A novel Twist1/hsa-miR138-5p/caspase-3 pathway regulates cell proliferation and apoptosis of human multiple myeloma.

The nuclear transcription factor twist-related protein 1 (Twist1) is associated with tumor malignant transformation and metastasis in various types of carcinomas. We found that Twist1 was highly expressed in clinical multiple myeloma (MM) cells, and explored its roles in proliferation and apoptosis in human MM cell lines U266 and RPMI-8226. In these cells, Twist1 transcriptionally regulated the miRNA hsa-miR138-5p, which targeted caspase-3 to control apoptosis. Silencing of Twist1 significantly suppressed cell proliferation and increased apoptosis, which was reversed by overexpression of hsa-miR138-5p or simultaneous silencing of caspase-3. This reversion was further substantiated by attenuated apoptotic signaling, including downregulated expression of the cleaved forms of caspase-3 and peroxisome proliferator-activated receptor 1 (PPAR1). We demonstrate here for the first time that the novel Twist1/hsa-miR138-5p/caspase-3 pathway contributes significantly to the proliferation and survival of human MM cells. Our study provides new insight for novel MM treatments by developing Twist1-targeted therapeutics.

[Establishment of A Myelodysplastic Syndrome Transformed to Leukemia Cell Line Stably Expressing Green Fluorescent Protein].

OBJECTIVE: To establish a myelodysplastic syndrome transformed to leukemia cell line stably expressing green fluorescent protein (GFP), and to evaluate its biological characteristics and applications. METHODS: SKM-1 cells were transfected by lentiviral particles with vector of GFP. The GFP positive single cell clone was isolated by limiting dilution and continued being cultured. The cells were injected into mice subcutaneously and were screened in vivo. Then SKM-1/GFP cells were obtained after tumour plaque was separated and cultivated. The cell morphology was observed by fluorescence microscopy. The GFP expression was further detected by flow cytometry. The cell proliferation was analysed by CCK-8 assay. SKM-1/GFP cells were inoculated to subcutaneous tissue of the immunodeficiency mice. The growth and invasion of the tumour were observed after tumour formation. RESULTS: No differences in cell morphology and growth characteristics were observed between SKM-1 cells and SKM-1/GFP cells. The rate of GFP expression was 100%. No differences in cell proliferation were observed between SKM-1 cells and SKM-1/GFP cells. The tumour mass was observed after 14 days of subcutaneous vaccination in NOD/SCID mice. Spontaneous fluorescence from plaque was observed by living fluorescence microscopy at 30th day after vaccination. Homogenous GFP positive cells were observed by fluorescence microscopy in the frozen section of tumour mass. The invasion of SKM-1/GFP cells was also detected in heart, liver, stomach and kidney of mice. CONCLUSION: A myelody-splastic syndrome transformed to leukemia cell line stably expressing green fluorescent protein has been established successfully, which can track tumor cell sensitively and can be applied to the research of minimal residual leukemia. The establishment of SKM-1/GFP cells may serve as a powerful means for studing myelodysplastic syndrome transformation.

Construction of a recombinant lentivirus-mediated shRNA expression vector targeting the human PSMD10 gene and validation of RNAi efficiency in RPMI­8226 multiple myeloma cells.

Multiple myeloma (MM) is one of the most common malignant blood cancers. Previous studies have reported that proteasome 26S subunit non-ATPase 10 (PSMD10) is an oncoprotein with complex roles in hepatocellular carcinoma and other malignant tumors. Notably, research on the relationship between PSMD10 and tumorigenesis of MM has rarely been reported. The present study was designed to explore the possibility of PSMD10 as a therapeutic target in the treatment of MM, and the use of RNA interference (RNAi) to determine the function PSMD10. A recombinant lentivirus-mediated short hairpin RNA (shRNA) targeting human PSMD10 mRNA was constructed and used to decrease endogenous PSMD10 expression in the MM RPMI-8226 cell line in vitro. Expression of the PSMD10-targeting shRNA in RPMI-8226 cells transduced with the recombinant vector could be tracked by observing the expression of green fluorescent protein after infection. A transient transgenic RPMI-8226 cell line was generated by transducing cells with the packaged viral particles. Western blot analysis indicated that the protein levels of PSMD10 in the PSMD10-shRNA MM cells were significantly lower than those in the cells transduced with the negative control shRNA. Notably, RT-qPCR analysis did not reveal a marked change in the PSMD10 mRNA level; thus, the knockdown effect of the PSMD10-shRNA may occur during translation. Furthermore, apoptosis of MM cells was increased by silencing PSMD10 expression. Overall, the results demonstrated that the lentivirus-mediated shRNA vector-based RNAi expression system is an efficient method to silence PSMD10 gene expression in the MM RPMI-8226 cell line. It may provide a basis to study the role of PSMD10 in tumor cells, and may be a reliable gene therapy strategy in the clinic.

Analyses on clinical characteristic and prognoses of 41 patients with chronic myelomonocytic leukemia in China.

PURPOSE: To investigate clinical characteristic and prognostic factors for chronic myelomonocytic leukemia (CMML). METHODS: A retrospective cohort study was used in the research. We investigated clinical and laboratory characteristics of CMML patients and survival status. Patients were followed up regularly through out the course of the research. RESULTS: Forty-one cases were diagnosed as CMML, including 27 male and 14 female patients. Median WBC was 13.7 x 10(9)/L. Five patients had leukocytopenia (1.92-3.46 x 10(9)/L). Median monocyte count in the peripheral blood was 2.13 x 10(9)/L. All patients presented with bone marrow dysplasia, and most showed hyperplasia, except 3 cases. Abnormal chromosome was detected in 34% cases. Median survival time for CMML-1 and CMML-2 was 20 and 12 months, respectively, but there were no statistical significance of survival duration between them. Univariable analysis showed that age (>60 years), neutrophil count (<2.0 x 10(9)/L), lymphocyte count (<1.0 x 10(9)/L), mature monocyte count (>or=5 x 10(9)/L) and anemia (Hb<60 g/L) were associated with poor prognosis for CMML. There was no statistical significance in LDH, gender, and abnormal chromosome for survival time. Only lymphocyte count and neutrophil count in peripheral blood were independent prognostic factors for CMML after multivariate analysis. CONCLUSION: CMML mainly occurs in elderly patients. Although most patients have leukocytosis and monocytosis at diagnosis, few cases show leucopenia and monocytopenia. Age, neutrophil, lymphopenia, monocytosis, and severe anemia are associated with inferior prognosis of CMML. Lymphocyte<1.0 x 10(9)/L and neutrophil count<2.0 x 10(9)/L are adversely independent prognostic factors for CMML.