Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive “core” sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA.
MicroRNA-200a (miR-200a) has been reported to regulate tumour progression in several tumours; however, little is known about its role in non-small cell lung cancer cells (NSCLCs). Here, we found that miR-200a was up-regulated in A549 and SK-MES-1 cells compared with normal lung cells HELF. By a series of gain-of-function and loss-offunction studies, over-expression of miR-200a was indicated to enhance cells migration, and its knock-down inhibited migration of cells in NSCLC cell lines. Furthermore, miR-200a was identified to induce TSPAN1 expression which was related to migration. TSPAN1 was proved to induce migration, and so up-regulation of TSPAN1 by miR-200a may explain why over-expressing miR-200a promotes NSCLC cells migration.
Glycogen synthase kinase-3β (GSK-3β) is required in the expression of epithelial junction proteins. It was found downregulated in hepatocellular carcinoma (HCC) tissues. The purpose of this study was to investigate the role of GSK-3β in modulating the metastatic behaviors of human HCC cell lines in vitro. In this study, the expression level of GSK-3β was measured in 4 human HCC cell lines, and the small interfering RNA (siRNA) vectors against or plasmids encoding GSK-3β were used to evaluate the responses of target cells to the knockdown or overexpression of this kinase, respectively. Our results showed that GSK-3β expression was significantly lower in human HCC cell lines with high metastatic potential than that in HCC cell lines without metastatic characteristics or in a normal human liver cell line. The knockdown of GSK-3β by siRNA led to a decreased expression of the epithelial junction molecules (ZO-1, E-cadherin) and an increase in the expression of a mesenchymal cell marker (α-SMA) and a gene transcription factor (β-catenin), resulting in enhanced tumor cell dissemination. In contrast, gain-of-function studies revealed that ectopic expression of GSK-3β reduced invasive and migratory abilities of HCC cells accompanied by decreased HCC cell proliferation and induced apoptosis. More importantly, downregulation of GSK-3β led to an increase in the expression and accumulation of β-catenin in the nuclei, promoting gene transcription. In conclusion, GSK-3β might play a vital role in suppressing HCC dissociation by preventing the disassembly of cancer cell epithelial junctional complex via the GSK-3β/β-catenin pathway.
Multiple targets RNAi strategy is a preferred way to treat multigenic diseases, especially cancers. In the study, multi-target siRNAs were designed to inhibit NET-1, EMS1 and VEGF genes in hepatocellular carcinoma (HCC) cells. And multi-target siRNAs showed better silencing effects on NET-1, EMS1 and VEGF, compared with single target siRNA. Moreover, multi-target siRNA showed greater suppression effects on proliferation, migration, invasion, angiogenesis and induced apoptosis in HCC cells. The results suggested that multi-target siRNA might be a preferred strategy for cancer therapy and NET-1, EMS1 and VEGF could be effective targets for HCC treatments.
RNA interference (RNAi) has been proven in recent years to be a newly advanced and powerful tool for development of therapeutic agents toward various unmet medical needs such as cancer, in particular, a great attention has been paid to the development of antineoplastic agents. Recent success in clinical trials related to RNAi-based therapeutics on cancer and ocular disease has validated that small interfering RNAs (siRNAs) constitute a new promising class of therapeutics. Currently, a great wealth of multi-target based siRNA structural modifications is available for promoting siRNA-mediated gene silencing with low side effects. Here, the latest developments in RNAi-based therapeutics and novel structural modifications described for siRNAs--in particular multi-target siRNAs--are reviewed.
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