Adipose Mesenchymal Stem Cell-Derived Exosomes Enhance PC12 Cell Function through the Activation of the PI3K/AKT Pathway

Transplantation of mesenchymal stem cells has been considered as an auspicious treatment for repairing nerve injuries. The rat adrenal pheochromocytoma cell line (PC12) is one of the traditional models for the study of neuronal differentiation and neuroregeneration in vitro. However, the effects of adipose mesenchymal stem cell-derived exosomes (ADSC-exo) on PC12 cells remain unclear and to be elucidated. In our study, the effects of ADSC-exo on PC12 cells were investigated. ADSC-exo were isolated by ultracentrifugation and characterized by transmission electron microscopy, flow nanoanalysis, and western blot. The effects of ADSC-exo on PC12 cell proliferation, migration, apoptosis, and the protein levels were analyzed using CCK-8 assay and EdU incorporation assay, transwell migration assay and scratch wound assay, flow cytometry, and western blot, respectively. We successfully isolated and purified exosomes from ADSC supernatant and found that ADSC-exo treatment significantly promoted PC12 cell proliferation and migration, inhibited their apoptosis, and activated the PI3K/AKT pathway, while PI3K/AKT signaling repression using LY294002 exhibited the opposite effects. The results showed that ADSC-exo promoted proliferation and migration and inhibited apoptosis of PC12 through the activation of the PI3K/AKT pathway. Thus, the effect of ADSC-exo on PC12 cells may suggest ADSC-exo may be a promising therapeutic for nerve damage.

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Seven Novel Genes Related to Cell Proliferation and Migration of VHL-Mutated Pheochromocytoma

Frontiers in Endocrinology ◽

10.3389/fendo.2021.598656 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shuai Gao ◽

Longfei Liu ◽

Zhuolin Li ◽

Yingxian Pang ◽

Jiaqi Shi ◽

...

Keyword(s):

Cell Proliferation ◽

Pc12 Cells ◽

Pc12 Cell ◽

Collagen Type ◽

Tissue Growth ◽

Cell Counting ◽

Type I ◽

Cell Proliferation And Migration ◽

And Migration ◽

Proliferation And Migration

Pheochromocytoma, as a neuroendocrine tumor with the highest genetic correlation in all types of tumors, has attracted extensive attention. Von Hipper Lindau (VHL) has the highest mutation frequency among the genes associated with pheochromocytoma. However, the effect of VHL on the proteome of pheochromocytoma remains to be explored. In this study, the VHL knockdown (VHL-KD) PC12 cell model was established by RNA interference (shRNA). We compared the proteomics of VHL-KD and VHL-WT PC12 cell lines. The results showed that the expression of 434 proteins (VHL shRNA/WT > 1.3) changed significantly in VHL-KD-PC12 cells. Among the 434 kinds of proteins, 83 were involved in cell proliferation, cell cycle and cell migration, and so on. More importantly, among these proteins, we found seven novel key genes, including Connective Tissue Growth Factor (CTGF), Syndecan Binding Protein (SDCBP), Cysteine Rich Protein 61 (CYR61/CCN1), Collagen Type III Alpha 1 Chain (COL3A1), Collagen Type I Alpha 1 Chain (COL1A1), Collagen Type V Alpha 2 Chain (COL5A2), and Serpin Family E Member 1 (SERPINE1), were overexpressed and simultaneously regulated cell proliferation and migration in VHL-KD PC12 cells. Furthermore, the abnormal accumulation of HIF2α caused by VHL-KD significantly increased the expression of these seven genes during hypoxia. Moreover, cell-counting, scratch, and transwell assays demonstrated that VHL-KD could promote cell proliferation and migration, and changed cell morphology. These findings indicated that inhibition of VHL expression could promote the development of pheochromocytoma by activating the expression of cell proliferation and migration associated genes.

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Long non-conding RNA LOXL1-AS1 sponges miR-589-5p to up-regulate CBX5 expression in renal cell carcinoma

Bioscience Reports ◽

10.1042/bsr20200212 ◽

2020 ◽

Vol 40 (11) ◽

Author(s):

Chunlei Wu ◽

Jiange Zhang

Keyword(s):

Renal Cell Carcinoma ◽

Cell Proliferation ◽

Western Blot ◽

Cell Carcinoma ◽

Renal Cell ◽

Cell Function ◽

Luciferase Reporter ◽

Cell Proliferation And Migration ◽

And Migration ◽

Proliferation And Migration

Abstract Background: Renal cell carcinoma (RCC) is a common malignant tumor that seriously endangers people’s health. In recent years, long non-coding RNAs (lncRNAs) have been discovered to play vital roles in diverse cancers, including RCC. LncRNA lysyl oxidase like 1 antisense RNA 1 (LOXL1-AS1) has been found to exert carcinogenic functions in several cancers, but its role and mechanism in RCC have not been investigated. Methods: qRT-PCR was utilized for testing RNA expression and Western blot for protein expression in RCC tissues or cells. Then, we assessed cell function by conducting a series of functional experiments, such as 5-ethynyl-2′-deoxyuridine staining, colony formation, flow cytometry, JC-1, Western blot and transwell migration experiments. Following, RNA immunoprecipitation, pull down and luciferase reporter experiments were carried out to explore the regulatory mechanisms of LOXL1-AS1 in RCC. Results: LOXL1-AS1 was highly expressed in RCC tissues and cells. Moreover, knockdown of LOXL1-AS1 hampered RCC cell proliferation and migration. Importantly, miR-589-5p that was lowly expressed and worked as a tumor-inhibitor in RCC was found to bind with LOXL1-AS1. Furthermore, chromobox 5 (CBX5) targeted by miR-589-5p could expedite cell proliferation and migration in RCC. Finally, overexpressed CBX5 or inhibited miR-589-5p reversed the repressive impacts of silenced LOXL1-AS1 on RCC malignant phenotypes. Conclusions: LncRNA LOXL1-AS1 sequestered miR-589-5p to augment CBX5 expression in RCC cells, opening a new way for potential development in RCC treatment.

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Extracellular matrix derived peptides and mesenchymal stem cell motility

10.26686/wgtn.17152088.v1 ◽

2021 ◽

Author(s):

◽

Sandi Grainne Dempsey

Keyword(s):

Extracellular Matrix ◽

Wound Healing ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Cell Motility ◽

Cell Attachment ◽

Mass Spectrometry Analysis ◽

And Migration ◽

Proliferation And Migration

<p>Biomaterials derived from decellularised extracellular matrices have shown promise as tools in tissue regeneration and wound healing. Such materials display biocompatibility as well as inherent bioactivity, promoting constructive remodelling in healing tissues. In this study, the bioactivity of ovine forestomach matrix (a decellularised extracellular matrix biomaterial) is assessed based on its ability to affect the proliferation and migration of wound healing cells. This material supported cell attachment and proliferation, but did not allow cell infiltration in vitro. Enzymatic digestion of the material rendered soluble components that were able to induce proliferation and migration of some cell types. Cell-mediated processing of the material generated a protein or proteins with chemotactic activity for mesenchymal stem cells in vitro. Mass spectrometry analysis indicated the bioactive component consisted of the proteoglycan decorin, or fragments thereof. Decorin has not previously been shown to induce mesenchymal stem cell motility, and these findings may add to what is known about decorin and its role in constructive remodelling. Furthermore, this cell-mediated approach for ECM breakdown could lead to the discovery of other bioactive peptides involved in ECM remodelling and wound healing.</p>

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Bone Marrow Mesenchymal Stem Cell-Derived Exosomal miR-25 Regulates the Ubiquitination and Degradation of Runx2 by SMURF1 to Promote Fracture Healing in Mice

Frontiers in Medicine ◽

10.3389/fmed.2020.577578 ◽

2020 ◽

Vol 7 ◽

Author(s):

Yikun Jiang ◽

Jun Zhang ◽

Zhengwei Li ◽

Guoliang Jia

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Fracture Healing ◽

Osteogenic Differentiation ◽

Bioinformatics Analysis ◽

Luciferase Reporter ◽

Gene Assay ◽

And Migration ◽

Proliferation And Migration

Recent evidence has demonstrated that mesenchymal stem cells (MSCs) can release a large number of functionally specific microRNA (miRNA) microvesicles that play a role in promoting osteogenic differentiation, but the specific mechanism is not yet clear. Under such context, this study aims to elucidate the mechanism of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exo) promoting fracture healing in mice. We isolated and identified the BMSC-Exo. Bioinformatics analysis predicted high expression of miRNA in exosomes and verified the transfer of miR-25 in exosomes by immunofluorescence. Targeting relationship between miR-25 and Smad ubiquitination regulatory factor-1 (SMURF1) was predicted and verified by dual-luciferase reporter gene assay. Immunoprecipitation and protein stability assays were used to detect Runt-related transcription factor 2 (Runx2) ubiquitination and the effect of SMURF1 on Runx2 ubiquitination, respectively. The effect of miR-25 in BMSC-Exo on fracture healing in mice was assessed using X-ray imaging. alkaline phosphatase, alizarin red staining, EdU, CCK-8, and Transwell were used to evaluate the effects of exosomes transferred miR-25 on osteogenic differentiation, proliferation, and migration of osteoblasts. Bioinformatics analysis predicted that miR-25 expression in exosomes increased significantly. Moreover, the targeted regulation of SMURF1 by miR-25 was verified. SMURF1 inhibited Runx2 protein expression by promoting ubiquitination degradation of Runx2. Notably, miR-25 secreted by BMSC-Exo can accelerate osteogenic differentiation, proliferation, and migration of osteoblasts through SMURF1/Runx2 axis. Our results demonstrate that miR-25 in BMSC-Exo regulates the ubiquitination degradation of Runx2 by SMURF1 to promote fracture healing in mice.

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MicroRNA-708 targeting ZNF549 regulates colon adenocarcinoma development through PI3K/AKt pathway

Scientific Reports ◽

10.1038/s41598-020-73929-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Zhidong Zhao ◽

Xianju Qin

Keyword(s):

Cell Proliferation ◽

Western Blot ◽

Cell Lines ◽

Colon Adenocarcinoma ◽

Signal Pathway ◽

Luciferase Reporter ◽

Mesenchymal Transition ◽

Cell Proliferation And Migration ◽

And Migration ◽

Proliferation And Migration

Abstract Colon adenocarcinoma (COAD) is the most common type of gastrointestinal cancer and is still the third leading cause of cancer-related mortality worldwide. Therefore, finding new and promising drugs to eradicate cancer may be a feasible method to treat COAD patients. Cys2-His2 zinc finger proteins (ZFPs) is one of the largest transcription factor family and many of them are highly involved in regulation of cell differentiation, proliferation, apoptosis, and neoplastic transformation. In this study, we identified a tumor-inhibiting factor, ZNF549, which expressed lowly in COAD tissues and COAD cell lines (HT29, HCT116, SW480, LoVo, and SW620). Overexpression of ZNF549 inhibit the ability of COAD cell proliferation and migration. On the contrary, decreasing the ZNF549 expression level promote the ability of COAD cell proliferation and migration. Through bioinformatics analysis, we found that ZNF549 was a potential target of hsa-miR-708-5p (miR-708-5p). Furthermore, we verified the possibility of miR-708-5p targeting the ZNF549 gene, and miR-708-5p inhibited the expression of ZNF549 by luciferase reporter assays, qRT-PCR and western blot assays. Moreover, the relationship between miR-708-5p and phosphatidylinositol 3-kinase/AKt (PI3K/AKt) signal pathway was elucidated. Overexpression and inhibition of miR-708-5p resulted in increased and decreased expression of p-AKt and p-PI3K in HCT116 cells, respectively. RT-qPCR and western blot assays results demonstrated that miR-708-5p regulated COAD cells development by promoting the process of Epithelial-mesenchymal transition (EMT) through PI3K/AKt signaling pathway. In summary, our findings demonstrated that ZNF549, the target gene of miR-708-5p, functions as a tumor suppressor to inhibit COAD cell lines proliferation and migration through regulate the PI3K/AKt signal pathway.

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