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:To explore the mechanisms underlying the radioresistance of colorectal cancer, we established specifically radioresistant HCT116 cell line (HCT116-R cells) and RKO cell line (RKO-R cells) derived from HCT116 and RKO cell lines by repeatedly exposing to multi-fractionated irradiation. Then, the aberrantly expressed miRNA were detected by miRNA sequencing and their bioinformatics were analyzed.
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).
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:Transcriptional profiling of hPTTG1-/- HCT116 human colorectal cancer cells comparing hPTTG1-/- HCT116 cells transfected with pcDNA3.1, and with hPTTG1-/- HCT116 cells transfected with pcDNA3.1-hPTTG1 plasmid.
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: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.
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).