Microarray gene expression analysis of tumorigenesis and regional lymph node metastasis in laryngeal squamous cell carcinoma
ABSTRACT: Objective: The purpose of this study was to investigate the molecular basis of tumorigenesis and regional lymph node metastasis in LSCC, and provide a set of genes that may be useful for the development of novel diagnostic markers and/or more effective therapeutic strategies. Methods: A total number of 10 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for microarray analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by Illumina mRNA microarrays,and LSCC tissues with regional lymph node metastasis and LSCC tissues without regional lymph node metastasis were analyzed in the same manner.The most frequently differently expressed genes screened by microarrays were also validated by qRT-PCR in another 42 patients diagnosed for LSCC . Results: Analysed by Illumina mRNA microarrays,there were 361 genes significantly related to tumorigenesis while 246 genes significantly related to regional lymph node metastasis in LSCC. We found that the six genes (CDK1,CDK2,CDK4,MCM2,MCM3,MCM4) were most frequently differently expressed functional genes related to tumorigenesis while eIF3a and RPN2 were most frequently differently expressed functional genes related to regional lymph node metastasis in LSCC. The expressions of these genes were also validated by qRT-PCR. Conclusions: The research revealed a gene expression signature of tumorigenesis and regional lymph node metastasis in laryngeal squamous cell carcinoma.Of the total, the deregulation of several genes (CDK1, CDK2, CDK4, MCM2, MCM3, MCM4, EIF3a and RPN2) were potentially associated with disease development and progression. The result will contribute to the understanding of the molecular basis of LSCC and help to improve diagnosis and treatment. A total number of 10 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for microarray analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by Illumina mRNA microarrays,and LSCC tissues with regional lymph node metastasis and LSCC tissues without regional lymph node metastasis were analyzed in the same manner.The most frequently differently expressed genes screened by microarrays were also validated by qRT-PCR in another 42 patients diagnosed for LSCC .
Project description:In this study, we aimed to explore the role of miR-145, which is downregulated in LSCC, on cancer stem cell potency of laryngeal cancer cells. Using microRNA expression profiling and experimental follow-up studies, we demonstrated the regulatory role of miR-145 in stem cell characteristics of Hep-2 cells. Based on these results, we propose that mir-145 might carry crucial roles in LSCC tumorigenesis, prognosis, metastasis, chemoresistance and recurrence through regulating stem cell properties of tumor cells. LSCC tissue samples and normal tissues were profiled for microRNA expression; cancer tissue profiles were compared with normal tissue profiles.
Project description:Objective: The purpose of this study was to investigate the miRNAs basis of tumorigenesis in LSCC, and analyze the physiological processes of target genes predicted by the screened miRNAs that may be useful for the effective therapeutic strategies. Methods: A total number of 6 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for miRNA array analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by the Affymetrix GeneChip miRNA Array 3.0 to screen effective miRNAs, then TargetScan,PITA and microRNAorg were used for target gene prediction in GeneSpring 12.5 software.Moreover, pathway-based methods were adopted to analyse physiological processes of the target genes. The screened miRNAs and the significant target genes were also validated by qRT-PCR in another 36 patients diagnosed for LSCC. Results: Analysed by the miRNAs arrays, there were seven miRNAs (hsa-miR-224, hsa-miR-183, hsa-miR-23a, hsa-miR-675, hsa-miR-27a, hsa-miR-503 and hsa-miR-4800-3p) significantly related to tumorigenesis ,and all the screened miRNAs in laryngeal squamous cell carcinoma tissues were significantly up-regulated(P<0.05). The expressions of these miRNAs were also validated by qRT-PCR. Then analysed by GeneSpring 12.5 software, there were 72 putative target genes corresponding to the seven significant miRNAs. Moreover,analysed by String database,the result indicated that most target genes could be composed of gene networks; Analysed by GO database, we observed that these target genes were involved in processes such as metabolic process, biological regulation,membrane,protein binding,ion binding and so on (P <0.05); Analysed by KEGG pathways database, MAPK signaling pathway, adherens junction and pathways in cancer played especially important role in tumorigenesis of LSCC (P<0.05). As the two important genes in MAPK signaling pathway which plays pivotal roles in tumorigenesis, FGF2 and MAP2K4 were validated by qRT-PCR, and they were significantly down-regulated(P<0.05). Conclusions: The research revealed seven miRNAs expression signature(hsa-miR-224, hsa-miR-183, hsa-miR-23a, hsa-miR-675, hsa-miR-27a, hsa-miR-503 and hsa-miR-4800-3p) of tumorigenesis in laryngeal squamous cell carcinoma,and analysed the physiological processes of the predicted target genes regulated by screened miRNAs in LSCC. The result will contribute to the understanding of the molecular basis of LSCC and help to improve the treatment. A total number of 6 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for miRNA array analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by the Affymetrix GeneChip miRNA Array 3.0 to screen effective miRNAs, then TargetScan,PITA and microRNAorg were used for target gene prediction in GeneSpring 12.5 software.Moreover, pathway-based methods were adopted to analyse physiological processes of the target genes. The screened miRNAs and the significant target genes were also validated by qRT-PCR in another 36 patients diagnosed for LSCC.
Project description:Tumor-adjacent noncancerous tissues often exhibited abnormalities on molecular levels, which is described as field effect of cancerization. Accumulated evidence demonstrated that filed effect may also play important role in cancer progression. In the present study, we found that the gene expression profile in noncancerous lung tissues adjacent to lung squamous cell carcinoma (SCC) was significantly associated with regional lymph node status of patients. Significance Analysis of Microarrays (SAM) showed that 121 genes were significantly associated with lymph node metastasis (delta=0.75, FDR=0.069). Interestingly, all of the significant genes were up-regulated in the lymph node positive samples. For these genes, the most significant biological GO terms were extracellular structure organization, cell adhesion, regulation of cell motion/migration, and vessel development, etc, which were also involved in EMT process supported by another experiment in vitro. Tumor-adjacent histologically normal lung tissues were collected from 60 primary lung SCC patients, of whom 34 (56.7%) suffered regional lymph node metastasis. Gene expression profiling analysis of these samples was performed using Agilent 4x44K human whole genome gene expression microarray (G4112F).
Project description:Larynx squamous cell carcinoma (LSCC) is the second most aggressive cancer from head and neck (HSCC). Survival of LSCC patients has been found worsening, albeit significant advances in technology and therapeutics were made. We sought to clarify new genetic targets driven the development of LSCC. Analyzing tumors revealed that eight members of the homeobox gene family (HOX) were expressed 200 times more in LSCC samples than in normal larynx tissue. qPCR analysis validated all transcriptomic findings for the HOX gene family. Receiver Operating Characteristic statistical method (ROC curve) predicted that 8 members of the HOX gene family differentiate tumors from their normal surrounding tissue. Enabling the comparison of patient clinics with gene expression data, ROC curve analysis yet revealed that HOXC8 and HOXD11 genes are related to the tumor differentiation degree and regional lymph node metastasis, respectively. From siRNA assay, we found that HOXC8 and HOXD11 genes are essential for the expansion of FADU cell colonies. Our findings strongly suggest that members of the HOX family might be associated with the development of LSCC. Twenty nine cancer samples and thirteen margin samples were collected from patients undergoing surgical ablation of LSCC according to Ethic Committee guideline and patient pre-informed signed consents. Whole human genome microarray analysis was applied to investigate new genetic targets in LSCC.
Project description:Lymph node metastasis is a poor prognosis indicator in esophageal cancer. Although tumor spreading currently forms the main basis for therapy selection, the molecular mechanisms underlying the metastatic pathway remain insufficiently understood. Several studies aimed to investigate these mechanisms but focused mainly on regulatory patterns in the tumors themselves and/or the invaded lymph nodes. To date no study has yet investigated the potential changes on transcription level, which take place within the yet non-invaded niche. Here we provide a comprehensive description of these regulations in patients. In this study the transcriptomic profiles of regional lymph nodes were determined for two patient groups: patients classified as pN1 (metastasis) or pN0 (no metastasis) respectively. All investigated lymph nodes, also those from pN1 patients, were still free of metastasis. The gene expression data was obtained via microarray analysis. Top candidates were validated via PCR and immunohistochemistry. The results show that regional lymph nodes of pN1 patients differ decisively from those of pN0 patients – even before metastasis has taken place. In the pN0 group distinct immune response patterns were observed. In contrast, lymph nodes of the pN1 group exhibited a clear profile of reduced immune response and reduced proliferation, but increased apoptosis, enhanced hypoplasia and morphological conversion processes. DKK1 was the most significant gene associated with the molecular mechanisms taking place in lymph nodes of patients suffering from metastasis (pN1). We assume that the two molecular profiles observed constitute two different stages of a progressive disease. Finally we suggest that DKK1 might play an important role within the mechanisms leading to lymph node metastasis. First, samples were classified according to the principal status of the patients exhibiting (pN1 group) or non-exhibiting (pN0 group) metastasis in regional lymph nodes. The comparison of pN1 and pN0 patients should highlight the general differences in regulations that might lead to or prevent from metastasis. Second, two lymph node samples were collected from each patient, one node located close to the tumor (regional) and the second node distant to the tumor. While the distant nodes served as reference sample the regional nodes were investigated for transcriptional regulations. Key to the work presented here is that all nodes investigated were made sure to be still metastasis free, independent of the patients’ pN1/pN0 classification and of the location relative to the tumor. This should allow the identification of early changes occurring prior to metastasis homing.
Project description:To investigate the aberrant expressed lncRNA and mRNA and identify the potential lncRNA correlated with core mRNA in LSCC, we screened lnRNA and mRNA expression profile in 9 pairs of primary Stage IV LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays, conducted in-depth integrated bioinformatics analysis of lncRNA and mRNA, and performed qRT-PCR assays to further validate their expressions in an independent cohort of 30 pairs tissue samples Overall design: lnRNA and mRNA expression profile in 9 pairs of primary Stage IV laryngeal squamous cell carcinoma (LSCC) cancer tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays
Project description:Lymph node involvement is a major prognostic variable in breast cancer. Whether the molecular mechanisms that drive breast cancer cells to colonize lymph nodes are shared with their capacity to form distant metastases is yet to be established. In a transcriptomic survey aimed at identifying molecular factors associated with lymph node involvement of ductal breast cancer, we found that luminal differentiation, assessed by the expression of estrogen receptor (ER) and/or progesterone receptor (PR) and GATA3, was only infrequently lost in node-positive primary tumors and in matched lymph node metastases. The transcription factor GATA3 critically determines luminal lineage specification of mammary epithelium and is widely considered a tumor and metastasis suppressor in breast cancer. Strong expression of GATA3 and ER in a majority of primary node-positive ductal breast cancer was corroborated by quantitative RT-PCR and immunohistochemistry in the initial sample set, and by immunohistochemistry in an additional set from 167 patients diagnosed of node-negative and positive primary infiltrating ductal breast cancer, including 102 samples from loco-regional lymph node metastases matched to their primary tumors, as well as 37 distant metastases. These observations suggest that loss of luminal differentiation is not a major factor driving the ability of breast cancer cells to colonize regional lymph nodes. The transcriptomic study comprises 16 samples from Lymph node metastasis from infiltrating ductal breast carcinoma, 18 samples from Primary node-positive infiltrating ductal,7 samples from Primary node-negative infiltrating ductal and 3 samples from Unaffected lymph node were included. Their RNA was isolated and prepared for hybridization to human Affymetrix GeneChip arrays.
Project description:Metastasis via the lymphatics is a major risk factor in squamous cell carcinoma of the oral cavity (OSCC). We sought to determine whether the presence of metastasis in the regional lymph node could be predicted by a gene expression signature of the primary tumor. A total of 18 OSCCs were characterized for gene expression by hybridizing RNA to Affymetrix U133A gene chips. Genes with differential expression were identified using a permutation technique and verified by quantitative RT-PCR and immunohistochemistry. A predictive rule was built using a support vector machine, and the accuracy of the rule was evaluated using crossvalidation on the original data set and prediction of an independent set of four patients. Metastatic primary tumors could be differentiated from nonmetastatic primary tumors by a signature gene set of 116 genes. This signature gene set correctly predicted the four independent patients as well as associating five lymph node metastases from the original patient set with the metastatic primary tumor group. We concluded that lymph node metastasis could be predicted by gene expression profiles of primary oral cavity squamous cell carcinomas. The presence of a gene expression signature for lymph node metastasis indicates that clinical testing to assess risk for lymph node metastasis should be possible.
Project description:Identifying the exact molecules associated with CRC metastasis may be crucial to understand the process, which might also be translated to the diagnosis and treatment of CRC. In this study, we investigate the association of microRNA expression patterns with the lymph node metastasis of colorectal cancer. To investigate the association of microRNA expression patterns with the lymph node metastasis of colorectal cancer, eight primary colorectal cancer tissues derived from stage II–III colorectal cancer patients with (n = 4) or without (n = 4) lymph node metastasis were collected and the miRNA expression profiles of them were determined using Agilent miRNA microarray. Different miRNA expression profiles were identified in CRC tissues between lymph node metastasis positive and negative group.