Poly(A)-specific ribonuclease protein promotes the proliferation,invasion and migration of esophageal cancer cells

BACKGROUND Bioinformatics analysis showed that the expression of the poly(A)-specific ribonuclease(PARN)gene in gastric cancer,head and neck squamous cell carcinoma,melanoma,cervical cancer and lung squamous cell carcinoma tissues was significantly higher than that in normal tissues and was associated with high stage and poor prognosis.The expression of the PARN gene in esophageal cancer(EC)tissue is also significantly higher than that in normal tissues,but the effect of PARN on the proliferation,migration and invasion of EC cells remains unclear.AIM To investigate the relationship between PARN and the proliferation,migration and invasion of EC cells.METHODS The EC tissues of 91 patients after EC surgery and 63 paired precancerous healthy tissues were collected.PARN mRNA levels were measured using a tissue microarray,and the PARN expression level was evaluated using immunohistochemistry to analyze the relationship between PARN expression and clinicopathologic features as well as the survival and prognosis of patients.In addition,the effects of PARN gene knockout on tumor cell proliferation,invasion and migration were studied by using shRNA during the in vitro culture of EC cell lines Eca-109 and TE-1,and the effects of the PARN gene on tumor growth in vivo were verified by a xenotransplantation nude mice model.RESULTS The expression of PARN in EC tissues was higher than that in adjacent normal tissues,and the level of PARN expression was significantly positively correlated with lymphatic metastasis.Patients with high PARN levels had poor overall survival.BIM,IGFBP-5 and p21 levels were significantly increased in the PARN knockout group,while the expression levels of the antiapoptotic proteins Survivin and sTNF-R1 were significantly decreased in the apoptotic antibody array data.In addition,the expression levels of Akt,p-Akt,PIK3CA and CCND1 in the downstream signaling pathway regulating EC progression were significantly decreased.The culture of EC cell lines confirmed that the apoptosis rate of EC cells was significantly increased,the growth and proliferation of tumor cells were significantly inhibited,and the invasion and migration ability of tumor cells were significantly decreased after PARN gene knockout.In vivo experiments of BALB/c nude mice transfected with Eca-109 cells expressing control shRNA(sh-NC)and PARN shRNA(sh-PARN)showed that the tumor volume and weight of nude mice treated with sh-PARN were significantly decreased compared with those of nude mice treated with sh-NC,indicating that PARN knockdown significantly inhibited tumor growth in vivo.CONCLUSION PARN has antiapoptotic effects on EC cells and promotes their proliferation,invasion and migration,which is associated with the development of EC and poor patient prognosis.PARN may become a potential target for the diagnosis,prognosis prediction and treatment of EC.

Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

甲基化酶DNMT3B、叉头框蛋白C1与宫颈癌超声特征的相关性分析

目的分析DNA甲基转移酶3B(DNA methyltransferase 3 beta,DNMT3B)、叉头框蛋白C1(forkhead box protein C1,FOXC1)与宫颈癌超声特征的相关性,以期为临床诊断宫颈癌提供更多的手段。方法选择我院收诊宫颈癌患者117例(研究组)与同期健康体检者50例(对照组)为研究对象,检测DNMT3B、FOXC1的表达,分析DNMT3B、FOXC1与彩色多普勒超声参数Adler分级、患者临床特征的关系,探究DNMT3B、FOXC1在宫颈癌中的临床意义。结果研究组DNMT3B、FOXC1高于对照组,且与Adler分级、糖类抗原125(carbohydrate antigen 125,CA125)、糖类抗原199(carbohydrate antigen 199,CA199)呈正相关(P<0.05)。DNMT3B与肿瘤大小、高危型人乳头瘤病毒(human papillomavirus,HPV)(-/+)、淋巴结是否发生转移、国际妇产科联盟(International Federation of Gynecology and Obstetrics,FIGO)分期有关,FOXC1则与肿瘤分化程度以及FIGO分期有关。结论宫颈癌中DNMT3B、FOXC1表达与宫颈癌动态超声结果有关,随着Adler分级的增加宫颈癌患者DNMT3B、FOXC1的表达水平也越来越高,且DNMT3B、FOXC1与CA125、CA199的表达均呈正相关,由此可见DNMT3B、FOXC1表达对宫颈癌的诊断有一定价值。

口蹄疫病毒非结构蛋白3B单克隆抗体的制备与鉴定

用E.coli原核表达并纯化的口蹄疫病毒(Foot-and-mouth disease virus,FMDV)非结构蛋白3B免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,间接ELISA筛选出分泌鼠IgG的杂交瘤细胞株,将该杂交瘤细胞注射小鼠产生的腹水,用间接ELISA法筛选获得6株能稳定分泌抗3B蛋白单克隆抗体的杂交瘤细胞株,分别命名为3E5、4B1、4D7、4E11、7B2、8B11。鉴定结果显示,4B1和4E11细胞分泌IgG1,其余4株细胞分泌IgG2b;纯化后6株腹水单抗的纯度达90%以上,对3B蛋白的ELISA滴度均可达到1∶100000以上;6株单抗均不与FMDV结构蛋白VP1和3D非结构蛋白发生反应;间接免疫荧光试验证明所制备的单抗能够识别3B蛋白;杂交瘤细胞株连续培养3个月以及冻存6个月后复苏,细胞生长良好,杂交瘤细胞分泌的抗体效价稳定。

口蹄疫病毒非结构蛋白3B基因的克隆表达及3B-EL ISA鉴别诊断方法的初步建立

从口蹄疫病毒(foot-and-m ou th d isease v irus,FM DV)细胞培养物中提取总RNA,经一步法RT-PCR获得了长约230 bp的3B基因片段,将PCR产物连接到pM D-18T载体上并测序,用B amHⅠ与H indⅢ双酶切后,将3B基因融合到pGEX-KG载体上形成表达质粒pGEX-KG-3B,转化到BL 21(DE 3)中在27℃诱导表达,将表达产物进行SDS-PAGE和蛋白质斑点EL ISA。结果表明3B基因主要以可溶形式高效表达,表达产物具有免疫原性。以纯化的表达产物为抗原建立了间接酶联免疫吸附试验(I-EL ISA)。方阵滴定其最佳包被浓度是6.1μg/孔,最佳血清稀释倍数为1∶80,通过测定36份FM DV阴性血清,确定了该方法的阳性判定标准。结果表明,该方法特异性强、重复性好;用3B-EL ISA方法与美国联合生物医学公司(U B I)生产的合成肽检测试剂盒U B I○RFM DV N S-EL ISA对比检测44份血清样品,符合率为93.1%。证明3B-EL ISA方法可用于口蹄疫的鉴别诊断。

SEMA3B基因在肺癌组织中的表达及其与p53表达、肿瘤血管新生的关系

目的:研究抑癌基因SEMA3B(semaphorin 3B)在肺癌组织中表达的临床意义及其与p53表达、肿瘤血管新生的关系。方法:利用RT-PCR检测46例肺癌及远癌正常肺组织中SEMA3BmRNA和VEGF165mRNA表达,免疫组化SP法检测p53蛋白表达和微血管密度(MVD)。结果:肺癌组织中SEMA3BmRNA表达缺失率显著高于正常肺组织(47.8%vs0%,P<0.01),其表达异常与肺癌组织分化程度、淋巴结转移和临床病理分期有关,而与性别、年龄和组织分型无关;肺癌组织中SEMA3BmRNA表达与VEGF165mRNA、p53蛋白表达及MVD均呈显著负相关(P<0.05)。结论:SEMA3B基因在肺癌组织中表达下调,并与肿瘤细胞凋亡、血管新生有密切关系,提示其表达异常对肺癌的发生、发展及预后起重要作用。

激光镊子拉曼光谱检测重组大肠杆菌表达蚕豆14-3-3b可溶性蛋白与包涵体蛋白

采用激光镊子拉曼光谱(LTRS)分析大肠杆菌(Escherichia.coli)表达蚕豆(Vicia faba L.)14-3-3b可溶性蛋白(Soluble protein)与包涵体蛋白(Inclusionbody protein)的结构差异以及两种蛋白在不同温度下在重组菌中的表达水平。结果表明,14-3-3b可溶性蛋白与包涵体蛋白有明显不同的拉曼光谱特征峰,说明LTRS技术可以鉴定14-3-3b的可溶性蛋白和包涵体蛋白。14-3-3b可溶性蛋白特征峰1002,1451和1665 cm!1在16℃下诱导的重组菌中明显增强,在28℃下诱导的重组菌中明显减弱,而反映14-3-3b包涵体蛋白的特征峰900和1446 cm!1在16℃下诱导的重组菌中明显减弱,在28℃下诱导的重组菌中明显增强,这几个峰强的变化反映14-3-3b可溶性蛋白与包涵体蛋白在大肠杆菌细胞中表达水平的变化和SDS-PAGE电泳分析的结果一致。说明LTRS是检测单个大肠杆菌细胞中可溶性重组蛋白和包涵体蛋白表达水平的一种快速有效的方法。

人自噬相关基因LC3B真核表达载体的构建及鉴定

目的:构建人自噬相关基因微管相关蛋白1轻链3B(microtubule-associated protein 1 light chain 3B,MAP1LC3B)真核表达栽体,并鉴定其生物学功能。方法:通过RT-PCR方法扩增得到人MAP1LC3B cDNA;应用基因重组技术构建pEGFP-N1-LC3B真核表达载体,通过酶切和测序进行鉴定;倒置荧光显微镜下观察EBBS诱导4 h后pEGFP-N1-LC3B表达载体转染的HepG2.2.15细胞质中GFP-LC3B分布的变化。结果:通过测序鉴定,pEGFP-N1-LC3B真核表达载体序列正确,编码框正确;转染后的HepG2.2.15细胞经EBBS诱导4 h后,倒置荧光显微镜检测发现GFP-LC3B由散在分布向点状分布改变。结论:成功构建了人自噬相关基因LC3B真核表达载体,为进一步研究自噬在乙型肝炎病毒中的作用机制奠定了基础。

CircFndc3b对乳腺癌细胞侵袭和迁移的影响及机制预测

为探讨CircFndc3b对乳腺癌细胞侵袭和迁移的影响,并预测其调控机制,利用实时荧光定量聚合酶链反应(qRT-PCR)检测CircFndc3b在不同乳腺癌细胞系中的表达;siRNA敲低CircFndc3b表达后,利用Transwell检测MCF-7和MDA-MB-231细胞侵袭能力变化,利用划痕实验检测MCF-7和MDA-MB-231细胞迁移能力变化,利用蛋白免疫印迹(Western blotting)检测侵袭迁移相关蛋白的表达变化;miRDB数据库预测CircFndc3b潜在靶点并通过qRT-PCR验证miRNA-5702和miRNA-7106-5p在乳腺癌中的表达水平。结果表明:CircFndc3b在人乳腺癌细胞中表达水平显著高于人正常乳腺上皮细胞(均P<0.01);敲低CircFndc3b表达后,侵袭的MCF-7和MDA-MB-231细胞显著降低(均P<0.01),同时MDA-MB-231细胞和MCF-7细胞的迁移距离显著降低(均P<0.01);Western blotting检测显示敲低CircFndc3b表达导致MDA-MB-231细胞和MCF-7细胞中E-cadherin表达明显升高,而N-cadherin表达显著降低(均P<0.01)。CircFndc3b具有47个潜在的靶点miRNAs,敲低CircFndc3b后,miRNA-5702和miRNA-7106-5p表达水平在MDA-MB-231细胞和MCF-7细胞中显著升高(均P<0.01)。综上所述,CircFndc3b促进了乳腺癌细胞侵袭和迁移,其作用可能与调控miRNAs表达有关。

AtGT-3b基因克隆及原核表达与纯化

GT-3b转录因子是一个受NaCl和病原体诱导表达的GT-1-like转录因子,它能与GT-1 cis-element(GAAAAA)相互作用,促进下游基因的表达,在植物耐盐中起着重要的调节作用.通过分离了拟南芥(Arabidopsis thaliana)AtGT-3b基因,克隆到原核表达载体pCold TF中,并在大肠杆菌(Escherichia coli)BL21中进行融合表达;通过纯化得到AtGT-3b融合蛋白,以期用于研究其与GT-1顺式作用元件在体外的相互作用.

Mechanism of stilbene glycosides on apoptosis of SH-SY5Y cells via regulating PI3K/AKT signaling pathway

Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.

自噬相关基因LC3B真核表达载体构建及胃癌细胞自噬的观察

目的:构建人自噬相关基因微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3B,LC3B)真核表达载体,并鉴定其生物学功能。方法:通过RT-PCR方法扩增得到人LC3B c DNA,应用基因重组技术构建p EGFP-C1-LC3B真核表达载体,通过酶切和测序进行鉴定。将p EGFP-N1-LC3B表达载体转染胃癌细胞MKN1,倒置荧光显微镜观察饥饿诱导后细胞中EGFP-LC3B的表达及分布变化。结果:通过测序鉴定,p EGFP-N1-LC3B真核表达载体序列正确,编码框正确。转染后的MKN1细胞经饥饿诱导后,检测发现EGFP-LC3B由散在分布向点状分布改变,即生成细胞自噬体。结论:成功构建了人自噬相关基因LC3B真核表达载体,并证实饥饿诱导胃癌细胞自噬的发生,为进一步研究自噬在肿瘤中的作用机制奠定了基础。

口蹄疫病毒非结构蛋白3B单克隆抗体(MAb)的制备及基于MAbELISA方法的初步建立

为建立区分口蹄疫病毒(FMDV)疫苗免疫和自然感染动物的血清学鉴别检测方法,本研究采用杆状病毒表达的FMDV非结构蛋白3ABC免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,并以原核表达的FMDV非结构蛋白3B作为抗原筛选融合细胞的培养上清液,获得一株稳定分泌抗3B蛋白单克隆抗体(MAb)的杂交瘤细胞。经间接免疫荧光鉴定其为一株抗FMDV非结构蛋白3B的特异性MAb,为IgG1/κ亚类,间接ELISA检测杂交瘤细胞上清液和腹水的抗体效价分别为1∶12 800和1∶106。硫氰酸盐洗脱法测定其相对亲和力常数为1.5 mol/L。采用该MAb作为包被抗体并特异性的结合原核表达的3B重组蛋白用于检测血清中抗FMDV的非结构蛋白抗体,初步建立了以MAb为基础的ELISA方法。其MAb包被浓度为5μg/mL,3B重组蛋白浓度为1.1μg/mL,被检血清1∶100稀释,通过检测猪血清并与3B间接ELISA和prioCHECKRNSP ELISA试剂盒进行比较,表明该方法具有更强的特异性。

达氏鲟自噬基因MAP1LC3B克隆及其组织表达分析

【目的】掌握达氏鲟微管相关蛋白1A/1B轻链3B基因(MAP1LC3B)的表达模式,为后续研究MAP1LC3B基因功能及揭示达氏鲟的抗病分子机制提供理论依据。【方法】采用RACE技术从达氏鲟组织中克隆MAP1LC3B基因,通过ProtParam、TMpred、Phyre2及Singal 4.1等在线软件进行生物信息学分析,同时借助实时荧光定量PCR检测MAP1LC3B基因在达氏鲟不同组织中的表达情况。【结果】达氏鲟MAP1LC3B基因cDNA序列全长2208 bp,包含378bp的开放阅读框(ORF)、202 bp的5’端非编码区(5’-UTR)和1628 bp的3’端非编码区(3’-UTR),编码125个氨基酸。MAP1LC3B氨基酸序列含有GABARAP泛素结构域、Agt7结合位点、脂化位点和维管束蛋白结合位点。达氏鲟MAP1LC3B蛋白由18种氨基酸组成,其中谷氨酸(Glu)含量最高(占9.6%)、丙氨酸(Ala)含量最低(占1.6%);蛋白分子量为14743.01 Da,理论等电点(pI)为8.92,不稳定系数为65.12,总平均亲水性为-0.484,是一种不稳定的疏水性蛋白,且不存在跨膜结构,也无信号肽序列。MAP1LC3B基因在达氏鲟鳃、脑、皮肤、头肾、前肾、脾脏、中肾、心脏和肠道等组织中均有表达,以在鳃和肠道组织中的相对表达量较高,显著高于在其他组织中的相对表达量(P<0.05)。【结论】达氏鲟MAP1LC3B基因具有较高的保守性,在鳃和肠道黏膜免疫组织中高表达,表明自噬参与达氏鲟黏膜免疫过程,调节机体免疫反应。

LC3B、HDAC6和Nrf2在慢性阻塞性肺疾病急性加重患者外周血中的表达

目的研究自噬相关基因微管相关蛋白1轻链3B(LC3B)、组蛋白去乙酰化酶6(HDAC6)和核因子E2相关因子2(Nrf2)在慢性阻塞性肺疾病急性加重(AECOPD)患者外周血中的表达水平及其临床意义。方法通过qRT-PCR检测21例健康体检组和36例AECOPD患者外周血单个核细胞(PBMC)中LC3B、HDAC6和Nrf2的表达水平,比较其在健康体检组和AECOPD患者间的差异;分析AECOPD患者外周血LC3B、HDAC6和Nrf2的表达水平与白细胞总数、单核细胞百分比、淋巴细胞百分比及中性粒细胞绝对值的相关性。结果 AECOPD患者外周血LC3B和HDAC6的表达水平均显著高于健康体检组(P<0.05),Nrf2的表达水平低于对照组(P<0.05);AECOPD患者外周血LC3B的表达水平与白细胞总数和中性粒细胞绝对值均呈正相关(P<0.05),AECOPD患者外周血Nrf2的表达水平与单核细胞百分比和淋巴细胞百分比均呈负相关(P<0.05)。结论 LC3B、HDAC6和Nrf2可能参与了AECOPD炎症反应的过程,这将为进一步研究自噬在AECOPD发病中的作用提供理论依据。

KIF3B基因对三阴性乳癌细胞生物学特性和阿霉素化疗敏感性的影响

目的研究KIF3B基因对三阴性乳癌MDA-MB-231细胞生物学特性和阿霉素化疗敏感性的影响。方法用Western blot技术检测KIF3B在人三阴性乳癌细胞系MDA-MB-231和MDA-MB-468中的表达,取KIF3B高表达细胞MDA-MB-231用于后续实验。用sh-NC质粒转染MDA-MB-231细胞作为对照组,用sh-KIF3B沉默质粒转染MDA-MB-231细胞作为实验组。应用Western blot方法检测基因沉默效率和基因沉默后上皮-间质转化(EMT)相关蛋白E-cadherin、基质金属蛋白酶2(MMP-2)和MMP-9表达变化,Transwell实验检测细胞迁移、侵袭能力变化,流式细胞术检测细胞周期变化,MTT实验检测细胞增殖能力和对阿霉素化疗敏感性的变化。结果KIF3B在三阴性乳癌细胞MDA-MB-231中高表达,而在MDA-MB-468中低表达(t=19.92,P<0.01)。沉默MDA-MB-231细胞KIF3B基因后,细胞迁移、侵袭数目明显减少(t=29.54、18.32,P<0.01),G_(0)/G_(1)期细胞比例降低而G_2/M期细胞比例增加(t=4.82、19.05,P<0.01),同时细胞增殖能力被显著抑制(F=7.56~270.09,P<0.01),对阿霉素化疗敏感性明显增加(F=26.37~167.11,P<0.01),E-cadherin表达升高(t=19.71,P<0.01),而MMP-2和MMP-9表达降低(t=26.57、16.11,P均<0.01)。结论沉默三阴性乳癌MDA-MB-231细胞KIF3B基因表达可以抑制细胞增殖、阻滞细胞周期并增加细胞对阿霉素化疗敏感性,并可以通过EMT抑制细胞迁移和侵袭。

Fenofibrate Pre-treatment Suppressed Inflammation by Activating Phosphoinositide 3 Kinase/Protein Kinase B(PI3K/Akt) Signaling in Renal Ischemia-Reperfusion Injury

The aim of this study was to investigate the possible beneficial effects of Fenofibrate on renal ischemia-reperfusion injury（IRI） in mice and its potential mechanism. IRI was induced by bilateral renal ischemia for 60 min followed by reperfusion for 24 h. Eighteen male C57BL/6 mice were randomly divided into three groups： sham-operated group（sham）, IRI＋saline group（IRI group）, IRI＋Fenofibrate（FEN） group. Normal saline or Fenofibrate（3 mg/kg） was intravenously injected 60 min before renal ischemia in IRI group and FEN group, respectively. Blood samples and renal tissues were collected at the end of reperfusion. The renal function, histopathologic changes, and the expression levels of pro-inflammatory cytokines [interleukin-8（IL-8）, tumor necrosis factor alpha（TNF-α） and IL-6] in serum and renal tissue homogenate were assessed. Moreover, the effects of Fenofibrate on activating phosphoinositide 3 kinase/protein kinase B（PI3K/Akt） signaling and peroxisome proliferator-activated receptor-α（PPAR-α） were also measured in renal IRI. The results showed that plasma levels of blood urea nitrogen and creatinine, histopathologic scores and the expression levels of TNF-α, IL-8 and IL-6 were significantly lower in FEN group than in IRI group. Moreover, Fenofibrate pretreatment could further induce PI3K/Akt signal pathway and PPAR-α activation following renal IRI. These findings indicated PPAR-α activation by Fenofibrate exerts protective effects on renal IRI in mice by suppressing inflammation via PI3K/Akt activation. Thus, Fenofibrate could be a novel therapeutic alternative in renal IRI.

早期食管鳞状细胞癌患者循环血清外泌体circFNDC3B表达水平及其临床意义

目的:分析癌前病变和早期食管鳞状细胞癌(ESCC)患者循环血清外泌体中Ⅲ型纤维连接蛋白域蛋白3B环状RNA(circFNDC3B)水平的差异,为ESCC发病机制的研究及早期筛查工作提供新的生物学标志分子。方法:收集2019年3月至2020年12月我院收治的74例早期ESCC患者、72例食管鳞状上皮内瘤变(EIN)患者和75例健康志愿者的血清样本,分别作为ESCC组、EIN组和正常组。提取并纯化血清外泌体,进行circRNAs微阵列分析,实时荧光定量PCR检测血清外泌体circFNDC3B表达水平。采用单因素和多因素Logistic模型分析早期ESCC发生的影响因素。采用受试者工作特征(ROC)曲线分析血清外泌体circFNDC3B水平对早期ESCC的诊断效能。结果:EIN组和ESCC组患者血清外泌体circFNDC3B表达水平均高于正常组,且ESCC组患者血清外泌体circFNDC3B表达水平高于EIN组(P<0.05)。经多因素Logistic回归分析,血清外泌体circFNDC3B表达水平升高是影响早期ESCC发生的独立危险因素(OR=4.221,95%CI 2.780~6.409,P<0.05)。经ROC曲线分析,血清外泌体circFNDC3B水平诊断早期ESCC的曲线下面积为0.870(95%CI 0.823~0.917),对应截断值为1.553,特异度为72.1%,敏感度为87.8%。结论:循环血清外泌体circFNDC3B高表达水平与ESCC上皮组织癌变有关,是发生ESCC的独立危险因素,对早期ESCC具有一定的诊断效能。

Y-box binding protein 1 augments sorafenib resistance via the PI3K/Akt signaling pathway in hepatocellular carcinoma

BACKGROUND Sorafenib is the first-line treatment for patients with advanced hepatocellular carcinoma(HCC).Y-box binding protein 1(YB-1)is closely correlated with tumors and drug resistance.However,the relationship between YB-1 and sorafenib resistance and the underlying mechanism in HCC remain unknown.AIM To explore the role and related mechanisms of YB-1 in mediating sorafenib resistance in HCC.METHODS The protein expression levels of YB-1 were assessed in human HCC tissues and adjacent nontumor tissues.Next,we constructed YB-1 overexpression and knockdown hepatocarcinoma cell lines with lentiviruses and stimulated these cell lines with different concentrations of sorafenib.Then,we detected the proliferation and apoptosis in these cells by terminal deoxynucleotidyl transferase dUTP nick end labeling,flow cytometry and Western blotting assays.We also constructed a xenograft tumor model to explore the effect of YB-1 on the efficacy of sorafenib in vivo.Moreover,we studied and verified the specific molecular mechanism of YB-1 mediating sorafenib resistance in hepatoma cells by digital gene expression sequencing(DGE-seq).RESULTS YB-1 protein levels were found to be higher in HCC tissues than in corresponding nontumor tissues.YB-1 suppressed the effect of sorafenib on cell proliferation and apoptosis.Consistently,the efficacy of sorafenib in vivo was enhanced after YB-1 was knocked down.Furthermore,KEGG pathway enrichment analysis of DGEseq demonstrated that the phosphoinositide-3-kinase(PI3K)/protein kinase B(Akt)signaling pathway was essential for the sorafenib resistance induced by YB-1.Subsequently,YB-1 interacted with two key proteins of the PI3K/Akt signaling pathway(Akt1 and PIK3R1)as shown by searching the BioGRID and HitPredict websites.Finally,YB-1 suppressed the inactivation of the PI3K/Akt signaling pathway induced by sorafenib,and the blockade of the PI3K/Akt signaling pathway by LY294002 mitigated YB-1-induced sorafenib resistance.CONCLUSION Overall,we concluded that YB-1 augments sorafenib resistance through the PI3K/Akt signaling pathway in HCC and suggest that YB-1 is a key drug resistance-related gene,which is of great significance for the application of sorafenib in advanced-stage HCC.

miR-409-3b通过下调表皮生长因子蛋白7抑制胃癌侵袭和转移的分子机制

目的:观察mi R-409-3b对胃癌细胞侵袭转移的影响,探讨mi R-409-3b通过下调表皮生长因子蛋白7(epidermal growth factor like domain protein 7,EGFL7)调节胃癌侵袭和转移的具体机制.方法:通过基因芯片技术筛选出胃癌及癌旁组织差异性表达的微小RNA(micro RNA m i R N A);采用生物学信息学技术预测E G F L7调控相关m i R N A,通过对比上述结果,筛选出mi R-409-3b;进一步以mi R-4093b慢病毒及mi R-409-3b mimics过表达mi R-409-3b后,用Western blot检测胃癌细胞中的EGFL7的表达变化;采用Transwell侵袭实验及划痕实验检测mi R-409-3b慢病毒感染后细胞体外侵袭能力的变化;q RT-PCR检测80例胃癌患者癌组织和癌旁组织中mi R-409-3b的表达差异,并分析mi R-409-3b的表达与胃癌患者临床病理之间的关系.结果:基因芯片及生物信息学预测发现m i R-409-3b在胃癌组织中低表达,同时又可能调控E G F L7,双荧光素酶实验证实m i R-409-3b可以结合在E G F L7上,m i R-409-3b慢病毒及mi R-409-3b mimics过表达m i R-409-3b都能在蛋白水平下调E G F L7;m i R-409-3b家族3条m i R N A的q RT-P C R结果表明癌旁组织相对含量高于胃癌组织;Tr a n s w e l l侵袭实验结果表明:m i R-409-3b与感染空载慢病毒相比感染能显著降低胃癌细胞穿的能力.体外划痕实验的结果表明,经LV-mi R-409-3b感染空载慢病毒比BGC-823细胞的迁移能力明显升高.进一步统计结果表明,mi R-409-3b与淋巴结转移有关(P<0.05),mi R-409-3b的C/P值在无远处转移的病例组织比具有远处转移的病例组织中明显升高(P<0.05).结论:mi R-409-3b可以在转录后水平调控EGFL7的表达,从而抑制胃癌细胞的侵袭和转移.