Expression and Clinical Significance of Exosome Component 4 in Newly Diagnosed Patients with Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To explore the expression of Exosome Component 4（EXOSC4） in the tissues of newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL) and its clinical significance.@*METHODS@#The expression of EXOSC4 protein in the tissues of 181 newly diagnosed DLBCL patients was analyzed by immunohistochemical staining. Clinical data were collected. The correlation between EXOSC4 protein expression in the tissues of newly diagnosed DLBCL patients and clinical features were analyzed and its prognostic significance.@*RESULTS@#The positive rate of EXOSC4 protein expression was 68.51% in the tissues of 181 newly diagnosed DLBCL patients. These patients were divided into two groups, with 44 cases in high expression group and 137 cases in low expression group. There were no significant differences in age, gender, B symptoms, serum lactate dehydrogenase (LDH) level, Eastern Cooperative Oncology Group (ECOG) score, Ann Arbor stage, extranodal disease, International Prognostic Index (IPI) score, National Comprehensive Cancer Network IPI (NCCN-IPI) score, and cell origin between the two groups (P>0.05). Cox multivariate regression analysis showed that high EXOSC4 protein expression in tissues was an independent poor prognostic factor for OS and PFS in newly diagnosed DLBCL patients (all P<0.05). K-M survival analysis showed that newly diagnosed DLBCL patients with high EXOSC4 protein expression had significantly shorter overall survival (OS) and progression free survival (PFS) than those patients with low EXOSC4 protein expression (all P<0.05).@*CONCLUSION@#High EXOSC4 protein expression in tissues of newly diagnosed DLBCL patients is an independent poor prognostic factor for survival.

Effect of Expression Regulation of Mitotic Control Protein DIS3 on Proliferation of 3 Cell Lines of Human Myeloma / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of the expression regulation of mitotic control protein DIS3 on the proliferation ability of 3 cell Lines of human myeloma.@*METHODS@#Human myeloma cell lines NCI-h929, RPMI 8226 and U266B1 were selected as study objects, and the over-expression vector of DIS3 gene and DIS3-siRNA were designed and constructed, respectively. The cell experiments were divided into 5 groups: control, DIS3 over-expression-empty vector, DIS3-siRNA negative control, DIS3 over-expression and DIS3-siRNA group. After culture for 24 h, 48 h and 72 h, the proliferation capacity of these three cell lines was measured by MTT assay. And cell samples were collected after culture for 72 h, the expression of proliferation cell nuclear antigen (PCNA) was detected by RT-qPCR and Western blot.@*RESULTS@#MTT assay results showed that the proliferation capacity of cells in DIS3 over-expression group was significantly reduced, as compared with the DIS3 over-expression-empty vector group at the same time point (24, 48 or 72 h) (P＜0.05, P＜0.01). Compared with DIS3-siRNA negative control group at the same time point (24, 48 or 72 h), the proliferation capacity of cells in the DIS3-siRNA group significantly increased (P＜0.05, P＜0.01). The results of RT-qPCR and Western blot showed that the mRNA and protein expression levels of PCNA in cells of DIS3 over-expression group were significantly reduced, as compared with DIS3 over-expression empty vector group (P＜0.01). Compared with the DIS3-siRNA negative control group, the mRNA and protein expression of PCNA in the cells of DIS3-siRNA group very significantly increased (P＜0.01).@*CONCLUSION@#Over expression of DIS3 can significantly reduce the proliferation ability of 3 cell lines of human myeloma, which may be closely related with reducing PCNA expression.

Detection of preoperative chemoradiotherapy sensitivity molecular characteristics of rectal cancer by transcriptome second generation sequencing / 北京大学学报(医学版)

OBJECTIVE@#To detect the preoperative chemoradiotherapy sensitivity molecular characteristics of rectal cancer by transcriptome second generation sequencing.@*METHODS@#The clinicopathological data of 30 patients with locally advanced rectal cancer were collected prospectively, including 9 indicators (general conditions, imaging data before radiotherapy and chemotherapy, pathological data of biopsy before radiotherapy and chemotherapy, and tumor differentiation degree, etc.), in order to analyze the correlation between them and tumor regression grading (TRG) after radiotherapy and chemotherapy for rectal cancer. At the same time, frozen specimens of colonoscopy biopsy before neoadjuvant therapy were collected from these 30 patients, and transcriptome second-generation sequencing was performed for bioinformatics analysis to screen out the genes that might drive the radio chemotherapy sensitivity of rectal cancer.@*RESULTS@#Among the 30 patients with rectal cancer, 9 had complete pathological remission, 12 had partial remission, and 9 had poor remission. The degree of pathological TRG remission after radiotherapy and chemotherapy for rectal cancer was negatively correlated with the preoperative MRI T stage (P=0.046), and positively correlated with preoperative MRI rectal cancer extravascular invasion (EMVI) (P=0.003). Transcriptome second-generation sequencing of the obtained 217 transcripts (P<0.05) for signal pathway enrichment analysis, and multiple cell signal transduction pathways related to antigen presentation could be found. The high expression of HSPA1A, HSPA1B and EXOSC2 was positively correlated with postoperative pathological remission (P<0.05). The high expression of DNMBP, WASH8P, FAM57A, and SGSM2 was positively correlated with postoperative pathological remission (P<0.05).@*CONCLUSION@#Preoperative NMR detection of extra-tumoral vascular invasion (EMVI-positive) in patients with rectal cancer was significantly better than that of EMVI-negative patients after chemoradiotherapy. Patients with high expressions of HSPA1A, HSPA1B and EXOSC2 had poor postoperative pathological remission, while patients with high expressions of genes, such as DMNMB, WASH8P, FAM57A, and SGSM2 had good postoperative pathological remission. Based on the molecular characteristics of rectal cancer radiotherapy and chemotherapy, attempts to block or enhance the molecular pathways associated with chemosensitivity of rectal cancer, are to be made to further explore new candidate therapeutic targets that can increase the sensitivity of radiotherapy and chemotherapy for rectal cancer.

Interaction of human genes WT1 and CML28 in leukemic cells / 华中科技大学学报（医学）（英德文版）

The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms' tumor 1 suppressor gene (WT1) and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the relationship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA (siRNA) against WT1 and CML28 in leukemic cell line K562 to examine the interaction between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was detected by using RQ-PCR and Western blotting. K562 cells transfected with WT1-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by using immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide important clues for further study on the role of CML28 and WT1 in leukemic cells.

Expression and purification of GST-CML28 fusion protein and preparation of its polyclonal antibody / 中国实验血液学杂志

This study was aimed to investigate the expression of GST-CML28 in Escherichia Coli and to prepare its antibody. The constructed recombinant expression vectors CML28-pGEX-3X were transformed into Escherichia Coli BL21 under IPTG induction. The protein was abstracted from the transformers, and purified by a GSTrap FF column. The rabbits were immunized by the purified fusion protein to produce serum with anti-CML28 antibody. The serum was purified by chromatographic column stuffed with glutathione Sephamse 4B to get the antibody. The specific antibody against CML28 was further identified by ELISA, Western blot, immunohistochemistry and quantum dot luminescence. The results indicated that GST-CML28 fusion protein was expressed in Escherichia coli and its specific polyclonal antibody was obtained. It is concluded that the anti-CML28 polyclonal antibodies with high titer and specificity are successfully prepared. These antibodies provide an useful experimental tool to profoundly research the physiological significance and biological function of the CML28 gene.

Construction and expression of dendritic cell nucleic acid vaccine containing CML28 gene in human dendritic cells / 中国实验血液学杂志

This study was aimed to construct nucleic acid vaccine containing the coding region of the CML28 gene and to express it in human dendritic cells. The full length of CML28 cDNA was amplified from K562 by RT-PCR and subcloned into pGEM-T vector. The CML28 fragment was digested and subsequently inserted into the EcoRI-Xba I sites of pcDNA3.1HisA to construct the recombinant expression vector pcDNA3.1HisA-CML28, which was identified by restrition analysis and sequencing. Human dendritic cells (DC) were separated from peripheral blood mononuclear cells (PBMC) by culture with rhGM-CSF, rhIL-4 and assessed by flow cytometry. The constructed plasmid pcDNA3.1 HisA-CML28 was transfected into DC by electroporation. Western blot was used to detect the expression of fusion protein His-CML28. The results showed that recombinant plasmid pcDNA3.1HisA-CML28 contained the correct full CML28 cDNA identified by restriction analysis and sequencing, and can express the fusion protein His-CML28 in DCs. It is concluded that nucleic acid vaccine containing CML28 gene was constructed and expressed in DC successfully.

Monitoring CML28 mRNA levels in patients before and after HSCT by real-time quantitative RT-PCR / 中国实验血液学杂志

The purpose of this study was to establish a SYBR Green I real-time quantitative RT-PCR method for investigating the correlation between CML28 mRNA expression levels and relapse of leukemia after allo-hematopoietic stem cell transplantation (HSCT). pcDNA3.1HisA-CML28 plasmid had been constructed as the standard template. Serial monitoring of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML, 1 patient with Ph(+) ALL. The results showed that the sensitivity of the established method was at 10(-4) (0.05 ng) level, with interassay variation and intraassay variation of standard samples both below 10%. The CML28 was highly expressed in AML and CML-BP or AP. CML28 level in newly diagnosed group was (6.58 +/- 2.34) x 10(-2), in pre-conditioning regimen group was (2.19 +/- 0.32) x 10(-2), in group that 1 month after HSCT was (1.35 +/- 1.28) x 10(-2), in group that 3 months after HSCT was (4.57 +/- 6.39) x 10(-3). CML28 can be detected 3 months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level (<2 x 10(-2)) survived without relapse, the other 2 case with high level (>2 x 10(-2)) relapsed within one year, 1 case died and 1 case received the second time HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2 x 10(-2) and relapse has taken place. The conclusions was made that CML28 mRNA level is obviously correlated with the development of leukemia. Serial quantification of CML28 mRNA levels are necessary for HSCT recipients, and more informative than a single detection. Using of this assay to evaluate MRD in the patients received HSCT is helpful for prediction of relapse.