Study On The Effects Of PiRNA-823on The Biological Behavior Of Multiple Myeloma And Correlated Mechanisms

Part1Expression of piRNAs in multiple myeloma patients and their relationship with clinical significanceObjective:To investigate the expression levels of piRNAs in multiple myeloma (MM) patients and evaluate their relationship with clinical significance.Methods:qRT-PCR analysis was used to analyze the levels of piRNA-823and piRNA-651in FFPE tissue samples of15bone marrow biopsy specimens from MM patients,10thoracic and lumbar tissues infiltrated by CD138+MM cells from surgical specimens of patients with MM as well as8bone marrow biopsy specimens from healthy individuals. Bone marrow derived CD138+cells from18healthy donors and43MM patients were obtained using CD138+microbead selection. Detection of piRNA-823、piRNA-651、piRNA-4987and piRNA-Hepl expression levels in these CD138+cells was carried out by qRT-PCR analysis and then their relationship with clinical significance of MM were evaluated.Results:As compared with normal controls, bone marrow biopsy specimens from newly diagnosed MM patients and CD138+MM cells-infiltrated thoracic and lumbar tissue from surgical patients with MM expressed high levels of piRNA-823, whereas no significant difference was found in piRNA-651.43MM patients were classified according to International Staging System (ISS). The expression of piRNA-823was remarkably higher in ISS Ⅱ and ISS III MM patients, whereas it was much lower in healthy individuals and ISS I MM patients. Moreover, the levels of piRNA-823were gradually increased with increasing stage of MM patients. piRNA-4987and piRNA-Hepl were upregulated in stage III MM patients compared with healthy individuals, whereas no significant difference was found in piRNA-651.Conclusion:Our data indicate that piRNAs are heterogeneous in MM patients and may play different roles in the pathogenesis of MM. piRNA-823is upregulated in MM patients, and positively correlates with the disease stage, indicating that it could be a poor prognostic index in MM. Part2Effects of piRNA-823on the biological behavior of multiple myeloma cells and correlated mechanismsObjective:To investigate the effects of piRNA-823on the biological behavior of multiple myeloma cells and correlated mechanisms in vitro and in vivo.Methods:qRT-PCR analysis was used to analyze the levels of piRNA-823in4MM cell lines, including RPMI8226、ARH-77、U266and KM3. Antagomir-823was designed and synthesized to silence endogenous piRNA-823and transfected into RPMI8226and ARH-77cells by Lipofectamine2000. MM cells were randomly allocated into3groups:blank group (non-transfected group), negative control transfected group and antagomir-823-transfected group. To assess the global effect of piRNA-823inhibition by antagomir which are relevant to MM cell growth, both antagomir-NC-transfected and antagomir-823-transfected RPMI8226cells were subjected to whole genome microarray analysis and pathway enrichment analysis was then carried out on the differentially expressed genes. CCK-8assay was performed to evaluate the effect of piRNA-823silencing on the proliferation of MM cells. Flow cytometry was used to measure the effects of piRNA-823silencing on the cell cycle and apoptosis of MM cells. Western blotting was used to observe the effect of piRNA-823silencing on the expression levels of apoptosis related proteins and cell cycle related proteins. Finally, subcutaneous xenografted mice model of MM lines were established to assess the effect of piRNA-823silencing on the growth of MM cells in vivo and the expression levels of piRNA-823and related proteins including Bcl-2、cyclinD1、Ki-67as well as caspase-3in retrieved xenografted tumor were analyzed by qRT-PCR and immunohistochemistry, respectively.Results:The expression levels of piRNA-823in4MM cells lines were significantly increased, which was down-regulated in RPMI8226and ARH-77cells after transfection with antagomir-823. After transfected with antagomir-823, we found a significant modulation of pathways such as apoptosis, cell cycle, MAPK, mTOR and IL-6signaling pathways, which may reveal the mechanism by which antagomir-823regulates MM cells growth. We next observed that the growth was significantly reduced in antagomir-823-transfected RMPI8226cells compared with controls at both48and72hours. By Annexin V-FITC/PI flow cytometry analysis, we observed that piRNA-823inhibition could trigger apoptosis, accompanied by reduced Bcl-2and Bcl-xl expression and increased cleaved caspase-3and PARP expression. Moreover, piRNA-823inhibition induced Gl/S cell cycle arrest, which was associated with the inhibition of cyclinD1, and CDK4expression, as well as the level of phosphorylated-Rb. We found that silencing piRNA-823also showed inhibition of pAKT and pERK. Finally, we found that antagomir-823could effectively repress tumor growth in vivo. Consistently, the reduced Ki-67, cyclinD1and Bcl-2expression and the increased cleaved caspase-3expression were found in antagomir-823-treatd tumor.Conclusion:The expression of piRNA-823was upregulated in multiple myeloma cell lines, and silencing piRNA-823in MM cells induced deregulation of cell cycle regulators and apoptosis-related proteins expression, accompanied by inhibition of tumorigenicity in vitro and in vivo. Part3Regulatory effect of piRNA-823on DNA methylation in multiple myelomaObjective:To investigate the regulatory effect of piRNA-823on DNA methylation in multiple myeloma and elucidate the relationship between anti-tumor effects of antagomir-823on MM and antagomir-823-induced changed in DNA methylation.Methods:Antagomir-823was designed and synthesized to silence endogenous piRNA-823and then transfected into RPMI8226and ARH-77cells by Lipofectamine2000. The expression of DNMT1, DNMT3A and DNMT3B in RPMI8226or ARH-77cells at mRNAs and proteins levels were measured by qRT-PCR and Western blotting, respectively. Then17primary CD138+MM cells were analyzed for expression levels of DNMT3A and3B mRNAs and for piRNA-823expression by qRT-PCR to explore the correlation between DNMT3A/3B and piRNA-823. Global DNA methylation was analyzed48hours after transfection by an ELISA-like assay, using an antibody against5-Methyl Cytosine. The effect of piRNA-823silencing on the methylation status of p16INK4A gene in RPMI8226cells was examined with MSP analysis. Finally, the expression of p16INK4A protein in antagomir-823-transfected RPMI8226cells and that in antagomir-823-treated tumor were examined by western blotting and immunohistochemistry, respectively.Results:piRNA-823inhibition by antagomir-823was found to lead to a marked reduction of DNMT3A and3B expression both at mRNA and protein levels in RMPI8226and ARH-77cells. However, DNMT1showed no significant changes. Integrated analysis highlighted a statistically significant direct correlation between DNMT3A mRNA levels and piRNA-823expression levels. A similar positive correlation was observed for DNMT3B mRNA levels and piRNA-823. We observed a reduction of approximately60%in global DNA methylation of RPMI8226cells after treated with antagomir-823, which was comparable with that achieved with decitabine treatment at the same time point. Similarly, a30%decreased of global DNA methylation was found in antagomir-823-treated ARH-77cells.72hours after transfection, reexpression of p16INK4A protein was observed in RPMI8226cells with antagomir-823treatment, which is coincided with reduction in the DNA methylation levels of this gene. In human myeloma xenograft model, compared with control group, antagomir-823-treated group had increased p16INK4A protein expression.Conclusion:we concluded that piRNA-823could inactivate p16INK4A involved in tumorigenesis by DNA hypermethylation in MM, whereas antagomir-823could partially revert aberrant methylation, with consequent reexpression of p16INK4A. Part4Effect of piRNA-823on angiogenesis in multiple myelomaObjective:To investigate the effect of piRNA-823on MM cell-induced proangiogenic activity on endothelial cells in vitro and in vivo.Methods:Antagomir-823was designed and synthesized to silence endogenous piRNA-823and then transfected into RPMI8226and ARH-77cells by Lipofectamine2000. VEGF secretion in the CM of antagomir-823-transfected RPMI8226and ARH-77cells was measured by ELISA. The expression levels of pAKT and pERK in HUVECs were observed by western blotting, after co-cultured with antagomir-823-transfected RPMI8226cells. After transfected with antagomir-823, the effects of the CM from RPMI8226and ARH-77cells on HUVECs angiogenesis in vitro were studied by Transwell migration assay and net-like formation assay. Finally, the effect of piRNA-823silencing on microvesseldensity (MVD) in human myeloma xenograft model was assessed by immunohistochemistry analysis for CD34and CD31 expression.Results:We first showed that VEGF secretion was significantly decreased in the CM of antagomir-823-transfected RPMI8226and ARH-77cells, compared with antagomir-NC-transfected or nontransfected cells. In addition, either antagomir-NC-transfected or nontransfected RPMI8226cells triggered activation of ERK and AKT pathways in endothelial cells, which was inhibited when ECs were cultured with antagomir-823-transfected RPMI8226cells. Regarding cell migration, CM of antagomir-823-transfected RPMI8226and ARH-77cells decreased the chemotactic motility of ECs as compared with CM of control groups. Moreover, we observed that ECs formed incomplete and fluffy tubular structures in the presence of CM obtained from antagomir-823-transfected MM cells. In contrast, the treatment with CM obtained from controls led to the formation of elongated and robust tubular structures. Finally, in human myeloma xenograft model, compared with control group, antagomir-823-treated group had decreased micro vessel density.Conclusion:We demonstrated that piRNA-823inhibitor reduced VEGF secretion from MM cells, thereby reducing MM cell-induced proangiogenic activity on endothelial cells in vitro and in vivo.