Downregulation of zinc finger protein 71 expression in oral squamous cell carcinoma tissues and its underlying molecular mechanism.

BACKGROUND: Patients with oral squamous cell carcinoma (OSCC) have poor prognoses due to a limited understanding of the pathogenesis of OSCC. Zinc finger protein (ZNF) is the largest transcription factor family in the human genome and exert diverse and important functions. Nevertheless, the exact expression status and molecular mechanism of ZNF71 have not been described in OSCC. Therefore, this study aimed to identify the specific expression level of ZNF71 in OSCC tissues and to further interpret the potential molecular mechanism of ZNF71 in the pathogenesis of OSCC. METHODS: In-house immunohistochemical staining of 116 OSCC samples and 29 non-OSCC samples was employed to detect the expression status of ZNF71 at the protein level of OSCC tissues. Single-cell RNA sequencing data from 7 OSCC samples was used to explore the expression landscape of ZNF71 in different cell types from OSCC tissues. High-throughput RNA sequencing data and gene chips data from 893 OSCC samples and 301 non-OSCC samples were utilized to identify the specific expression level of ZNF71 at the bulk mRNA level of OSCC tissues. Here, standardized mean difference (SMD) value was applied to calculate the expression differences between OSCC group and non-OSCC group. Multiple datasets were included; hence, the results were considered to be more reliable. Sensitivity analysis was conducted to evaluate the stability of the results. Enrichment analysis and immune infiltration analysis were used to explore the underlying molecular mechanism of ZNF71 in OSCC. RESULTS: ZNF71 was significantly downregulated in OSCC tissues at the protein level (SMD = -1.96, 95 % confidence interval [95 % CI]: -2.43 to -1.50). ZNF71 was absent in various cell types from OSCC tissues including cancerous epithelial cells and tumor-infiltrating immune cells. ZNF71 was downregulated in OSCC tissues at the bulk mRNA level (SMD = -0.38, 95 % CI: -0.75 to -0.02). Enrichment analysis showed that positively and differentially co-expressed genes mainly concentrated on "herpes simplex virus 1 infection" and "regulation of plasma membrane bounded cell projection organization", and negatively and differentially co-expressed genes mainly participated in "cell cycle" and "DNA metabolic process". Moreover, the putative target genes of ZNF71 mainly participated in "cellular respiration" and "protein catabolic process". Finally, immune infiltration analysis revealed that ZNF71 expression was positively correlated with multiple immune cells including activated B cells, memory B cells, and natural killer (NK) cells, and negatively correlated with various immune cells, including CD56 bright NK cells, neutrophil, and immature dendritic cells. CONCLUSION: The downregulation of ZNF71 may influence the initiation and promotion of OSCC by reducing immune infiltration, accelerating cell cycle progression, and affecting metabolic process, and this requires further research.

Signature microRNAs and long noncoding RNAs in laryngeal cancer recurrence identified using a competing endogenous RNA network.

The aim of the present study was to identify novel microRNA (miRNA) or long noncoding RNA (lncRNA) signatures of laryngeal cancer recurrence and to investigate the regulatory mechanisms associated with this malignancy. Datasets of recurrent and nonrecurrent laryngeal cancer samples were downloaded from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus database (GSE27020 and GSE25727) to examine differentially expressed miRNAs (DE­miRs), lncRNAs (DE­lncRs) and mRNAs (DEGs). miRNA­mRNA and lncRNA­miRNA networks were constructed by investigating the associations among these RNAs in various databases. Subsequently, the interactions identified were combined into a competing endogenous RNA (ceRNA) regulatory network. Feature genes in the miRNA­mRNA network were identified via topological analysis and a recursive feature elimination algorithm. A support vector machine (SVM) classifier was established using the betweenness centrality values in the miRNA­mRNA network, consisting of 32 optimal feature­coding genes. The classification effect was tested using two validation datasets. Furthermore, coding genes in the ceRNA network were examined via pathway enrichment analyses. In total, 21 DE­lncRs, 507 DEGs and 55 DE­miRs were selected. The SVM classifier exhibited an accuracy of 94.05% (79/84) for sample classification prediction in the TCGA dataset, and 92.66 and 91.07% in the two validation datasets. The ceRNA regulatory network comprised 203 nodes, corresponding to mRNAs, miRNAs and lncRNAs, and 346 lines, corresponding to the interactions among RNAs. In particular, the interactions with the highest scores were HLA complex group 4 (HCG4)­miR­33b, HOX transcript antisense RNA (HOTAIR)­miR­1­MAGE family member A2 (MAGEA2), EMX2 opposite strand/antisense RNA (EMX2OS)­miR­124­calcitonin related polypeptide α (CALCA) and EMX2OS­miR­124­Î³­aminobutyric acid type A receptor Î³2 subunit (GABRG2). Gene enrichment analysis of the genes in the ceRNA network identified that 11 pathway terms and 16 molecular function terms were significantly enriched. The SVM classifier based on 32 feature coding genes exhibited high accuracy in the classification of laryngeal cancer samples. miR­1, miR­33b, miR­124, HOTAIR, HCG4 and EMX2OS may be novel biomarkers of recurrent laryngeal cancer, and HCG4­miR­33b, HOTAIR­miR­1­MAGEA2 and EMX2OS­miR­124­CALCA/GABRG2 may be associated with the molecular mechanisms regulating recurrent laryngeal cancer.

Drug repositioning in head and neck squamous cell carcinoma: An integrated pathway analysis based on connectivity map and differential gene expression.

The severe damage to health and social burden caused by head and neck squamous cell carcinoma (HNSCC) generated an urgent need to develop novel anti-cancer therapy. Currently, drug repositioning has risen in responses to the proper time as an efficient approach to invention of new anti-cancer therapies. In the present study, we aimed to screen candidate drugs for HNSCC by integrating HNSCC-related pathways from differentially expressed genes (DEGs) and drug-affected pathways from connectivity map (CMAP). We also endeavored to unveil the molecular mechanism of HNSCC through creating drug-target network and protein-to-protein (PPI) network of component DEGs in key overlapping pathways. As a result, a total of 401 DEGs were obtained from TCGA and GTEx mRNA-seq data. Taking the intersection part of 27 HNSCC-related Kyoto Encyclopedia of Genes and Genomes pathways and 33 drug-affected pathways, we retained 22 candidate drugs corresponding to two key pathways (cell cycle and p53 signaling pathways) of the five overlapping pathways. Two of the hub genes (PCNA and CCND1) identified from the PPI network of component DEGs in cell cycle and p53 signaling pathways were defined as the critical targets of candidate drugs with increased protein expression in HNSCC tissues, which was reported by the human protein atlas (HPA) database and cBioPortal. Finally, we validated via molecular docking analysis that two drugs with unknown effects in HNSCC: MG-262 and bepridil might perturb the development of HNSCC through targeting PCNA. These candidate drugs possessed broad application prospect as medication for HNSCC.

Expression level and prospective mechanism of miRNA-99a-3p in head and neck squamous cell carcinoma based on miRNA-chip and miRNA-sequencing data in 1, 167 cases.

BACKGROUND: The role of miR-99a-3p in Head and neck squamous cell carcinoma (HNSCC) has not been reported. Therefore, in this study, we examined the expression level and its molecular mechanisms of miR-99a-3p in HNSCC. MATERIALS AND METHODS: MiR-99a-3p-related miRNA-chip and miRNA-sequencing data were collected. We then carried out meta-analyses to pool the standard mean difference (SMD) value and generate a summarized receiver operating characteristic (sROC) curve. MiR-99a-3p mimic was transfected into FaDu cells and those genes influenced by miR-99a-3p were gathered. The target genes were also predicted from 12 tools through miRwalk2.0, and combined with differentially expressed genes in HNSCC from the The Cancer Genome Atlas and Genotype-Tissue Expression sequencing databases. FunRich and DAVID were used for the pathway signaling analyses for the potential targets of miR-99a-3p in HNSCC. RESULTS: The SMD was -0.30 (95% CI: -0.51, -0.08) in the fixed-effect model and -0.28 (95% CI: -0.67, 0.10) in the random-effect model (I2 = 60%), indicating a reduced expression level of miR-99a-3p in HNSCC tissues based on 1167 cases. In the sROC curve, the area under the curve (AUC) was 0.77 (95% CI: 0.73, 0.81). The 251 potential targets of miR-99a-3p were enriched in several pathways related to cancer, with the "Pathways in cancer" standing at the top. vascular endothelial growth factor A was selected as an example with up-regulated trend in HNSCC tissues. CONCLUSION: MiR-99a-3p exhibits a significant lower expression status in HNSCC, and this reduced or deletion status promotes the malignant progression of HNSCC. However, its molecular mechanism is still unclear and requires further investigation.

A regulatory mutant on TRIM26 conferring the risk of nasopharyngeal carcinoma by inducing low immune response.

The major histocompatibility complex (MHC) is most closely associated with nasopharyngeal carcinoma (NPC), but the complexity of its genome structure has proven challenging for the discovery of causal MHC loci or genes. We conducted a targeted MHC sequencing in 40 Cantonese NPC patients followed by a two-stage replication in 1065 NPC cases and 2137 controls of Southern Chinese descendent. Quantitative RT-PCR analysis (qRT-PCR) was used to detect gene expression status in 108 NPC and 43 noncancerous nasopharyngeal (NP) samples. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) were used to assess the transcription factor binding site. We discovered that a novel SNP rs117565607_A at TRIM26 displayed the strongest association (OR = 1.909, Pcombined = 2.750 × 10-19 ). We also observed that TRIM26 was significantly downregulated in NPC tissue samples with genotype AA/AT than TT. Immunohistochemistry (IHC) test also found the TRIM26 protein expression in NPC tissue samples with the genotype AA/AT was lower than TT. According to computational prediction, rs117565607 locus was a binding site for the transcription factor Yin Yang 1 (YY1). We observed that the luciferase activity of YY1 which is binding to the A allele of rs117565607 was suppressed. ChIP data showed that YY1 was binding with T not A allele. Significance analysis of microarray suggested that TRIM26 downregulation was related to low immune response in NPC. We have identified a novel gene TRIM26 and a novel SNP rs117565607_A associated with NPC risk by regulating transcriptional process and established a new functional link between TRIM26 downregulation and low immune response in NPC.

Up-regulation of Polo-like Kinase 1 in nasopharyngeal carcinoma tissues: a comprehensive investigation based on RNA-sequencing, gene chips, and in-house tissue arrays.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a highly invasive malignancy which has unique characteristics when found among individuals from certain ethnic or geographic populations. The role and molecular mechanism of Polo-like kinase 1 (PLK1) in NPC remain yet to be clarified. Hence, the aim of this study is to identify the clinical implications of PLK1 in NPC based on gene chip, tissue microarray, and other silico approaches. METHODS: Relevant data related to PLK1 levels in NPC was screened for by searching in SRA, GEO, ArrayExpress, Oncomine and throughout the existing literature on this topic. The raw data about gene chips were normalized by using an RMA algorithm provided by "Limma" package. Furthermore, the "SVA" package of R software was used to remove the batch effect and data from the same platform were merged into one part. The differential expression levels of PLK1 between NPC and non-NPC tissues were extracted and analyzed with the Student's t-test. Meta-analyses were used to calculate the standard mean difference and sROC. Furthermore, in-house immunohistochemistry was performed with tissue microarrays. Weighted correlation network analysis was used to identify the PLK1-related genes. Several bioinformatic evaluations, including the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interactions, were also performed to assess the PLK1-related pathways. RESULTS: The tissue microarray and gene chips indicated that the PLK1 levels clearly had an up-regulating trend as compared to the non-cancerous controls. These trends were observed in both the single study and the comprehensive meta-analysis. The area under the sROC curve in the NPC tissues was 0.87, with pooled sensitivity and specificity at 0.950 and 0.710, respectively, based on 393 NPC tissues and 83 non-cancerous controls. A total of 144 genes were identified as co-expressed genes of PLK1 in NPC and were mainly enriched in the "cell cycle" pathway. Among the genes related to the cell cycle, CDK1, CCNA2 and CCNB2 were all closely related to PLK1 expression level. CONCLUSIONS: PLK1 may play a potential oncogenic role in the tumorigenesis and development of NPC. Since several PLK1 inhibitors have been developed, it is believed that the PLK1 inhibitors have great therapeutic potential in clinic applications for NPC patients.

Further evidence for the existence of major susceptibility of nasopharyngeal carcinoma in the region near HLA-A locus in Southern Chinese.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a multi-factorial malignancy closely associated with environmental factors, genetic factors and Epstein-Barr virus infection. Human leukocyte antigen (HLA) complex, specially the region near HLA-A locus, was regarded as a major candidate region bearing NPC genetic susceptibility loci in many previous studies including two recent genome-wide association (GWA) studies. To provide further evidence for the NPC susceptibility in the region near HLA-A locus based on other previous studies, we carried out a two-stage hospital-based case control association study including 535 sporadic NPC patients and 525 cancer-free control subjects from Guangdong, a high prevalence area of NPC in China. METHODS: 38 tag SNPs were initially selected by Heploview from the segment around HLA-A locus (from D6S211 to D6S510) and genotyped on GenomeLab SNPstream platform in 206 cases and 180 controls in the stage 1. Subsequently, the stage 1 significant SNPs and 17 additional SNPs were examined on another platform (Sequenom iPlex Assay) in another independent set of study population including 329 cases and 345 controls. RESULTS: Totally eight SNPs from the segment from D6S211 to D6S510 within HLA complex were found to be significantly associated with NPC. Two of the most significant SNPs (rs9260734 and rs2517716) located near to HLA-A and HCG9 respectively were in strong LD with some other SNPs of this region reported by two previous GWA studies. Meanwhile, Meanwhile, novel independent susceptibility loci (rs9404952, Pcombined = 6.6 × 10-5, OR combined = 1.45) was found to be close to HLA-G. CONCLUSION: Therefore, our present study supports that the segment from D6S211 to D6S510 in HLA complex region might contain NPC susceptibility loci which indeed needs to be fully investigated in the future.

[The combined application of dissociate skin flap and vacuum sealing drainage on the defect of the large neck neoplasms after surgical procedures].

OBJECTIVE: To evaluate the effect of the combined application of dissociate skin flap and vacuum sealing drainage (VSD) for the repairing for defect after surgical management of huge neck neoplasms. METHOD: Nineteen patients with huge neck malignant tumor involving the skin of the neck were given radical operation, making use of VSD covering the wound surface. After giving 6.65-7.98 mm Hg continuous negative pressure drainage for 72 h, the patients turned to be treated by intermittent negative pressure therapy with 2 min free interval after each treatment period for 5 min. After dismantling the VSD at 7th to 10th day postoperatively, the good wounds covered by granulation tissue were treated by the skin graft operation with dissociate skin flap from thighs; as for the wounds of which the granulation tissue didn't grow well and important cervical tissues was not fully covered by the granulation tissue, VSD was applied again for 1 week, followed by the skin graft operation. RESULT: Nineteen patients have received a total of 23 times of VSD wound treatment, one-stage operation time was significantly shortened. The granulation tissue grew faster on the wound after VSD treatment, and the important cervical tissues such as great vessels could be well covered. The infection and tumor recurrence were observed directly after dismantling the VSD. The skin graft transplantation would be performed after 1-3 weeks. CONCLUSION: The treatment by vacuum sealing drainage combined with skin graft for surgical wounds of huge neck tumor postoperatively has the advantages of simple operation, little injury and promotion of the wound healing, which is an effective way for treatment of neck skin defect by surgical operation for the huge tumor.