Indole-Terpenoids With Anti-inflammatory Activities From Penicillium sp. HFF16 Associated With the Rhizosphere Soil of Cynanchum bungei Decne.

Four new indole-terpenoids (1-4) named encindolene A, 18-O-methyl-encindolene A, encindolene B, and encindolene C, as well as three known analogs (5-7), were isolated from the fungus Penicillium sp. HFF16 from the rhizosphere soil of Cynanchum bungei Decne. The structures of compounds including absolute configurations were elucidated by spectroscopic data and electronic circular dichroism (ECD) analysis. Anti-inflammatory activity evaluation revealed that compounds 1-7 inhibit the production of nitric oxide with IC50 values of 79.4, 49.7, 81.3, 40.2, 86.7, 90.1, and 54.4 µM, respectively, and decrease the levels of tumor necrosis factor-α, interleukin-6 contents in lipopolysaccharide-induced RAW264.7 macrophages.

Extracellular vesicle-mediated delivery of miR-101 inhibits lung metastasis in osteosarcoma.

Rationale: Extracellular vesicles (EVs) have emerged as novel mediators of cell-to-cell communication that are capable of the stable transfer of therapeutic microRNAs (miRNAs), and thus, EVs hold immense promise as a miRNA delivery system for cancer therapy. Additionally, as miRNA-containing EVs are secreted into circulation, miRNAs contained within plasma EVs may represent ideal biomarkers for diseases. The objective of this study was to characterize a potential tumor suppressor miRNA, miR-101, and explore the potential of miR-101 delivery via EVs for in vivo therapy of metastatic osteosarcoma as well as the potential value of plasma EV-packaged miR-101 (EV-miR-101) level for predicting osteosarcoma metastasis. Methods: The relationship of miR-101 expression and osteosarcoma progression was investigated in osteosarcoma specimens by in situ hybridization (ISH), and the potential inhibitory effect of miR-101 was further investigated using in vivo models. Using prediction software analysis, the mechanism of action of miR-101 in osteosarcoma was explored using quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting and dual-luciferase assay. Adipose tissue-derived mesenchymal stromal cells (AD-MSCs) were transduced with lentiviral particles to obtain miR-101-enriched EVs. A Transwell assay and lung metastasis models of osteosarcoma were used to observe the effect of miR-101-enriched EVs on osteosarcoma invasiveness and metastasis. Detection of plasma EV-miR-101 levels was carried out in osteosarcoma patients and healthy controls by qRT-PCR. Results: miR-101 expression was markedly lower in metastatic osteosarcoma specimens compared to non-metastatic specimens. Significantly fewer metastatic lung nodules were formed by Saos-2 cells overexpressing miR-101 and SOSP-9607 cells overexpressing miR-101 injected into mice. With increased miR-101 expression, B cell lymphoma 6 (BCL6) mRNA and protein expression levels were reduced, and miR-101 was found to exert its effects by directly targeting BCL6. AD-MSCs were successfully engineered to secrete miR-101-enriched EVs. Once taken up by osteosarcoma cells, these EVs showed suppressive effects on cell invasion and migration in vitro, and systemic administration of these EVs effectively suppressed metastasis in vivo with no significant side effects. Finally, the EV-miR-101 level was lower in osteosarcoma patients than in healthy controls and even lower in osteosarcoma patients with metastasis than in those without metastasis. Conclusion: Our data support the function of miR-101 as a tumor suppressor in osteosarcoma via downregulation of BCL6. AD-MSC derived miR-101-enriched EVs represent a potential innovative therapy for metastatic osteosarcoma. EV-miR-101 also represents a promising circulating biomarker of osteosarcoma metastasis.

Downregulation of the long non­coding RNA FOXD2­AS1 inhibits cell proliferation, migration and invasion in 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 (FOXD2­AS1) has been reported to be involved in the progression of several types of human cancer. However, the function and mechanism of FOXD2­AS1 in osteosarcoma (OS) are currently unclear. The present study aimed to investigate the function and mechanism of FOXD2­AS1 in OS. Firstly, it was revealed that the expression levels of FOXD2­AS1 were significantly upregulated in OS tissues and cells, compared with in adjacent tissues and normal cells, as determined using reverse transcription­quantitative polymerase chain reaction. Notably, the overall survival of patients with relatively high FOXD2­AS1 expression in OS tissues was significantly lower than that of patients with relatively low expression, as determined using Kaplan­Meier analysis. In addition, loss­of­function experiments were performed in vivo and in vitro to study the biological effects of FOXD2­AS1. The SOSP­9607 and U2OS OS cell lines were infected with lentivirus­mediated FOXD2­AS1 short hairpin RNA; subsequently, the alterations in cell phenotype and downstream molecules were evaluated. Knockdown of FOXD2­AS1 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 FOXD2­AS1 knockdown. Finally, in a nude mouse model of tumorigenesis, it was revealed that, when FOXD2­AS1 expression was downregulated, tumor growth was significantly reduced and pulmonary metastatic nodules were markedly reduced. The results of the present study suggested that decreased FOXD2­AS1 expression may inhibit the growth, migration and invasion of tumor cells, and it may regulate downstream gene expression. In conclusion, these findings indicated that FOXD2­AS1 may be used as a potential therapeutic target and early tumor marker for the diagnosis and prognosis of OS.

Microwave Ablation of Primary Malignant Pelvic Bone Tumors.

Background: En bloc tumor resection followed by reconstruction is a widely used surgical treatment for malignant pelvic bone tumors. High rates of complications and mechanical instability often contribute to poor postoperative results. We attempted en bloc microwave ablation (MWA) in situ to improve the outcome. Methods: From May 1995 to December 2015, 104 patients with primary pelvic malignancy received radical MWA in our department. After careful dissection of the tumor-bearing bone from surrounding normal tissues with safe margins, a microwave antenna array was inserted into the tumor mass to emit electromagnetic energy, inducing tumor cellular death via thermocoagulation. The loose, devitalized tumor tissues were removed by cutting or curettage, leaving a defective bone scaffold. Re-strengthening by autograft or allograft was needed in most patients. Results: The over 3 years survival rate was 51.5% for high-grade malignancies (among them, 26.9% were osteosarcoma) and 94.8% for low-grade malignancies (chondrosarcoma). In most of the living patients, cosmetic and useful limbs were preserved. The mean functional score (Musculoskeletal Tumor Society) was 27 or 90% (range: 25-30, 75-100%). Among the 56 patients who belonged to the excellent function group, 11 were followed up for more than 10 years. The local recurrence rate was 8.6%. Among the 9 patients with recurrence, 5 died from disease, 2 were treated by hemipelvic amputation, and 2 underwent revision surgery with MWA and gained local control. The deep infection rate was 5.6%. All six patients with infection were healed by irrigation, debridement, and systemic antibiotic administration. Conclusion: Local, microwave-induced hyperthermia for treating malignant pelvic bone tumors is an effective alternative method. The oncological and functional results are encouraging. The use of MWA should be continued to evaluate and improve this new therapeutic system.

Resveratrol counteracts bone loss via mitofilin-mediated osteogenic improvement of mesenchymal stem cells in senescence-accelerated mice.

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.

Is Limb Salvage With Microwave-induced Hyperthermia Better Than Amputation for Osteosarcoma of the Distal Tibia?

BACKGROUND: Amputation has been the standard surgical treatment for distal tibia osteosarcoma owing to its unique anatomic features. Preliminary research suggested that microwave-induced hyperthermia may have a role in treating osteosarcoma in some locations of the body (such as the pelvis), but to our knowledge, no comparative study has evaluated its efficacy in a difficult-to-treat location like the distal tibia. QUESTIONS: Does microwave-induced hyperthermia result in (1) improved survival, (2) decreased local recurrence, (3) improved Musculoskeletal Tumor Society (MSTS) scores, or (4) fewer complications than amputation in patients with a distal tibial osteosarcoma? METHODS: Between 2000 and 2015, we treated 79 patients for a distal tibia osteosarcoma without metastases. Of those, 52 were treated with microwave-induced hyperthermia, and 27 with amputation. Patients were considered eligible for microwave-induced hyperthermia if they had an at least 20-mm available distance from the tumor edge to the articular surface, good clinical and imaging response to neoadjuvant chemotherapy, and no pathologic fracture. Patients not meeting these indications were treated with amputation. In addition, if neither the posterior tibial artery nor the dorsalis pedis artery was salvageable, the patients were treated with amputation and were not included in any group in this study. A total of 13 other patients were treated with conventional limb-salvage resections and reconstructions (at the request of the patient, based on patient preference) and were not included in this study. All 79 patients in this retrospective study were available for followup at a minimum of 12 months (mean followup in the hyperthermia group, 79 months, range 12-158 months; mean followup in the amputation group, 95 months, range, 15-142 months). With the numbers available, the groups were no different in terms of sex, age, tumor grade, tumor stage, or tumor size. All statistical tests were two-sided, and a probability less than 0.05 was considered statistically significant. Survival to death was evaluated using Kaplan-Meier analysis. Complications were recorded from the patients' files and graded using the classification of surgical complications described by Dindo et al. RESULTS: In the limb-salvage group, Kaplan Meier survival at 6 years was 80% (95% CI, 63%-90%), and this was not different with the numbers available from survivorship in the amputation group at 6 years (70%; 95% CI, 37%-90%; p = 0.301).With the numbers available, we found no difference in local recurrence (six versus 0; p = 0.066). However mean ± SD MSTS functional scores were higher in patients who had microwave-induced hyperthermia compared with those who had amputations (85% ± 6% versus 66% ± 5%; p = 0.008).With the numbers available, we found no difference in the proportion of patients experiencing complications between the two groups (six of 52 [12%] versus three of 27 [11%]; p = 0.954). CONCLUSIONS: We were encouraged to find no early differences in survival, local recurrence, or serious complications between microwave-induced hyperthermia and amputation, and a functional advantage in favor of microwave-induced hyperthermia. However, these findings should be replicated in larger studies with longer mean duration of followup, and in studies that compare microwave-induced hyperthermia with conventional limb-sparing approaches. LEVEL OF EVIDENCE: Level III, therapeutic study.

Microwave ablation of malignant extremity bone tumors.

BACKGROUND: The current application of limb salvage process has some unsolved problems, such as prosthesis loosening, which severely limits the function of the preserved limbs. Innovative approaches are needed to further improve functional outcome. PATIENTS AND METHODS: Instead of en-bloc resection of tumor-bearing bone, it is dissected from the surrounding normal tissues, followed by devitalizing the bone segment and the extra-cortical bulk by microwave induced hyperthermia in situ through the antenna array. From May 1999 to March 2012, 544 patients with malignant bone tumors of the extremities were treated by the novel method. RESULTS: The over 3-year survival rate was 59.1 % for high-grade malignancy, 88.7 % for low-grade malignancy. In the majority of the patients, cosmetic and useful limbs were preserved. Local recurrence rate was 9.8 % for the high grade malignancy (mainly occurred at the early stage of the research). The overall fracture rate was 2.6 %. Deep infection rate was 1.8 %. The complication rate is lower than the literature reports. After heat necrosis, the dead bone maintains both the osteoconduction and osteoinduction properties. CONCLUSIONS: The application of microwave induced hyperthermia for treatment of malignant bone tumors, except the late diagnosed cases who's tumor-bearing bone was destroyed too severe to do biological reconstruction, is an effective, simple, and inexpensive method. The oncological and functional results are encouraging.

MicroRNA profiling identifies MiR-195 suppresses osteosarcoma cell metastasis by targeting CCND1.

Metastasis is a leading cause of mortality for osteosarcoma patients. The molecular pathological mechanism remains to be elucidated. In the previously study, we established two osteosarcoma cell lines with different metastatic potentials. Differential expressed genes and proteins regarding metastatic ability have been identified. MicroRNAs are important regulators in tumorigenesis and tumor progression. In this study, microRNA microarray was used to assess the differential expressed miRNAs level between these two cell lines. One of the top ranked miRNAs-miR-195 was identified highly expressing in lowly metastatic cells. It was showed that over-expression of miR-195 substantially inhibits migration and invasion of osteosarcoma cells in vitro and pulmonary metastasis formation in vivo. Meanwhile, CCND1 was identified as the target gene of miR-195 and further studied. More importantly, using real-time PCR, we evaluated the expression of miR-195 and CCND1 in osteosarcoma samples from 107 frozen biopsy tissues and 99 formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Results indicated lowly expressed miR-195 or highly CCND1 correlated with positive overall survival and their expression inversely related to each other. In summary, our study suggests miR-195 functions as a tumor metastasis suppressor gene by down-regulating CCND1 and can be used as a potential target in the treatment of osteosarcoma.

microRNA-194 suppresses osteosarcoma cell proliferation and metastasis in vitro and in vivo by targeting CDH2 and IGF1R.

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.

Homologous mesenchymal stem cells promote the emergence and growth of pulmonary metastases of the rat osteosarcoma cell line UMR-106.

Osteosarcoma (OS) is the most frequent primary bone sarcoma and tends to develop pulmonary metastasis. Studies have shown that mesenchymal stem cells (MSCs) are involved in OS growth and metastasis, but the mechanism remains unclear. The aim of the present study was to identify whether homologous MSCs could promote the growth and metastasis of OS in rats with a normal immune system. The OS cell line, UMR-106, which originally derives from a Sprague-Dawley (SD) rat-transplantable osteogenic sarcoma with an osteoblastic phenotype, has a strong carcinogenic capability and a high lung metastasis. Xenotransplanted models of UMR-106 with or without MSCs injected through the tibia (IT) or caudal vein (IV) were established. SD rats were randomly divided into six groups: Control, UMR-106 (IT), MSCs (IV), UMR-106 (IT) + MSCs (IV), UMR-106 (IV) and UMR-106 (IV) + MSCs (IV). Following injection, all rats were sacrificed at week 5, and the volume and quantity of metastatic sarcoma and the serum alkaline phosphatase levels were measured. There was no metastatic sarcoma in the liver, spleen and kidney in all groups. The rats in the MSCs (IV) + UMR-106 (IV) group showed a significantly higher volume and number of pulmonary metastatic tumors than those of the UMR-106 (IV) group. In pulmonary metastatic tissues, MSCs were found in the MSCs (IV) + UMR-106 (IV) group, but not in the UMR-106 (IT) + MSCs (IV) group. Notably, the expression of vascular endothelial growth factor (VEGF) was increased in the MSCs + UMR-106 cells co-culture system. The present study indicated that MSCs can significantly promote the pulmonary metastasis of the rat OS cell line, UMR-106, with a normal immune system, and VEGF was involved in MSC-promoted UMR-106 emergence and growth of pulmonary metastasis.

Proteomic profiling of osteosarcoma cells identifies ALDOA and SULT1A3 as negative survival markers of human osteosarcoma.

Osteosarcoma (OSA) is the most common primary malignancy of bone. Molecular mechanism underlying OSA remains to be fully elucidated. It is critical to identify reliable diagnostic and prognostic markers for OSA at the molecular levels. This study is designed to investigate possible molecular mechanisms behind OSA development and to identify novel prognostic markers related to OSA survival. We conduct a comprehensive proteomic profiling analysis of human OSA cell lines with differential metastatic potential. Through comprehensive combinatorial analyses of the proteomic data and the previously obtained cDNA microarray results, we identify 37 candidate proteins which are differentially expressed in OSA sublines. Among them, ALDOA and SULT1A3 are selected for further investigation. The expressions of protein are confirmed by Western blotting analysis. We further analyze the expression levels of ALDOA and SULT1A3 from 40 clinical cases of OSA. The results demonstrate that the expression of ALDOA and/or SULT1A3 is significantly higher in patients with worse survival time than patients with better survival time. Five-year survival analysis shows there is a statistically significant difference between two patient populations. The data strongly suggest that ALDOA and/or SULT1A3 expression level in biopsy samples may predict the clinical outcomes of OSA patients. Furthermore, the biological functions of ALDOA and SULT1A3 may be implicated in OSA development and/or progression.

CXCR4-mediated osteosarcoma growth and pulmonary metastasis is promoted by mesenchymal stem cells through VEGF.

Chemokines and chemokine receptor 4 (CXCR4) play an important role in metastasis. CXCR4 is also expressed in the human osteosarcoma cell line 9607-F5M2 (F5M2), which has a high tumorigenic ability and potential for spontaneous pulmonary metastasis. Mesenchymal stem cells (MSCs) contribute to the formation of the tumor stroma and promote metastasis. However, mechanisms underlying the promotion of osteosarcoma growth and pulmonary metastasis by MSCs are still elusive. Our study co-injected the human MSCs and F5M2 cells into the caudal vein of nude mice. The total number of tumor nodules per lung was significantly increased in the F5M2+MSC group compared to the other groups (control, F5M2 cells alone and MSCs alone) at week six. Moreover, a high number of Dil-labeled MSCs was present also at the osteosarcoma metastasis sites in the lung. Using Transwell assays, we found that F5M2 cells migrate towards MSCs, while the CXCR4 inhibitor AMD3100 decreased the migration potential of F5M2 cells towards MSCs. Furthermore, upon treatment with F5M2-conditioned medium, MSCs expressed and secreted higher levels of VEGF as determined by immunohistochemistry, western blotting and ELISA, respectively. Importantly, co-cultured with F5M2 cells, MSCs expressed and secreted higher VEGF levels, while AMD3100 dramatically decreased the VEGF secretion by MSCs. However, CXCR4 expression on F5M2 cells was not significantly increased in the co-culture system. Additionally, VEGF increased the proliferation of both MSCs and F5M2 cells. These findings suggest that CXCR4-mediated osteosarcoma growth and pulmonary metastasis are promoted by MSCs through VEGF.

MicroRNA-221 induces cell survival and cisplatin resistance through PI3K/Akt pathway in human 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.

miR-15a and miR-16-1 downregulate CCND1 and induce apoptosis and cell cycle arrest in osteosarcoma.

Osteosarcoma, the most common primary tumor of the bones, causes many deaths due to its rapid proliferation and drug resistance. Recent studies have shown that cyclin D1 plays a key regulatory role during cell proliferation, and non-coding microRNAs (miRNAs) act as crucial modulators of cyclin D1 (CCND1). The aim of the current study was to determine the role of miRNAs in controlling CCND1 expression and inducing cell apoptosis. CCND1 has been found to be a target of miR-15a and miR-16-1 through analysis of complementary sequences between microRNAs and CCND1 mRNA. The upregulation of miR-15a and miR-16-1 in the cell line SOSP-9607 induces apoptosis and cell cycle arrest. Osteosarcoma cells transfected with miR-15a and miR-16-1 show slower proliferation curves. Moreover, the transcription of CCND1 is suppressed by miR-15a and miR-16-1 via direct binding to the CCND1 3'-untranslated region (3'-UTR). The data presented here demonstrate that the CCND1 contributes to osteosarcoma cell proliferation, suggesting that repression of CCND1 by miR-15a and miR-16-1 could be used for osteosarcoma therapy.

MicroRNA-34a inhibits the proliferation and metastasis of osteosarcoma cells both in vitro and in vivo.

BACKGROUND: MicroRNAs (miRNAs) are a class of endogenously expressed, small noncoding RNAs, which suppress its target mRNAs at the post-transcriptional level. Studies have demonstrated that miR-34a, which is a direct target of the p53 tumor suppressor gene, functions as a tumor suppressor and is associated with the tumor growth and metastasis of various human malignances. However, the role of miR-34a in osteosarcoma has not been totally elucidated. In the present study, the effects of miR-34a on osteosarcoma and the possible mechanism by which miR-34a affected the tumor growth and metastasis of osteosarcoma were investigated. METHODOLOGY/PRINCIPAL FINDING: Over-expression of miR-34a partially inhibited proliferation, migration and invasion of osteosarcoma cells in vitro, as well as the tumor growth and pulmonary metastasis of osteosarcoma cells in vivo. c-Met is a target of miR-34a, and regulates the migration and invasion of osteosarcoma cells. Osteosarcoma cells over-expressing miR-34a exhibited a significant decrease in the expression levels of c-Met mRNA and protein simultaneously. Finally, the results from bioinformatics analysis demonstrated that there were multiple putative targets of miR-34a that may be associated with the proliferation and metastasis of osteosarcoma, including factors in Wnt and Notch signaling pathways. CONCLUSION/SIGNIFICANCE: The results presented in this study demonstrated that over-expression of miR-34a could inhibit the tumor growth and metastasis of osteosarcoma probably through down regulating c-Met. And there are other putative miR-34a target genes beside c-Met which could potentially be key players in the development of osteosarcoma. Since pulmonary metastases are responsible for mortality of patient carrying osteosarcoma, miR-34a may prove to be a promising gene therapeutic agent. It will be interesting to further investigate the mechanism by which miR-34a functions as a tumor suppressor gene in osteosarcoma.

MicroRNA expression during osteogenic differentiation of human multipotent mesenchymal stromal cells from bone marrow.

MicroRNAs comprise a group of non-coding small RNAs (17-25 nt) involved in post-transcriptional regulation that have been identified in various plants and animals. Studies have demonstrated that miRNAs are associated with stem cell self-renewal and differentiation and play a key role in controlling stem cell activities. However, the identification of specific miRNAs and their regulatory roles in the differentiation of multipotent mesenchymal stromal cells (MSCs) have so far been poorly defined. We isolated and cultured human MSCs and osteo-differentiated MSCs from four individual donors. miRNA expression in MSCs and osteo-differentiated MSCs was investigated using miRNA microarrays. miRNAs that were commonly expressed in all three MSC preparations and miRNAs that were differentially expressed between MSCs and osteo-differentiated MSCs were identified. Four underexpressed (hsa-miR-31, hsa-miR-106a, hsa-miR-148a, and hsa-miR-424) and three novel overexpressed miRNAs (hsa-miR-30c, hsa-miR-15b, and hsa-miR-130b) in osteo-differentiated MSCs were selected and their expression were verified in samples from the fourth individual donors. The putative targets of the miRNAs were predicted using bioinformatic analysis. The four miRNAs that were underexpressed in osteo-differentiated MSCs were predicted to target RUNX2, CBFB, and BMPs, which are involved in bone formation; while putative targets for miRNAs overexpressed in osteo-differentiated MSCs were MSC maker (e.g., CD44, ITGB1, and FLT1), stemness-maintaining factor (e.g., FGF2 and CXCL12), and genes related to cell differentiation (e.g., BMPER, CAMTA1, and GDF6). Finally, hsa-miR-31 was selected for target verification and function analysis. The results of this study provide an experimental basis for further research on miRNA functions during osteogenic differentiation of human MSCs.

Gene expression profiles of human osteosarcoma cell sublines with different pulmonary metastatic potentials.

AIM: to screen the pulmonary metastasis-associated molecules of Osteosarcoma and evaluate their functions concerning prognosis prediction. METHODS: cDNA microarray analysis has been applied to 2 pairs of osteosarcoma cell sublines with differential metastatic potentials to the lung. Immunohistochemistry and survival analysis have been performed to clinical samples of osteosarcoma patients. RESULT: Analysis detected 484 differentially expressed genes between the high metastatic subline, F5M2, and the low metastatic subline, F4. There were 1257 genes differentially expressed between newly established high-metastatic sublines named Saos-2M2 and its parental cell line Saos-2. Furthermore, 16 commonly up-regulated genes and 5 commonly down-regulated genes were identified by clustering analysis. EREG and CHST2, two genes not previously described in osteosarcoma, were finally seen to be differentially expressed in all examined osteosarcoma cell lines and in samples between the different prognosis sample groups. Survival analysis also confirmed these two molecules could be used to predict the outcome of OSA patients. CONCLUSION: This work represents a rationale approach to the evaluation of microarray data and will be useful to identify genes that may be causally associated with metastasis. EREG and CHST2 will be likely considered as clinical molecular markers to predict the outcome of OSA.

Specific microRNA expression during chondrogenesis of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have the capacity to self-renew and differentiate into multiple cell types, but little is known about the precise mechanism of this procedure. Recent studies show that a group of short noncoding RNAs called microRNAs (miRNAs) play a key role in this procedure. However, little work has been done to determine the miRNAs which specifically regulate the differentiation of MSCs. In this study, we cultured human MSCs and chondrogenic differentiation MSCs of 3 donors, and investigated the miRNA expression profiles of MSCs and chondrogenic differentiated MSCs from 2 donors by using miRNA microarrays. We found 5 miRNAs were significantly overexpressed in chondrogenic differentiated MSCs in each sample, and 4 were further confirmed by real-time RT-PCR assay in the sample from the third independent donor. We also predicted the confirmed miRNAs corresponding to putative target genes by online software. The results indicated that the overexpressed miRNAs in chondrogenic differentiated MSCs may play a role in the procedure of MSC chondrogenesis, which offers us guidance for further studies.

[ShRNA of Cyclin D1 decreased the proliferation of human osteosarcoma cell line SOSP-9607.].

AIM: To investigate the effect of Cyclin D1 shRNA on the apoptosis and proliferation of human osteosarcoma cell line SOSP-9607. METHODS: Human Cyclin D1 shRNA vector was stably transfected into SOSP-9607 osteosarcoma cells. The mRNA and protein cxpression levels of Cyclin D1 were detected by semiquantitative RT-PCR and Western blot respectively. The cell cycle and pretiferation of osteosarcoma cells were examined by FCM analysis and CCK-8 method, respectively. RESULTS: After stable transfection of Cyclin D1 shRNA, the expression of Cyclin D1 were inhibited at mRNA and protein levels. The proliferation of SOSP-9607 osteosarcoma cells was inhibited. The difference was significant compared with control groups (P<0.05). At the same time, Cyclin D1 shRNA transfection increased G0/1 phage content and decreased S phage content. CONCLUSION: Cyclin D1 shRNA could down-regulate the expression of Cyclin D1, effectively inhibit the proliferation of osteosarcoma cells, and have significant effect on the cell cycle.

Establishment and characterization of human osteosarcoma cell lines with different pulmonary metastatic potentials.

To develop an investigative tool for the study of human osteosarcoma (OSA), we established a human OSA cell line, namely, SOSP-9607, which exhibits a potential for spontaneous pulmonary metastasis. Subsequently, we screened two related sublines (F5M2 and F4) that have different pulmonary metastatic potentials. An in vivo orthotopic transplantation assay confirmed spontaneous pulmonary metastasis in all mice (100%) transplanted with the more aggressive OSA cells (F5M2) and a lesser degree of metastases with smaller nodules in 33.3% mice transplanted with the less aggressive OSA cell subline (F4). In mice transplanted with F5M2 cells, death from metastasis occurred at a median of 71 days; however, in mice transplanted with F4, no death occurred even after 120 days. Therefore, the F5M2 and F4 sublines, which originated from the same parent cell line, differed with respect to metastasis-related properties such as proliferating ability and invasiveness. Hence, these well-characterized human OSA sublines can be used as valuable models for comparative studies of genetic determinants of OSA in the future.