Genome-wide gene expression analysis of HCT116 cells subjected to stable gelsolin overexpression
ABSTRACT: Analysis of gene expression levels altered by stable gelsolin overexpression. Results provide important information of the modulation of various genes by gelsolin. Total RNA obtained from stable gelsolin-overexpressing HCT116 clones (C1, C2, C3, C8) as well as emtpy vector control HCT116 cell lines (V4, V5)
Project description:HCT116 parental, HCT116 5-FU resistant and HCT116 oxaliplatin resistant cells have been transiently treated with with their respective drug (5-FU or oxaliplatin) for 0, 6 12 or 24h in 3 independent experiments.
Project description:HCT116 cells transfected with lentiviral vectors expressing two different N-BLR shRNAs (clones #3-1 and #4-7) and empty vector control Overall design: Two HCT116 stable clones expressing N-BLR shRNAs (named clones #3-1 and #4-7) compared to empty vector control. Biological replicate: two stable clones vs empty vector controls
Project description:X-linked inhibitor of apoptosis (XIAP) is the most potent endogenous caspase inhibitor preventing cell death via caspase-9, -7 and -3 (initiator and executioner caspase pathways). Using short hairpin RNA (shRNA) against XIAP, stably expressed in a parent HCT116 human colon cancer cell line, a series of clones have been developed. XIAP mRNA levels were established by RT-PCR, the four X (XIAP knockdown) clonal cell lines show 82-93% reduction in XIAP mRNA when compared to the four L (luciferase control) cell lines. Immunoblot analysis showed a 67-89% reduction in XIAP protein in X cell lines compared to L. RNA was analysed by microarray and XIAP knockdown was confirmed in 7 probe sets, there was no significant compensation of other IAP family members. XIAP knockdown induced a 2-fold increase in the basal level of apoptosis without modification of caspase 3/7 activity. Finally, XIAP knockdown sensitises cells to radiotherapy by 20%, to recombinant TRAIL by a 3-fold factor, and to paclitaxel and docetaxel by >2 fold factor. Future work should focus on targeted agents such as rhTRAIL in combination with strategies to down regulate XIAP. XIAP antisense is now in clinical development in oncology. Overall design: Human HCT116 colon cancer cell lines were transfected with either XIAP (X) or luciferase (L) shRNA. The expression profiles of both were compared at early (p4) and late (p8) passage.
Project description:In an effort to identify genes whose expression is regulated by activated PI3K signaling, we performed microarray analysis and subsequent qRT-PCR on an isogenic set of PTEN gene-targeted human cancer cells. Numerous p53 effectors were upregulated following PTEN deletion, including p21, GDF15, PIG3, NOXA, and PLK2. Stable depletion of p53 led to reversion of the gene expression program. Western blots revealed that p53 was stabilized in HCT116 PTEN-/- cells via an Akt1-dependent and p14ARF-independent mechanism. Stable depletion of PTEN in untransformed human fibroblasts and epithelial cells also led to upregulation of p53 and senescent-like growth arrest. Simultaneous depletion of p53 rescued this phenotype, enabling PTEN-depleted cells to continue proliferating. Next, we tested whether oncogenic PIK3CA, like inactivated PTEN, could activate p53. Retroviral expression of oncogenic human PIK3CA in MCF10A cells led to activation of p53 and upregulation of p53-regulated genes. Stable depletion of p53 reversed these PIK3CA-induced expression changes and synergized with oncogenic PIK3CA in inducing anchorage-independent growth. Finally, targeted deletion of an endogenous allele of oncogenic but not wild-type PIK3CA in a human cancer cell line led to a reduction in p53 levels and a decrease in the expression of p53-regulated genes. These studies demonstrate that activation of PI3K signaling by mutations in PTEN or PIK3CA can lead to activation of p53-mediated growth suppression in human cells, indicating that p53 can function as a brake on PIP3-induced mitogenesis during human cancer pathogenesis. Experiment Overall Design: Two HCT116 PTEN+/+ cell lines (parental cells and a clone with random integration of the targeting vector) and three independently-derived HCT116 PTEN-/- cell lines were studied
Project description:Global mRNA expression profiling of HCT116 cells overexpressing miR30a-5p were collected using Agilent human whole genome array (G4845A AMADID 026652, cRNA 4x44k V2) . Two different sources of RNA were analyzed: 1.) HCT116 colon cancer cells overexpressing human mir-30a-5p together with a sponge vector expressing binding sites for the antisense sequence of sh30a-5p vector construct to compete out possible miRNAs derived from the anti-guide strand of this vector. 2.) HCT116 colon cancer cells expressing control vector (pLKO.1-LV) and the sponge vector described above. Two conditions (sponge-LV vs sponge-sh30a-5p), each condition is represented by 3 biological replicates
Project description:The gain of Protocadherin LKC (PCDH24) expression in colon carcinoma cell line HCT116 has been shown to induce contact inhibition, thereby completely abolishing tumor formation in vivo. To clarify the molecular mechanism, we performed DNA microarray analysis and compared gene-expression pattern between control and PCDH24-expressing HCT116 cells. Approximately 2000 genes were apparently changed their expression. Further proteomics analysis using 2-DE/MS confirmed the dramatic changes and provided additional information. We were aware that these changes are quite similar to the changes observed in epithelial-mesenchymal transition (EMT), most drastic changes in development and cancer metastasis. We thus further analyzed these changes using specific antibodies, and found distinct difference between these two phenomena. Among the differences, nuclear translocation of catenin beta 1 (CTNNB1) was inhibited by PCDH24-expression, subsequently some of the downstream nodes were suppressed. Although contact inhibition and cancer metastasis are completely opposite aspect of the cells, we expect that the identified differences will be key nodes to understand the relationship. We also expect that the nodes will be a target to modulate tumors arising stem cell transplantation (SCT), as well as a therapeutic target for cancer metastasis. Keywords: dose response Overall design: Three HCT116 PCLKC cell lines (parental cells and two stable expression clones with random integration of the targeting vector, PCLKC-GFP, GFP) were studied.
Project description:The experiment was to study the gene expression changes in human colorectal cell HCT116 DICER Exon5 knockout cells comparing to that in parental HCT116 cellls. Experiment Overall Design: Experiment includes using of two Agilent human 44K microarrays with dye-swap replication.
Project description:Expression of a panel of miRNAs was analyzed in three different cell lines comparing stable knock down of ZEB1 (shZEB) to control transfected cell lines. The analyzed cell lines derived of mammary (MDA MB 231) or colon cancer (SW480, HCT116).
Project description:VGLL4, a tumor suppressor, is negative regulator of Hippo/YAP signaling. To explore the role of VGLL4 during colorectal cancer cell line, we explore microarray analysis of HCT116 cells after stable transfection of VGLL4 and shVGLL4 for 48 h. Overall design: Scr-1, Scr-2, shVGLL4-1, shVGLL4-2