Human bone marrow mesenchymal stem cells (hMSCs) are promising candidates for cell therapy and tissue engineering. The life span of hMSCs during in vitro culture is limited. Human telomerase catalytic subunit (hTERT) gene transduction can prolong the life span of hMSCs and maintain their potential of osteogenic differentiation. We established a line of hMSCs transduced with exogenous hTERT (hTERT-hMSCs) and investigated its sustaining cellular properties in a long-term culture. This line of hTERT-hMSCs was cultured for 290 population doublings (PDs) without loss of contact inhibition. Under adipogenic, chondrogenic and osteogenic induction, hTERT-hMSCs at PD 95 and PD 275 could differentiate respectively into adipocytes, chondrocytes, and osteocytes. hTERT-hMSCs at these PDs showed no transforming activity through both in vitro assay of cell growth in soft agar and in vivo assay of tumorigenicity in NOD-SCID mice. Karyotype analyses showed no significant chromosomal abnormalities in hTERT-hMSCs at these PDs. These results suggested that the hTERT-hMSCs at lower population doubling levels (PDLs) should be considered as a cell model for studies of cellular senescence, differentiation and in vitro tissue engineering experiment because of its prolonged life span and normal cellular properties.
Cancer-secreted exosomes are critical mediators of cancer-host crosstalk. In the present study, we showed the delivery of miR-21-5p from colorectal cancer (CRC) cells to endothelial cells via exosomes increased the amount of miR-21-5p in recipient cells. MiR-21-5p suppressed Krev interaction trapped protein 1 (KRIT1) in recipient HUVECs and subsequently activated β-catenin signaling pathway and increased their downstream targets VEGFa and Ccnd1, which consequently promoted angiogenesis and vascular permeability in CRC. A strong inverse correlation between miR-21-5p and KRIT1 expression levels was observed in CRC-adjacent vessels. Furthermore, miR-21-5p expression in circulating exosomes was markedly higher in CRC patients than in healthy donors. Thus, our data suggest that exosomal miR-21-5p is involved in angiogenesis and vascular permeability in CRC and may be used as a potential new therapeutic target.
Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have been found to enhance fracture healing. In addition, microRNAs contributing to the healing of various bone fractures have attracted widespread attention in recent years, but knowledge of the mechanisms by which they act is still very limited. In this study, we clarified the function of altered microRNA-19b (miR-19b) expression in BMSCs in fracture healing. We modulated miR-19b expression via mimics/inhibitors in BMSCs and via agomirs in mice to explore the effects of these changes on osteogenic factors, bone cell mineralization and the healing status of modeled fractures. Through gain- and loss-of function assays, the binding affinity between miR-19b and WWP1/Smurf2 was identified and characterized to explain the underlying mechanism involving the KLF5/β-catenin signaling pathway. miR-19b promoted the differentiation of human BMSCs into osteoblasts by targeting WWP1 and Smurf2. Overexpression of WWP1 or Smurf2 degraded the target protein KLF5 in BMSCs through ubiquitination to inhibit fracture healing. KLF5 knockdown delayed fracture healing by modulating the Wnt/β-catenin signaling pathway. Furthermore, miR-19b enhanced fracture healing via the KLF5/β-catenin signaling pathway by targeting WWP1 or Smurf2. Moreover, miR-19b was found to be enriched in BMSC-derived exosomes, and treatment with exosomes promoted fracture healing in vivo. Collectively, these results indicate that mesenchymal stem cell-derived exosomal miR-19b represses the expression of WWP1 or Smurf2 and elevates KLF5 expression through the Wnt/β-catenin signaling pathway, thereby facilitating fracture healing.
Objective: This study aimed to investigate the effect of long non-coding TDRG1 on proliferation and migration of osteosarcoma cells through PI3K/AKT signaling pathway. Materials and Methods: Altogether 87 cases of osteosarcoma tissues and adjacent tissues were collected, and osteosarcoma cells and osteoblasts were purchased. The expression of LncRNA TDRG1 in tissues and cells was detected by RT-PCR. Si-NC, si-TDRG1, and Sh-TDRG1 were transfected into osteosarcoma cells. L740Y-P (activator of PI3K/AKT pathway) and LY294002 (inhibitor of PI3k/AKT pathway) were used to interfere with PI3k/Akt signaling pathway in osteosarcoma cells. qRT-PCR was used to detect the expression of TDRG1 in osteosarcoma tissues and cells. WB was used to detect the expression of p-PI3K, p-AKT, N-cadherin, E-Cadherin, vimentin, Bax, Caspase-3, and Bcl-2 in cells. CCK-8, Transwell and cell scratch tests were used to detect cell proliferation, invasion and migration, and flow cytometry was used to detect cell apoptosis. Results: TDRG1 was highly expressed in osteosarcoma, and the levels of p-PI3K and p-AKT were also up-regulated. Cell experiments showed that inhibiting the expression of TDRG1 could inhibit the proliferation, invasion, migration and EMT of osteosarcoma cells, promote the apoptosis of cells, and up-regulating the expression of TDRG1 could promote the proliferation, invasion, migration and EMT of osteosarcoma cells and inhibit the apoptosis of cells. The 740Y-P intervention could reverse the inhibition of Si-TDRG1 on osteosarcoma cell proliferation, invasion, migration and EMT and the promotion of cell apoptosis. LY294002 intervention could reverse the promotion of Sh-TDRG1 on osteosarcoma cell proliferation, invasion, migration and EMT and the inhibition of cell apoptosis. Conclusion: TDRG1 is highly expressed in osteosarcoma tissue. Silencing the expression of osteosarcoma can inhibit the proliferation, invasion, migration and EMT of osteosarcoma cells by inhibiting PI3K/AKT signaling pathway, which may be a new target for diagnosis and treatment of osteosarcoma.
Total sleep deprivation (TSD) is common in modern society leading to deterioration of multiple aspects of cognition. Dynamic interaction effect of circadian rhythmicity and homeostatic sleep pressure on sustained attention have been intensively investigated, while how this effect was represented on performance and cerebral responses to working memory, another important element of many neurobehavioral tasks, was not well elucidated. Thirty-six healthy subjects with intermediate chronotype performed the Sternberg working-memory task (SWMT) while undergoing functional magnetic resonance imaging every 2 hr from 10:00 p.m. on the first day to 6:00 a.m. on the second day. Using data from three imaging sessions (10:00 p.m., 04:00 a.m., and 06:00 a.m.), we found that the slowest SWMT reaction time and weakest cerebral responses were not at the end of TSD (06:00 a.m.) but during the early morning (04:00 a.m.) hours of the TSD. In addition, during this worst period of TSD, reaction time for the SWMT were found to be negatively correlated with task-related activation in the angular gyrus and positively correlated with the degree of negative correlation between the control and default networks. Our results revealed a rebound of SWMT reaction time and cerebral responses after the mid-time point of regular biological sleep night and provided more evidence that different cognitive tasks are differentially affected by sleep loss and circadian rhythmicity. K E Y W O R D S dynamic changes, rebound response, sleep deprivation, working memory 1 | INTRODUCTION A single night of total sleep deprivation (TSD) produces a range of fundamental neurocognitive deficits and is associated with serious outcomes (Basner, Rao, Goel, & Dinges, 2013; Lim & Dinges, 2010). To understand the neural underpinnings of TSD-related deterioration within a specific cognitive domain, most imaging studies typically conduct two imaging sessions for each participant, one after TSD and the other after rested wakefulness (RW). The neurocognitive consequences of TSD were largely explained by comparing the differences in brain activity between these two sessions (Durmer & Dinges, 2005; Goel, Rao, Durmer, & Dinges, 2009). However, these types of observations describe modulated cerebral responses rather than the process through which the modulation occurs. A recent study exploring 13 repeated functional magnetic resonance imaging (fMRI) sessions during 42 hr of TSD found differential results for cortical and subcortical responses during a psychomotor vigilance task (PVT) and greatly expanded our understanding of how sustained attention deteriorates during TSD (Muto et al., 2016). Thus, to better understand how sleep deprivation impairs cognitive performance, increasing the numbers of sessions and investigating dynamic changes in behavioral and cerebral responses is critical. Yuanqiang Zhu, Yibin Xi, Jinbo Sun, Fan Guo authors contributed equally to this work.
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