肝脏特异性miR-122的分子生物学特性及功能

miR-122是在肝脏特异高表达的一种microRNA。研究表明：生理状态下,miR-122 在调控肝脏的细胞发育、诱导细胞分化、调节细胞代谢、参与肝细胞应急应答等生命活动过程中发挥重要作用;而在病理状态下,miR-122 与丙型肝炎病毒（HCV）和肝细胞肝癌（HCC）密切相关,可能促进HCV RNA 复制,并在HCC发生、发展过程中发挥抑癌基因样作用,可能对HCC 临床诊断和预后具有重要价值。鉴于miR-122 参与调控肝脏生理及肝脏重大疾病的发生、发展等过程,文章详细阐述并讨论miR-122 在肝脏中的生物学特性和功能,以及可能的作用机制。肝脏特异性miR-122 有可能作为治疗人类肝脏疾病的关键靶点。     

人肝癌细胞系的糖蛋白质组学研究

糖基化是最重要的蛋白质翻译后形式之一,糖基化蛋白的糖链部分影响着蛋白质的折叠和稳定性以及其生物学功能.许多恶性肿瘤组织与正常组织相比已显示出蛋白质糖基化的差异.采用蛋白质组学分析方法结合先进的糖蛋白荧光染色技术,研究了正常人肝细胞系(ChangLiver)和人肝癌细胞系(Hep3B)糖蛋白糖基化的差异.首先用细胞裂解法提取细胞总蛋白质,进行双向电泳(2-DE),然后用pro-QEmerald488糖蛋白荧光染料进行糖蛋白染色,得到两种细胞系糖基化蛋白表达谱,经2-DE分析软件Dymension分析2-DE图像,比较糖蛋白的糖基化程度,并对糖基化蛋白进行质谱鉴定.结果显示正常人肝细胞表达(74±2)个(n=3),而人肝癌细胞系表达(78±3)个糖蛋白(n=3).两者匹配的糖蛋白质点31个,Hep3B表达而ChangLiver不表达的糖蛋白质点47个,ChangLiver表达而Hep3B不表达的糖蛋白质点43个.两种细胞系糖基化程度存在明显差异,与正常人肝细胞相比,肝癌细胞发生糖基化改变的糖蛋白有25个,其中糖基化水平上调的有10个,下调的有15个,质谱鉴定出12个发生糖基化改变的糖蛋白.这些结果显示蛋白质糖基化改变可能在肝癌的发生和发展中起一定作用.     

阿霉素诱导下的肝癌细胞HepG2中微小RNA差异表达分析

旨在研究阿霉素诱导引起的DNA损伤压力下,肝癌细胞Hep G2中参与DNA损伤应答的mi RNA,并分析这些mi RNA靶基因参与肝癌DNA损伤应答相关的生物学进程与通路。通过小RNA测序检测阿霉素处理肝癌细胞Hep G2前后mi RNA的差异表达情况,使用GO与KEGG通路富集方法对差异表达mi RNA靶基因进行功能富集分析。结果显示,共检测出显著表达差异mi RNA 68个,其中上调13个,下调55个。mi RNA靶基因的功能分析结果显示,53条mi RNAs靶基因显著富集于调控细胞增殖、细胞凋亡、细胞迁移和细胞周期等与DNA损伤应答以及肿瘤相关的生物进程和信号通路,包括p53信号通路、癌症通路、Wnt信号通路和MAPK信号通路等。研究表明,在阿霉素诱导下,Hep G2中的差异表达mi RNAs与DNA损伤相关的肿瘤生物学进程以及信号通路显著相关,预示这些mi RNAs在阿霉素引发的肝细胞癌DNA损伤应答中起着重要的作用。     

转录因子XBP1S基因表达谱差异分析

目的 应用基因表达谱芯片技术了解XBP1S在肝细胞中可能上调或下调的基因,了解其可能的调节功能线索.方法 构建pcDNA3.1（-）-XBP1S真核表达载体,转染HepG2细胞,同时以空载体pcDNA3.1（-）处理相同细胞系作为对照.48 h后制备细胞裂解液,提取mRNA,应用基因表达谱芯片技术对差异表达mRNA进行检测和分析.结果 构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误,提取高质量的总mRNA并进行逆转录成为cDNA,进行基因表达谱芯片技术分析.经过差异基因表达谱的筛选,发现HepG2细胞转染XBP1S以后,有38个基因表达水平显著上调,30个基因表达水平显著下调.结论 成功构建XBP1S的真核表达载体pcDNA3.1（-）-XBP1S,运用基因表达谱芯片技术成功筛选了XBP1S转染细胞后的差异表达基因,这些差异表达基因包括细胞周期、蛋白质的翻译合成及运输、能量代谢、体内免疫调节、细胞凋亡及细胞内的信号转导等方面起重要作用及肿瘤发生相关的基因,为进一步阐明XBP1S可能存在的调控机制及XBP1S蛋白可能的生物学功能提供理论依据.     

NOR1基因转染对肝癌细胞HepG2基因表达谱的影响

NOR1基因是一在正常组织中广泛表达且在肿瘤组织中表达下调的新基因.为进一步研究NOR1基因的功能和寻找其下游基因,利用脂质体技术将NOR1基因转染进HepG2细胞,采用cDNA微阵列技术分析其基因表达谱的改变.试验表明NOR1基因的转染能使Grb2,HBP17,TNFRSF11B等59个基因上调,同时也下调Bik,MAp2K6,ZFP95等103个基因.随后用实时荧光定量PCR对cDNA 微阵列结果中上述3个上调表达基因进行验证,结果表明,基因表达差异具有统计学意义(P<0.05),荧光定量PCR结果与微阵列结果相符.这些结果提示,NOR1基因对肝癌HepG2细胞的生物学行为的影响可能与它对细胞信号转导,细胞周期调控,转录、翻译调控相关基因的表达影响有关.     

人肝脏特异性miR-122表达载体的构建及鉴定

人肝脏特异性miRNA-122是肝脏中表达丰度最高的miRNA。为研究该miR-122的生物学功能,从HepG2细胞基因组中用PCR的方法扩增了miR-122的前体,构建了miR-122的表达载体pLMP-miR-122。pLMP-miR-122质粒转染人正常肝细胞系L-O2和肝癌细胞系HepG2后,细胞内成熟miRNA-122的表达量显著增加。该质粒与HBV1.3共转染HepG2细胞72h后,HBV的HBs和HBe蛋白水平的表达量均下降,说明miRNA-122参与了HBV基因的复制和表达的调控,为进一步研究miRNA-122的功能和其他一些肝病如HCC的调控机制打下基础。     

电脉冲刺激法提高小鼠miR-122表达的研究

目的:电脉冲能够改变细胞膜的通透性,促进外源DNA进入细胞内。通过基因导入仪电脉冲刺激已注入质粒pcDNA3.0-miR-122的小鼠肌肉细胞,检测对小鼠miR-122表达的影响。方法:将扩增得到的pre-miR-122克隆到真核表达载体pcDNA3.0上,构建pcDNA3.0-miR-122表达质粒。利用基因导入仪分别将生理盐水、pcDNA3.0、pcDNA3.0-miR-122质粒DNA导入小鼠腿部肌肉,然后进行电脉冲刺激。qRT-PCR测定各组小鼠血清及肝脏中的miR-122水平,同时检测肝癌组织和经Cecropin XJ处理的肝癌组织中的miR-122水平。结果:基因导入仪电脉冲刺激已注入pcDNA3.0-miR-122的腿部肌肉组织后,小鼠血清和肝脏中miR-122的表达分别提高了23和11倍,经Cecropin XJ处理的小鼠肝癌组织中miR-122的表达比对照高1.6倍。结论:电脉冲刺激能够显著提高小鼠体内miR-122的表达,为探讨miR-122表达对肝癌生长影响的调控作用奠定基础。     

miR-203在肝细胞肝癌中的表达及其对肿瘤细胞增殖及侵袭能力的影响

目的:探讨miR-203在肝细胞肝癌(Hepatocellular carcinoma,HCC)患者血浆中的表达及其对肝癌细胞增殖和侵袭能力的影响。方法:选择2013年5月-2015年7月在我院确诊为肝细胞肝癌的患者57例作为研究对象,另选取同期在我院接受健康体检的志愿者49例作为对照组。采用实时荧光定量RT-PCR法检测两组血浆中miR-203的表达水平;将过表达的miR-203转染至人肝癌细胞系Hep 3B和JHH-1;采用CCK-8法检测miR-203对肝癌细胞增殖能力的影响;采用transwell法检测miR-203对肝癌细胞迁移及侵袭能力的影响。结果:miR-203在肝癌患者血浆中的表达水平显著低于对照组,差异具有统计学意义(P0.01);与未转染的肝癌细胞比较,Hep 3B和JHH-1细胞转染miR-203mimic后,肿瘤细胞的增殖、迁移及侵袭能力受到明显抑制,差异具有统计学意义(P0.05)。结论:miR-203在肝细胞肝癌中呈低表达,而过表达miR-203能够抑制肿瘤细胞的增殖及侵袭能力,提示miR-203可以作为一种新的肿瘤抑制性micro RNA。     

红色毛癣菌不同生长阶段的基因表达谱分析

红色毛癣菌是最重要的浅表感染真菌.目前对红色毛癣菌分子和遗传学方面的研究还比较有限.应用cDNA芯片技术对红色毛癣菌不同生长阶段的基因表达谱进行了分析,获得了2044个在不同生长阶段差异表达的基因.聚类分析表明,这些基因可以分为3种不同的表达模式.本研究验证了先前的结论,即在红色毛癣菌中存在预储存的mRNA.对不同生长状态下转录谱和基因功能分析表明,糖酵解过程中的一些重要的酶具有相似的表达模式,说明在红色毛癣菌的生长过程中,这些酶可能具有共同的调控模式.同时,在红色毛癣菌生长过程中,一些参与诸如小GTPase调控、cAMP依赖的调控途径以及MAPK信号传导途径的基因表达也发生了变化.尽管这些基因和相关的调控途径在红色毛癣菌中的具体生物学功能还不清楚,但它们可能在其生长过程中起了调控作用.     

miR-93通过靶定PTEN基因增加肝癌细胞对奥沙利铂的耐药性

微小RNA（miRNA）参与了肿瘤的耐药过程.本研究通过建立对奥沙利铂 (oxaliplatin,Oxa)耐药的肝癌细胞系BEL-7402/Oxa和Hep-3B/Oxa,利用miRNA芯片结合实时荧光定量PCR的方法,筛选得到数个参与肝癌细胞对奥沙利铂耐药的miRNA 分子,其中miR-93表达增加最为明显.MTT实验发现,增加肝癌细胞株中miR-93的表达可以增强其对奥沙利铂的耐药性.进一步结合生物信息学、荧光报告载体及免疫印迹实验,证实miR-93通过靶定抑癌基因PTEN增加肝癌细胞对奥沙利铂的耐药性.总之,肝癌耐药细胞系的建立及其miRNA差异表达谱的分析,以及miRNA分子对肝癌细胞发生奥沙利铂耐药的具体作用及其分子机制的研究,不仅有助于理解肝癌细胞发生耐药的分子机制,而且为探索克服肝癌对奥沙利铂耐药性的有效途径提供可靠依据.     

肝细胞癌患者血清外泌体microRNA表达鉴定

目的:探讨肝癌细胞外泌体中差异表达的microRNAs(miRNAs)在肝细胞癌(HCC)诊断中的应用价值。方法:通过高通量测序筛选肝癌细胞外泌体中差异表达的miRNAs。实时定量PCR验证差异表达分子;检测差异表达的miRNAs在健康人(Health)、慢性乙型肝炎患者(CHB)、肝硬化患者(LC)及乙型肝炎病毒阳性的肝细胞癌患者(HCC)血清外泌体中的表达。结果:高通量测序筛选到肝癌细胞外泌体中差异表达的miRNA共88种,其中58种表达上调,30种表达下调。选择其中8种差异表达的miRNAs进行q RT-PCR验证,结果显示,此8种miRNAs在细胞上清外泌体、细胞内、癌与癌旁组织中的表达趋势与测序结果一致。miR-221-3p和miR-224-5p在HCC组外泌体中的表达水平显著高于Health组、CHB组和LC组(P0.01),miR-124-3p和let-7a-5p在HCC组外泌体中的表达水平显著低于其他各组(P0.05)。四个组中,miR-21-5p、miR-191-5p、miR-34a-5p和miR-122-5p的表达水平不存在显著性差异(P0.05)。结论:血清外泌体中的miR-221-3p、miR-224-5p、miR-124-3p和let-7a-5p可能成为肝细胞癌的候选标志物。     

Investigation of key microRNAs associated with hepatocellular carcinoma using small RNA-seq data

To identify key microRNAs (miRNAs) associated with hepatocellular carcinoma (HCC) using small RNA-seq data. Small RNA-seq data for two HCC samples and two normal samples were downloaded from NCBI Gene Expression Omnibus. MiRNAs were identified through database search. Differentially expressed miRNAs were screened out with t test and their target genes were retrieved. Functional enrichment analysis was performed to uncover their biological functions. Regulatory networks and core metabolic networks were also constructed to present the global patterns. In addition, new miRNAs and their target genes were predicted. A total of 59 differentially expressed miRNAs were obtained, 12 up-regulated and 47 down-regulated. A total of 3,306 target genes were retrieved for eight miRNAs. Pathway enrichment analysis for the target genes showed that “pathways in cancer” and “MAPK signaling pathway” were significantly over-represented. Functional enrichment analysis found that “biological regulation” and “macromolecule modification” were significantly related to the target genes. Two regulatory networks were constructed for up- and down-regulated differentially expressed miRNAs with information from Ingenuity Pathway Analysis database. Two metabolic networks were also established based upon “pathways in cancer” and “MAPK signaling pathway”, consisting of miRNAs, target genes, compounds and others genes. Moreover, a number of new miRNAs and relevant target genes were predicted. Our study discloses a number of miRNAs as well as genes which may be involved in the development of HCC and these findings are beneficial in guiding future researches.     

MiR-199a Regulates Cell Proliferation and Survival by Targeting FZD7

A growing amount of evidence indicates that miRNAs are important regulators of multiple cellular processes and, when expressed aberrantly in different types of cancer such as hepatocellular carcinoma (HCC), play significant roles in tumorigenesis and progression. Aberrant expression of miR-199a-5p (also called miR-199a) was found to contribute to carcinogenesis in different types of cancer, including HCC. However, the precise molecular mechanism is not yet fully understood. The present study showed that miR-199a is frequently down-regulated in HCC tissues and cells. Importantly, lower expression of miR-199a was significantly correlated with the malignant potential and poor prognosis of HCC, and restoration of miR-199a in HCC cells led to inhibition of the cell proliferation and cell cycle in vitro and in vivo. Furthermore, Frizzled type 7 receptor (FZD7), the most important Wnt receptor involved in cancer development and progression, was identified as a functional target of miR-199a. In addition, these findings were further strengthened by results showing that expression of FZD7 was inversely correlated with miR-199a in both HCC tissues and cells and that over-expression of miR-199a could significantly down-regulate the expression of genes downstream of FZD7, including β-catenin, Jun, Cyclin D1 and Myc. In conclusion, these findings not only help us to better elucidate the molecular mechanisms of hepatocarcinogenesis from a fresh perspective but also provide a new theoretical basis to further investigate miR-199a as a potential biomarker and a promising approach for HCC treatment.     

miRNA expression profile during osteogenic differentiation of human adipose-derived stem cells

Human adipose-derived stem cells (hADSC) are capable of differentiating into an osteogenic lineage. It is believed that microRNAs (miRNAs) play important roles in regulating this osteogenic differentiation of human adipose-derived cells, although its molecular mechanism remains unclear. We investigated the miRNA expression profile during osteogenic differentiation of hADSCs, and assessed the roles of involved miRNAs during the osteogenic differentiation. We obtained and cultured human adipose-derived stems cells from donors who underwent elective liposuction or other abdominal surgery at our institution. miRNA expression profiles pre- and post-osteogenic induction were obtained using microarray essay, and differently expressed miRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of osteogenic proteins was detected using an enzyme-linked immunosorbent assay. Putative targets of the miRNAs were predicted using online software MiRanda, TargetScan, and miRBase. Eight miRNAs were found differently expressed pre- and post-osteogenic induction, among which four miRNAs (miR-17, miR-20a, miR-20b, and miR-106a) were up-regulated and four miRNAs (miR-31, miR-125a-5p, miR-125b, and miR-193a) were down-regulated. qRT-PCR analysis further confirmed the results. Predicted target genes of the differentially expressed miRNAs based on the overlap from three public prediction algorithms: MiRanda, TargetScan, and miRBase Target have the known functions of regulating stem cell osteogenic differentiation, self-renewal, signal transduction, and cell cycle control. We identified a group of miRNAs that may play important roles in regulating hADSC cell differentiation toward an osteoblast lineage. Further study of these miRNAs may elucidate the mechanism of hADSC differentiation into adipose tissue, and thus provide basis for tissue engineering.     

Retracted: Long noncoding LINC01551 promotes hepatocellular carcinoma cell proliferation,migration, and invasion by acting as a competing endogenous RNA of microRNA-122-5p to regulate ADAM10 expression

Hepatocellular carcinoma (HCC) is a severe disease with high mortality in the world. It has been shown that long noncoding RNA (lncRNA) might play a role in HCC. The aim of the present study was to identify the role of long intergenic noncoding RNA 01551 (LINC01551) in the HCC development and explore the underlying mechanism of LINC01551/miR-122-5p/ADAM10 axis. The differentially expressed lncRNAs associated with HCC were screened out by a microarray analysis. The expression of LINC01551, miR-122-5p, and ADAM10 was determined in HCC tissues and cells. The potential miRNA (miR-122-5p) regulated by LINC01551 was explored, and the target relationship between miR-122-5p and ADAM10 was confirmed. To evaluate the effect of LINC01551 and miR-122-5p on proliferation, migration, invasion, and apoptosis of HCC, different plasmids were delivered into MHCC97-H cells. High expression of LINC01551 and ADAM10 yet low-expression of miR-122-5p were revealed in HCC tissues and cells. Overexpression of miR-122-5p could downregulate ADAM10. Biological prediction websites and fluorescence in situ hybridization assay verified that LINC01551 was mainly expressed in the cytoplasm. Silencing LINC01551 reduced HCC cell viability, proliferation, migration, invasion, and cell cycle entry yet induce cell apoptosis. Upregulation of LINC01551 increased its ability of competitively binding to miR-122-5p, thus reducing miR-122-5p and upregulating ADAM10 expression, as well as promoting the proliferative, migrative, and invasive ability. Taken together the results, it is highly possible that LINC01551 functions as an competing endogenous RNA (ceRNA) to regulate the miRNA target ADAM10 by sponging miR-122-5p and therefore promotes the development of HCC, highlighting a promising competitive new target for the HCC treatment.     

miR-150-5p Inhibits Hepatoma Cell Migration and Invasion by Targeting MMP14

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Despite progress in diagnostics and treatment of HCC, its prognosis remains poor because the molecular mechanisms underlying hepatocarcinogenesis are not well understood. In the study, we focused on identifying the role of miRNAs in HCC progression. miRNA microarray was used to analyze the differentially expressed miRNAs, and the results were validated by qPCR. We found that the miR-150-5p expression is down-regulated in HCC tissues compared with pair non-tumor tissues. miR-150-5p expression is also decreased in metastatic cancer tissues compared with pair primary tissues, indicating that miR-150-5p may be involved in HCC metastasis. Functionally, miR-150-5p inhibition significantly promotes hepatoma cell migration and invasion, whereas miR-150-5p overexpression suppresses cancer cell migration and invasion in vitro. The matrix metalloproteinase 14 (MMP14) is identified as a new target gene of miR-150-5p. miR-150-5p markedly inhibits MMP14 expression in hepatoma cells, and miR-150-5p expression is negative correlation with MMP14 expression in vivo. More important, re-expression of MMP14 in hepatoma cells partially reverses the effect of miR-150-5p in inhibiting cell invasion.