Tongtao Yang

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.

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. J. Cell. Biochem. 112: 1844–1856, 2011.

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.

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.

A Caspase-6 and Anti-HER2 Antibody Chimeric Tumor-Targeted Proapoptotic Molecule Decreased Metastasis of Human Osteosarcoma

Human growth factor receptor-2 (HER2), overexpressed as a result of gene amplification, is detected in 20–40% of patients with breast, ovarian, endometrial, gastric, bladder, prostate, or lung cancers, correlated to metastasis of many tumors, and considered to be a poor prognostic indicator for these tumors. However, the data was controversial for HER2 overexpression and the prognosis of osteosarcoma, which is the most common primary malignant bone tumor, presents a therapeutic challenge in medical oncology due to its metastasis and poor response to current treatments. Previously, we reported that the immunocasp-6 gene fused by a HER2-specific single-chain antibody with domain II of Pseudomonas exotoxin A (PEA) and the 5′ end of the truncated active caspase-6 could selectively suppress the HER2-positive tumor growth. In this study, we extend its application. We first confirmed the higher HER2 expression on the surface of metastatic osteosarcoma SOSP-9607(E10) cells, which then be proved specifically addicted to immunocasp-6-induced cells killing in vitro. Thereafter, the efficacy of immunocasp-6 was tested in an osteosarcoma lung metastasis mouse model using intramuscular (i.m.) injections of liposome-encapsulated vectors. Our results showed that the expression of the immunocasp-6 gene not only significantly prolonged animals survival, but also greatly inhibited tumor metastasis. Thereby, our strategy suggests an alternative approach to treating HER2/neu-positive osteosarcoma.

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.

Specific antitumor effects of tumor vaccine produced by autologous dendritic cells transfected with allogeneic osteosarcoma total RNA through electroporation in Rats

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.

scFv-mediated delivery of truncated BID suppresses HER2-positive osteosarcoma growth and metastasis.

Osteosarcoma is the most common primary malignant bone tumor, with high rates of metastasis. Here, we examined the expression of human epidermal growth factor receptor-2 (HER-2) in osteosarcoma cell lines with different metastatic potential, finding that the expression was correlated with metastasis of implanted tumors. We then introduced an expression vector encoding the e23sFv-PEA II-Bid ∆1-60 gene, composed of a HER2-specific single-chain antibody fused with domain II of Pseudomonas exotoxin A (PEA) and the carboxy end of truncated active Bid. We demonstrated that the e23sFv-PEA II-Bid ∆1-60 molecule selectively recognized and killed HER2-overexpressing osteosarcoma cells in vitro. Subsequently, we introduced the e23sFv-PEA II-bid ∆1-60 gene into BALB/c athymic mice bearing HER2-positive osteosarcomas using i.m. injections of liposome-encapsulated vector. Expression of the e23sFv-PEA II-Bid ∆1-60 gene suppressed tumor growth, significantly prolonged animal survival, and inhibited metastasis, thereby suggesting it may represent a competitive approach to treating HER2/neu-positive osteosarcoma.

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 microarrray 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.