The basal transcriptional activity of the murine Klf10 gene is regulated by the transcriptional factor JunB.

BACKGROUND: Krüppel-like factor 10 (KLF10) belongs to the Sp1-like transcription factor family, which plays an important role in many directions, e.g., cell proliferation, apoptosis, and differentiation. Its 5' upstream regions are conserved across mammalian species. However, the regulatory mechanism has not been elucidated yet. OBJECTIVE: Nonetheless the basal transcriptional regulation mechanisms of these regions are unknown. Here, we characterized it which is indispensable for the basal transcription of the Klf10 gene. METHODS: Seven deletions of 5' upstream DNA fragments from the 10 kb mKlf10 genomic DNA were produced by PCR and cloned into the upstream of the luciferase (Luc) reporter gene in the pGL3 basic plasmid. RESULT: The luciferase reporter assay showed that the DNA sequence at positions from -101 to +68 was required for a principle activity in the promoter of mKlf10 gene, in which transcriptional factor binding motifs, one JunB and two Sp1 sites, are included. Mutations at the sequence of JunB motif, but not at the two Sp1, abrogated the promoter activity completely, suggesting the indispensable role of JunB site for basal transcription of mKlf10 gene. Moreover, electrophoretic mobility and supershift assays (EMSA) uncovered that JunB protein bound to this region specifically. CONCLUSION: Taken together, our study revealed that the JunB but not Sp1 at mKlf10 promoter functions as a positive basic factor for the transcriptional activity of the gene.

MicroRNA-196b enhances the radiosensitivity of SNU-638 gastric cancer cells by targeting RAD23B.

Gastric cancer is characterized by resistance to ionizing radiation. The development of resistance to radiotherapy in gastric cancer patients is one of the obstacles to effective radiotherapy. MicroRNAs are small well-conserved non-coding RNA species that regulate post-transcriptional activation. Our study aimed to investigate the role of miR-196b in radiation-induced gastric cancer. In the present study, we found that miR-196b expression was significantly reduced following radiation. The ectopic miR-196b expression sensitized SNU-638 gastric cancer cells and increased γ-H2AX foci upon radiation treatment. Bioinformatics analysis suggested that the DNA repair protein RAD23B was a putative target gene of miR-196b. Overexpression of miR-196b suppressed RAD23B expression in SNU-638 cells. Reporter assays further showed that miR-196b inhibited RAD23B 3'-UTR luciferase activity. Knockdown of RAD23B by small interfering RNA transfection closely mimicked the outcomes of miR-196b transfection, leading to impaired DNA damage repair in gastric cancer cells. Our results show that miR-196b improved radiosensitivity of SNU-638 cells by targeting RAD23B. Our data indicate that miR-196b is a potential target to enhance the effect of radiation treatment on gastric cancer cells. These findings will provide evidence for a new therapeutic target in radiotherapy.

Identification of reference genes for relative quantification of circulating microRNAs in bovine serum.

Circulating microRNAs in body fluids have been implicated as promising biomarkers for physiopathology disorders. Currently, the expression levels of circulating microRNAs are estimated by reverse transcription quantitative real-time polymerase chain reaction. Use of appropriate reference microRNAs for normalization is critical for accurate microRNA expression analysis. However, no study has systematically investigated reference genes for evaluating circulating microRNA expression in cattle. In this study, we describe the identification and characterization of appropriate reference microRNAs for use in the normalization of circulating microRNA levels in bovine serum. We evaluated the expression stability of ten candidate reference genes in bovine serum by using reverse transcription quantitative real-time polymerase chain reaction. Data were analyzed using geNorm, NormFinder, and BestKeeper statistical algorithms. The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference. The suitability of these microRNAs was validated, and even when compared among different genders or breeds, the combination of miR-93 and miR-127 was ranked as the most stable microRNA reference. Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum. The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle.

Procedure used for denuding pig oocytes influences oocyte damage, and development of in vitro and nuclear transfer embryos.

The effects of different denuding procedures used during the in vitro culture of porcine embryos on oocyte damage and aspects of porcine embryo development were investigated in a series of studies. Oocytes were denuded by vortexing or pipetting after 44h in vitro maturation (IVM) or pre-denuded after 22h IVM. The total oocyte death rate was significantly (P<0.05) higher for pre-denuded (27.3±1.4%) than for vortexed (20.3±1.2%) or pipetted (16.2±2.2%) oocytes. There was no significant difference between the treatments in the percentage of oocytes that extruded the first polar body. The type I cortical granule distribution (reflecting complete maturity) and normal spindle formation rates were significantly lower in the pre-denuding than in the vortexing and pipetting treatments. Blastocyst formation rates were significantly lower for the pre-denuding treatment in PA (25.7±4.5%) and IVF (6.1±1.5%) culture than in the vortexing (PA 42.0±4.5%; IVF 11.2±0.5%) and pipetting (PA 43.4±3.1%; IVF 9.4±1.6%) treatments. The proportion of oocytes developing to blastocysts in SCNT culture was not significantly different between treatments ranging from 9.9±1.8% for pre-denuding to 12.3±2.7% for vortexing. No significant differences in apoptosis or embryonic fragmentation were observed. This study shows that the denuding procedure used for porcine oocytes during the in vitro production of embryos can significantly affect oocyte damage, spindle patterns, oocyte maturation, embryo development but not embryonic apoptosis or the frequency of fragmentation.