Effects of Interferons and Double-Stranded RNA on Human Prostate Cancer Cell Apoptosis

Prostate cancer is the second most commonly diagnosed cancer among men in the United States. Prostate cancer therapy is severely hampered by lack of response and development of resistance to conventional chemotherapeutic drugs in patients. Therefore, the development and discovery of new drugs have become an urgent clinical need. Interferons (IFNs), a family of pleiotropic cytokines, exert antitumor activities due to their anti-proliferative, immunomodulatory and proapoptotic functions. Here, we report that pretreatment of prostate cancer PC-3 cells with IFNs sensitized these cells to double-stranded RNAs (dsRNAs)-induced apoptosis. The enhancement effect of IFN treatment was dependent on IFN subtypes, in particular, IFN γ. In comparison with IFN α or β, IFN γ treatment remarkably augmented apoptosis in PC-3 cells induced with polyinosinic:polycytidylic acid (poly I:C), a synthesized form of dsRNA. We demonstrated that IFN-signaling was necessary for these effects by using mutant cell lines. Transfection of 2–5A, the activator of RNase L, or silencing of dsRNA-dependent protein kinase R (PKR) by siRNA did not have any significant impact on this event, suggesting that neither RNase L nor PKR was involved in poly I:C/IFN γ-induced apoptosis in the cells. Further investigation of the apoptotic pathway revealed that Bak, a pro-apoptotic member of the Bcl-2family, was synergistically up-regulated by IFN γ and poly I:C, whereas other members of the family were not affected. Knocking down of Bak demonstrated its contribution to poly I:C/IFN γ-induced apoptosis in the cells. We believeour findings will precipitate the design of novel therapeutic strategies for prostate cancer

Stability in and Correlation between Factors Influencing Genetic Quality of Seed Lots in Seed Orchard of Pinus tabuliformis Carr. over a 12-Year Span

Coniferous seed orchards require a long period from initial seed harvest to stable seed production. Differential reproductive success and asynchrony are among the main factors for orchard crops year-to-year variation in terms of parental gametic contribution and ultimately the genetic gain. It is fundamental in both making predictions about the genetic composition of the seed crop and decisions about orchard roguing and improved seed orchard establishment. In this paper, a primary Chinese pine seed orchard with 49 clones is investigated for stability, variation and correlation analysis of factors which influence genetic quality of the seed lots from initial seed harvest to the stable seed production over a 12 years span. Results indicated that the reproductive synchrony index of pollen shedding has shown to be higher than that of the strobili receptivity, and both can be drastically influenced by the ambient climate factors. Reproductive synchrony index of the clones has certain relative stability and it could be used as an indication of the seed orchard status during maturity stage; clones in the studied orchard have shown extreme differences in terms of the gametic and genetic contribution to the seed crop at the orchard's early production phase specifically when they severe as either female or male parents. Those differences are closely related to clonal sex tendency at the time of orchard's initial reproduction. Clonal gamete contribution as male and female parent often has a negative correlation. Clone utilization as pollen, seed or both pollen and seed donors should consider the role it would play in the seed crop; due to numerous factors influencing on the mating system in seed orchards, clonal genetic contribution as male parent is uncertain, and it has major influence on the genetic composition in the seed orchard during the initial reproductive and seed production phase

Regulation of autophagy and lipid accumulation under phosphate limitation in Rhodotorula toruloides

BackgroundIt is known that autophagy is essential for cell survival under stress conditions. Inorganic phosphate (Pi) is an essential nutrient for cell growth and Pi-limitation can trigger autophagy and lipid accumulation in oleaginous yeasts, yet protein (de)-phosphorylation and related signaling events in response to Pi limitation and the molecular basis linking Pi-limitation to autophagy and lipid accumulation remain elusive.ResultsHere, we compared the proteome and phosphoproteome of Rhodotorula toruloides CGMCC 2.1389 under Pi-limitation and Pi-repletion. In total, proteome analysis identified 3,556 proteins and the phosphoproteome analysis identified 1,649 phosphoproteins contained 5,659 phosphosites including 4,499 pSer, 978 pThr, and 182 pTyr. We found Pi-starvation-induced autophagy was regulated by autophagy-related proteins, but not the PHO pathway. When ATG9 was knocked down, the engineered strains produced significantly less lipids under Pi-limitation, suggesting that autophagy required Atg9 in R. toruloides and that was conducive to lipid accumulation.ConclusionOur results provide new insights into autophagy regulation under Pi-limitation and lipid accumulation in oleaginous yeast, which should be valuable to guide further mechanistic study of oleaginicity and genetic engineering for advanced lipid producing cell factory

Sensitization of Gastric Cancer Cells to 5-FU by MicroRNA-204 Through Targeting the TGFBR2-Mediated Epithelial to Mesenchymal Transition

Background/Aims: Gastric cancer (GC) is the most common gastrointestinal malignancy, causing cancer-related deaths in East Asia. MicroRNAs (miRNAs) are small non-coding RNAs aberrantly expressed in human tumors. In this study, we aim to investigate the roles of miR-204 in the epithelial to mesenchymal transition (EMT)-associated chemosensitivity. Methods: The expression of miR-204 was detected in clinical tumor samples and GC cell lines by real time PCR. Tumor cell’s growth, invasion, and migration were measured by MTT assay, wound healing assay, and transwell invasion assay, respectively. Western blot method was used to detect the protein levels of indicated genes. Luciferase reporter assay was performed to validate the target gene of miR-204. The in vivo role of miR-204 was measured using a xenograft mouse model of GC. Results: By comparing the expressions of miR-204 in human gastric tumors and their adjacent normal tissues, it was disclosed that miR-204 was significantly downregulated in gastric tumors. Moreover, miR-204 was downregulated in multiple GC cell lines compared with normal gastric epithelial cells. Overexpression of miR-204 suppressed GC cells’ proliferation, invasion, and migration. It is noteworthy that 5-FU treatments induced miR-204 expression and suppressed TGF-β pathway. By establishment of 5-FU resistant GC cell line, it was revealed that miR-204 was significantly downregulated in 5-FU resistant GC cells, representing mesenchymal features with downregulation of epithelial marker, while mesenchymal markers were upregulated. We identified TGFBR2 as a direct target of miR-204 by Western blot method and luciferase assay in GC cells and tumor samples as well. In addition, overexpression of miR-204 sensitized GC cells to 5-FU in vitro. Xenograft experiments demonstrated that the combination of miR-204 and 5-FU efficiently inhibited tumor growth and improved survival rate of mice as well. Eventually, we illustrated the restoration of TGFBR2 in miR-204 overexpression GC cells, which recovered resistance to 5-FU treatments compared with miR-204 overexpression GC cells. Conclusion: This study describes a miRNA-based therapeutic strategy against 5-FU resistance in GC, contributing to the development of anti-chemoresistance therapeutic agents

Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

BACKGROUND: Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). RESULTS: We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. CONCLUSIONS: These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components