Prediction of Tumor Microenvironment Characteristics and Treatment Response in Lung Squamous Cell Carcinoma by Pseudogene OR7E47P-related Immune Genes

Objective Pseudogenes are initially regarded as nonfunctional genomic sequences,but some pseudogenes regulate tumor initiation and progression by interacting with other genes to modulate their transcriptional activities.Olfactory receptor family 7 subfamily E member 47 pseudogene(OR7E47P)is expressed broadly in lung tissues and has been identified as a positive regulator in the tumor microenvironment(TME)of lung adenocarcinoma(LUAD).This study aimed to elucidate the correlation between OR7E47P and tumor immunity in lung squamous cell carcinoma(LUSC).Methods Clinical and molecular information from The Cancer Genome Atlas(TCGA)LUSC cohort was used to identify OR7E47P-related immune genes(ORIGs)by weighted gene correlation network analysis(WGCNA).Based on the ORIGs,2 OR7E47P clusters were identified using non-negative matrix factorization(NMF)clustering,and the stability of the clustering was tested by an extreme gradient boosting classifier(XGBoost).LASSO-Cox and stepwise regressions were applied to further select prognostic ORIGs and to construct a predictive model(ORPScore)for immunotherapy.The Botling cohorts and 8 immunotherapy cohorts(the Samstein,Braun,Jung,Gide,IMvigor210,Lauss,Van Allen,and Cho cohorts)were included as independent validation cohorts.Results OR7E47P expression was positively correlated with immune cell infiltration and enrichment of immune-related pathways in LUSC.A total of 57 ORIGs were identified to classify the patients into 2 OR7E47P clusters(Cluster 1 and Cluster 2)with distinct immune,mutation,and stromal programs.Compared to Cluster 1,Cluster 2 had more infiltration by immune and stromal cells,lower mutation rates of driver genes,and higher expression of immune-related proteins.The clustering performed well in the internal and 5 external validation cohorts.Based on the 7 ORIGs(HOPX,STX2,WFS,DUSP22,SLFN13,GGCT,and CCSER2),the ORPScore was constructed to predict the prognosis and the treatment response.In addition,the ORPScore was a better prognostic factor and correlated positively with the immunotherapeutic response in cancer patients.The area under the curve values ranged from 0.584 to 0.805 in the 6 independent immunotherapy cohorts.Conclusion Our study suggests a significant correlation between OR7E47P and TME modulation in LUSC.ORIGs can be applied to molecularly stratify patients,and the ORPScore may serve as a biomarker for clinical decision-making regarding individualized prognostication and immunotherapy.

不同地区HPV16 E7基因的克隆及序列差异分析

采用PCR扩增、pGEM T载体克隆和核苷酸序列分析的方法对一例武汉地区及两例五峰县高发区宫颈癌患者体内HPV16型的E7基因编码区进行序列分析并与野生型 (德国标准株 )及已发表的HPV16湖北株 (HPVHB)进行了比较。结果发现武汉地区HPV16型E7基因仅第 5 4位出现一个同义突变 ,而高发区HPV16型E7基因存在差异 ,第 77位氨基酸由精氨酸 (Arg)变为半胱氨酸 (Cys) ,第 96位由谷氨酰氨酸 (Gln)变为精氨酸 (Arg) ,E7蛋白的二级结构及亲、疏水性也相应改变 。

HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

Using human papillomavirus type 16 （HPV16） E7 as an antigen and Heat Shock Protein 70 as adjuvant, we constructed a DNA vaccine by linking HSP70 gene to E7^C91G; gene. Mice, after being immunized with E7^C91G;-HSP70, E7^C91G/HSP70, E7^C91G, and wild E7 DNA vaccines respectively, produced E7 specific CD8^＋ T-cell precursor frequencies of 280.33±2.52, 144.34±4.04, 164.34±5.13 and 82.33±3.51 respectively within every 1 × 10^5 mouse splenocytes. This proves that E7^C91G-HSP70 fusion vaccine can significantly enhance the E7 specific cellular immunity within the mice body（p〈0. 01）. After being immunized with E7^C91G-HSP70 fusion vaccine, tumor-bearing mice of the group being treated have significantly longer latency and survival periods, comparing with other three categories of E7 vaccines. Experiment shows that this vaccine has a significant effect on enhancing E7 positive tumor-treatment within mice body. After being immunized with E7^C91G-HSP70 vaccine, there were no pathological changes found in livers, kidneys and spleens of the mice, which proves that the vaccine is quite safe. After all, E7^C91G-HSP70 fusion vaccine has a much stronger tumor- treatment effect than thai of wild type E7 DNA vaccine.

大肠杆菌O_(157)∶H_7的分离鉴定及应用复合PCR法同时检测其三个毒力因子

〔目的〕对驻滇部队感染性腹泻患者、猪和污水进行大肠杆菌O157∶H7(EO157∶H7)的分离鉴定。〔方法〕用O157免疫磁珠富集后 ,接种S -MAC琼脂平板培养 ,用MUG试验初筛 ,VIATEK3 2 全自动微生物鉴定仪鉴定 ,应用复合PCR法同时检测其三个毒力因子。〔结果〕从腹泻患者和猪中检出EcoliO157∶H7,该菌具有三个毒办因子。〔结论〕云南战区部队存在EcoliO157∶H7感染。复合PCR法的建立 。

Induction of SiHa Cells Apoptosis by Nanometer Realgar Suspension and Its Mechanism

The effects of nanometer realgar uterine cervix cancer cell line SiHa cells and suspension on proliferation and apoptosis of human oncogenic genes HPV16E6/E7 were investigated. A ＂micro-jet effiux＂ strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations （6.25, 12.5, 25 and 50 mg/L） for different durations （12, 24, 48 and 72 h）. The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy （TEM） and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry （FCM）. The expression of HPV 16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25--50 mg/L nanometer realgar suspension for 48 h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time- and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group （P〈0.01）, and G0/G1 phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50 mg/L for 48 h. RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

人乳头瘤病毒52型E 6和E 7基因突变与宫颈癌及癌前病变关系的研究

目的分析人乳头瘤病毒(human papillomavirus,HPV)52型E 6和E 7基因突变位点分布,评估基因突变与宫颈癌及癌前病变的相关性。方法收集2016年1月至2017年5月经HPV DNA检测为HPV 52单独阳性的DNA样本120例。应用Generunner V 3.01软件设计扩增E 6/E 7区域的1对PCR引物和Sanger测序的4条延伸引物。以HPV 52原型(Gen Bank Accession No.X 74481.1)作为参考序列,应用MEGA 5.05软件分析每个样本的碱基序列和蛋白序列的变异;应用SPSS 17.0进行一般线性相关检验和Fisher's检验或者Pearson Chi-square(χ~2)检验,分析E 6和E 7基因突变与宫颈病变的关系。结果在104例样本中,共发现HPV 52 E 6基因的16个突变位点和E 7基因的17个突变位点。HPV 52 E 6和E 7基因中不同突变位点的突变率差异无统计学意义(χ~2=14.829、12.548,P=0.464、0.766)。在HPV 52 E 6和E 7基因的33种突变中,无突变位点的突变率随着宫颈病变等级恶化有升高或降低的趋势(P>0.05)。HPV 52 E 6基因在CIN 1+样本中的突变频率(14.0%)显著低于正常样本中突变频率(34.4%),(OR=0.31,95%CI=0.11~0.85,χ~2=5.500,P=0.019)。E 7基因的突变频率在CIN 1+样本中的突变频率(16.3%)也低于在正常样本中的突变频率(27.9%),但差异无统计学意义(OR=0.50,95%CI=0.19~1.35,χ~2=1.908,P=0.167)。结论在单独感染HPV 52的条件下,E 6基因突变是阻碍宫颈病变和宫颈癌发生发展的因素。

孕马尿提取物中4种化学成分的雌激素活性研究

目的 研究孕马尿提取物中四种化学成分的雌激素活性。方法 研究四种成分3β-羟基-5α-16-孕甾烷-20-酮（3β-hydroxy-5α-pregn-16-en-20-one,HP）、5β-孕甾烷-3β,16α,20α-醇（5β-pregnane-3β,16α,20α-triol,PT）、3β,16α-脱氢-5α-孕甾烷-20-酮（3β,16α-dihydroxy-5α-pregnan-20-one,DHP）、3β-羟基-5α-雄甾烷-17-酮（3β-hydroxy-5α-androstan-17-one,HA）对人乳腺癌细胞MCF-7的细胞增殖效应（E-screen实验）;利用Luciferase报告基因与雌激素反应元件重组,与转染对照质粒pRL-cmv、雌激素受体ERα或ERβ质粒共同瞬时转染中国仓鼠卵巢细胞CHO,构建雌激素活性筛选模型,检测四种成分的雌激素活性。结果 E-screen实验结果显示,HP、PT、DHP、HA在1~50μmol·L-1内均能诱导MCF-7细胞增殖,显示出雌激素活性。与雌二醇（17α-estro-diol,E2）的增殖效应（proliferativeeffect,PE）、相对增殖效应（relativeproliferativeeffect,RPE）和EC50相比,HP、PT、DHP、HA的雌激素活性均小于E2的雌激素活性。Luciferase报告基因实验结果显示,HP、PT、DHP、HA在1~50μmol·L-1内均能诱导Luciferase表达,显示雌激素活性。与E2的EC50值相比,HP、PT、DHP、HA的雌激素活性均弱于E2的雌激素活性。结论 HP、PT、DHP、HA均具有雌激素活性,与E2的雌激素活性相比,四种化合物均发挥弱雌激素效应。

雌激素通过其受体α上调LRP16 mRNA表达并促进MCF-7细胞增殖

目的 探讨雌激素对人乳腺癌MCF 7细胞LRP16mRNA表达的调控作用以及过表达LRP16对MCF 7细胞生长特性的影响。方法 ( 1)用Northern印迹方法检测 17β 雌二醇 ( 17β E2 )对LRP16mRNA表达水平的作用 ;( 2 )构建LRP16启动子 ( -2 .6kb)调控的荧光素酶报告子 ( pGL3 S0 ) ,并与各种核受体包括雌激素受体α和 β(ERα和ERβ)、糖皮质激素受体α(GRα)、雄激素受体 (AR)和过氧化物酶体增殖物激活的受体γ和α(PPARγ和PPARα)表达载体共转染COS 7细胞 ,测定荧光素酶活性 ;( 3 )将LRP16表达载体转染MCF 7细胞 ,测定过表达LRP16对细胞的生长特性和细胞周期的影响 ,并用Western印迹方法测定周期素E、p5 3和 p2 1WAF1/CIP1蛋白的变化。结果 ( 1) 17β E2 使MCF 7细胞的LRP16mRNA表达水平增加 5～ 8倍 ,增加幅度未显示出 β E2 培养时间和剂量的依赖性 ;( 2 ) pGL3 S0 与ERα或AR共转染细胞的相对荧光素酶活性较单独转染 pGL3 S0 细胞 (对照组 )分别升高 11和 7.8倍 ;( 3 )LRP16过表达促进MCF 7细胞的生长且伴有周期素E显著升高 ,S期细胞的数量较对照组增加约 10 %。结论 ( 1)雌激素通过激活ERα增加MCF 7细胞LRP16mRNA的表达 ;( 2 )过表达LRP16可能通过上调周期素E促进MCF 7细胞生长。

CPC酰化酶基因的人工合成与重组表达

7-氨基头孢烷酸(7-ACA)是头孢菌素类抗生素的中间体,在医药工业中具有重要的应用价值。该文通过人工设计、合成并在重组大肠杆菌中表达头孢菌素C(CPC)酰化酶基因来实现7-ACA的一步酶法催化合成。以Pseudomonassp.SE83菌株的CPC酰化酶蛋白质序列为模板,对CPC酰化酶基因分两段进行了全局优化设计,包括密码子替换、鸟嘌呤和胞嘧啶含量调整以及酶切位点修改等。通过装配聚合酶链式反应方法最终获得了目标基因,并克隆至表达载体pET-28a,成功构建了诱导型重组大肠杆菌E.coli BL21(DE3)/pET28-acy。经异丙基-β-D-硫代半乳糖苷诱导,在Luria-Bertani培养基中,新建重组大肠杆菌所表达的可溶CPC酰化酶占菌体总蛋白的75%以上;经优化培养基摇瓶发酵,重组菌的CPC酰化酶酶活高达2956U/L。采用上述方法获得的CPC酰化酶,在一步酶法合成7-ACA的工业化生产中具有广阔的应用前景。