Project description:We have used RNA-seq to examine gene expression from paired human CRC/control mucosa samples and identified CRC-specific expressed long noncoding RNAs
Project description:We have used RNA-seq to examine gene expression from paired human CRC/control mucosa samples and identified CRC-specific expressed long noncoding RNAs To identify CRC-specific expressed genes, including long noncoding RNAs
Project description:Inflammation has been identified as an important factor in cancer development and progression, including colorectal cancer [CRC]. To determine the role of inflammation and the specific contribution of activated fibroblasts in cancer development and progression we analyzed six human CRC tissue specimens and paired normal adjacent mucosa samples by LS/MS-MS. Amongst other, indeed several inflammation associated proteins were found differentially expressed compared to normal colonic mucosa. Two candidate molecules, SPARC and THBS2 were recently identified as proteins of a fibroblast inflammation signature. Analysis of public available RNA expression datasets revealed, that the mRNA of both SPARC and THBS2 are upregulated in CRC, typically in association with the CRC consensus molecular subtype 4 [CMS4]. Immunohistochemistry staining also demonstrated upregulation of SPARC and THBS2 in the tumor stroma compared to normal adjacent mucosa and indicated co-localization with the mesenchymal marker [alpha]SMA. In vitro 3D co-culture experiments with human colon derived fibroblasts and CRC cell lines resulted in enhanced SPARC and THBS2 protein levels when both cell types were cultivated in direct physical contact, compared to fibroblasts or cancer cells alone. This study demonstrates the feasibility of detecting tumor-specific signatures by LC-MS/MS and compatibility with RNA expression datasets. We identified an inflammation signature in CRC tissue and the data emphasized the contribution of activated fibroblasts in these events.
Project description:Colorectal cancer (CRC) is one of the most prevalent cancers, with over one million new cases per year. Overall, prognosis of CRC largely depends on the disease stage and metastatic status. As precision oncology for patients with CRC continues to improve, this study aimed to integrate genomic, transcriptomic, and proteomic analyses to identify significant differences in expression during CRC progression using a unique set of paired patient samples while considering tumour heterogeneity.We analysed fresh-frozen tissue samples prepared under strict cryogenic conditions of matched healthy colon mucosa, colorectal carcinoma, and liver metastasis from the same patients. Somatic mutations of known cancer-related genes were analysed using Illumina's TruSeq Amplicon Cancer Panel; the transcriptome was assessed comprehensively using Clariom D microarrays. The global proteome was evaluated by liquid chromatography-coupled mass spectrometry (LC‒MS/MS) and validated by two-dimensional difference in-gel electrophoresis. Subsequent unsupervised principal component clustering, statistical comparisons, and gene set enrichment analyses were calculated based on differential expression results.Although panomics revealed low RNA and protein expression of CA1, CLCA1, MATN2, AHCYL2, and FCGBP in malignant tissues compared to healthy colon mucosa, no differentially expressed RNA or protein targets were detected between tumour and metastatic tissues. Subsequent intra-patient comparisons revealed highly specific expression differences (e.g., SRSF3, OLFM4, and CEACAM5) associated with patient-specific transcriptomes and proteomes.Our research results highlight the importance of inter- and intra-tumour heterogeneity as well as individual, patient-paired evaluations for clinical studies. In addition to changes among groups reflecting CRC progression, we identified significant expression differences between normal colon mucosa, primary tumour, and liver metastasis samples from individuals, which might accelerate implementation of precision oncology in the future.
Project description:Colorectal cancer (CRC) is a genetically heterogeneous disease with several distinct morphological growth patterns. This study was aimed to investigate genes differentially expressed between ulcerative and polypoid colorectal CRC. cDNA microarray was first employed to compare the gene expression profiling of ulcerative and polypoid CRC with paired normal mucosa. Potential candidates identified by data filtering were further validated using quantitative real-time polymerase chain reaction, western blot and immunohistochemistry. Epigenetic regulation of gene expression was investigated using methylation-specific PCR (MSP). cDNA microarray identified 11 up-regulated and 14 down-regulated genes differentially expressed in both types of tumor compared to matched normal mucosa. Among them, S100P was the only upregulated gene preferentially associated with polypoid CRC (p = 0.032), whereas no genes demonstrated significantly differential association with ulcerative CRC. S100P protein and mRNA expression level of polypoid CRC was significantly higher than that of ulcerative CRC (p < 0.05, respectively). Immunoreactivity of S100P protein was localized predominantly in the nuclei and to a less extent in the cytoplasm. Overexpression of S100P occurred early in the adenoma stage. 80% (24/30) and 20% (6/30) polypoid CRC showed diffusely strong and moderate overexpression, respectively. In contrast, S100P was diffusely and strongly expressed in 15% (6/40) ulcerative CRC, with 52.5% (21/40) and 32.5% (13/40) tumors having moderate and weak overexpression, respectively. S100P overexpression was preferentially associated with polypoid CRC (p < 0.001). The relative methylation level determined by MSP was not statistically different between polypoid and ulcerative CRC (43.36% vs. 49.10%, p = 0.168), indicating that promoter hypomethylation was directly related to upregulation of S100P mRNA. The gene expression profiling of ulcerative and polypoid CRC with paired normal mucosa were compared. Dye swapping experiments with ulcerative CRC vs normal mucosa or polypoid CRC vs normal mucosa were performed and we averaged the log ratios of the duplicated spots on each slide.
Project description:Dysregulated miRNA in human colorectal cancer (CRC) were identified through comparison between 4 CRC tumors and their adjacent normal tissues by miRNA array. Histologically-confirmed CRC were included in this study. CRC tissues and paired adjacent normal tissues were obtained from the resected surgical specimens. The adjacent normal tissue is composed of normal colonic mucosa located at approximately 10 cm away from the cancer tissue. miRNA profiling of 754 human miRNAs was performed using TaqMan Human MiRNA Array Set v3.0. Quantitative real-time polymerase chain reaction (Q-PCR) was performed using Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems). Results were analyzed by the SDS RQ Manager 1.2 software (Applied Biosystems). 4 CRC tissues and 4 adjacent normal tissues were subjucted to qPCR based miRNA expression profiling. Equal amount of total RNA were used for analysis.
Project description:We generated reduced representation bisulfite sequencing (RRBS) and RNA sequencing (RNA-Seq) data of 20 samples (paired primary and liver metastases from 10 CRC patients) to understand key molecular events involved in tumor progression and metastasis.
Project description:Dysregulated miRNA in human colorectal cancer (CRC) were identified through comparison between 4 CRC tumors and their adjacent normal tissues by miRNA array. Histologically-confirmed CRC were included in this study. CRC tissues and paired adjacent normal tissues were obtained from the resected surgical specimens. The adjacent normal tissue is composed of normal colonic mucosa located at approximately 10 cm away from the cancer tissue. miRNA profiling of 754 human miRNAs was performed using TaqMan Human MiRNA Array Set v3.0. Quantitative real-time polymerase chain reaction (Q-PCR) was performed using Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems). Results were analyzed by the SDS RQ Manager 1.2 software (Applied Biosystems).
Project description:Unearthing of silenced genes in colorectal cancer (CRC) is of great importance. We employed oligonucleotide microarray to find changes in global gene expression of five CRC cell lines. These were analyzed before and after treatment with the 5-aza-2'-Deoxycitidine. Expression of the responding genes was integrated with gene expression profiling generated by microarray analysis of matched colorectal tissue samples. Selected candidates were subjected to methylation-specific PCR (MSP) and real-time quantitative reverse transcription-PCR using CRC cell lines and paired tumor and normal samples from CRC patients. Sixty eight genes were re-expressed after 5-aza-2'-Deoxycitidine treatment and over-expressed in normal colorectal mucosa, including genes that were known to be methylated in CRC. After applying study selection criteria, we identified 16 potential genes. Two candidates were selected (ASPP1 and SCARA5). Among 15 CRC cell lines, methylation was identified in SCARA5 (20%). The methylation status of SCARA5 was subsequently investigated in 23 paired colorectal tissue samples; methylation was detected in 17%, respectively. Observed promoter methylation showed a tendency towards methylation in tumor-derived samples, in SCARA5 gene. Significant down expression of SCARA5 mRNA was observed in CRC cell lines and tumor tissues compared to adjacent normal tissues (P < 0.001 and P = 0.001, respectively). The use of genome-wide screening led to the identification of a group of candidate genes. Among them, SCARA5 was methylated and markedly down-regulated in CRC. SCARA5 gene may have a role in CRC tumorigenesis.