Stathmin inhibits proliferation and differentiation of dental pulp stem cells via sonic hedgehog/Gli

The mineralization of dental pulp stem cells is an important factor in the tissue engineering of teeth, but the mechanism is not yet obvious. This study aimed to identify the effect of Stathmin on the proliferation and osteogenic/odontoblastic differentiation of human dental pulp stem cells (hDPSCs) and to explore whether the Shh signalling pathway was involved in this regulation. First, Stathmin was expressed in the cytoplasm and on the cell membranes of hDPSCs by cell immunofluorescence. Then, by constructing a lentiviral vector, the expression of Stathmin in hDPSCs was inhibited. Treatment with Stathmin shRNA (shRNA‐Stathmin group) inhibited the ability of hDPSCs to proliferate, as demonstrated by a CCK8 assay and flow cytometry analysis, and suppressed the osteogenic/odontoblastic differentiation ability, as demonstrated by alizarin red S staining and osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) activity, compared to that of hDPSCs from the control shRNA group. Molecular analyses showed that the Shh/GLI1 signalling pathway was inhibited when Stathmin was silenced, and purmorphamine, the Shh signalling pathway activator, was added to hDPSCs in the shRNA‐Stathmin group, real‐time PCR and Western blotting confirmed that expression of Shh and its downstream signalling molecules PTCH1, SMO and GLI1 increased significantly. After activating the Shh signalling pathway, the proliferation of hDPSCs increased markedly, as demonstrated by a CCK8 assay and flow cytometry analysis; osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) expression also increased significantly. Collectively, these findings firstly revealed that Stathmin‐Shh/GLI1 signalling pathway plays a positive role in hDPSC proliferation and osteogenic/odontoblastic differentiation.     

Mineral trioxide aggregate promotes odontoblastic differentiation via mitogen-activated protein kinase pathway in human dental pulp stem cells

Mitogen-activated protein kinase (MAPK) pathways are involved in stem cell differentiation. However, the odontoblastic differentiation-inducing effects by mineral trioxide aggregate (MTA) via MAPK pathways have not been clarified in human dental pulp stem cells (DPSCs). In this study we investigated the effects of MTA on cell viability and production of differentiation markers, and the involvement of MAPK signaling pathways in cultured human DPSCs. Cells were cultured with MTA, and the viability and differentiation productions of the cells were determined using the MTT assay and real-time PCR analysis, respectively. MAPK activation was measured by western blotting. MTA at concentrations of 20 and 10 mg/ml was toxic for human DPSCs. MTA significantly increased the expression of alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP), type I collagen (COLI), osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs and induced the phosphorylation of p42 and p44 (p42/44), p38 and c-Jun N-terminal kinases 1 and 2 (JNK1/2) MAPK. Furthermore, the inhibitor of p42/44 MAPK attenuated the MTA-induced odontoblastic differentiation. These data indicated that MTA-induced odontoblastic differentiation of human DPSCs was via MAPK pathways, which may play a key role in the repair responses of dentin-pulp-like complexes.     

Retracted: microRNA-542-5p protects against acute lung injury in mice with severe acute pancreatitis by suppressing the mitogen-activated protein kinase signaling pathway through the negative regulation of P21-activated kinase 1

Severe acute pancreatitis (SAP) is a condition associated with high rates of mortality and lengthy hospital stays. In the current study, SAP mouse models were established in BALB/c wild-type and P21-activated kinase 1 (PAK1) knockdown mice with the objective of determining the expression of microRNA-542-5p (miR-542-5p) and the subsequent elucidation of the mechanism by which it influences acute lung injury (ALI) by mediating mitogen-activated protein kinase (MAPK) signaling and binding to PAK1. The targeting relationship between miR-542-5p and PAK1 was verified using the bioinformatics prediction website and by the means of a dual-luciferase reporter assay. Following the SAP model establishment, the mice were assigned into various groups with the introduction of different mimic and inhibitors in an attempt to investigate the effects involved with miR-542-5p on inflammatory reactions among mice with SAP-associated ALI. Our results indicated that PAK1 was targeted and negatively mediated by miR-542-5p. Mice with SAP-associated ALI exhibited an increased wet-to-dry weight ratio, myeloperoxidase activity, serum amylase activity, TNF-α, interleukin-1 beta (IL-1β), and intercellular adhesion molecule-1 (ICAM-1) contents, p-p38MAPK, p-ERK1/2, and p-JNK protein levels as well as PAK1 positive expression, while decreased miR-542-5p levels were observed. Functionally, overexpression of miR-542-5p improves ALI in mice with SAP via inhibition of the MAPK signaling pathway by binding to PAK1.Based on the evidence from experimental models, miR-542-5p was shown to improve ALI among mice with SAP, while suggesting that the effect may be related to the inactivation of the MAPK signaling pathway and downregulation of PAK1 gene. Thus, miR-542-5p could serve as a promising target for ALI treatment.     

MiR-21协同BMP9促进间充质干细胞C3H10T1/2成骨分化

目的:探讨miR-21与BMP9之间的关系,明确miR-21在BMP9诱导间充质干细胞成骨分化中的作用。方法:(1)Ad-BMP9感染C3H10T1/2细胞,Real-time-PCR检测miR-21表达。RT-PCR检测ALP的表达。(2)MiR-21转染C3H10T1/2细胞,Real-time-PCR检测miR-21和BMP9表达。(3)MiR-21和BMP9-CM处理C3H10 T1/2细胞,ALP活性和染色实验检测C3H10 T1/2细胞早期成骨能力。茜素红S染色实验检测钙盐沉积情况。(4)MiR-21和BMP9-CM处理C3H10 T1/2细胞,Real-time-PCR检测成骨分化相关因子ALP,OCN的表达。(5)MiR-21和BMP9-CM处理C3H10T1/2细胞,Western blot检测p-Smad1/5蛋白水平的表达。结果:(1)BMP9暂时降低miR-21的表达。MiR-21也可以暂时降低BMP9的表达。(2)MiR-21可以协同BMP9增强ALP和钙盐沉积。(3)MiR-21协同BMP9增加了p-Smad1/5蛋白水平的表达。结论:MiR-21与BMP9存在相互关系,两者可以互相调节表达。MiR-21可以协同BMP9促进间充质干细胞C3H10T1/2细胞成骨分化,这一过程与增强BMP9/Smad信号的激活程度有关。     

LncRNA CALB2 sponges miR-30b-3p to promote odontoblast differentiation of human dental pulp stem cells via up-regulating RUNX2

Illuminating the mechanisms of odontoblast differentiation of human dental pulp stem cells (hDPSCs) is the key to find therapeutic clues to promote odontogenesis. LncRNAs play a regulatory role in odontoblast differentiation. Here, we identified a novel lncRNA, named lncRNA CALB2. It was up-regulated in odontoblast-differentiated hDPSCs and potentially interacted with miR-30b-3p and RUNX2. Via gain- and loss-of-function approaches, we found lncRNA CALB2 significantly promoted the odontoblast differentiation of hDPSCs. Then, dual luciferase reporter assay and RNA immunoprecipitation assay revealed that both lncRNA CALB2 and RUNX2 mRNA could directly bind to miR-30b-3p via the same binding sites. Interestingly, miR-30b-3p in hDPSCs was down-regulated and RUNX2 was up-regulated during odontoblast differentiation. Moreover, lncRNA CALB2 knockdown significantly reduced the protein level of RUNX2, DSPP and DMP-1, while miR-30b-3p inhibitor rescued the reduction. Furthermore, miR-30b-3p exerted an inhibitory effect on odontoblast differentiation, which could be reversed by lncRNA CALB2. Collectively, these findings indicate that the newly identified lncRNA CALB2 acts as a miR-30b-3p sponge to regulate RUNX2 expression, thus promoting the odontoblast differentiation of hDPSCs. LncRNA CALB2/miR-30b-3p/RUNX2 axis could be a novel therapeutic target for accelerating odontogenesis.     

The role of lysyl oxidase-like 2 in the odontogenic differentiation of human dental pulp stem cells

Adult human dental pulp stem cells (hDPSCs) are a unique population of precursor cells those are isolated from postnatal dental pulp and have the ability to differentiate into a variety of cell types utilized for the formation of a reparative dentin-like complex. Using LC-MS/MS proteomics approaches, we identified the proteins secreted from the differentiating hDPSCs in mineralization media. Lysyl oxidase-like 2 (LOXL2) was identified as a protein that was down-regulated in the hDPSCs that differentiate into odontoblast-like cells. The role of LOXL2 has not been studied in dental pulp stem cells. LOXL2 mRNA levels were reduced in differentiating hDPSCs, whereas the levels of other LOX family members including LOX, LOXL1, LOXL3, and LOXL4, are increased. The protein expression and secretion levels of LOXL2 were also decreased during odontogenic differentiation. Recombinant LOXL2 protein treatment to hDPSCs resulted in a dose-dependent decrease in the early differentiation and the mineralization accompanying with the lower levels of odontogenic markers such as DSPP, DMP-1 and ALP. These results suggest that LOXL2 has a negative effect on the differentiation of hDPSCs and blocking LOXL2 can promote the hDPSC differentiation to odontoblasts.     

Dexamethasone suppresses osteogenesis of osteoblast via the PI3K/Akt signaling pathway in vitro and in vivo

The hypofunction of osteoblasts induced by glucocorticoids (GCs) has been identified as a major contributing factor for GC-induced osteoporosis (GIO). However, the biological mechanism underlying the effect of GC in osteoblasts are not fully elucidated. Recent studies implicated an important role of phosphoinositide 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway in the regulation of bone growth. We propose that the PI3K/Akt signaling may be implicated in the process of GC-induced osteogenic inhibition in osteoblasts. In this study, primary osteoblasts were used in vitro and in rats in vivo to evaluate the biological significance of the PI3K/Akt pathway in GC-induced bone loss. In vivo, dexamethasone (Dex)-treated rats had low bone mineral density and decreased expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and phosphorylated Akt (p-Akt) in bone tissue. In vitro study shows that Dex over the dose of 10^–8 M remarkably inhibited cellular osteogenesis, as represented by decreased cell viability, lessened ALP activity, and suppressed osteogenic protein expressions including ALP and OCN. Meanwhile, a dramatic downregulation in the PI3K/Akt pathway phosphorylation was also observed in Dex-treated osteoblasts. These changes were marked rescued by treatment with a PI3K agonist 740Y-P. Moreover, downregulation of ALP and OCN expressions by LY294002 can mimic the suppressive effects of Dex. These data together reveal that the suppressed PI3K/Akt pathway is involved in the regulatory action of Dex on osteogenesis.     

MicroRNA-329-mediated PTTG1 downregulation inactivates the MAPK signaling pathway to suppress cell proliferation and tumor growth in cholangiocarcinoma

Cholangiocarcinoma (CCA) is a severe malignancy usually producing a poor prognosis and high mortality rate. MicroRNAs (miRNAs) have been reported in association with CCA; however, the role miR-329 plays in the CCA condition still remains unclear. Therefore, this study was conducted to explore the underlying mechanism of which miR-329 is influencing the progression of CCA. This work studied the differential analysis of the expression chips of CCA obtained from the Gene Expression Omnibus database. Next, to determine both the expression and role of pituitary tumor transforming gene-1 (PTTG1) in CCA, the miRNAs regulating PTTG1 were predicted. In the CCA cells that had been intervened with miR-329 upregulation or inhibition, along with PTTG1 silencing, expression of miR-329, PTTG1, p-p38/p38, p-ERK5/ERK5, proliferating cell nuclear antigen (PCNA), Cyclin D1, Bcl-2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and caspase-3 were determined. The effects of both miR-329 and PTTG1 on cell proliferation, cell-cycle distribution, and apoptosis were also assayed. The miR-329 was likely to affect the CCA development through regulation of the PTTG1-mediated mitogen-activated protein kinase (MAPK) signaling pathway. The miR-329 targeted PTTG1, leading to inactivation of the MAPK signaling pathway. Upregulation of miR-329 and silencing of PTTG1 inhibited the CCA cell proliferation, induced cell-cycle arrest, and subsequently promoted apoptosis with elevations in Bax, cleaved caspase-3, and total caspase-3, but showed declines in PCNA, Cyclin D1, and Bcl-2. Moreover, miR-329 was also found to suppress the tumor growth by downregulation of PTTG1. To summarize, miR-329 inhibited the expression of PTTG1 to inactivate the MAPK signaling pathway, thus suppressing the CCA progression, thereby providing a therapeutic basis for the CCA treatment.     

Retracted: Effects of microRNA-494 on proliferation,migration, invasion,and apoptosis of medulloblastoma cells by mediating c-myc through the p38 MAPK signaling pathway

Medulloblastoma (MB) is the most prevalent brain tumor that occurs during childhood and originates from cerebellar granule cell precursors. Based on recent studies, the differential expression of several microRNAs is involved in MB, while the role of microRNA-494 (miR-494) in MB remains unclear. Therefore, we conducted this study to investigate the regulative role of miR-494 in MB cells via the p38 mitogen-activated protein kinase (MAPK) signaling pathway by mediating c-myc. In the current study, MB cells were collected and transfected with miR-494 mimic, miR-494 inhibitor, siRNA- c-myc, and miR-494 inhibitor + siRNA-c-myc. The expressions of miR-494, c-myc, p38 MAPK, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), interleukin-6 (IL-6), metadherin (MTDH), phosphatase and tensin homolog (PTEN) and survivin were determined. Cell proliferation, cell-cycle distribution, apoptosis, migration, and invasion were evaluated. The results revealed that there was a poor expression of miR-494 and high expression of c-myc in MB tissues. C-myc was determined as the target gene of miR-494. In response to miR-494 mimic, MB cells were found to have increased Bax and PTEN expressions, as well as cell number in G1 phase and cell apoptosis and decreased c-myc, p38 MAPK, Bcl-2, MTDH, IL-6, and survivin expression and cell number count in the S phase, cell proliferation, migration, and invasion. In conclusion, the results demonstrated that the upregulation of miR-494 results in the suppression of cell proliferation, migration, and invasion, while it promotes apoptosis of MB cells through the negative mediation of c-myc, which in turn inactivates the p38 MAPK pathway.     

Lentiviral-mediated RNAi targeting p38MAPK ameliorates high glucose-induced apoptosis in osteoblast MC3T3-E1 cell line

The p38 mitogen activated protein kinase (p38MAPK) pathway is an important signaling cascade involved in cell growth, differentiation and apoptosis. High glucose activates p38MAPK pathway in different cells, including osteoblasts. In the present study, role of p38MAPK in high glucose induced osteoblast apoptosis and potential of RNA interference (RNAi) targeting p38MAPK as a therapy strategy have been reported. Lentiviral-mediated RNAi effectively reduced p38MAPK and p-p38MAPK expressions in osteoblastic cell line (MC3T3-E1) following high glucose (22 mM) induction. Inhibition of p38MAPK activity significantly suppressed high glucose induced apoptosis of MC3T3-E1 cell and was confirmed by flow cytometry and ultra-structural examination by transmission electronic microscope. Inhibition of p38MAPK also significantly attenuates caspase-3 and bax protein expressions, but increased significantly bcl-2 expression as determined by Western blot analysis. The results suggested that p38MAPK mediates high glucose induced osteoblast apoptosis, partly through modulating the expressions of caspase-3, bax and bcl-2. Inhibition of p38MAPK with lentiviral-mediated RNAi or its specific inhibitor provides a new strategy to treat high glucose induced osteoblast apoptosis.