STAT proteins play critical roles in the signal transduction pathways for various cytokines. The type I interferons (IFN␣͞␤) promote the DNA-binding activity of the transcription factors STAT1, STAT2, and STAT3. Although the requirement for STAT1 and STAT2 in IFN␣͞␤ signaling and action is well documented, the biological importance of STAT3 to IFN action has not yet been addressed. We found that STAT3 plays a critical role in signal transduction by IFN␣͞␤. A human cell line that is resistant to the antiviral and antiproliferative activities of IFN but is still IFN-responsive by virtue of STAT1 and STAT2 activation was found to be defective in STAT3 activation and in induction of NF-B DNA-binding activity. Expression of STAT3 in these resistant cells complemented these signaling defects and also markedly increased cellular sensitivity to the antiviral and antiproliferative effects of IFN. Because STAT3 is involved in the induction of NF-B DNA-binding activity and in the induction of antiviral and antiproliferative activity, our results place STAT3 as an important upstream element in type I IFN signal transduction and in the induction of biological activities. Therefore, our results indicate that STAT1 and STAT2 are not the only STATs required for the expression of the key biological activities of IFN␣͞␤.Interferons (IFNs) are cytokines that block the viral infection of cells, inhibit cell proliferation, and modulate cell differentiation. Type I IFNs (IFN ␣, ␤, and ) compete with each other for binding to a common cell surface receptor, whereas the receptor for type II IFN (IFN␥) is a distinct entity (1). The type I IFN receptor is composed of IFNAR1 and IFNAR2 chains (2-4), which undergo rapid, ligand-dependent tyrosine phosphorylation. Although IFNAR2 is the ligand-binding subunit, IFNAR1 acts as a species-specific transducer for the actions of type I IFN (5-7). IFNs transduce signals from the cell surface resulting in selective gene activation (8-10) through the activation of JAK tyrosine kinases and signal transducers and activators of transcription (STAT) factors (11,12). Upon their tyrosine phosphorylation, IFN-activated STATs (STAT1, STAT2, and STAT3) dimerize and translocate to the nucleus.The crucial role that STAT1 and STAT2 play in the transcriptional response to IFN␣͞␤ and in the induction of antiviral activity has been demonstrated in knockout mice and in mutant human cell lines deficient in these proteins. However, the importance of STAT3 in IFN␣͞␤ action has been unresolved. For example, knockout of the STAT3 gene in mice leads to early embryonic lethality and STAT3-deficient cell lines could not be isolated (13). We recently reported that STAT3 acts as a bridge (adapter) for the IFN-dependent interaction of the IFNAR1 receptor chain and the regulatory 85-kDa (p85) subunit of phosphatidylinositol-3Ј (PI-3) kinase (14). PI-3 kinase is important in the regulation of many cellular events involving protein tyrosine kinases and is an upstream element in a serine kinase transduction cascade (15, 1...

Based on the reports of the activation of the transcription factor known as STAT3 (for signal transducers and activators of transcription) or APRF (for acute phase response factor) by various cytokines, we investigated the possible role of STAT3 in type I interferon (IFN) receptor signaling. We show that STAT3 undergoes IFNalpha-dependent tyrosine phosphorylation and IFNalpha treatment induces protein-DNA complexes that contain STAT3. In addition, STAT3 associates with the IFNAR-1 chain of the type I receptor in a tyrosine phosphorylation-dependent manner upon IFNalpha addition. The binding of STAT3 to the IFNAR-1 chain occurs through a direct interaction between the SH2 domain-containing portion of STAT3 and the tyrosine-phosphorylated IFNAR-1 chain. Furthermore, tyrosine-phosphorylated STAT3 bound to the IFNAR-1 chain also undergoes a secondary modification involving serine phosphorylation. This phosphorylation event is apparently mediated by protein kinase C, since it was blocked by low concentrations of the protein kinase inhibitor H-7. The biological relevance of IFN activation of STAT3 is further illustrated by the finding that STAT3 is not activated by IFN in a cell line resistant to the antiviral and antiproliferative actions of IFN alpha but in which other components of the JAK-STAT pathway are activated by IFNalpha.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.

Background:The role of DGCR8 in VSMCs is not known. Results: Loss of DGCR8 in VSMCs results in embryonic mortality by inhibiting cell proliferation and differentiation and promoting apoptosis. Conclusion: DGCR8 is required for vascular development. Significance: Elucidation of the role of DGCR8 in VSMCs will reveal the significance of DGCR8-mediated miRNA maturation in vascular diseases.

The transcription factor Krüppel-like factor 4 (KLF4) has been implicated in regulating cell proliferation, migration and differentiation in a variety of human cells and is one of four factors required for the induction of pluripotent stem cell reprogramming. However, its role has not been addressed in ocular neovascular diseases. This study investigated the role of KLF4 in angiogenesis and underlying molecular mechanisms in human retinal microvascular endothelial cells (HRMECs). The functional role of KLF4 in HRMECs was determined following lentiviral vector mediated inducible expression and shRNA knockdown of KLF4. Inducible expression of KLF4 promotes cell proliferation, migration and tube formation. In contrast, silencing KLF4 inhibits cell proliferation, migration, tube formation and induces apoptosis in HRMECs. KLF4 promotes angiogenesis by transcriptionally activating VEGF expression, thus activating the VEGF signaling pathway in HRMECs.