No abstract

Elevation of Hemoglobin F ameliorates symptoms of β-thalassemia, a common autosomal recessive disorder. The transcription factor SOX6 plays a key role in the γ to β-globin gene switching. In the current investigation, a mutation was induced using the CRISPR/Cas9 technology in the binding domain region of SOX6 to reactivate γ-globin expression. Three CRISPR/Cas9 cassettes were provided, whose single-guide RNAs targeted different regions in the SOX6 gene-binding domain. After transfection of K562 cells with CRISPR a, b and c, and subsequent erythroid differentiation, the indel percentage of the cells was about 30%, 25%, and 24%, respectively. Relative quantification showed that the γ-globin mRNA level increased to 1.3-, 2.1-, and 1.1-fold in the cells treated with CRISPR/Cas9 a, b, and c, respectively, compared with untreated cells. Our results show that mutation induction in the binding site of the SOX6 gene leads to γ-globin reactivation. These findings support the idea that CRISPR interrupts the SOX6 binding site, and, as a result, SOX6 is incapable of binding the γ-globin promoter. In conclusion, SOX6 disruption could be considered as a therapeutic approach for β-thalassemia treatment. CRISPR/Cas9 was selected for this purpose as it is the most rapidly evolving technology.

Blockade of α4 integrin by antibodies could be an appropriate treatment strategy in multiple sclerosis and Crohn's disease. Considering disadvantages of antibodies, other elements (e.g. aptamers) have been proposed for antibodies replacement. Isolation of aptamers through cell-SELEX (systematic evolution of ligands by exponential enrichment) method requires positive and negative expressing α4 integrin cell lines. For a better isolation, we intended to construct a negative cell line lacking of specific ligand binding site of α4 integrin. Escherichia coli strain top 10 was used for truncated integrin subunit α4 (TITGA-4) expression vector. Human embryonic kidney (HEK)-293T cell was transfected with linearized TITGA-4 plasmid and subsequently screened for stable truncated TITGA-4 expressing cells. Chromosomal DNA of truncated TITGA-4-transfected cells was extracted and the presence of truncated TITGA-4 gene in HEK-293T genome was confirmed by polymerase chain reaction (PCR). The expression level of truncated TITGA-4 on HEK-293T cells was also analysed by real-time PCR and flow cytometry. Real-time PCR and flow cytometric analysis showed significant difference of truncated TITGA-4 expression between untransfected HEK-293T cells compared to transfected cells. The results suggest that we have successfully constructed the truncated integrin α4 expressing HEK-293T cell, which will facilitate further research into the production of antibody, nanobody, and aptamer against α4 integrin.

Background. Long noncoding RNAs (lncRNAs) perform as competing endogenous RNAs (ceRNAs) to sponge microRNAs (miRNAs) lead to the advancement of cancer. In this work, we attempted to identify possible long noncoding RNA biomarkers as well as a new regulatory axis in colorectal cancer. This investigation was carried out in consideration of the critical oncogenic function played by miR-616-3p in many malignancies, as well as its regulatory involvement in several signaling pathways.Material and Methods: Differentially expressed lncRNAs (DELs) were identified from Gene Expression Omnibus (GEO) database. Targeted mRNAs were recognized by the Targetscan database. ciBioPortal database and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also were applied to identify mRNAs. CeRNA networks constructed via Cytoscape 3.7.1. QRT-PCR was utilized to verify these RNA molecules' expression levels in 30 CRC tissues compared to 30 adjacent tissue samples. Results: Three lncRNAs and three mRNAs were discovered to have the greatest interaction with miR-616-3p. QRT-PCR revealed overexpression of (Linc01282, lnc-MYADM-1:1, ZNF347, XIAP) and downregulation of (Lnc-atp12a-1:1, SIK1 and miR-616-3p). In CRC, bioinformatic research revealed many unique ceRNA networks centered on miR-616-3p that were previously unknown.Conclusion: This work reveals novel, previously unreported lncRNAs as prognostic biomarkers for CRC, as well as potential mRNAs as new therapeutic targets and predictive biomarkers for CRC. Furthermore, our analysis identified novel ceRNA networks that need be investigated further in CRC.