Search

To investigate the effect of RITA on TP53 mutant human mantle cell lymphoma (MCL) cell line Mino and its possible mechanism.Mino cells were cultured in RPMI-1640 and treated with RITA at a concentration of 0-16 μmol/L for 24,48,72 hours. Cell viability was assessed by CCK-8 assay. The cells were treated by RITA (0-8 μmol/L) for 48 h, the cell apoptosis induced by RITA was detected by annexin V/PI flow cytometry. Western blot was performed to evaluate the expression of protein BCL-2, Caspase-3, Cleaved Caspase-3, PARP, MDM2, and P53 in Mino cells.After treatment with 0.5, 1, 2, 4, 8, and 16 μmol/L RITA for 48 h, the proliferation inhibition rate of Mino cells was (1.2±5.6)%, (14.9±4.9)%, (41.7±5.0)%, (61.8±2.4)%, (70.2±2.8)%, and (70.8±2.4)%, respectively. RITA could inhibit the proliferation of Mino cells significantly, and statistical analysis showed that the inhibition rate was increased with the increasing of RITA concentration (r=0.767). After the cells were treated by 4 μmol/L RITA for 24, 48, and 72 h, the proliferation inhibition rate was (25.2±3.8)%, (61.8±2.4)%, and (87.0±0.7)%, respectively. Satistical analysis showed that the inhibition rate was also increased with the increasing of treatment time (r=0.978). The apoptosis rate of Mino cells treated by 0, 2, 4, and 8 μmol/L RITA for 48 h was (5.4±0.4)%, (15.3±0.6)%, (38.7±1.7)%, and (50.8±1.1)%, respectively, and it showed dose-dependent manner (r=0.961). Western blot showed that with the increasing of RITA concentration, the BCL-2 protein expression was decreased in a dose-dependent manner (r=0.932), moreover, PARP cleavage and Caspase-3 activation were found, while the protein expression of MDM2 and P53 showed no change.RITA can inhibit the proliferation and induce the apoptosis of Mino cells significantly. The mechanism may be dependent on the Caspase pathway, but independent on the P53 pathway.RITA对TP53突变人套细胞淋巴瘤细胞株的作用及调控机制研究.研究RITA对TP53突变的人套细胞淋巴瘤细胞株Mino的作用及其调控机制.体外培养Mino细胞，不同浓度（0-16 μmol/L）RITA作用于Mino细胞24、48、72 h后，采用CCK-8法检测RITA对Mino细胞的增殖抑制作用； 0-8 μmol/L浓度RITA作用于Mino细胞48 h，采用Annexin V/PI流式细胞术检测RITA对Mino细胞的凋亡诱导作用，采用Western blot法检测蛋白BCL-2、Caspase-3、Cleaved Caspase-3、PARP、MDM2、P53的表达.RITA能显著抑制TP53突变细胞株Mino增殖，经0.5、1、2、4、8、16 μmol/L的RITA作用Mino细胞48 h后，细胞的增殖抑制率分别为（1.2±5.6）%、（14.9±4.9）%、（41.7±5.0）%、（61.8±2.4）%、（70.2±2.8）%和（70.8±2.4）%，随着RITA作用浓度的增加，抑制率也增加（r=0.767）。Mino细胞经4 μmol/L的RITA作用24、48和72 h后的增殖抑制率分别为（25.2±3.8）%、（61.8±2.4）%和（87.0±0.7）%，随着RITA作用时间增加，抑制率亦增加（r=0.978）。细胞凋亡检测结果显示，Mino细胞经0、2、4、8 μmol/L的RITA作用48 h后的凋亡率分别为（5.4±0.4）%、（15.3±0.6）%、（38.7±1.7）%和（50.8±1.1）%，随着RITA作用浓度增加，诱导Mino细胞凋亡的作用增强（r=0.961）。Western blot检测结果显示，随着RITA浓度的增加，BCL-2蛋白表达下降（r=0.932），出现PARP切割和Caspase-3激活，而MDM2、P53蛋白表达无变化.P53小分子RITA对套细胞淋巴瘤TP53突变细胞株Mino有显著的增殖抑制和诱导凋亡的作用，其机制可能依赖于Caspase途径，而与P53通路无关.

BACKGROUND We investigated the relationship of the polymorphisms of SET and MYND domain-containing protein 3 (SMYD3) with risk and prognosis of ovarian cancer. MATERIAL AND METHODS The polymerase chain reaction (PCR) amplification method was applied to detect the polymorphisms of variable number of tandem repeats (VNTR) in the SMYD3 gene promoter region for 156 patients with ovarian cancer (case group) and 174 healthy people (control group). Quantitative reverse transcription polymerase chain reaction and Western blot were applied to detect SMYD3 mRNA and protein expressions. RESULTS The frequencies of VNTR genotype 3/3 and allele genotype 3 in the case group were significantly higher than those in the control group, while the frequency of genotype 2/2 in the control group was significantly higher than that in case group (all P

To investigate miR-181a function and regulation mechanism by identifying miR-181a target genes in acute myeloid leukemia (AML).The HL-60 cells of human AML was transfected by small molecular analog miR-181a, the cell proliferation was detected by CCK-8 method after electroporation in HL-60 cell lines. Target genes of miR-181a were predicted and analyzed by the bioinformatics software and database. Target genes were confirmed by HL-60 cell line and the patient leukemia cells.Overexpressed miR-181a in HL-60 cell line significantly enhanced cell proliferation compared with that in control (P0.05). Dual luciferase reporter gene assay showed that miR-181a significantly suppressed the reporter gene activity containing ATM 3'-UTR by about 56.8% (P0.05), but it didn't suppress the reporter gene activity containing 3'-UTR ATM mutation. Western blot showed that miR-181a significantly downregulated the expression of ATM in human leukemia cells. It is also found that miR-181a was significantly increased in AML, which showed a negative correlation with ATM expression.miR-181a promotes cell proliferation in AML by regulating the tumor suppressor ATM, thus it plays the role as oncogene in pathogenesis of AML.

Objective:The epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (type 1 IGF receptor or IGF1R) have played an important role in the growth and apoptosis of cancer. The RNA interference (RNAi) technique can suppress gene expression, but the effects of dual silencing of EGFR and type 1 IGF receptor have not been well understood.Methods:pU6-EGFR-shRNA-1, pU6-EGFR-shRNA-2, pU6-IGF1R-shRNA-1 and pU6-IGF-1R-shRNA-2 plasimd vectors were transfected to the nasopharyngeal cancer cells. Seven groups were selected for the study. The protein and downstream protein expression were assessed by Western blot. Apoptosis was determined via flow cytometry. Meanwhile, chemosensitivity of nasopharyngeal cancer cell lines transfeced to chemotherapeutic drugs were carried out by MTT.Results:In dual silencing of EGFR and IGF-1R, the protein expression much more was decreased than single silencing of EGFR or IGF-1R, but the cell apoptosis much more is increased than single silencing EGFR or IGF-1R. Dual silencing of EGFR and IGF-1R enhanced chemosensitivity to anticancer drugs, compared with single silencing of EGFR or IGF-1R.Conclusion:Dual silencing of EGFR and IGF-1R are capable of suppressing EGFR and IGF-1R expression of the nasopharyngeal cancer cell and can promote apoptosis and increase the cell sensitivity of anticancer drug. The dual silencing of genes RNAi technique is significantly better than a single gene.

To study the relationship between the donor and recipient serum interleukin 2 (IL-2) and tumor necrosis factor alpha (TNF-alpha) levels and the occurrence of acute graft-versus-host disease (aGVHD) following hematopoietic stem cell transplantation.Twenty-two leukemic patients undergoing allo-hematopoietic stem cells transplantation and their donors were examined for serum levels of IL-2 and TNF-alpha using ELISA during conditioning and after the transplantation. IL-2 and TNF-alpha levels were also detected in the donors during mobilization to analyze the relationship between the cytokines and aGVHD.Five recipients showed no aGVHD, 10 developed grade I aGVHD, and 7 developed grade II-IV aGVHD. The serum levels of IL-2 and TNF-alpha after conditioning and post-transplantation were significantly higher in the recipients with grade II-IV aGVHD than in those with grade 0-I aGVHD, but no difference was found before the pre-conditioning. The serum IL-2 levels in the mobilized donors for the recipients with grade II-IV aGVHD were significantly higher than that in donors for recipients with grade 0-I aGVHD, whereas the levels of TNF-alpha showed no such a difference.Serum IL-2 and TNF-alpha levels in the leukemic patients after conditioning and post-transplantation, and the donor serum IL-2 level after mobilization may be correlative to the severity of aGVHD and can be used as indicators for predicting the severity of aGVHD after the transplantation.

To develop an RNA-interference (RNAi) approach involving the hitting of multiple targets by a recombinant plasmid and evaluate its antitumor effect on laryngeal squamous carcinoma in vitro and in vivo.A plasmid containing 3 different short hairpin RNA (shRNA) segments termed pEGFPshVEGF-shTERT-shBcl-xl was constructed. Plasmids containing single shRNA against each target (VEGF, TERT, BCL-xl alone) individually were also constructed as control. Cells were treated with these plasmids. The expression of targeted genes as well as apoptosis of tumor cells were evaluated after treatment with multiple shRNA vectors or control vectors. The mRNA and protein expression were determined by RT-PCR and western blotting. Cell viability was examined using the MTT assay. Apoptotic morphological alterations were observed by Hoechst staining and electron microscopy. The in vivo antitumor effect was characterized in a nude mice model of laryngeal squamous carcinoma.We demonstrated that a recombinant plasmid containing multiple shRNAs could effectively and simultaneously inhibit VEGF, TERT and Bcl-xl mRNA and protein expression in the HEp-2 cells; the plasmid containing the 3 different shRNAs exhibited a potent antitumor effect on LSCC both in vitro and in vivo, and could much more effectively induced cell apoptosis than each single shRNA. We also demonstrated that the simultaneous blockage of these 3 genes have a better inhibitory effect on human HEp-2 cells than the blockage of each single shRNA.Our study demonstrates that the application of vector-based RNAi technology that involves hitting multiple targets will be a promising therapeutic modality in the gene therapy of human laryngeal cancers; furthermore, it provides experimental evidence for the clinical application of this technology in the future.

To identify the antithrombin (AT) phenotype and gene mutation of a kindred with hereditary antithrombin deficiency.Plasma AT activity and AT antigen level of the propositus and his kindred members were determined with chromogenic substrate method and immunoassay, respectively. All the seven exons and intron-exon boundaries of antithrombin gene were analyzed by PCR and direct sequencing of amplified PCR products from the propositus.The propositus AT antigen level was normal but his AT activity was only 65% of normal value suggesting that he had type II AT deficiency. A heterozygous G13830A mutation in exon 6 resulting in Arg393His missense mutation in his AT polypeptide was identified in the propositus. The same phenotype and gene mutation were found in other 3 kindred members.The type II AT deficiency found in this kindred is caused by heterozygous G13830A mutation in AT gene.

There are few ideal predictors used to evaluate the prognosis of laryngeal squamous cell carcinoma (LSCC). This study was designed to investigate the expression of p73 and PTEN (phosphates and tension homolog deleted on chromosome ten) and their association with clinical, histologic characteristics and prognosis in LSCC.p73 protein and PTEN protein were examined by using immunochistochemical SABC staining method in 65 cases of LSCC and in 23 cases of para-tumor tissues.p73 protein and PTEN protein in LSCC showed positive expression of about 58.5% (38/65) and 49.2% (32/65), compared to para-tumor tissues of about 17.4% (4/23) and 95.7% (22/23) with statistical significance (P0.05). p73 protein positive expression showed stronger in stage III-IV of LSCC than that in stage I-II (P0.05); it more often appeared in recurrent cases than in primary cases (P0.05). And p73 protein positive expression with distant metastasis was stronger than that in LSCC without distant metastasis (P0.05). PTEN protein positive expression was stronger in stage I-II of LSCC than that in stage III-IV (P0.05); PTEN protein positive expression appears less frequently in poor differentiation of LSCC, compared to well/moderate differentiation (P0.05); PTEN positive expression in cases with cervical lymph node metastasis and distant metastasis was lower than that in those without metastasis (P0.05). PTEN expression showed significantly stronger in the patients whose survival time over 5 years (66.7%) than in the patients whose survival time was less than 5 years (27.3%) (P0.05).PTEN positive expression may be useful for predicting the prognosis of LSCC.

The aim was to study the morphological, histochemical and immunological characteristics of less-differentiated acute myeloid leukemic cells, and their diagnostic significance. Wright-Giemsa and histochemical staining were used to stain bone marrow smears from 2 case of AML-Mo. Immunological phenotypes were determined with flow cytometry. The results showed that myeloperoxidase stainings of both cases were negative, PAS was positive with fine particles, CD33/CD13 were positive, CD2/CD3/CD10/CD19/CD22 were negative. It is concluded that morphology, histochemistry and immunological phenotype on bone marrow smears are the main diagnostic basis for AML-Mo. The use of multiple monoclonal antibodies for staining may improve the accuracy.