微小RNA-194-5p与cullin4A在肺结核病人中的表达及意义

目的探讨微小RNA-194-5p(miR-194-5p)与泛素连接酶cullin4A(CUL4A)在肺结核病人中的表达及其意义。方法选取2016年12月至2018年12月中国平煤神马集团职业病防治院结核病病人为研究对象,确诊为肺结核病人50例作为肺结核组,潜伏结核感染病人50例作为潜伏结核感染组,同期该院体检健康者50例作为对照组。实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测miR-194-5p、CUL4A mRNA、鸟苷酸结合蛋白5(GBP5)mRNA的表达;酶联免疫吸附测定(ELISA)检测可溶性Tim-3(sTim-3)、可溶性Gal-9(sGal-9)水平;采用Pearson相关性分析检验肺结核病人miR-194-5p、CUL4A表达量与GBP5、sTim-3、sGal-9的相关性。结核分枝杆菌(Mtb)感染人巨噬细胞系U937,ELISA检测白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)水平。蛋白质印迹法(Western blotting)检测Wnt/β-连环素(Wnt/β-catenin)信号通路相关蛋白T细胞因子(TCF)、糖原合成酶激酶-3(GSK-3β)、β-catenin表达量。结果与对照组相比,潜伏结核感染组与肺结核组病人血清miR-194-5p、GBP5 mRNA的表达水平升高(P<0.05),其中miR-194-5p:(1.01±0.23)比(2.32±0.53)比(3.35±0.20),sTim-3、sGal-9水平升高(P<0.05),CUL4A mRNA的表达水平显著降低[(1.03±0.11)比(0.73±0.12)比(0.41±0.15),P<0.05],潜伏结核感染组与肺结核组各指标间相比差异有统计学意义(P<0.05);miR-194-5p与CUL4A呈负相关(r=-0.421,P<0.001),miR-194-5p与GBP5、sTim-3、sGal-9呈正相关(P<0.05);Mtb感染细胞后,IL-6、TNF-α的表达水平升高(P<0.05),TCF、GSK-3β、β-catenin表达量升高(P<0.05)。结论miR-194-5p在肺结核病人中表达上调,CUL4A表达下调,二者可能参与肺结核发生及发展过程。

泛素连接酶Cullin4A与恶性肿瘤关系的研究进展

Cullin4A(Cul4A)是新近发现的一类E3泛素连接酶,在底物蛋白的泛素化过程中发挥重要作用。Cul4A在多种恶性肿瘤中异常表达,且与恶性肿瘤的浸润、转移密切相关。本文就Cul4A的基本结构、功能作用及其与恶性肿瘤关系的研究进展作一综述。

CUL4B、TRPM2在胰腺癌组织中的表达和临床意义

目的探讨胰腺癌组织中E3泛素连接酶Cullin4B(CUL4B)、人瞬时受体电位M2(TRPM2)的表达及临床意义。方法收集2019年3月—2020年3月在西安交通大学医学院附属3201医院接受手术治疗胰腺癌患者86例的癌组织和癌旁组织。采用免疫组织化学检测组织中CUL4B、TRPM2蛋白表达。采用实时荧光定量PCR检测组织中CUL4B、TRPM2 mRNA水平。Spearman秩相关分析CUL4B与TRPM2蛋白表达的关系。Kaplan-Meier曲线分析CUL4B、TRPM2蛋白表达对患者预后的影响。Cox比例风险模型分析胰腺癌预后影响因素。结果胰腺癌组织中CUL4B、TRPM2蛋白阳性率及mRNA基因的相对表达量高于癌旁组织(χ^(2)/t=70.245、60.224、15.741、10.976,P均<0.001)。胰腺癌组织中CUL4B与TRPM2蛋白表达呈显著正相关(r=0.720,P<0.001)。TNM分期ⅡB~Ⅲ期、淋巴结转移胰腺癌中CUL4B、TRPM2蛋白阳性率高于Ⅰ~ⅡA期、无淋巴结转移癌组织(χ^(2)=16.511、14.834,15.576、15.007,P均<0.001)。CUL4B阳性组和阴性组3年总生存率分别为9.68%(6/62)、41.67%(10/24),TRPM2阳性组和阴性组3年总生存率分别为8.33%(5/60)、42.31%(11/26),CUL4B阳性组、TRPM2阳性组患者3年累积生存率低于CUL4B阴性组、TRPM2阴性组患者(Log-Rankχ^(2)/P=9.933/0.002、11.102/0.001)。TNM分期ⅡB~Ⅲ期、淋巴结转移、CUL4B阳性、TRPM2阳性是影响胰腺癌不良预后的独立危险因素[HR(95%CI)=1.781(1.199~2.646),1.962(1.172~3.285),2.482(1.445~4.263),1.733(1.223~2.457)]。结论胰腺癌组织中CUL4B、TRPM2表达升高,两者表达与胰腺癌不良临床病理特征有关,是胰腺癌预后相关肿瘤标志物。

Cullin蛋白4B在非小细胞肺癌中的表达及其临床意义和细胞功能分析

目的探讨Cullin蛋白4B(CUL4B)在非小细胞肺癌(NSCLC)组织中的表达及其对NSCLC细胞生物功能的影响。方法采用免疫组化、实时荧光定量PCR(qPCR)检测77例NSCLC组织及42例癌旁正常组织中的CUL4B表达。qPCR检测常见NSCLC细胞株(H358、H460、H1299、A549和SK-MES-1)中的CUL4B mRNA水平,选取表达最高的细胞株分别转染CUL4B siRNA(CUL4B干扰组)和CUL4B阴性对照siRNA(阴性对照组)的慢病毒载体,qPCR验证转染效率;采用MTT法检测干扰CUL4B表达24、48、72、96 h对细胞增殖的影响,流式细胞术和Transwell法检测干扰CUL4B表达48 h对细胞凋亡及侵袭能力的影响。结果 CUL4B蛋白在NSCLC组织及癌旁正常组织中的高表达率分别为63.6%(49/77)和38.1%(16/42),差异有统计学意义(P<0.05);CUL4B表达与肿瘤直径、淋巴结转移及临床分期均有关(P<0.05),而与性别、年龄、病理分型及组织分级无关(P>0.05);qPCR检测NSCLC组织中CUL4B mRNA相对水平为2.713±0.246,经干扰CUL4B表达,NSCLC细胞增殖能力明显减弱,细胞凋亡率增加且侵袭转移能力降低。结论 NSCLC组织及细胞株中CUL4B蛋白表达均明显升高,NSCLC组织CUL4B表达与淋巴结转移、临床分期及细胞分化有关;沉默CUL4B表达可影响NSCLC细胞的增殖、凋亡与侵袭。

宫颈上皮内瘤变及宫颈鳞癌组织中p16、CUL4B表达观察

目的观察宫颈上皮内瘤变(CIN)及宫颈鳞癌组织中p16、Cullin4B(CUL4B)的表达变化。方法选取正常宫颈组织、CIN1、CIN2、CIN3、宫颈鳞癌组织标本共计173例份,分为正常宫颈组30例、CIN1组33例、CIN2组38例、CIN3组40例、宫颈鳞癌组32例。采用免疫组化法检测各组中的p16、CUL4B。结果正常宫颈组、CIN1组、CIN2组、CIN3组、宫颈鳞癌组p16蛋白表达阳性分别为1(3.33%)、13(39.39%)、30(78.95%)、40(100%)、32(100%)例;CUL4B表达阳性分别为0、11(33.33%)、29(76.32%)、40(100%)、32(100%)例。CIN各组和宫颈鳞癌组p16、CUL4B蛋白阳性表达率高于正常宫颈组,其中p16、CUL4B在CIN1、2、3组中的阳性表达率依次增高,在宫颈鳞癌组中的阳性表达率高于CIN1组和CIN2组(P均<0.05)。p16、CUL4B在宫颈病变组织中的表达呈正相关(r=0.969,P<0.01)。结论 CIN及宫颈鳞癌组织中p16、CUL4B表达高于正常宫颈组织,且随着宫颈病变程度加重而增高。p16、CUL4B表达可能与宫颈病变的发生和进展有关,二者之间可能存在相互作用。

Cullin 4A is associated with epithelial to mesenchymal transition and poor prognosis in perihilar cholangiocarcinoma

AIM To explore the functional role of cullin 4A(CUL4A), a core subunit of E3 ubiquitin ligase, in perihilar cholangiocarcinoma(PHCC).METHODS The expression of CUL4 A in PHCC cell lines was evaluated by Western blot and quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry(IHC) was adopted to investigate the relationship between CUL4 A expression and clinicopathological characteristics of PHCC. Univariate analysis and multivariate regression analysis were performed to analyze the risk factors related to overall survival(OS) and progression-free survival(PFS) of PHCC patients. Wound healing, Transwell and Matrigel assays were utilized to explore the function of CUL4 A in PHCC metastasis. Furthermore, expression of epithelial to mesenchymal transition(EMT) markers was verified in cells with CUL4 A knockdown or overexpression. The relationship between CUL4 A expression and E-cadherin expression was also analyzed by IHC assay. Finally, the role of ZEB1 in regulating CUL4 A mediated PHCC was detected by IHC, Western blot, Transwell and Matrigel assays.RESULTS CUL4 A overexpression was detected in PHCC cell lines and clinical specimens. Clinicopathological analysis revealed a close correlation between CUL4 A overexpression and tumour differentiation, T, N and TNM stages in PHCC. Kaplan-Meier analysis revealed that high CUL4 A expression was correlated with poor OS and PFS of PHCC patients. Univariate analysis identified the following four parameters as risk factors related to OS rate of PHCC: T, N, TNM stages and high CUL4 A expression; as well as three related to PFS: N stage, TNM stage and high CUL4 A expression. Further multivariate logistic regression analysis identified high CUL4 A expression as the only independent prognostic factor for PHCC. Moreover, CUL4 A silencing in PHCC cell lines dramatically inhibited metastasis and the EMT. Conversely, CUL4 A overexpression promoted these processes. Mechanistically, ZEB1 was discovered to regulate the function of CUL4 A in promoting the EMT and metastasis.CONCLUSION CUL4 A is an independent prognostic factor for PHCC, and it can promote the EMT by regulating ZEB1 expression. CUL4 A may be a potential therapeutic target for PHCC.

Lentivirus-mediated short hairpin RNA interference of CENPK inhibits growth of colorectal cancer cells with overexpression of Cullin 4A

BACKGROUND Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative.Immunohistochemical analysis has revealed high expression of centromere protein K(CENPK)in CRC.However,the role of CENPK in the progression of CRC is not well characterized.AIM To evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A(CUL4A)in RKO and HCT116 cells.METHODS Human colon cancer samples were collected and tested using a human gene expression chip.We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis.In vitro experiments verified the function of this gene.We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction(qPCR),western blot,and flow cytometry.The effect of short hairpin RNA(shRNA)virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging.To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells,we performed a series of in vitro experiments,using qPCR,western blot,MTT assay,and flow cytometry.RESULTS We demonstrated overexpression of CENPK in human colon cancer samples.CENPK was an independent risk factor in patients with CRC.The downstream genes FBX32,CUL4A,and Yesassociated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells.Significantly delayed xenograft tumor emergence,slower growth rate,and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group.The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo.The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells,with overexpression of the CUL4A.CONCLUSION We indicated a potential role of CENPK in promoting tumor proliferation,and it may be a novel diagnostic and prognostic biomarker for CRC.

Cullin 4B在结直肠癌中表达与β-catenin关系及临床意义

目的研究Cullin 4B(Cul4B)在结直肠癌中表达情况及其临床意义,探讨其表达与β-catenin表达的相关性。方法应用免疫组织化学方法检测83例结直肠癌组织、正常结直肠组织中Cul4B的表达,同时检测83例结直肠癌组织中β-catenin的表达情况。结合临床病理情况进行统计学分析。结果 83例结直肠癌组织中Cul4B高表达率为74.7%,显著高于正常结直肠组织中的高表达率(39.8%,P<0.05),且Cul4B高表达与肿瘤TNM分期及淋巴结转移密切相关。β-catenin在结直肠癌中阳性率为66.3%,与Cul4B表达呈正相关(r=0.464,P<0.01)。结论 Cul4B在结直肠癌中表达与β-catenin呈正相关。

MACC1与CUL4B蛋白在子宫内膜样腺癌中的表达及临床意义

目的探讨子宫内膜癌组织中结肠癌转移相关基因-1(MACC1)及Cullin蛋白4B(CUL4B)的表达及其临床意义。方法应用免疫组织化学(S-P)法检测正常子宫内膜组织及子宫内膜癌组织中MACC1和CUL4B蛋白的表达,分析其与子宫内膜癌患者临床病理特征之间的关系。结果子宫内膜癌组织中MACC1蛋白的表达水平与正常子宫内膜组织之间无明显差异(P>0.05)。CUL4B蛋白在子宫内膜癌组织中的表达明显高于正常内膜,差异有统计学意义(P=0.007),其阳性表达率与子宫内膜癌患者的FIGO分期、组织分化程度相关(P<0.001)。结论 MACC1基因可能不是子宫内膜癌变的重要影响因子。CUL4B基因的表达与子宫内膜癌患者的临床分期、组织分化程度和淋巴结转移相关,CUL4B可能参与了子宫内膜癌的发生和发展。