Cloning and characterization of the human USP22 gene promoter.

Ubiquitin-specific processing enzyme 22 (USP22) plays a direct role in regulating cell cycle, and its overexpression has been reported to be involved in tumor progression. However, little is known about the regulation of USP22 transcription. In this study, we cloned and characterized the human USP22 promoter. Using 5' RACE (rapid amplification of cDNA ends) analysis, the transcriptional initiation site was identified. Promoter deletion analysis showed that the sequence between -210 and -7 contains the basal promoter for USP22 in human fibroblast and tumor cells. Surprisingly, mutations in a putative Sp1 binding site immediately upstream of the USP22 transcriptional start site (-13 to -7) resulted in a significant induction of promoter activity. Further study revealed that Sp1 binds to this site in human normal fibroblast cells, and treatment with the Sp1 inhibitor mithramycin A led to a marked increase in USP22 transcript levels. Forced expression of exogenous Sp1 repressed the USP22 promoter activity in HeLa cells. In contrast, knockdown of Sp1 enhanced USP22 promoter activity and mRNA levels. These data suggest that Sp1 is a crucial regulator of USP22 transcription

The synergistic effect of dietary cholesterol with fruit tannins in forming kidney stones

Prevalence of kidney stones has increased continously over several decades worldwide, the major causes of which are largely unknown. To explore the dietary causes of kidney stones, and reveal mechanisms underlying dietary risk factors inducing kidney stones, animal experiments using mice as the disease model were performed. Eight-week old male CD-1 mice were treated by ethylene glycol, cholesterol or/and apple tannins for 3 d, respectively. In the present study, the crystalline analysis in urine and kidney tissues, HE staining kidney sections as well as observation of micro-stones, tannins and cholesterol deposition in kidneys of mice in different groups were conducted. We found that gavage with ethylene glycol, cholesterol and tannins resulted in mice urine solute supersaturation in renal tubules and forming kidney stones. Significant cholesterol and tannin deposits in mouse kidney were observed by laser confocal microscopy and crystals were shown either adhered with or co-deposited with cholesterol and tannin deposits. The primary crystals were found in renal cortex, medullar, especially papilla in the kidney sections under polarized microscope. These findings demonstrate that interaction of cholesterol and tannins in kidney plays a critical role in the formation of kidney stones

Candidate genes responsible for lipid droplets formation during adipogenesis simultaneously affect osteoblastogenesis

Introduction. With cellular lipid storage varying, the balance between lipid intake and lipid degradation was a must to keep healthy and determined the level of lipid droplets. Although lipid droplets accumulation had been well demonstrated in adipocytes, gene expression profiling and gene function during adipogenesis and osteoblastogenesis remain unknown. Material and methods. Here, this work profiled gene transcriptional landscapes of lipid droplets formation during adipogenesis from human mesenchymal stem cells (hMSCs) using RNA-Seq technique. By using RNA interference (RNAi) we investigated the function of candidate genes during adipogenesis and osteoblastogenesis using Oil Red/Alizarin Red/alkaline phosphatase (ALPL) staining and qRT-PCR (quantitative real-time PCR). Results. Eleven differentially up-regulated genes associated with lipid droplets formation were identified at 3, 5, 7, 14, 21, and 28 days during adipogenesis. Unexpectedly, APOB per se inhibiting adipogenesis weakened osteoblastogenesis and METTL7A facilitating adipogenesis negligibly inhibited osteoblastogenesis according to the phenotypic characterization of adipocytes and osteoblasts and transcriptional condition of biomarkers through lentivirus transfection assays. Conclusions. The establishment of the gene transcriptional profiling of lipid droplets formation would providethe molecular switches of hMSCs cell fate determination and the study targets for fat metabolic diseases

Osteogenesis of Human iPSC-Derived MSCs by PLLA/SF Nanofiber Scaffolds Loaded with Extracellular Matrix

Bone defects that arise from trauma, skeletal diseases, or tumor resections have become the commonest and most thorny problems in orthopedic clinics. Recently, biocomposite materials used as artificial bone repair materials have provided a promising approach for bone regeneration. In this study, poly (l-lactide acid) (PLLA) and silk fibroin (SF) were used to fabricate nanofiber scaffolds by electrospinning technology. In order to simulate a biomimetic osteoblast microenvironment, decellularized extracellular matrix from osteoblasts was loaded into the biocomposite scaffolds (O-ECM/PLLA/SF). It was found that the O-ECM/PLLA/SF scaffolds were nontoxic for L929 cells and had good cytocompatibility. Their effects on mesenchymal stem cells derived from human-induced pluripotent stem cell (iPSC-MSC) behavior were investigated. As a result, the scaffolds with the addition of O-ECM showed enhanced alizarin red S (ARS) activity. In addition, higher expression of osteogenic gene markers such as runt-related transcription factor 2 (Runx2), collagen type I (Col-1), and osteocalcin (OCN) as well as upregulated expression of osteogenic marker protein osteopontin (OPN) and Col-1 further substantiated the applicability of O-ECM/PLLA/SF scaffolds for osteogenesis. Furthermore, the in vivo study also indicated maximal new bone formation in the skull defect model of Sprague Dawley (SD) rats treated with the O-ECM/PLLA/SF carried by human iPSC-MSCs. Hence, this study suggests that O-ECM/PLLA/SF scaffolds have a potential application in bone tissue engineering.</jats:p