Project description:Purpose: Focal adhesion kinase (FAK), hyaluronan (HA), and hyaluronan synthase-3 (HAS3) have been implicated in cancer growth and progression. FAK inhibition with the small molecule inhibitor Y15 decreases colon cancer cell growth in vitro and in vivo. HAS3 inhibition in colon cancer cells decreases FAK expression and activation, and exogenous HA increases FAK activation. We sought to determine the genes affected by HAS and FAK inhibition and hypothesized that dual inhibition would synergistically inhibit viability. Methods: We treated SW620 colon cancer cells with Y15 to inhibit FAK. We used two strategies to inhibit HAS: (1) cells were transfected with siRNA (HAS3 inhibited); a scrambled sequence was used as a control (HAS3 scrambled), and (2) cells were treated with the HAS inhibitor 4-methylumbelliferone (4-MU). To determine the effect on viability, MTT assays were performed on transfected cells treated with Y15, and wild type cells treated with Y15 alone, 4-MU alone or Y15+4-MU. Treated and untreated cells were submitted to the gene microarray facility for expression profiling. RT-PCR was done to confirm the results. Results: HAS and FAK inhibition affected cell viability. Y15 and 4-MU decreased viability in a dose-dependent manner; viability was further inhibited by treatment with Y15+4-MU in combination (p<0.05). HAS-inhibited cells treated with as little as 2 M of Y15 showed significantly decreased viability compared to HAS scrambled cells treated with the same dose (p<0.05), suggesting synergistic inhibition of viability with dual FAK/HAS inhibition. Microarray analysis showed more than 2-fold up- or down-regulation of 121 genes by HAS inhibition, and 696 genes by FAK inhibition (p<0.05). Of 29 genes that were common to both groups, 9 were down-regulated (CBS, DHRS3, EEPD1, ESPN, FAM46C, GRTP1, IL20RA, INHBE, SCNN1A) and 4 were up-regulated (ANXA1, MALL, RGS2, SNAI2). RT-PCR confirmed these findings. Among the genes affected by FAK or HAS3 inhibition were FOX genes (apoptosis, cell cycle regulation), ANXA1 (apoptosis, proliferation), IL8 (cell cycle regulation, adhesion, proliferation), RGS2 (cell cycle regulation), CEACAM6 (adhesion), SNAI2 (transcription regulation), and SFRP5 (apoptosis). Several genes were specific to either FAK or HAS3 inhibition and several were common to both. Gene expression profiles of samples isolated from human colorectal cancer cells (SW620). A comparison of gene expression between untreated cells and cells treated with 4mcM of Y15. A second comparison between cells transfected with siRNA to HAS3 (HAS3-silenced) and cells transfected with a scrambled control sequence (sc). Two replicates each.
Project description:Purpose: Focal adhesion kinase (FAK), hyaluronan (HA), and hyaluronan synthase-3 (HAS3) have been implicated in cancer growth and progression. FAK inhibition with the small molecule inhibitor Y15 decreases colon cancer cell growth in vitro and in vivo. HAS3 inhibition in colon cancer cells decreases FAK expression and activation, and exogenous HA increases FAK activation. We sought to determine the genes affected by HAS and FAK inhibition and hypothesized that dual inhibition would synergistically inhibit viability. Methods: We treated SW620 colon cancer cells with Y15 to inhibit FAK. We used two strategies to inhibit HAS: (1) cells were transfected with siRNA (HAS3 inhibited); a scrambled sequence was used as a control (HAS3 scrambled), and (2) cells were treated with the HAS inhibitor 4-methylumbelliferone (4-MU). To determine the effect on viability, MTT assays were performed on transfected cells treated with Y15, and wild type cells treated with Y15 alone, 4-MU alone or Y15+4-MU. Treated and untreated cells were submitted to the gene microarray facility for expression profiling. RT-PCR was done to confirm the results. Results: HAS and FAK inhibition affected cell viability. Y15 and 4-MU decreased viability in a dose-dependent manner; viability was further inhibited by treatment with Y15+4-MU in combination (p<0.05). HAS-inhibited cells treated with as little as 2 M of Y15 showed significantly decreased viability compared to HAS scrambled cells treated with the same dose (p<0.05), suggesting synergistic inhibition of viability with dual FAK/HAS inhibition. Microarray analysis showed more than 2-fold up- or down-regulation of 121 genes by HAS inhibition, and 696 genes by FAK inhibition (p<0.05). Of 29 genes that were common to both groups, 9 were down-regulated (CBS, DHRS3, EEPD1, ESPN, FAM46C, GRTP1, IL20RA, INHBE, SCNN1A) and 4 were up-regulated (ANXA1, MALL, RGS2, SNAI2). RT-PCR confirmed these findings. Among the genes affected by FAK or HAS3 inhibition were FOX genes (apoptosis, cell cycle regulation), ANXA1 (apoptosis, proliferation), IL8 (cell cycle regulation, adhesion, proliferation), RGS2 (cell cycle regulation), CEACAM6 (adhesion), SNAI2 (transcription regulation), and SFRP5 (apoptosis). Several genes were specific to either FAK or HAS3 inhibition and several were common to both.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of Homo sapiens inflammatory skin diseases (whole skin biospies): Psoriasis (Pso), vs Atopic Dermatitis (AD) vs Lichen planus (Li), vs Contact Eczema (KE), vs Healthy control (KO) In recent years, different genes and proteins have been highlighted as potential biomarkers for psoriasis, one of the most common inflammatory skin diseases worldwide. However, most of these markers are not psoriasis-specific but also found in other inflammatory disorders. We performed an unsupervised cluster analysis of gene expression profiles in 150 psoriasis patients and other inflammatory skin diseases (atopic dermatitis, lichen planus, contact eczema, and healthy controls). We identified a cluster of IL-17/TNFα-associated genes specifically expressed in psoriasis, among which IL-36γ was the most outstanding marker. In subsequent immunohistological analyses IL-36γ was confirmed to be expressed in psoriasis lesions only. IL-36γ peripheral blood serum levels were found to be closely associated with disease activity, and they decreased after anti-TNFα-treatment. Furthermore, IL-36γ immunohistochemistry was found to be a helpful marker in the histological differential diagnosis between psoriasis and eczema in diagnostically challenging cases. These features highlight IL-36γ as a valuable biomarker in psoriasis patients, both for diagnostic purposes and measurement of disease activity during the clinical course. Furthermore, IL-36γ might also provide a future drug target, due to its potential amplifier role in TNFα- and IL-17 pathways in psoriatic skin inflammation. In recent years, different genes and proteins have been highlighted as potential biomarkers for psoriasis, one of the most common inflammatory skin diseases worldwide. However, most of these markers are not psoriasis-specific but also found in other inflammatory disorders. We performed an unsupervised cluster analysis of gene expression profiles in 150 psoriasis patients and other inflammatory skin diseases (atopic dermatitis, lichen planus, contact eczema, and healthy controls). We identified a cluster of IL-17/TNFα-associated genes specifically expressed in psoriasis, among which IL-36γ was the most outstanding marker. In subsequent immunohistological analyses IL-36γ was confirmed to be expressed in psoriasis lesions only. IL-36γ peripheral blood serum levels were found to be closely associated with disease activity, and they decreased after anti-TNFα-treatment. Furthermore, IL-36γ immunohistochemistry was found to be a helpful marker in the histological differential diagnosis between psoriasis and eczema in diagnostically challenging cases. These features highlight IL-36γ as a valuable biomarker in psoriasis patients, both for diagnostic purposes and measurement of disease activity during the clinical course. Furthermore, IL-36γ might also provide a future drug target, due to its potential amplifier role in TNFα- and IL-17 pathways in psoriatic skin inflammation.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.