Antisense oligonucleotides targeting seed sequence of miR-155 and its applications in multiple myeloma

AIM：To investigate the role of tiny antisense nucleic acid against miR-155 (tiny antimiR-155, t-antimiR-155) in multiple myeloma cells. METHODS：According to the seed sequence of miR-155, t-antimiR-155 was designed and synthesized. t-antimiR-155 was transfected by LipofectamineTM 2000 into RPMI-8266 cells. The cells were divided into t-antimiR-155 group, scrambled control (SCR) group and blank control group. The growth-inhibitory potencies were measured by MTT assay. The ability of cell colony formation was detected by cell colony formation assay. The cell apoptosis was assessed by flow cytometry with annexin V/PI double staining. RESULTS：The best concentration and time were 0.4 μmol/L and 48 h, respectively. The cell colony forming experiment showed that the circumstances of forming cell community in t-antimiR-155 group was weaker than that in SCR group, and the colony formation inhibitory rate of former was significant higher than the latter. Compared with SCR group, the cell apoptosis in t-antimiR-155 group significantly increased. CONCLUSION：The t-antimiR-155 inhibits the progression of multiple myeloma cells by interfering with miR-155. miR-155 may serve as a potential target in gene therapy for treating multiple myeloma.     

Progression of cutaneous plasmacytoma to plasma cell leukemia in a dog

A 5‐year‐old male neutered Bernese Mountain Dog was presented for cutaneous plasmacytoma, which was treated by surgical excision. Four months later, the dog developed multiple skin masses, hyphema, pericardial and mild bicavitary effusions, myocardial masses, and marked plasmacytosis in the peripheral blood. Circulating plasma cells expressed CD34 and MHC class II by flow cytometry. Immunocytochemistry demonstrated that these cells were strongly positive for multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM‐1) and weakly to moderately positive for Pax5. The dog was hypoglobulinemic but had a monoclonal IgA gammopathy detected by serum immunofixation electrophoresis. The PCR analysis of antigen receptor gene rearrangements (PARR) by fragment analysis using GeneScan methodology revealed that plasmacytoid cells in the original cutaneous plasmacytoma and peripheral blood had an identical immunoglobulin heavy chain gene (IgH) rearrangement, indicating that both populations were derived from the same neoplastic clone. Canine cutaneous plasmacytoma rarely progresses to a malignant form and plasma cell leukemia is rarely diagnosed in the dog. This report describes a case of cutaneous plasmacytoma progressing to plasma cell leukemia with a rapid and aggressive clinical course. This report also highlights the utility of flow cytometry, immunocytochemistry, immunofixation electrophoresis, and PARR by fragment analysis using GeneScan methodology in the diagnosis of this hematopoietic neoplasm.     

Changes in TCR Vβ subfamily nave T cells in peripheral blood of patients with multiple myeloma

AIM: To detect the existence of signal joint T-cell receptor excision DNA circles (sjTRECs) of 23 TCR Vβ subfamilies in mononuclear cells of patients with multiple myeloma (MM), and to evaluate the recent thymic emigrants of corresponding Vβ subfamily nave T cells in MM patients. METHODS: 23 TCR Vβ subfamily sjTRECs were amplified in genomic DNA from 5×104 PBMCs of 12 cases in MM patients by using semi-nest PCR.10 normal individuals served as controls. RESULTS: The number of detectable Vβ subfamily sjTRECs was 5.00±2.45 from MM patients, as compared with 9.60±5.48 from normal individuals, the difference was significant (P<0.05). The frequencies of Vβ2-, Vβ10-, Vβ16-, Vβ17-, and Vβ21-Dβ1 sjTRECs were significantly lower than those from normal individuals. 2-9 Vβ subfamily sjTRECs were detected from 12 cases of MM patients. It was negative correlation between age and the number of detectable Vβ subfamily sjTRECs in MM patients (r=-0.892; P<0.01). CONCLUSION: It has been found that some of 23 Vβ subfamily nave T cells are absent or lower level of recent thymic output function in MM patients, suggesting that MM patients have severe cellular immunodeficiency and the capacity and potential of long-term TCR Vβ repertoire reconstitution are dramatically lowered.     

Effects of curcumin on migration and invasion of myeloma cells

AIM：To explore the effect of curcumin on the migratory and invasive abilities of myeloma cells. METHODS：shRNA expression plasmid was transfected into RPMI8226 cells to knock down IQ motif-containing GTPase-activating protein 1 (IQGAP1). The expression of IQGAP1 in RPMI8226 cells tranfected with shIQGAP1 or shRNA negative control, and in un-transfected RPMI8226 cells was detected by Western blotting. All the cells in RPMI8226-shIQGAP1 group, RPMI8226-shRNA negative control group and un-transfected RPMI8226 group were treated with curcumin at various concentrations. The migratory and invasive abilities of the RPMI8226 cells were measured by Transwell chamber and Matrigel assays. The expression of IQGAP1 at mRNA and protein levels in the RPMI8226 cells treated with curcumin was also determined by RT-PCR and Western blotting. RESULTS：The expression of IQGAP1 decreased when IQGAP1 gene was knocked down by shRNA. The migration and Matrigel invasion tests showed that the number of cells moving into under chamber of Transwell decreased in RPMI8226-shIQGAP1 group in comparison with the other 2 groups. Curcumin decreased the migratory and invasive abilities of RPMI8226-shRNA negative control cells and un-transfected RPMI8226 cells, which was not related to the curcumin concentratory. The migratory and invasive abilities of RPMI8226-shIQGAP1 cells showed no significant difference when treated with curcumin at various concentrations. The expression of IQGAP1 at mRNA and protein levels decreased in the RPMI8226 cells treated with curcumin. CONCLUSION：Curcumin decreases the migratory and invasive abilities of myeloma cells via inhibition of IQGAP1 expression.     

Detection of biclonal gammopathy by capillary zone electrophoresis in a cat and a dog with plasma cell neoplasia

Gammopathies associated with plasma cell neoplasms in a 15-year-old female spayed domestic shorthaired cat and a 9-year-old female spayed Rottweiler dog were evaluated by serum protein electrophoresis. In the cat, the plasma cell neoplasm was found in the liver and spleen, and an evaluable sample of bone marrow was not obtained. Some of the plasma cells had the morphologic appearance of flame cells. The paraprotein was confirmed as IgG based on agar gel immunodiffusion precipitation and both immunocytochemical and immunohistochemical staining. The dog had multiple myeloma with production of IgG and IgA paraproteins. In both cases, serum proteins were evaluated by 2 methods of protein electrophoresis: cellulose acetate electrophoresis (CAE) and capillary zone electrophoresis (CZE). In the cat and the dog, CAE showed a single large oligoclonal-like peak, which occurred in the γ-region in the cat and the β-γ-region in the dog, whereas CZE showed a biclonal gammopathy with 2 very close narrow spikes in the γ- and β-γ-regions in the cat and dog, respectively. In selected cases, CZE may be more effective than routine CAE in distinguishing oligoclonal from monoclonal or biclonal paraproteinemia.     

Phagocytic plasmacytoma in a dog

A 4-year-old neutered male Golden Retriever was presented to the oncology service of the North Carolina State University Veterinary Teaching Hospital for staging of a histiocytic sarcoma of the left forelimb, diagnosed on the basis of biopsies submitted by the referring veterinarian. Cytologic assessment of aspirates of 2 splenic nodules identified on ultrasonographic examination of the abdomen revealed a highly phagocytic population of neoplastic round cells morphologically suggestive of plasma cells. Histologic assessment of the forelimb mass after amputation of the limb revealed a neoplastic round cell population demonstrating extensive cytophagia and erythrophagia. Immunohistochemical analysis of the tumor population revealed it to be negative for BLA.36 with sporadic positivity for lysozyme and CD79a. Immunofluorescent evaluation revealed weak tumor cell positivity for immunoglobulin (Ig) A and IgM, but extensive strong positivity for IgG, confirming the plasma cell origin of the tumor. Although extensive phagocytic activity may strongly suggest histiocytic origin, plasma cell origin must also be considered among the differential diagnoses for phagocytic round cell tumors.     

Celastrol induces apoptosis of multiple myeloma H929 cells

AIM: To investigate the effect of celastrol on the apoptosis of human multiple myeloma H929 cells and its molecular mechanism. METHODS: The H929 cells were cultured in vitro and treated with celastrol at different concentrations (0.5, 1, 5 and 10 mg/L). The viability of H929 cells was analyzed by CCK8 assay. Annexin V-PE/7-AAD staining was used to analyzed the effect of celastrol on apoptosis of H929 cells, and mitochondrial membrane potential was observed by flow cytometry. The effect of celastrol on DNA damage was detected by comet assay. The protein levels of apoptosis-related molecules P53, XIAP, cleaved PARP-1 and cleaved caspase-3, and the release of mitochondrial cytochrome C in the H929 cells treated with celastrol were determined by Western blot. RESULTS: The viability of H929 cells was significantly inhibited by different concentrations of celastrol in a concentration-dependent and time-dependent manner. Apoptosis and decreased mitochondrial membrane potential of H929 cells in a concentration-dependent manner were observed after treatment with celastrol (P<0.05). The results of comet assay showed that celastrol induced DNA damage in the H929 cells. The protein levels of apoptotic molecules P53, cleaved PARP-1 and cleaved caspase-3 were significantly increased and the expression level of anti-apoptotic protein XIAP was significantly decreased in the H929 cells treated with celastrol (P<0.05). Celastrol promoted the release of cytochrome C in mitochondria, and activated caspase-3 in dependence on caspase-9. CONCLUSION: Celastrol has an apoptosis-inducing effect on multiple myeloma H929 cells. Its mechamism may be related to activation of mitochondrial apoptosis pathway by inducing DNA damage.     

Regulatory effects of DIS3 expression on colony formation ability in human myeloma cells and tube structure formation of endothelial cells

AIM:To investigate the effects of DIS3 expression on the colony formation ability of 3 kinds of human myeloma cells and tube structure formation of the endothelial cells. METHODS:Human myeloma cell lines NCI-H929, RPMI-8226 and U266 were selected as the study objects, and DIS3 gene over-expression vector and DIS3-siRNA were designed and constructed respectively. The cell experiments were divided into 5 groups:control group, siRNA negative control (siRNA-NC) group, siRNA-DIS3 group, empty vector group and DIS3 over-expression group. The colony formation ability was tested by the plate colony formation assay. Western blot was used to detect the protein expression of hypoxia inducible factor-1α (HIF-1α) and HIF-3α. The expression of angiogenesis-related molecules angiogenin 1 (Ang1), Ang2 and vascubar enelothelial growth factor-A(VEGF-A) at the mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. Matrigel method was used to detect the effect of supernatant from each group of the cells on the tube structure formation of HUVECs. RESULTS:The trends of the following indexes in NCI-H929 cells, RPMI-8226 cells and U266 cells were similar. Compared with empty vector group, the colony formation ability of the cells in DIS3 over-expression group was significantly inhibited (P<0.05). Compared with siRNA-NC group, siRNA-DIS3 significantly enhanced the colony formation ability of the cells (P<0.05). DIS3 over-expression significantly reduced the expression of HIF-1α and HIF-3α (P<0.05), while knock-down of DIS3 expression significantly increased the protein levels of HIF-1α and HIF-3α (P<0.05). In addition, DIS3 over-expression significantly reduced the expression of Ang1, Ang2 and VEGF-A at mRNA and protein levels (P<0.05), while siRNA-DIS3 significantly promoted the expression of Ang1, Ang2 and VEGF-A (P<0.05). Compared with empty vector group, the supernatant from DIS3 over-expression group significantly inhibited the tube structure formation of HUVECs (P<0.01). Compared with siRNA-NC group, the supernatant from siRNA-DIS3 group significantly promoted the tube structure formation of HUVECs (P<0.05). CONCLUSION:DIS3 over-expression significantly inhibits the colony formation ability of human myeloma cells and tube structure formation of HUVECs, which may be closely related to the regulation of the expression of HIF-1α and HIF-3α.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells

AIM:To explore the effect of pyrrolidine dithiocarbamate (PDTC),an NF-κB inhibitor,on the proliferation and apoptosis of human multiple myeloma U266 cells and its mechanisms.METHODS:The U266 cells were treated with PDTC at different concentrations (0,25,50,100 and 200 μmol/L)in vitro.The growth inhibitory rate of the U266 cells was detected by CCK-8 assay and cell counting.The cell cycle of the U266 cells was determined by flow cyto-metry,and the apoptosis was examined by flow cytometry with Annexin V-FITC/PI staining.The effect of PDTC on the expression of DNA methyltransferase 1(DNMT1) at mRNA and protein levels was measured by RT-qPCR and Western blot,respectively.The effects of PDTC on the protein levels of NF-κB (P65),DNMT1,Bcl-2,cyclin D1,cleaved caspase-3 and cleaved caspase-8 were determined by Western blot.RESULTS:The protein level of NF-κB (P65) was decreased after treatment with PDTC for 48 h or 72 h.PDTC inhibited the proliferation of U266 cells in both dose-and time-dependent manners.After treatment with PDTC for 48 h,the percentage of U266 cells in G₂ phase increased compared with control group (P<0.05).PDTC induced the apoptosis of U266 cells in a dose-dependent manner.The expression of DNMT1 at mRNA and protein levels decreased (P<0.05).The results of Western blot showed that the expression of Bcl-2 in PDTC groups decreased,while the protein levels of cyclin D1,cleaved caspase-3 and cleaved caspase-8 were higher than those in control group (P<0.05).CONCLUSION:The NF-κB inhibitor PDTC inhibits the proliferation of U266 cells by inducing cell apoptosis.It may be related to the down-regulated expression of DNMT1,cell cycle arrest and activation of the apoptotic pathways.