Systematic gene regulation involving miRNAs during neuronal differentiation of mouse P19 embryonic carcinoma cell.

MicroRNAs (miRNAs) are small noncoding RNA and play an essential role in gene regulation. In this study, we investigated regulation of gene expression during neuronal differentiation of mouse P19 embryonic carcinoma cells and described a systematic pathway of gene regulation involving miRNAs. In the pathway, downregulation of Lin28 involved in blocking the let-7 maturation and upregulation of let-7 occur following induction of the differentiation, thereby triggering suppression of the downstream High Mobility Group A2 (Hmga2) gene expression via activation of gene silencing mediated by let-7. Our data further suggest that miR-9, as well as miR-125b, participate in the reduction of the Lin28 expression. The gene regulation involving miRNAs likely contributes to a rapid and programmed change in gene expression in neuronal differentiation of P19 cells.

Neurite outgrowths of neurons on patterned self-assembled monolayers.

The effects of specific chemical functionalities on the neurite outgrowths of embryonic chick dorsal root ganglia (DRG) neurons and PC12h cells were investigated using a set of chemically functionalized surfaces prepared by self-assembled monolayers (SAMs) of alkanethiolates with R = NH2, COOH, and CH3 on patterned gold surfaces. The numbers of neurons with neurite outgrowths were compared in the course of a two-week cultivation period. Neurons with neurite outgrowths were observed predominantly on a patterned SAM of long-chain alkanethiolates with amino groups. After about two weeks, the neurons detached from the patterned SAM. However, the activity of beta-galactosidase immobilized via a patterned SAM did not decrease over a 13-d period, reflecting the long-term stability of the SAM. Therefore, the neurons became detached upon cell death. These results demonstrate that the patterned SAM of 11-amino-1-undecanethiolate is a scaffold suitable for making cell chips.

Gene silencing mediated by endogenous microRNAs under heat stress conditions in mammalian cells.

Heat shock, sudden change in temperature, triggers various responses in cells for protecting the cells from such a severe circumstance. Here we investigated gene silencing mediated by endogenous microRNAs (miRNAs) in mammalian cells exposed to a mild hyperthermia, by means of miRNA activity assay using a luciferase reporter gene as well as miRNA expression analysis using a DNA microarray. Our findings indicated that the gene silencing activities involving miRNAs were enhanced without increasing in their expression levels under heat-stress conditions. Additionally, the gene silencing activity appeared to be independent of the cytoprotective action involving heat shock proteins that are immediately activated in heat-shocked cells and that function as molecular chaperons for restoring heat-denatured proteins to normal proteins. Our current findings suggested the possibility that gene silencing involving endogenous miRNAs might play a subsidiary role in heat-shocked cells for an aggressive inhibition of the expression of heat-denatured proteins.

Reduction of type IV collagen by upregulated miR-29 in normal elderly mouse and klotho-deficient, senescence-model mouse.

MicroRNA (miRNA), a small non-coding RNA that functions as a mediator in gene silencing, plays important roles in gene regulation in various vital functions and activities. Here we show that the miR-29 members are upregulated in klotho-deficient [klotho(-/-)] mice, a senescence-model animal, and also in normal elderly ICR mice relative to wild-type littermates and young ICR mice. In addition, levels of type IV collagen, a major component of basement membranes and a putative target of miR-29, were lower in klotho(-/-) and elderly ICR mice than in wild-type littermates and young ICR mice. RNA degradation mediated by miR-29 may participate in the suppression of type IV collagen, both in vivo and in vitro. Taken together, our current findings suggest that the miR-29 upregulated in aging may be involved in the downregulation of type IV collagen, leading to a possible weakening of the basal membrane in senescent tissues, and miR-29 may be a useful molecular marker of senescence.

Alteration of microRNA expression in the process of mouse brain growth.

MicroRNAs (miRNAs) are a functional small non-coding RNA and play essential roles in gene regulation in development, differentiation and proliferation. In this study, we investigated expression profiles of miRNAs in the process of normal mouse brain growth from embryonic day 16.5 to ~19 months old by means of DNA microarray and reverse-transcription quantitative polymerase chain reaction, and we examined whether there was any association between the expression of miRNAs and brain growth. The results indicated that a major change in the expression of miRNAs occurred in the brain within the first week to the fourth week postnatally. The data also exhibited the miRNAs that gradually increased and decreased in their levels, over the course of brain growth. Therefore, the current study suggests that miRNAs are capable of becoming a useful biological marker for study of brain growth, and leads to the possibility that gene silencing involving miRNAs may participate in the process of brain growth and perhaps brain aging.

Production of brain-derived neurotrophic factor in Escherichia coli by coexpression of Dsb proteins.

When brain-derived neurotrophic factor (BDNF) is produced in the Escherichia coli periplasm, insoluble BDNF proteins with low biological activity and having mismatched disulfide linkages are formed. The coexpression of cysteine oxidoreductases (DsbA and DsbC) and membrane-bound enzymes (DsbB and DsbD), which play an important role in the formation of disulfide bonds in the periplasm, was investigated to improve the production of soluble and biologically active BDNF. The expression levels of Dsb proteins changed when the growth medium and the Dsb expression plasmids were changed, and the production rate of soluble BDNF was almost proportional to the expression level of DsbC protein with disulfide isomerase activity in the case of a low expression level of BDNF. The rate of soluble BDNF production with coexpression of DsbABCD was as high as 35%. These results show that coexpression of BDNF and Dsb proteins can effectively increase the production of soluble and biologically active BDNF.