METHODS: We performed trio-based whole-exome sequencing (WES) in 210 families and case-only WES in 489 individuals with epileptic encephalopathies. The functional effect of CYFIP2 variants on WAVE signaling was evaluated by computational structural analysis and in vitro transfection experiments.
RESULTS: We identified three de novo CYFIP2 variants at the Arg87 residue in 4 unrelated individuals with early-onset epileptic encephalopathy. Structural analysis indicated that the Arg87 residue is buried at an interface between CYFIP2 and WAVE1, and the Arg87 variant may disrupt hydrogen bonding, leading to structural instability and aberrant activation of the WAVE regulatory complex. All mutant CYFIP2 showed comparatively weaker interactions to the VCA domain than wild-type CYFIP2. Immunofluorescence revealed that ectopic speckled accumulation of actin and CYFIP2 was significantly increased in cells transfected with mutant CYFIP2.
INTERPRETATION: Our findings suggest that de novo Arg87 variants in CYFIP2 have gain-of-function effects on the WAVE signaling pathway and are associated with severe neurological disorders. Ann Neurol 2018;83:794-806.
MATERIALS AND METHODS: MicroRNA software predicted that miR21 targets VCL while miR29a targets CX3CL1. Twenty benign prostatic hyperplasia (BPH) and 16 high grade CaP formalinfixed paraffin embedded (FFPE) specimens were analysed. From the bone scan results, high grade CaP samples were further classified into CaP with no BM and CaP with BM. Transient transfection with respective microRNA inhibitors was done in both RWPE1 (normal) and PC3 cell lines. QPCR was performed in all FFPE samples and transfected cell lines to measure VCL and CX3CL1 levels.
RESULTS: QPCR confirmed that VCL messenger RNA (mRNA) was significantly down regulated while CX3CL1 was upregulated in all FFPE specimens. Transient transfection with microRNA inhibitors in PC3 cells followed by qPCR of the targeted genes showed that VCL mRNA was significantly up regulated while CX3CL1 mRNA was significantly downregulated compared to the RWPE1 case.
CONCLUSIONS: The downregulation of VCL in FFPE specimens is most likely regulated by miR21 based on the in vitro evidence but the exact mechanism of how miR21 can regulate VCL is unclear. Upregulated in CaP, CX3CL1 was found not regulated by miR29a. More microRNA screening is required to understand the regulation of this chemokine in CaP with bone metastasis. Understanding miRNAmRNA interactions may provide additional knowledge for individualized study of cancers.
RESULTS: In this study, L-cells were isolated from a primary intestinal cell line to create suitable target cells for insulin expression studies. The isolated cells displayed L-cell properties and were therefore used as an L-cell surrogate. Next, the isolated L-cells were transfected with the recombinant plasmid consisting of an insulin gene located downstream of the GLP-1 promoter. The secretion tests revealed that an increase in glucose concentration from 5 mM to 25 mM induced insulin gene expression in the L-cells by 2.7-fold. Furthermore, L-cells quickly responded to the glucose stimulation; the amount of insulin protein increased 2-fold in the first 30 minutes and then reached a plateau after 90 minutes.
CONCLUSION: Our data showed that L-cells efficiently produced the mature insulin protein. In addition, the insulin protein secretion was positively regulated with glucose induction. In conclusion, GLP-1 promoter and L-cell could be potential candidates for diabetes gene therapy agents.
METHODS: In this study, transfection of VP3 and silencing of CD147 genes was achieved through the treatment of tumors with pVIVO1-GFP/VP3 (VP3), psiRNA-CD147/2 (shCD147/2), and their combination of CT26 colon cancer cell-induced in mice. The effectiveness of tumor-treatment was ascertained by electrophoresis, TUNEL assay, and flow cytometry analysis. While histopathological and biochemical analysis were used as toxic side effect identification.
RESULTS: The tumor growth delay index (TGDI) after treatment with VP3, shCD147/2, and their combination treatments increased by 1.3-, 1.2-, 2.0- and 2.3-fold respectively, over untreated control. The VP3-shCD147/2 combination treatment was more efficacious then either VP3 or shCD147/2 alone in the retardation of mouse CT26 colorectal cell tumor allograft.
CONCLUSION: The antitumor effect of the combination treatment is the result of synergistic effects of VP3 and shCD147/2 on the tumor cells resulting in apoptosis. Thus, the study shows that combination of VP3 and shCD147/2 treatment can be developed into a potential approach for anticolorectal cancer treatment regimen.