ABSTRACT
Background: Insulin-degrading enzyme [IDE] is a conserved zinc metallopeptidase. Here, we have evaluated the effect of passage number and culture time on IDE expression and activity in colorectal adenocarcinoma cell line [Caco-2]
Methods: Caco-2 cells were cultured with different passage ranges of 5-15, 25-35, 52-63 for 48, 72, and 120 hours. Subsequently, IDE expression and enzyme activity were assessed by Western blot analysis and fluorescent assay, respectively
Results: Our results confirmed that the amount of IDE was higher in cell extract compared to supernatant, and different passage numbers and culture times had small effect on IDE expression. However, when cells were cultured in the passage number range of 5-15 for 72 hours, the IDE activity was 35% higher compared to other passage numbers [p < 0.05]
Conclusion: The use of Caco-2 cells at passage number range of 5-15 and culture time of 72 hours provides proper conditions for IDE-related studies
ABSTRACT
Objective: In this study, we describe an efficient approach for stable knockdown of adenosine kinase [ADK] using lentiviral system, in an astrocytoma cell line and in human Wharton's jelly mesenchymal stem cells [hWJMSCs]. These sources of stem cells besides having multilineage differentiation potential and immunomodulatory activities, are easily available in unlimited numbers, do not raise ethical concerns and are attractive for gene manipulation and cell-based gene therapy
Materials and Methods: In this experimental study, we targeted adenosine kinase mRNA at 3' and performed coding sequences using eight miR-based expressing cassettes of anti-ADK short hairpin RNA [shRNAs]. First, these cassettes with scrambled control sequences were cloned into expressing lentiviral pGIPZ vector. Quantitative real time-polymerase chain reaction [qRT-PCR] was used to screen multi-cassettes anti-ADK miR-shRNAs in stably transduced U-251 MG cell line and measuring ADK gene expression at mRNA level. Extracted WJMSCs were characterized using flow cytometry for expressing mesenchymal specific marker [CD44+] and lack of expression of hematopoietic lineage marker [CD45-]. Then, the lentiviral vector that expressed the most efficient anti-ADK miR-shRNA, was employed to stably transduce WJMSCs
Results: Transfection of anti-ADK miR-shRNAs in HEK293T cells using CaPO4 method showed high efficiency. We successfully transduced U-251 cell line by recombinant lentiviruses and screened eight cassettes of anti-ADK miR- shRNAs in stably transduced U-251 MG cell line by qRT-PCR. RNAi-mediated down-regulation of ADK by lentiviral system indicated up to 95% down-regulation of ADK. Following lentiviral transduction of WJMSCs with anti-ADK miR- shRNA expression cassette, we also implicated, down-regulation of ADK up to 95% by qRT-PCR and confirmed it by western blot analysis at the protein level
Conclusion: Our findings indicate efficient usage of shRNA cassette for ADK knockdown. Engineered WJMSCs with genome editing methods like CRISPR/cas9 or more safe viral systems such as adeno-associated vectors [AAV] might be an attractive source in cell-based gene therapy and may have therapeutic potential for epilepsy
ABSTRACT
Background and study aims: Homeobox-containing genes are composed of a group of regulatory genes encoding transcription factors involved in the control of developmental processes. The homeodomain proteins could activate or repress the expression of downstream target genes. This study was conducted to in vivo identify the potential target gene[s] of TGIF2LX in colorectal adenocarcinoma
Methods: A human colorectal adenocarcinoma cell line, SW48, was transfected with the recombinant pEGFPN1-TGIF2LX. The cells were injected subcutaneously into the flank of the three groups of 6-week-old female athymic C56BL/6 nude mice [n = 6 per group]. The transcript profiles in the developed tumours were investigated using the cDNA amplified fragment length polymorphism [cDNA-AFLP] technique
Results: The real-time RT-PCR and DNA sequencing data for the identified genes indicated that the N-terminal domain-interacting receptor 1 [Nir1] gene was suppressed whereas Nir2 and fragile histidine triad [FHIT] genes were upregulated followed by the overexpression of TGIF2LX gene
Conclusion: Downregulation of Nir1 and upregulation of Nir2 and FHIT genes due to the overexpression of TGIF2LX suggests that the gene plays an important role as a suppressor in colorectal adenocarcinoma