Project description:Patients with advanced colorectal cancer (CRC) are commonly treated with systemic combination therapy but suffer eventually from drug resistance. MicroRNAs (miRNAs) are suggested to play a role in treatment resistance of CRC. We studied whether restoring downregulated miR-195-5p and 497-5p sensitize CRC cells to currently used chemotherapeutics 5-fluorouracil, oxaliplatin and irinotecan. Sensitivity to 5-FU, oxaliplatin and irinotecan before and after transfection with miR-195-5p and miR-497-5p mimics was analyzed in CRC cell lines HCT116, RKO, DLD-1 and SW480. Mass spectrometry based proteomic analysis of transfected and wild-type cells was used to identify targets involved in sensitivity to chemotherapy. Proteomic analysis revealed 181 proteins with significantly altered expression after transfection with miR-195-5p mimic in HCT116 and RKO, including 118 downregulated and 63 upregulated proteins. After transfection with miR-497-5p mimic, 130 proteins were significantly downregulated and 102 were upregulated in HCT116 and RKO (P<0.05 and FC<-3 or FC>3). CHUK and LUZP1 were coinciding downregulated proteins in sensitized CRC cells after transfection with either mimic. Resistance mechanisms of these two proteins may be related to nuclear factor kappa-B signaling and G1 cell cycle arrest, respectively. Restoring miR-195-5p and miR-497-5p expression enhanced sensitivity to chemotherapy, mainly oxaliplatin, in CRC cells and could be a promising treatment strategy for patients with mCRC. Proteomics revealed potential targets of these miRNAs involved in sensitivity to chemotherapy.
Project description:Understanding of molecular system how tumor-suppressor p53 selectively activates and/or completes appropriate tumor-suppressor pathway in response to various stresses is still one of the most important issues to be clarified. We analyzed a molecular mechanism for the completion of p53-dependent ribosome stress responsible pathway by miR-101. To this end, expression microarray analyses were conducted in the presence or absence of miR-101. We found that miR-101 induces the activation of p53-dependent G2 phase cell cycle arrest through the increased phosphorylation of ATM, leading to the p53-depdent apoptosis. Overall design: HCT116 or RKO cells were transfected with a Pre-miR-101 miRNA Precursor Molecule (Ambion) or Pre-miR miRNA Molecules Negative Control #2 (Ambion) at a final concentration of 5 nM. After incubtion for 3 days, total RNAs were extracted and gene expression signatures were determined. Three independent experiments were performed for each transfection.
Project description:We found frequent epigenetic silencing of microRNA-34b/c in human colorectal cancer. Introduction of miR-34b/c into a colorectal cancer cell line induced significant changes in gene expression profile. We also found overlap between the genes downregulated by miR-34b/c and those downregulated by DAC. Keywords: dose response A colorecal cancer cell line HCT116 was transfected with miR-34b or -c precursor or negative control. Also, HCT116 was treated with 5-aza-2'-deoxycytidine (DAC) or mock. Genes up- or downregulated by miR-34b/c and those by DAC was compared.
Project description:We found frequent epigenetic silencing of microRNA-34b/c in human colorectal cancer. Introduction of miR-34b/c into a colorectal cancer cell line induced significant changes in gene expression profile. We also found overlap between the genes downregulated by miR-34b/c and those downregulated by DAC. Keywords: dose response Overall design: A colorecal cancer cell line HCT116 was transfected with miR-34b or -c precursor or negative control. Also, HCT116 was treated with 5-aza-2'-deoxycytidine (DAC) or mock. Genes up- or downregulated by miR-34b/c and those by DAC was compared.
Project description:The Arraystar Human LncRNA Array v2.0 was designed for researchers who were interested in profiling both LncRNAs and protein-coding RNAs in human genome. 33,045 LncRNAs were collected from the authoritative data sources including RefSeq, UCSC knowngenes, Ensembl and many related literatures. This experiment is to profile lncRNAs and protein-coding RNAs using Arraystar Human LncRNA Array v2.0. Identification of coding RNAs and lncRNAs that are diffrentially expressed in colorectal cancer by comparing sample A-E (normal colorectal cells) vs sample F-J (colorectal tumor cells) and c-MYC-regulating lncRNAs by comparing sample 1-3 (triplicate of HCT116 cells treated with control siRNA) vs sample 4-6 (triplicate of HCT116 cells treated with siRNA targeting MYC) and sample 7-9 (triplicate of RKO cells treated with control siRNA) vs sample 10-12 (triplicate of RKO cells treated with siRNA targeting MYC) .
Project description:We identified that miR-1 is silenced in association with CpG island hypermethylation in a colorectal cancer (CRC) cell line, HCT116. To determine whether miR-1 serves as a tumor suppressor in CRC, we transfected CRC cell lines with a miR-1 precursor molecule or a negative control, and carried then out a series of MTT assays. Forty-eight hours after transfection, we observed that ectopic expression of miR-1 moderately suppressed growth in all three cell lines. To further clarify the effect of the miRNA, we next performed gene expression microarray analysis in HCT116 cells transfected with a miR-1 precursor molecule or a negative control. We found that 2769 probe sets were downregulated (> 1.5-fold) by ectopic miR-1 expression, and gene ontology analysis revealed that “extracellular regions”, “membrane” and “response to wound healing” genes were significantly enriched among the downregulated genes. HCT116 cells were transfected with a Pre-miR-1 miRNA Precursor Molecule (Ambion) or Pre-miR miRNA Molecules Negative Control #1 (Ambion). Forty-eight hours after transfection, total RNA extraction were carried out, and gene expression signatures were analyzed.
Project description:We have developed a novel analysis method that can interrogate the authenticity of biological samples used for generation of transcriptome profiles in public data repositories. The method uses RNA sequencing information to reveal mutations in expressed transcripts and subsequently confirms the identity of analysed cells by comparison with publicly available cell-specific mutational profiles. Cell lines constitute key model systems widely used within cancer research, but their identity needs to be confirmed in order to minimise the influence of cell contaminations and genetic drift on the analysis. Using both public and novel data, we demonstrate the use of RNA-sequencing data analysis for cell line authentication by examining the validity of COLO205, DLD1, HCT15, HCT116, HKE3, HT29 and RKO colorectal cancer cell lines. We successfully authenticate the studied cell lines and validate previous reports indicating that DLD1 and HCT15 are synonymous. We also show that the analysed HKE3 cells harbour an unexpected KRAS-G13D mutation and confirm that this cell line is a genuine KRAS dosage mutant, rather than a true isogenic derivative of HCT116 expressing only the wild type KRAS. This authentication method could be used to revisit the numerous cell line based RNA sequencing experiments available in public data repositories, analyse new experiments where whole genome sequencing is not available, as well as facilitate comparisons of data from different experiments, platforms and laboratories. Overall design: RNA-sequencing of three colorectal cancer cell lines (HCT116, HKE3 and RKO) with either three (HCT116, RKO) or four (HKE3) replicates each.
Project description:We used expression profiling of colorectal cancer and endometrial cancer cell lines treated with demethylating agents to search for epigenetically regulated miRNAs. The study included three MMR-deficient colorectal cancer cell lines (HCT116, HCT15, and RKO), two MMR-proficient colorectal cancer cell lines (SW480, and T84) and two MMR-deficient endometrial cancer cell lines (AN3CA and HEC59). Overall design: Cancer cell lines were cultured and divided to two parts. One part of cell culture was treated with demethylating agents (5 uM 5-aza-2' deoxycytidine, Sigma A3656, for 96 hours and with 300 nM trichostatin, Sigma T1952, for 18 hours). The other part of cancer cell line was cultured without treatments. All treatments were performed in duplicates.
Project description:To screen for epigenetically silenced miRNAs, wecarried out miRNA microarray analysis in three colorectal cancer (CRC) cell lines (HCT116, DLD-1 and RKO) treated with or without 5-aza-2'-deoxycytidine (aza). HCT116 and RKO cells were also treated with aza plus 4-phenylbutyric acid (PBA). In addition, we analyzed HCT116 cells in which the DNA methyltransferase genes DNMT1 and DNMT3B were genetically disrupted (double knockout; DKO cells), thereby abrogating DNA methylation. Expression of a majority of miRNAs was downregulated in all three CRC cell lines tested, as compared to normal colonic mucosa. DAC treatment upregulated expression of a large number of miRNAs in all three CRC cell lines, and combination treatment with DAC plus PBA induced even greater numbers of miRNAs in CRC cells. The most profound effect on the miRNA expression profile was induced by genetic disruption of DNMT1 and DNMT3B in HCT116 cells. CRC cells were treated with 5-aza-2’-deoxycytidine (aza) or aza plus 4-phenylbutyrate (PBA). Nomal colon RNA was purchased from BioChain. Expression of 470 miRNAs was analyzed using Human miRNA Microarray V1 (G4470A; Agilent technologies, Santa Clara, CA, USA).
Project description:This study is to identify downstream targets of homeobox gene CDX1. The study assayed the expression of 2 pairs of stably transfected colorectal cancer cell lines: The CDX1 nonexpressing CRC cell line HCT116 was stably transfected with either CDX1 cDNA in the pRC/CMV expression vector (HCT116-CDX1) or with vector control (HCT116-Vec). The CDX1-expressing CRC cell line LS174T was similarly transfected with either a pSilencer vector containing a short sequence of CDX1 siRNA (LS174T-siRNA) , or a pSilencer vector containing a scrambled siRNA sequence as a control (LS174T-Vec). Experiment Overall Design: 2 pairs of colorectal cancer cell lines were used for comparison