Rational: Senescence of mesenchymal stem cells (MSCs) and the related functional decline of osteogenesis have emerged as the critical pathogenesis of osteoporosis in aging.Resveratrol (RESV), a small molecular compound that safely mimics the effects of dietary restriction, has been well documented to extend lifespan in lower organisms and improve health in aging rodents.However, whether RESV promotes function of senescent stem cells in alleviating age-related phenotypes remains largely unknown.Here, we intend to investigate whether RESV counteracts senescence-associated bone loss via osteogenic improvement of MSCs and the underlying mechanism.Methods: MSCs derived from bone marrow (BMMSCs) and the bone-specific, senescence-accelerated, osteoblastogenesis/osteogenesis-defective mice (the SAMP6 strain) were used as experimental models.In vivo application of RESV was performed at 100 mg/kg intraperitoneally once every other day for 2 months, and in vitro application of RESV was performed at 10 μM.Bone mass, bone formation rates and osteogenic differentiation of BMMSCs were primarily evaluated.Metabolic statuses of BMMSCs and the mitochondrial activity, transcription and morphology were also examined.Mitofilin expression was assessed at both mRNA and protein levels, and short hairpin RNA (shRNA)-based gene knockdown was applied for mechanistic experiments.Results: Chronic intermittent application of RESV enhances bone formation and counteracts accelerated bone loss, with RESV improving osteogenic differentiation of senescent BMMSCs.Furthermore, in rescuing osteogenic decline under BMMSC senescence, RESV restores cellular metabolism through mitochondrial functional recovery via facilitating mitochondrial autonomous gene transcription.Molecularly, in alleviating senescence-associated mitochondrial disorders of BMMSCs, particularly the mitochondrial morphological alterations, RESV upregulates Mitofilin, also known as inner membrane protein of mitochondria (Immt) or Mic60, which is the core component of the mitochondrial contact site and cristae organizing system (MICOS).Moreover, Mitofilin is revealed to be indispensable for mitochondrial homeostasis and osteogenesis of BMMSCs, and that insufficiency of Mitofilin leads to BMMSC senescence and bone loss.More importantly, Mitofilin mediates resveratrol-induced mitochondrial and osteogenic improvements of BMMSCs in senescence.Conclusion: Our findings uncover osteogenic functional improvements of senescent MSCs as critical impacts in anti-osteoporotic practice of RESV, and unravel Mitofilin as a novel mechanism mediating RESV promotion on mitochondrial function in stem cell senescence.
Studies have shown that miR-194 functions as a tumor suppressor and is associated with tumor growth and metastasis. We studied the effects of miR-194 in osteosarcoma and the possible mechanism by which miR-194 affected the survival, apoptosis and metastasis of osteosarcoma. Both human osteosarcoma cell lines SOSP-9607 and U2-OS were transfected with recombinant lentiviruses to regulate miR-194 expression. Overexpression of miR-194 partially inhibited the proliferation, migration, and invasion of osteosarcoma cells in vitro, as well as tumor growth and pulmonary metastasis of osteosarcoma cells in vivo. Potential miR-194 target genes were predicted using bioinformatics. Luciferase reporter assay, real-time quantitative PCR and western blotting confirmed that CDH2 (N-cadherin) and IGF1R were targets of miR-194. Using real-time quantitative PCR, we evaluated the expression of miR-194 and two miR-194 target genes, CDH2 and IGF1R in osteosarcoma samples from 107 patients and 99 formalin- or paraformalin-fixed paraffin-embedded tissues. The expressions of the target genes were also examined in osteosarcoma samples using immunohistochemistry. Overexpression of miR-194 inhibited tumor growth and metastasis of osteosarcoma probably by downregulating CDH2 and IGF1R. miR-194 may prove to be a promising therapeutic agent for osteosarcoma.
Background MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional and/or translational level and are deeply involved in the pathogenesis of several types of cancers. Specifically, microRNA-221 (miR-221) is overexpressed in many human cancers, wherein accumulating evidence indicates that it functions as an oncogene. However, the function of miR-221 in human osteosarcoma has not been totally elucidated. In the present study, the effects of miR-221 on osteosarcoma and the possible mechanism by which miR-221 affected the survival, apoptosis, and cisplatin resistance of osteosarcoma were investigated. Methodology/Principal Findings Real-time quantitative PCR analysis revealed miR-221 was significantly upregulated in osteosarcoma cell lines than in osteoblasts. Both human osteosarcoma cell lines SOSP-9607 and MG63 were transfected with miR-221 mimic or inhibitor to regulate miR-221 expression. The effects of miR-221 were then assessed by cell viability, cell cycle analysis, apoptosis assay, and cisplatin resistance assay. In both cells, upregulation of miR-221 induced cell survival and cisplatin resistance and reduced cell apoptosis. In addition, knockdown of miR-221 inhibited cell growth and cisplatin resistance and induced cell apoptosis. Potential target genes of miR-221 were predicted using bioinformatics. Moreover, luciferase reporter assay and western blot confirmed that PTEN was a direct target of miR-221. Furthermore, introduction of PTEN cDNA lacking 3′-UTR or PI3K inhibitor LY294002 abrogated miR-221-induced cisplatin resistance. Finally, both miR-221 and PTEN expression levels in osteosarcoma samples were examined by using real-time quantitative PCR and immunohistochemistry. High miR-221 expression level and inverse correlation between miR-221 and PTEN levels were revealed in osteosarcoma tissues. Conclusions/Significance These results for the first time demonstrate that upregulation of miR-221 induces the malignant phenotype of human osteosarcoma whereas knockdown of miR-221 reverses this phenotype, suggesting that miR-221 could be a potential target for osteosarcoma treatment.
OBJECTIVE: Transfection of dendritic cells with tumor-derived RNA has recently been shown to elicit tumor-specific CTL capable of recognizing and lysing a variety of tumor cells. However, the integration of allogeneic osteosarcoma mRNA and autologous DCs has not been fully examined. This study was designed to investigate the antitumor effects of tumor vaccine produced by autologous dendritic cells transfected with allogeneic osteosarcoma total RNA through electroporation in tumor-bearing rats model. METHODS: In the present study, we transfected Wistar rat osteosarcoma cells derived total RNA to SD rat bone marrow-derived DCs through electroporation. The tumor vaccine was applied to in tumor-bearing rats model and the specific antitumor effects of the tumor vaccine were observed. Then CTL activity was evaluated one week after the first immunization of SD rats with electroporated DCs and the specificity of the cytotoxic activity was confirmed in cold target inhibition assays and using mAbs blocking MHC class I or CD8 molecules. RESULTS: The immunization using autologous DCs electrotransfected with allogeneic osteosarcoma total RNA induced UMR106-specific CTL responses which were statistically significant and the cytotoxic activity was inhibited by the treatment with anti-CD8 and anti-MHC-class I monoclonal antibodies. In in vivo experiments, 80% of the rats immunized with allogeneic osteosarcoma RNA transfected to DCs were typically able to reject tumor challenge and remained tumor-free. Vaccinated survivors developed long immunological memory and were able to reject a subsequent rechallenge with the same tumor cells but not a syngeneic unrelated tumor line. CONCLUSION:The present study provided valid evidence of integration of rat allogeneic tumor-derived mRNA and autologous DCs through electroporation and confirmed this novel tumor vaccine have the potential to induced osteosarcoma-specific CTL response and reject osteosarcoma challege. This technique and its products may thus represent a promising strategy for DC-based immunotherapy of patients with osteosarcoma.
Increasing amounts of long noncoding RNAs (lncRNAs) have been shown to be involved in the development of cancer. Recently, aberrant expression of the lncRNA forkhead box D2 adjacent opposite strand RNA1 (FOXD2‑AS1) has been reported to be involved in the progression of several types of human cancer. However, the function and mechanism of FOXD2‑AS1 in osteosarcoma (OS) are currently unclear. The present study aimed to investigate the function and mechanism of FOXD2‑AS1 in OS. Firstly, it was revealed that the expression levels of FOXD2‑AS1 were significantly upregulated in OS tissues and cells, compared with in adjacent tissues and normal cells, as determined using reverse transcription‑quantitative polymerase chain reaction. Notably, the overall survival of patients with relatively high FOXD2‑AS1 expression in OS tissues was significantly lower than that of patients with relatively low expression, as determined using Kaplan‑Meier analysis. In addition, loss‑of‑function experiments were performed in vivo and in vitro to study the biological effects of FOXD2‑AS1. The SOSP‑9607 and U2OS OS cell lines were infected with lentivirus‑mediated FOXD2‑AS1 short hairpin RNA; subsequently, the alterations in cell phenotype and downstream molecules were evaluated. Knockdown of FOXD2‑AS1 inhibited the proliferation, migration and invasion of OS cells. Furthermore, the number of cells in the S phase was significantly decreased, which was consistent with the results of the Cell Counting kit 8 proliferation assay. The expression levels of ribonucleotide reductase regulatory subunit M2 and phosphoglycerate dehydrogenase were decreased, as determined by western blotting, following FOXD2‑AS1 knockdown. Finally, in a nude mouse model of tumorigenesis, it was revealed that, when FOXD2‑AS1 expression was downregulated, tumor growth was significantly reduced and pulmonary metastatic nodules were markedly reduced. The results of the present study suggested that decreased FOXD2‑AS1 expression may inhibit the growth, migration and invasion of tumor cells, and it may regulate downstream gene expression. In conclusion, these findings indicated that FOXD2‑AS1 may be used as a potential therapeutic target and early tumor marker for the diagnosis and prognosis of OS.
Objective:AIM To obtein a recombinant replication-deficient adenovirus expressing LacZ protein. Methods:The recombinant adenovirus vector engineered to express LacZ gene(Ad-LacZ) was constructed by recombination in E.coli-BJ5183.The replication-deficient adenovirus was then packed in HEK293 cells after transfection with the construct and subsequently identified by X-gal staining,electron microscopic observation and dot blotting.The expression of LacZ in HeLa cells infected by Ad-LacZ could be detected by X-gal stain. Results:Recombinant adenovirus expression vector of LacZ was successfully reconstructed.And the titer of Ad-LacZ virus reached 1.58×10~(9) pfu/ml.Under transmission(electron) microscope,adenoviral inclusions could be found in the nucleus of the infected HEK293 cells. Ad-LacZ virus was comfirmed to be replication-deficient by dot blotting.And the expression of LacZ was observed in the infected HeLa cells.The gene transfer capability of the recombinant virus was correlated with multiplicity of infection and the infection time. Conclusion:Recombinant adenovirus expression vector of LacZ was successfully reconstructed.
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