Briefly, the adaptor sequences were trimmed from all reads generated and sequences were then aligned to the GRCh38 (Genome Reference Consortium Human Build 38)78 for the HeLa cell samples and GRCm38 (Genome Reference Consortium Mouse Build 38)79 for NIH/3T3 cell samples using Bowtie 2 algorithm. than mir-29b-2, to be the main source of Rabbit polyclonal to PDCD6 generating mature miR-29b. The editing of miR-29b decreased expression levels of its family members miR-29a/c via changing the tertiary structures of surrounding nucleotides. Comparing transcriptome profiles of human and mouse cell lines, miR-29b displayed common regulation pathways involving distinct downstream targets in macromolecular complex assembly, cell cycle regulation, and LCI-699 (Osilodrostat) Wnt and PI3K-Akt signalling pathways; miR-29b also demonstrated specific functions reflecting cell characteristics, LCI-699 (Osilodrostat) including fibrosis and neuronal regulations in NIH/3T3 cells and tumorigenesis and cellular senescence in HeLa cells. miR-29b knockdown effect in both cell lines for target and pathway analysis. In NIH/3T3 cells, there are 120, 271, 139 and 117 genes with over 1.5-fold changes detected in clones cas1-1, cas1-2, cas2-1 and cas2-2, respectively, compared to px458 (Fig.?6a). 23 genes were found to be dysregulated among all clones compared to px458, including upregulated Col6a1, Col6a2, Cst3, F3, 2410006H16, Ywhag, Canx, Ppp2ca, Serpinh1 and Saa3, and downregulated Ybx1, Mt2, S100a10, S100a11, Fkbp1a, Anxa5, Tubb4b, Tuba1b, Tagln2, Tubb6, Bgn, Lrp1, and Fbln2 (Fig.?6a,b). Open in a separate window Figure 6 Differential gene expressions following miR-29b knockdown in NIH/3T3 and LCI-699 (Osilodrostat) HeLa clones. DGEs assay was performed using Partek Genome Suite platform, with p value set less than 0.05. (a) The venn diagram displayed the numbers of DGEs in NIH/3T3 clones compared to px458. 23 genes were overlapped from all clones. (b) Heatmap showing the overlapped genes expressions in NIH/3T3 cell clones. (c) The venn diagram displayed the numbers of DGEs in HeLa clones compared to px458. 25 genes were overlapped from all clones. (d) Heatmap showing the overlapped genes expression across all HeLa cell clones. The miRNA target prediction database www.microrna.org was used to assess whether these DEGs were targeted by miR-29b in their 3 UTRs; mirSVR score represents the effect of a miRNA on target downregulation, combining both non-canonical and non-conservative binding sites, with a lower value represents a strong repression from miRNA on the target38. PhastCons score is the conservative score for the target and binding sites among species39. Among these genes, upregulated Canx, Ppp2ca, 2410006H16, Cst3, Col6a1, and Col6a2 were predicted to have potential binding sites for miR-29b in their 3 UTRs (Table?4); downregulated Fkbp1a and Ybx1 were also on the list (Table?4), suggesting that miR-29b may function to activate the expression of these two genes. Table 4 The DEGs potentially targeted by miR-29b in NIH/3T3 and HeLa cells. strain Stbl3 (ThermoFisher Scientific), and LCI-699 (Osilodrostat) the colony growth was inspected the next day. For each construction, two or three colonies were picked to check for the correct insertion of the gRNAs. Cell transfection Cells were plated at a density of 1 1.5??105 cells per well (12-well plate) the day before transfection, reaching approximately 80% confluence prior to transfection. Reconstructed CRISPR/Cas9 plasmids were transfected into cells using Lipofectamine 3000 transfection reagents (ThermoFisher Scientific) according to manufacturers instructions. Microscope imaging Cells transfected with the CRISPR/Cas9 plasmids were imaged using a Leica AF6000 widefield epi-fluorescence microscope (Leica Microsystems) using 10x and 20x objectives. Bright field images were taken at the same time with the same magnification power. The exposure time for all samples was set to be the same in each experiment. The images were annotated with micron scales and exported using Leica AF6000 imaging software. Fluorescence Activated.