Project description:Purpose: The goal of this study is to compare Next Generation Sequencing-derived transcriptome profiling (RNA-seq) of a negative control cell line to two independent shDANCR cell lines. Methods: mRNA profiles of HCT116 colorectal cancer cell lines transfected with lentiviruses containing a negative control vector and two independent shRNA vectors for DANCR knockdown were generated by deep sequencing using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed at the transcript level with the method of HISAT2, and StringTie followed by FPKM calculation. Results: Using the described data analysis workflow, we mapped about 40 million sequence reads per sample to the human genome (GRCh38) and identified about 130,000 transcripts (transcribed from about 40,000 genes) per sample with StringTie. Approximately 2.5% of the genes showed differential expression between the Control and shDANCR cell lines, with a fold change ≥1.5 and p value <0.05. Conclusions: Our study represents a detailed analysis of gene expression changes affected by DANCR knockdown in colorectal cancer cell line HCT116.
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
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 Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)