Objective: To investigate the effects of long non-coding RNA (lncRNA) LINC01133 on the cementogenic differentiation of human periodontal ligament stem cells (hPDLSC) and the underlying mechanism. Methods: A total of 12 teeth were harvested from 10 patients aged 17-30 years in the Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University for impacted or orthodontic reasons from September 2021 to January 2022. The hPDLSCs were isolated from the teeth and transfected with small interfering RNA-LINC01133 (si-LINC01133) or small interfering RNA-negative control (si-NC). The si-LINC01133 was regarded as the experimental group, and the si-NC was regarded as the control one. The silencing efficiency of LINC01133 in the hPDLSCs was evaluated by real-time quantitative PCR (RT-qPCR). Western blotting was used to detect the protein expression levels of cementogenic differentiation-related factors including bone sialoprotein (BSP), cementum attachment protein (CAP), and cementum protein-1 (CEMP-1). Mitochondrial reactive oxygen species (mtROS) production was assessed using the MitoSox by flow cytometry. Mitochondrial membrane potential (MMP) was detected by JC-1 fluorescence staining. Mitochondrial respiratory chain complexes proteins including NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 (NDUFB8), succinate dehydrogenase complex flavoprotein subunit A (SDHA), ubiquinol-cytochrome c reductase core protein 1 (UQCR1), cytochrome c oxidase subunit 4 isoform 1 (COXⅣ), and ATP synthase F1 subunit alpha (ATP5A) were evaluated by Western blotting. Results: The expression levels of LINC01133 could be suppressed by more than 60% with si-LINC01133 (control group: 1.000±0.000, experimental group: 0.385±0.128) (t=10.72, P<0.01). Suppression of LINC01133 in hPDLSCs decreased the levels of cementogenic differentiation-related proteins including BSP (control group: 1.000±0.000, experimental group: 0.664±0.179) (t=4.62, P<0.01) and CAP (control group: 1.000±0.000, experimental group: 0.736±0.229) (t=2.83, P<0.05). Suppression of LINC01133 in hPDLSCs increased the production of mtROS (control group: 1.000±0.000, experimental group: 1.458±0.185) (t=4.96, P<0.05) and the expression of NDUFB8 (control group: 1.000±0.000, experimental group: 1.683±0.397) (t=3.45, P<0.05), as well as decreased MMP levels (control group: 1.000±0.000, experimental group: 0.209±0.029) (t=53.99, P<0.01) and the expression of SDHA (control group: 1.000±0.000, experimental group: 0.428±0.228) (t=5.02, P<0.05). No significant changes in the UQCR1, COXⅣ, and ATP5A expression levels were found between the control group and exprimental group (P>0.05). Conclusions: LINC01133 regulates the cementogenic differentiation of hPDLSCs possibly via modulating the mitochondrial functions.
Humans ; Periodontal Ligament ; RNA, Long Noncoding/metabolism* ; Cells, Cultured ; Stem Cells ; Cell Differentiation ; Integrin-Binding Sialoprotein/metabolism* ; Mitochondrial Proteins/metabolism* ; Mitochondria/genetics* ; RNA, Small Interfering/metabolism* ; Osteogenesis

OBJECTIVETo investigate the roll of bone sialoprotein (BSP), a secreted glycoprotein, found in mineralized tissues in the development and progression of human esophageal squamous cell carcimoma (ESCC), and explore its association with clinicopathological characteristics and five-year survival of the patients.

METHODSThe expression of BSP was determined in 211 primary ESCC tumors and their paired nontumorous tissues using tissue-array, RT-PCR and immunohistochemistry.

RESULTSPrimary ESCC tissues showed a significantly higher expression rate of BSP mRNA than their paired nontumorous tissues (93.8% vs. 16.6%, P < 0.001), the same with BSP protein (56.9% vs. 31.3%, P < 0.001). The expression rate of BSP protein was correlated to lymph node metastasis and TNM stage (P < 0.05). The 5-year survival rate of BSP protein-positive ESCC patients was significantly lower than that of BSP protein-negative ESCC patients (P < 0.05). Multivariate analysis showed that tumor differentiation, TNM staging and BSP protein expression were independent factors affecting the prognosis of ESCC patients (P < 0.05).

CONCLUSIONSThe abnormal expression of BSP may play a significant role in the malignant progression and prognosis of ESCC, and BSP might be a marker reflecting the biologial behavior of ESCC.

Blotting, Western ; Carcinoma, Squamous Cell ; diagnosis ; metabolism ; Esophageal Neoplasms ; diagnosis ; metabolism ; Humans ; Immunohistochemistry ; Integrin-Binding Sialoprotein ; genetics ; metabolism ; Lymphatic Metastasis ; Neoplasm Staging ; Prognosis ; RNA, Messenger ; Survival Rate

We performed this research mainly to explore the effect of bone sialoprotein (BSP) silence by siRNA on the adhesion ability to bone matrix of bone-seeking breast cancer cells (MDA-MB-231BO). Also we aimed to provide experimental data for prevention and treatment of breast cancer bone metastasis by targeting BSP. We explored the effects of BSP gene silence on characteristics of bone-seeking breast cancer cells: proliferation by MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay, bone adhesion ability by a mouse bone adhesion model in vitro, morphology of the cells by SEM, and secretion of transforming growth factor-beta1 (TGF-beta1) and receptor activator of nuclear factor-kappa B ligand (RANKL) by ELISA kits. We performed intra-cardiac injection in nude mice to explore bone metastatic ability of different cell lines. The results showed that knockdown of BSP significantly inhibited the proliferation of MDA-MB-231BO cells and their adhesion to bone matrix. We also observed bone destruction caused by bone resorption around some adhering cells. The appearances of the cells changed in BSP gene silenced group, and the secretion of TGF-beta1 and RANKL decreased. The results showed BSP gene silence can partial inhibition bone metastasis of breast cancer cells in nude mice by X-ray assay and hematoxylin-eosin staining. Based on our research, siRNA-mediated BSP silencing can inhibit proliferation and adhesion to bone matrix of bone-seeking breast cancer cells and change their surface structure, thus inhibits their bone metastatic ability.
Animals ; Bone Matrix ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; Cell Adhesion ; Female ; Gene Silencing ; Humans ; Integrin-Binding Sialoprotein ; genetics ; pharmacology ; Mice ; Mice, Nude ; Neoplasm Metastasis ; genetics ; prevention & control ; RNA, Small Interfering ; genetics ; Tumor Cells, Cultured

OBJECTIVETo investigate the effect of stem cell factor on the proliferation and osteogenic differentiation of human deciduous dental pulp stem cells.

METHODHuman dental pulp tissues were harvested from extracted deciduous teeth and digested by collagenase and dispase. The stem cells from human exfoliated deciduous teeth (SHED) obtained were cultured in the presence of 3 or 10 µmol/L stem cell factor, and the proliferation of the cells was assessed by MTT assay. The influence of stem cell factor on alkaline phosphatase (ALP) activity was evaluated using ALP kit. Bone sialoprotein and osteocalcin mRNA expression in the treated cells were examined by real-time PCR.

RESULTMTT assay indicated that both 3 and 10 µmol/L stem cell factor promoted the proliferation of SHED. Stem cell factor enhanced ALP activity in the SHED, and the effect was more obvious at 10 µmol/L. Treatment of the cells with stem cell factor up-regulated the mRNA expressions of bone sialoprotein and osteocalcin.

CONCLUSIONStem cell factor can promote the proliferation and osteogenic differentiation SHED, suggesting the effect of stem cell factor in promoting tooth regeneration.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dental Pulp ; cytology ; Humans ; Integrin-Binding Sialoprotein ; metabolism ; Osteocalcin ; metabolism ; Osteogenesis ; Stem Cell Factor ; pharmacology ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology

OBJECTIVETo investigate the role of transcription factor special AT-rich binding protein 2 (SATB2) in the osteoblasts differentiation of bone marrow stromal cells (BMSC) in vitro.

METHODSRats bone marrow stromal cells were isolated by Percoll sedimentation and the cells were placed and allowed to attach for three times. After passages, expression plasmid pBABE-hygro-satb2 was constructed, then transfected into BMSC. BMSCs were inoculated in conditioned medium and osteogenic factors were detected by western blotting and reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe morphological observation of BMSC showed either spindle or polygonal pattern. The cellular phenotypic marker of the third passage was CD29 positive and CD34 negative. The growth curve possessed "S" pattern. The intensity of calfilication in BMSC was higher in SATB2 transfection group (IA value 125974 ± 241) than that in the control groups (IA value 178486 ± 406). Moreover, cell migration rate increased in SATB2 transfection group [width of scratch (0.72 ± 0.01) mm] compared with control group [width of scratch (0.83 ± 0.03) mm]. In addition, the mRNA expression of osteogenic factors runt-related transcription factor 2, Osterix, activating transcription factor 4, integrin-binding sialoprotein were upregulated.

CONCLUSIONSCells cultured with this method have general biological characteristics and osteogenic differentiation potential in vitro. SATB2 can promote osteoblasts differentiation of BMSC.

Activating Transcription Factor 4 ; metabolism ; Animals ; Bone Marrow Cells ; metabolism ; pathology ; Cell Differentiation ; Cell Movement ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Integrin-Binding Sialoprotein ; metabolism ; Male ; Matrix Attachment Region Binding Proteins ; genetics ; metabolism ; Osteoblasts ; cytology ; Osteogenesis ; Plasmids ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stromal Cells ; metabolism ; pathology ; Thy-1 Antigens ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transfection

Amelogenin (AMG) is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG (rhAMG) on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1, 1, 10, 100, 1000, 10,000, 100,000 ng · mL) of recombinant human AMG (rhAMG) and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type I (COL I), osteopontin (OPN), runt-related transcription factor 2 (Runx2), cementum attachment protein (CAP), and alkaline phosphatase (ALP) genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer. Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomal-associated membrane protein-1 (LAMP-1), which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100,000 ng · mL increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng · mL) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng · mL remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng · mL rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.
Alkaline Phosphatase ; metabolism ; Amelogenin ; physiology ; Animals ; Biomarkers ; metabolism ; Calcification, Physiologic ; Cell Adhesion Molecules ; metabolism ; Cell Proliferation ; Cementogenesis ; physiology ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Gene Expression Regulation ; In Vitro Techniques ; Integrin-Binding Sialoprotein ; metabolism ; Mice ; Microscopy, Confocal ; Osteocalcin ; metabolism ; Osteopontin ; metabolism ; Real-Time Polymerase Chain Reaction

OBJECTIVETo analyze the effects of calcium hydroxide [Ca(OH)2] on transcription of the bone sialoprotein (BSP) gene in human dental pulp cells.

METHODSHuman dental pulp tissues were collected from extracted teeth for orthodontic reason. In cell culture media, different dose (0.012, 0.120, 0.400 and 1.200 mmol/L) of Ca(OH)2 was added. Total RNA of cells were extracted. The best dose of Ca(OH)2 on human BSP was determined with the real-time polymerase chain reaction (PCR). Further, the time (0, 3, 6, 12, 24 h) effects of the best dose Ca(OH)2 on human BSP, runt-related transcription factor-2 (Runx-2) and osterix (OSX) mRNA levels were determined with PCR. Further method included transient transfection assays, linking chimeric constructs of the human BSP gene promoter to a luciferase reporter gene, then ransfected using lipofectamine in cells and measured the luciferase activities of BSP gene promoter.

RESULTSWith the real-time PCR, the optimal Ca(OH)2 concentration was determined as 1.200 mmol/L. With this concentration at different time points (0, 3, 6, 12 and 24 h), the levels of BSP mRNA increased at 6 h (1.45 ± 0.36), reached maximal at 12 h (2.66 ± 0.18); the levels of Runx-2mRNA increased at 6 h (2.38 ± 0.08), at 12 h (2.73 ± 0.16), and decreased at 24 h. OSX mRNA could be recognized at 12 h, reached maximal levels at 24 h (3.30 ± 0.062). Transient transfection assays showed that treatment of human dental pulp cells with Ca(OH)2 (1.200 mmol/L) increased the luciferase activities of the constructs between -84LUC and -868LUC at 12 h (2.00 ~ 2.60 fold).

CONCLUSIONSThis study demonstrate that Ca(OH)2 could stimulate BSP transcription between -84LUC and -868LUC in the human BSP gene promoter in human dental pulp cells.

Calcium Hydroxide ; pharmacology ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Dental Pulp ; cytology ; metabolism ; Humans ; Integrin-Binding Sialoprotein ; genetics ; metabolism ; Luciferases ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Sp7 Transcription Factor ; Transcription Factors ; genetics ; metabolism ; Transcription, Genetic ; drug effects ; Transfection

OBJECTIVETo find an ideal method inducing dental pulp stem cells (DPSC) osteogenic differentiation. To compare the effect of co-culture method and that of mineralizing culture medium.

METHODSDPSC were co-cultured with osteoblasts using cell culture inserts system as experiment group, and DPSC were cultured in mineralizing culture medium as control group. The cell morphology and ultrastructure and mineralized nodes were analyzed under phase contrast microscope, transmission electron microscope, and alizarin red S staning. Bone sialoprotein (BSP), Runx-2, osteocalcin, and collagen-1 (Col-1) osteoblastic genes expressions of DPSC cultivated in special niche of osteoblasts were assayed by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe mineralization nudoles of experiment group were more than control group. Fifteen days later, BSP and Col-1 genes in the DPSC of co-cultures were 9.807 ± 1.135 and 2.913 ± 0.310, respectively. And those in the DPSC of mineralizing culture medium were 6.478 ± 0.781 and 1.703 ± 0.184, respectively. Co-cultures and mineralizing were significantly different (P < 0.05).

CONCLUSIONSAs osteoblasts can secret lots of osteogenic cell cytokines, they have more significant effect than mineralizing culture medium on osteogenesis of DPSC.

Cell Differentiation ; Coculture Techniques ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Dental Pulp ; cytology ; Gene Expression Regulation, Developmental ; Humans ; Integrin-Binding Sialoprotein ; metabolism ; Microscopy, Electron, Transmission ; Microscopy, Phase-Contrast ; Osteoblasts ; cytology ; Osteocalcin ; metabolism ; Osteogenesis ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; ultrastructure

BACKGROUNDTissue engineering techniques combined with gene therapy have been recently used to improve osteogenesis. NEL-like molecule-1 (Nell-1), a novel growth factor, has been reported to have specificity for osteochondral lineage. The study assessed the osteogenic differentiation of rat bone marrow stromal cells (bMSCs) after Nell-1 gene modification and examined its ectopic bone formation ability in a nude mice model with tissue engineering technique.

METHODSbMSCs obtained from Fischer 344 rats were transduced with either AdNell-1 (Nell-1 group) or Ad-beta-galactosidase (AdLacZ, LacZ group) or left untransduced (untransduced group). The expression of Nell-1 protein was determined by Western blotting and transfer efficiency was assessed. mRNA expressions of osteopontin (OP), bone sialoprotein (BSP) and osteocalcin (OC) were assessed by real-time PCR 0, 3, 7, 14, and 21 days after gene transfer. Alkaline phosphatase (ALP) activity was measured and von Kossa test was also conducted. Finally, with a tissue engineering technique, gene transduced bMSCs, combining with beta-tricalcium phosphate (beta-TCP) at a concentration of 2 x 10(7) cells/ml, were implanted at subcutaneous sites on the back of nude mice. Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation.

RESULTSUnder current transduction conditions, gene transfer efficiency reached (57.9 +/- 6.8)%. Nell-1 protein was detected in Nell-1 group but not in untransduced group and LacZ group. Induced by Nell-1, BSP and OP expression were increased at intermediate stage and OC expression was increased at later stage. ALP activity and the number of calcium nodules were highest in Nell-1 group. Four weeks after implanted into nude mice subcutaneously, the percentage of new bone area in Nell-1 group was (18.1 +/- 5.0)%, significantly higher than those of untransduced group (11.3 +/- 3.2)% and LacZ group (12.3 +/- 3.1)% (P < 0.05).

CONCLUSIONSThis study has demonstrated the ability of Nell-1 to induce osteogenic differentiation of rat bMSCs in vitro and to enhance bone formation with a tissue engineering technique. The results suggest that Nell-1 may be a potential osteogenic gene to be used in bone tissue engineering.

Alkaline Phosphatase ; metabolism ; Animals ; Blotting, Western ; Bone Marrow Cells ; cytology ; metabolism ; ultrastructure ; Integrin-Binding Sialoprotein ; Male ; Mice ; Mice, Nude ; Microscopy, Electron, Scanning ; Nerve Tissue Proteins ; metabolism ; Osteocalcin ; genetics ; Osteogenesis ; Osteopontin ; genetics ; Rats ; Rats, Inbred F344 ; Reverse Transcriptase Polymerase Chain Reaction ; Sialoglycoproteins ; genetics ; Stromal Cells ; cytology ; metabolism ; ultrastructure ; Tissue Engineering