PHOSPHO1 Serves as a Key Metabolism-Related Biomarker in the Tumorigenesis of Diffuse Large B-cell Lymphoma

Objective:Diffuse large B-cell lymphoma(DLBCL)is an aggressive type of non-Hodgkin lymphoma.Due to its genetic heterogeneity and abnormal metabolism,many DLBCL patients have a poor prognosis.This study investigated the key metabolism-related genes and potential mechanisms.Methods:Differentially expressed genes,differentially expressed transcription factors(TFs),and differentially expressed metabolism-related genes(DEMRGs)of glucose and lipid metabolic processes were identified using the edgeR package.Key DEMRGs were screened by Lasso regression,and a prediction model was constructed.The cell type identification by estimating relative subsets of RNA transcripts algorithm was utilized to assess the fraction of immune cells,and Gene Set Enrichment Analysis was used to determine immune-related pathways.A regulatory network was constructed with significant co-expression interactions among TFs,DEMRGs,immune cells/pathways,and hallmark pathways.Results:A total of 1551 DEMRGs were identified.A prognostic model with a high applicability(area under the curve=0.921)was constructed with 13 DEMRGs.Tumorigenesis of DLBCL was highly related to the neutrophil count.Four DEMRGs(PRXL2AB,CCN1,DECR2 and PHOSPHO1)with 32 TF-DEMRG,36 DEMRG-pathway,14 DEMRG-immune-cell,9 DEMRG-immune-gene-set,and 67 DEMRG-protein-chip interactions were used to construct the regulatory network.Conclusion:We provided a prognostic prediction model based on 13 DEMRGs for DLBCL.We found that phosphatase,orphan 1(PHOSPHO1)is positively regulated by regulatory factor X5(RFX5)and mediates MYC proto-oncogene(MYC)targeting the V2 pathway and neutrophils.

DETECTION OF BCL-2 GENE MAJOR BREAKPOINT REGION REARRANGEMENT IN HUMAN B-CELL LYMPHOMAS

Objective To investigate the frequency of t(14; 18) in different subtypes of B-cell lymphomas and the ability or the polymerase chain reaction(PCR) to detect this rearrangement in frozen samples. Methods 1o7 cases of B-cell lymphomas were studied uslng DNA extracted from rresh-frozen tissues. The DNA samples were amplified by PCR for bcl-2 MBR/JH. The products of bcl-2/JH rearrangement were hybridized with an internal olignucleotide probe or bcl-2 MBR. Results The rearranged bcl-2MBR/JH gene was detected in 13 of the 25(52. o% ) follicular center lymphomas, according to REAL classification: 8 of 11 (72. 7%) grade 1, 2 of 5(40. 0%) grade I, and 3 of 90 (33. 3%) grade, 17 of 82(2o. 8%) cases or difruse large B-cell lymphomas were found to have detectable bel-2 MBR/J. rearrangement- Conclusion The rrequency or bcl-2 MBR/JH rearrangement in diffuse large B-cell lymphomas is significantly lower than those in follicular center lympkomas(X2= 9. 28, P <o. oo5), suggesting that bcl2/JH rearrangements occur mainly in follicular center lymphomas. in addition, the result of reconstruction experiments suggest that amplification or bcl-2 MBR/JH rearrangements by PCR is both sensitive and specific for detection of t (14; 18 ) translocation.

Primary anaplastic lymphoma kinase-positive large B-cell lymphoma of the left bulbar conjunctiva: A case report

BACKGROUND Anaplastic lymphoma kinase(ALK)-positive large B-cell lymphoma(LBCL)is an aggressive and rare variant of diffuse LBCL.Herein,we report an uncommon case of stage IE extranodal ALK-positive LBCL initially originating in the bulbar con-junctiva.CASE SUMMARY A 63-year-old woman presented with a mass in the left bulbar conjunctiva that had persisted for six months,accompanied by swelling and pain that had per-sisted for 3 d.Eye examination revealed an 8 mm slightly elevated pink mass in the lower conjunctival sac of the left eye.Microscopically,the tumor was com-posed of large immunoblastic and plasmablastic large lymphoid cells with scattered anaplastic or multinucleated large cells.Immunophenotypically,the neoplastic cells were positive for ALK,CD10,CD138,Kappa,MUM1,BOB.1,OCT-2,CD4,CD45,EMA,CD79a,CD38,and AE1/AE3,and negative for CD20,PAX5,Lambda,BCL6,CD30 and all other T-cell antigens.The results of gene rearrangement tests showed monoclonal IGH/IGK/IGL and TCRD rearran-gements.Fluorescence in situ hybridization studies did not reveal any BCL2,BCL6 or MYC rearrangements.Furthermore,Epstein-Barr virus was not detected by in situ hybridization in the lesions.Based on the histopathological and imaging examinations,the neoplasm was classified as stage IE ALK-positive LBCL.No further treatments were administered.At the 6,15,and 21 mo postoperative follow-up visits,the patient was in good condition,without obvious discomfort.This case represents the first example of primary extranodal ALK-positive LBCL presenting as a bulbar conjunctival mass,which is extremely rare and shares morphological and immunohistochemical features with a variety of other neo-plasms that can result in misdiagnosis.CONCLUSION Awareness of the condition presented in this case report is necessary for early and accurate diagnosis and appropriate treatment.

Prognostic role of sensitive-to-apoptosis gene expression in rectal cancer

AIM:To investigate the association between prognosis of rectal cancer treated with chemoradiotherapy(CRT) and expression of sensitive-to-apoptosis(SAG),B-cell lymphoma-extra large(Bcl-X L) and Bcl-2 homologous antagonist/killer(Bak).METHODS:Real-time quantitative polymerase chain reaction was used to determine the expression of proteins of interest,namely SAG,Bcl-X L,Bak and β-actin,in rectal carcinoma patients who had a follow-up period of 3 years after CRT.Biopsy specimens were excised from the rectal tumor preceding CRT.RESULTS:SAG,Bcl-X L and Bak proteins showed significant correlations with each other.In multivariate analysis,patients with high vs low SAG expression showed a statistically significant difference in 2-year survival rates:56% vs 73%,respectively(P = 0.056).On the other hand,there were no significant correlations between the expression levels of all three genes and metastatic rates or tumor responses to CRT.Mean overall survival in the patients with elevated SAG expression was 27.1 mo ± 3.9 mo [95% confidence interval(CI):19.3-34.9],and in patients with reduced expression,it was 32.1 mo ± 2.5 mo(95% CI:27.3-36.9).The corresponding values for Bcl-X L were 28.0 mo ± 4.1 mo(95% CI:19.9-36.1) and 31.7 mo ± 2.9 mo(95% CI:26.0-37.5),and those for Bak were 29.8 mo ± 3.7 mo(95% CI:22.5-37.2) and 30.6 mo ± 2.4 mo(95% CI:25.5-35.0),respectively.CONCLUSION:Two-year survival rates significantly correlated with low SAG expression,and SAG may be a candidate gene for good prognosis,independent of therapeutic response of different individuals.

低剂量辐射对人卵巢癌顺铂耐药细胞耐药性的影响

目的探讨低剂量辐射是否可通过Bcl-2相关x(Bax)基因、B细胞淋巴瘤-xL(Bcl-xL)参与逆转人卵巢癌顺铂耐药细胞(SKOV3/DDP)的耐药性,为耐药性卵巢癌的治疗提供新的思路。方法将细胞密度为80%的SKOV3/DDP随机分为对照组、低剂量辐射组及常规剂量辐射组3组,对照组仅摆位(0 Gy),低剂量辐射组每次照射0.5 Gy,共4次,常规剂量辐射组一次性照射2.0 Gy,均照射后12 h进行后续实验。采用细胞增殖及毒性检测试验(CCK-8)检测顺铂对SKOV3/DDP细胞增殖抑制率,计算各组顺铂的IC 50;采用流式细胞术和Annexin V-FITC/PI双染色检测各组SKOV3/DDP细胞凋亡率;采用RT-PCR技术检测各组SKOV3/DDP细胞中Bax、Bcl-xL基因的扩增情况。结果与对照组和常规剂量辐射组相比较,低剂量辐射组顺铂IC 50明显降低(F=16551.931,P<0.05),而细胞凋亡率则显著升高(F=696.081,P<0.05);低剂量辐射组SKOV3/DDP细胞中Bax mRNA相对表达量明显升高(F=11.811,P<0.05);Bcl-xL mRNA相对表达量明显降低(F=54.538,P<0.05)。结论低剂量辐射可以促进SKOV3/DDP细胞凋亡,增加SKOV3/DDP细胞对顺铂的敏感性,此可能与细胞凋亡调控因子Bax、Bcl-xL的参与有关。

Impaired PI3K/Akt signal pathway and hepatocellular injury in high-fat fed rats

AIM:To determine whether mitochondrial dysfunction resulting from high-fat diet is related to impairment of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt,also known as PKB) pathway. METHODS:Rat models of nonalcoholic fatty liver were established by high-fat diet feeding. The expression of total and phosphorylated P13K and Akt proteins in hepatocytes was determined by Western blotting. Degree of fat accumulation in liver was measured by hepatic triglyceride. Mitochondrial number and size were determined using quantitative morphometric analysis under transmission electron microscope. The permeability of the outer mitochondrial membrane was assessed by determining the potential gradient across this membrane.RESULTS:After Wistar rats were fed with high-fat diet for 16 wk,their hepatocytes displayed an accumulation of fat (103.1 ± 12.6 vs 421.5 ± 19.7,P < 0.01),deformed mitochondria (9.0% ± 4.3% vs 83.0% ± 10.9%,P < 0.05),and a reduction in the mitochondrial membrane potential (389.385% ± 18.612% vs 249.121% ± 13.526%,P < 0.05). In addition,the expression of the phosphorylated P13K and Akt proteins in hepatocytes was reduced,as was the expression of the anti-apoptotic protein Bcl-2,while expression of the pro-apoptotic protein caspase-3 was increased. When animals were treated with pharmacological inhibitors of P13K or Akt,instead of high-fat diet,a similar pattern of hepatocellular fat accumulation,mitochondrial impairment,and change in the levels of PI3K,Akt,Bcl-2 was observed. CONCLUSION:High-fat diet appears to inhibit the PI3K/Akt signaling pathway,which may lead to hepa-tocellular injury through activation of the mitochondrial membrane pathway of apoptosis.

携带FLAG标签的人BTG2真核表达载体的构建及其在HeLa细胞中的表达

目的:构建人B细胞易位基因2(BTG2)真核表达载体,并在HeLa细胞中表达携带FLAG标签的BTG2蛋白,为阐明BTG2基因的功能提供实验工具。方法:利用PCR法获得全长BTG2片段,将扩增得到的片段插入真核细胞表达载体pcDNA3.1(+)的多克隆位点;然后按照FLAG序列设计并合成寡核苷酸片段,插入pcDNA3.1(+)-BTG2载体,构建pcDNA3.1(+)-FLAG-BTG2载体;将以上重组质粒转染HeLa细胞,将细胞分为转染pcDNA3.1(+)空载体组、转染pcDNA3.1(+)-BTG2组和转染pcDNA3.1(+)-FLAG-BTG2组,采用抗FLAG抗体的Western blotting法,检测FLAG-BTG2融合蛋白在HeLa细胞中的表达水平。结果:将全长BTG2片段链接于pcDNA3.1(+)质粒,经限制性核酸内切酶BamHⅠ酶切分析及DNA测序证实载体序列准确;该载体转染HeLa细胞后用抗FLAG抗体进行Western blotting法检测,在空载体组和pcDNA3.1(+)-BTG2组HeLa细胞中检测不到FLAG融合蛋白的表达,而在转染pcDNA3.1(+)-FLAG-BTG2组中检测到FLAG-BTG2融合蛋白的表达。结论:成功构建了pcDNA3.1(+)-FLAG-BTG2真核表达载体,并能在HeLa细胞中有效表达携带FLAG标签的BTG2蛋白。

Role of hydrogen sulfide in portal hypertension and esophagogastric junction vascular disease

AIM: To investigate the association between endogenous hydrogen sulfide (H2S) and portal hypertension as well as its effect on vascular smooth muscle cells.

Identification of BTG1 Status in Solid Cancer for Future Researches Using a System Review and Meta-analysis

Abundant studies have been conducted to identify how B-cell translocation gene 1 protein(BTG1)gene affects the differentiation,proliferation,metastasis of cancer cells,and how it further regulates the generation or development of diseases to influence the prognosis of patients.However,the data from single research were not powerful enough.The correlations between BTG1 expression and mechanisms of tumorigenesis or prognosis of patients are still in controversial.Our system review and meta-analysis provided a complete explanation about the association between BTG1 expression and clinicopathological features or prognosis of patients,which further laid a foundation for future research on BTG1.Fifteen eligible studies consisting of 1992 participants were included.We uncovered that BTG1 expression in solid tumors was associated w让h lymph node status(RR=0.66,95%CI:0.58-0.75,P=0.142),TMN stage status(RR=2.13,95%CI:1.71-2.65,P=0.001),T category(RR=1.90,95%CI:1.20-3.00,P=0.000),histological differentiation(RR-1.91,95%CI:1.55-2.37,7=0.012),vascular invasion(RR=0.90,95%CI:0.57-1.41,P=0.001).BTG1 low expression was significantly associated with overall survival(OS)(HR=0.47,95%CI:0.38-0.67,P=0.000).It concluded that BTG1 possessed the potential value for future research and could be recommended as a significant biomarker in solid tumor.

A bendamustine resistance gene signature in diffuse large B-cell lymphoma and multiple myeloma

Aim:Bendamustine is primarily used for treatment of indolent lymphomas but has shown efficacy in some patients with diffuse large B-cell lymphoma(DLBCL)and multiple myeloma(MM).Molecular-based patient stratification for identification of resistant patients,who will benefit from alternative treatments,is important.The aim of this study was to develop a resistance gene signature(REGS)from bendamustine dose-response assays in cultures of DLBCL and MM cell lines,enabling prediction of bendamustine response in DLBCL and MM patients.Methods:Bendamustine response was determined in 14 DLBCL and 11 MM cell lines.Using baseline gene expression profiles and degree of growth inhibition after bendamustine exposure,a bendamustine REGS was developed and examined for the risk stratification potential in DLBCL(n=971)and MM(n=1,126)patients divided into prognostic subtypes.Results:Bendamustine resistance significantly correlated with resistance to cyclophosphamide in DLBCL and melphalan in MM cell lines.The bendamustine REGS showed significantly lower bendamustine resistance probabilities in DLBCL patients with GCB subtype tumors and in tumors of the differentiation dependent centrocyte and plasmablast subtypes.In MM patients,pre-BII classified tumors displayed high bendamustine resistance probabilities and the plasma cell subtype had lower bendamustine resistance probability than memory cells.Furthermore,tumors belonging to the 4p14,MAF,and D2 TC subclasses consistently displayed high bendamustine resistance probabilities.Conclusion:Significant differences in predicted response to bendamustine were found in molecular subtypes of DLBCL and MM,encouraging validation in prospective bendamustine-treated cohorts with available gene expression profiles and follow-up data.

B-cell Ligand Processing Pathways Detected by Large-scale Comparative Analysis

The initiation of B-cell ligand recognition is a critical step for the generation of an immune response against foreign bodies. We sought to identify the biochemical pathways involved in the B-cell ligand recognition cascade and sets of ligands that trigger similar immunological responses. We utilized several comparative approaches to analyze the gene coexpression networks generated from a set of microarray experiments spanning 33 different ligands. First, we compared the degree distributions of the generated networks. Second, we utilized a pairwise network alignment algorithm, BiNA, to align the networks based on the hubs in the networks. Third, we aligned the networks based on a set of KEGG pathways. We summarized our results by constructing a consensus hierarchy of pathways that are involved in B cell ligand recognition. The resulting pathways were further validated through literature for their common physiological responses. Col- lectively, the results based on our comparative analyses of degree distributions, alignment of hubs, and alignment based on KEGG path- ways provide a basis for molecular characterization of the immune response states of B-cells and demonstrate the power of comparative approaches （e.g., gene coexpression network alignment algorithms） in elucidating biochemical pathways involved in complex signaling events in ceils.

丫蕊花甾体皂苷YB16对人前列腺癌细胞PC-3增殖与凋亡的影响

目的:探讨丫蕊花甾体皂苷YB16对人前列腺癌细胞PC-3增殖与凋亡的影响,并探讨其作用机制。方法:体外培养PC-3细胞,给予不同浓度的YB16(0.125~16μmol·L-1),以噻唑蓝(MTT)比色法检测YB16对PC-3的细胞毒性,倒置相差显微镜观察细胞形态变化,吖啶橙(AO)染色、流式细胞术检测YB16对PC-3的凋亡影响,逆转录-聚合酶链式反应(RTPCR)检测YB16对PC-3细胞B淋巴细胞瘤-2(Bcl-2),B细胞淋巴瘤-xl(Bcl-xl),Bcl-2相关X蛋白(Bax),半胱氨酸蛋白酶-3(Caspase-3)mRNA的表达;蛋白免疫印迹法(Western blot)检测Bcl-2,Bcl-xl,Bax,激活型Caspase-3(cleaved-Caspase-3)蛋白表达,并对结果进行分析。结果:YB16能显著抑制PC-3细胞的生长,具有剂量依赖性(P<0.05);YB16能促进细胞的凋亡,相差显微镜,AO染色法观察可见细胞具凋亡特征性改变;YB16能下调Bcl-2,Bcl-xl,上调Bax,Caspase-3 mRNA和蛋白的表达(P<0.05)。结论:YB16能抑制PC-3细胞增殖,促进PC-3发生凋亡,其机制可能与促进Caspase-3表达有关,具有良好的抗肿瘤作用。