Insulin-like growth factor binding protein 5 accelerate the senescence of periodontal ligament stem cells.

Evidences have showed stem cell mediated tissue regeneration is a promising method for the treatment of periodontitis. Insulin-like growth factor binding proteins-5 (IGFBP5) is a member of the insulin growth factor (IGFs) family and plays a regulatory role in cell proliferation and differentiation. Our previous study showed that IGFBP5 can promote osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and enhance periodontal tissue regeneration mediated by PDLSCs. However, the function of IGFBP5 in the process of PDLSCs senescence remains unclear. The present study showed IGFBP5 mRNA level was highly expressed in passage-induced aged PDLSCs cells. IGFBP5 knockdown decreased the ratio of senescence associated ß-galactosidase (SA-ß-Gal) positive cells, enhanced the activity of TERT, and down-regulated the expression levels of P16, P21, P53 mRNA and protein. Overexpression of IGFBP5 increased the ratio of SA-ß-Gal positive staining PDLSCs, decreased the activity of telomerase TERT, and up-regulated the expression levels of P16, P21, P53 mRNA and protein related to PDLSCs senescence. In conclusion, IGFBP5 can accelerate the senescence of PDLSCs, indicating the potential target for maintaining the "young state" of stem cells.

MicroRNA-196a-5p overexpression in Wharton's jelly umbilical cord stem cells promotes their osteogenic differentiation and new bone formation in bone defects in the rat calvarium.

The peri-tooth root alveolar loss often does not have sufficient space for repair material transplantation and plasticity. Mesenchymal stem cell (MSC) sheets have an advantage in providing more extracellular matrix (ECM) and may prove to be a new therapeutic consideration for this bone defect repair. The identification of key regulators that stimulate MSCs' osteogenic potential and sheet-derived ECM deposition is the key to promoting its application. In this study, we found that inhibition or overexpression of miR-196a-5p led to a decline or enhancement, respectively, in the alkaline phosphatase (ALP) activity, mineralization, and the levels of osteogenic markers, Osteocalcin (OCN), Dentin Matrix Protein 1 (DMP1), Bone Sialoprotein (BSP), and Dentin Sialophosphoprotein (DSPP) of Wharton's jelly of umbilical cord stem cells (WJCMSCs) in vitro. Moreover, the 5,6-Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) analysis revealed inhibition of the WJCMSCs' proliferative ability upon miR-196a-5p overexpression. Characterization of the sheet formation by picrosirius red and Masson staining indicated that miR-196a-5p overexpression significantly promoted the collagen content in whole WJCMSC sheet-derived ECM. Furthermore, micro-CT and histopathology results indicated that the miR-196a-5p-overexpressed WJCMSC sheets significantly promoted new bone regeneration and rat calvarial bone defect closure 12 weeks following transplantation. The mRNA microarray analysis of miR-196a-5p-overexpressed WJCMSCs revealed 959 differentially expressed genes (DEGs) (34 upregulated and 925 downregulated). Moreover, 241 genes targeted by miR-196a-5p were predicted by using miRNA function websites of which only 19 predicted genes were consistent with the microarray revealed DEGs. Hence, one unrevealed downregulated DEG Serpin Family B Member 2 (SERPINB2) was investigated. And the deletion of SERPINB2 enhanced the ALP activity and mineralization of WJCMSCs in vitro. In conclusion, our study found that miR-196a-5p, as a key regulator, could repress the proliferation tendency, while stimulating osteogenic ability and WJCMSC sheet-derived ECM deposition, thus promoting new bone formation and rat calvarial bone defect closure. Furthermore, SERPINB2 is a key downstream gene involved in the miR-196a-5p-promoted WJCMSC osteogenesis.

miR-663a inhibits hepatocellular carcinoma cell proliferation and invasion by targeting HMGA2.

Hepatocellular carcinoma (HCC) is a highly aggressive solid malignancy throughout the world. Dysregulation of miRNAs play essential roles in HCC progression via aberrant regulation of cell proliferation, apoptosis, as well as metastasis. miR-663a is a poorly investigated miRNA. Whether miR-663a regulates HCC development remains unknown. The aim of the study was to explore the role of miR-663a in HCC development. To determine the expression level of miR-663a in HCC, we analyzed the data from GSE21362 and TCGA. The results showed that miR-663a was significantly down-regulated in HCC tissue compared with adjacent non-tumor tissue. Gain of function and loss of function assays revealed that miR-663a distinctly inhibited cell proliferation, migration and invasion. Mechanistic investigations demonstrated that miR-663a modulated cell functions through targeting and suppressing high mobility group A2 (HMGA2). In addition, overexpression of HMGA2 remarkably attenuated the tumor repressive effect of miR-663a. Taken together, miR-663a inhibits HCC cell proliferation and motility by targeting HMGA2.

Transient collagen triple helix binding to a key metalloproteinase in invasion and development.

Skeletal development and invasion by tumor cells depends on proteolysis of collagen by the pericellular metalloproteinase MT1-MMP. Its hemopexin-like (HPX) domain binds to collagen substrates to facilitate their digestion. Spin labeling and paramagnetic nuclear magnetic resonance (NMR) detection have revealed how the HPX domain docks to collagen I-derived triple helix. Mutations impairing triple-helical peptidase activity corroborate the interface. Saturation transfer difference NMR suggests rotational averaging around the longitudinal axis of the triple-helical peptide. Part of the interface emerges as unique and potentially targetable for selective inhibition. The triple helix crosses the junction of blades I and II at a 45° angle to the symmetry axis of the HPX domain, placing the scissile Gly∼Ile bond near the HPX domain and shifted ∼25 Å from MMP-1 complexes. This raises the question of the MT1-MMP catalytic domain folding over the triple helix during catalysis, a possibility accommodated by the flexibility between domains suggested by atomic force microscopy images.

Apparent tradeoff of higher activity in MMP-12 for enhanced stability and flexibility in MMP-3.

The greater activity of MMP-12 than MMP-3 toward substrates from protein fibrils has been quantified. Why is MMP-12 the more active protease? We looked for behaviors associated with the higher activity of MMP-12 than MMP-3, using nuclear magnetic resonance to monitor backbone dynamics and residue-specific stabilities of their catalytic domain. The proteolytic activities are likely to play important roles in inflammatory diseases of arteries, lungs, joints, and intestines. Nuclear magnetic resonance line broadening indicates that regions surrounding the active sites of both proteases sample conformational substates within milliseconds. The more extensive line broadening in MMP-3 suggests greater sampling of conformational substates, affecting the full length of helix B and beta-strand IV forming the active site, and more remote sites. This could suggest more excursions to functionally incompetent substates. MMP-3 also has enhanced subnanosecond fluctuations in helix A, in the beta-hairpin of strands IV and V, and before and including helix C. Hydrogen exchange protection in the EX2 regime suggests that MMP-3 possesses 2.8 kcal/mol higher folding stability than MMP-12(E219A). The beta-sheet of MMP-3 appears to be stabilized still more. The higher stability of MMP-3 relative to MMP-12 coincides with the former's considerably lower proteolytic activity. This relationship is consistent with the hypothesis that enzymes often trade stability for higher activity.