expression of their corresponding proteins. The Cox regression model for survival analysis showed that increases in CDH1 methylation had a greater impact on the prognosis than tumor clinical stage. These findings suggest that CDH1 gene silencing by promoter hypermethylation and the resultant reduction of CDH1 expression may play an important role in the progression of ESCC. CDH1 methylation was a significant predictor of survival in ESCC patients after surgery.