Project description:Methylation analysis of normal lymphocytes, HCT116, RKO and SW480 In vitro methylated DNA (IVD) generated from normal lymphocytes, HCT116, RKO and SW480 genomic DNA was subjected to sodium bisulfite treatment and the DNA was analyzed for CpG methylation using the Infinium Methylation Array.
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:DNA hypermethylated genes undergo silencing and show re-expression in response to 5-Aza-CdR treatment. Agilent arrays were used to determine the genes that get reactivated in response to the drug treatment. To determine expression levels of genes in SW480, HCT116 and RKO.
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.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
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:We detected whole genome microarray expression profiling in three pairs of Dicer knockout coloretal cell lines and their normal controls, including DLD-1, RKO and HCT116.
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.
