MicroRNA-27a通过靶向HBx mRNA抑制HBV复制

目的 探究微小RNA-27a(microRNA-27a,miR-27a)在HBV感染中的作用及分子机制.方法 实时定量PCR(quantitative real-time PCR,qRT-PCR)方法检测miR-27a对HBV DNA和HBV RNA的影响.ELISA法检测miR-27a对HBeAg和HBsAg的影响.生物信息学预测miR-27a的靶基因,然后用双荧光素酶报告基因检测、qRT-PCR和western blot检测miR-27a是否能够直接靶向靶基因以及对靶基因mRNA和蛋白表达的影响.最后用qRT-PCR检测HBV蛋白表达对miR-27a的影响.结果 MiR-27a能抑制HBV DNA、HBV RNA、HBeAg和HBsAg等HBV复制和表达的指标.生物信息学预测和双荧光素酶报告基因检测表明miR-27a能够直接靶向HBx,抑制HBx mRNA和蛋白表达.反之,HBx蛋白表达能抑制细胞内miR-27a的表达.结论 MiR-27a通过直接靶向HBx进而抑制HBV的复制和表达,可以作为一种新的治疗HBV感染的潜在靶点.     

miR-302 b-3 p靶向AKT1调节皮肤成纤维细胞衰老的作用机制

目的 探讨miR-302b-3p靶向丝/苏氨酸蛋白激酶(AKT)1调节皮肤成纤维细胞衰老的作用及其分子机制.方法 建立复制性细胞衰老模型后,采用real-time PCR法检测细胞miR-302b-3p的表达情况;采用生物信息学软件分析miR-302b-3p的靶基因;分别将miR-302b-3p模拟物(mimic)及抑制剂(inhibitor)转染皮肤成纤维细胞后,β-半乳糖苷酶染色分析细胞衰老情况,qRT-PCR法检测AKT1 mRNA表达水平,Western印迹法检测AKT1蛋白表达情况.结果 与对照组比较,复制性衰老皮肤成纤维细胞中miR-302b-3p显著升高.转染miR-302b-3p mimic后,细胞衰老阳性染色细胞数目显著增加(P<0.01).AKT1为miR-302b-3p的预测靶基因.当细胞上调miR-302b-3p表达时,靶基因AKT1 mRNA及蛋白水平显著降低(P<0.05).反之,当细胞下调miR-302b-3p表达时,靶基因AKT1 mRNA及蛋白水平显著升高(P<0.05).结论 miR-302b-3p可能通过靶向抑制AKT1表达调节皮肤成纤维细胞的衰老.     

miR-515-5p通过靶向Wnt3抑制胃癌细胞增殖和侵袭

目的旨在研究miR-515-5p在调控胃癌进展过程中的作用及机制。方法通过qRT-PCR实验检测胃癌患者标本和胃癌细胞系中miR-515-5p的表达情况。将miR-515-5p模拟物转染至人MGC-803和SGC-7901细胞系。采用CCK-8和transwell实验检测细胞增殖和侵袭能力。qRT-PCR和Western blot实验检测相关mRNA和蛋白表达情况。荧光素酶报告基因实验证实miR-515-5p和Wnt3的靶向关系。结果 miR-515-5p在人胃癌组织和细胞系中的表达水平下调。过表达miR-515-5p可显著抑制胃癌细胞的增殖和侵袭能力。双荧光素报告实验证明Wnt3是miR-515-5p在胃癌细胞中的直接作用靶点。同时,拯救实验证明过表达Wnt3可以逆转miR-515-5p抑制胃癌细胞增殖和侵袭的能力。结论 miR-515-5p可以通过靶向Wnt3抑制胃癌细胞增殖和侵袭。     

UCA1-miR-582-EZH2轴对肺癌细胞A549生物学功能的影响机制

目的 探究尿路上皮癌相关1基因(UCA1)调控miR-582-5p-Zeste增强子同源物(EZH)2信号轴在非小细胞肺癌(NSCLC)中的调控机制。方法 逆转录-聚合酶链式反应(qRT-PCR)检测UCA1、miR-582-5p和EZH2在NSCLC组织及细胞系(NCI-H460、A549和NCI-H1299)中的表达;Western印迹检测EZH2在NSCLC组织中的蛋白表达;原位杂交(FISH)检测UCA1和miR-582-5p的信号强度;双荧光素酶报告基因检验miR-582-5p抑制剂靶向UCA1和EZH2的调控机制;qRT-PCR检测下调UCA1/miR-582-5p对EZH2 mRNA的调控关系;Transwell、TUNEL细胞凋亡和CCK8分别检测下调UCA1-miR-582-5p-EZH2轴对侵袭、凋亡和增殖的调控能力。结果 UCA1和EZH2在NSCLC组织和细胞系中高表达,miR-582-5p则相反为低表达。UCA1竞争性结合miR-582-5p,且miR-582-5p抑制剂可以回补siUCA1负调控EZH2。siUCA1-In-miR-582-5p-siEZH2...     

靶向ASGPR1的外源性microRNA对HBV表达和复制的抑制作用

目的:设计并构建ASGPR1靶向的mieroRNA表达载体,观察其对ASGPR1基因的抑制作用及其在HBV感染基因治疗中的应用价值.方法:以ASGPR1为靶基因.设计并构建3个针对ASGPR1不同位点的microRNA表达栽体.通过脂质体方法转染HepG2.2.15细胞,RTPCR和Western blot检测其对ASGPR1 mRNA和蛋白的抑制作用,乙肝五项定量和HBVDNA检测其对HBV的抑制作用.结果:ASGPR1 mRNA和蛋白的平均水平分别下降了57.3%和49.8%(P＜0.01);在病毒水平3种amiRNA均能明显抑制相应细胞株中HBsAg和HBeAg的分泌,其中以amiRNAASGPR1-610抑制作用最强,对培养72 h的细胞上清中的HBsAg和HBeAg抑制率分别为31.3%和33.6%(P＜0.01),HBV DNA的抑制率为29.7%(P＜0.01).结论:靶向ASGPR1的外源性microRNA能显著抑制靶基因的表达,进而抑制HBV的复制和表达.ASGPR1可以作为慢性HBV感染基因治疗的候选靶点.     

miR-142-5p靶向调控TGF-β2促进人巨噬细胞凋亡

目的探讨miR-142-5p在动脉粥样硬化组织中的表达及对人巨噬细胞凋亡的作用。方法构建动脉粥样硬化大鼠模型,qRT-PCR检测动脉粥样硬化组织中miR-142-5p的表达水平。50μg/L氧化型低密度脂蛋白(ox-LDL)刺激人巨噬细胞、血管平滑肌细胞(VSMC)、内皮细胞24 h后,提取细胞RNA,qRT-PCR检测细胞中miR-142-5p的表达水平。靶基因预测软件预测miR-142-5p的靶基因,双荧光素酶报告基因鉴定靶基因的正确性。细胞转染miR-142-5p mimic、mimic control、miR-142-5p inhibitor、inhibitor control,Western blot和qRT-PCR检测miR-142-5p对靶基因的调控作用,流式细胞仪检测miR-142-5p和靶基因对巨噬细胞凋亡的作用。结果 miR-142-5p在动脉粥样硬化组织中表达上调,与正常组织相比差异显著(P0.01)。血管平滑肌细胞和内皮细胞经ox-LDL刺激后miR-142-5p的表达水平与刺激前没有明显变化,而巨噬细胞经ox-LDL刺激后miR-142-5p的表达水平较刺激前明显升高(P0.01)。预测miR-142-5p的靶基因为转化生长因子β2(TGF-β2)。miR-142-5p mimic与TGF-β2共转染后荧光素酶活性最低;miR-142-5p mimic组TGF-β2蛋白和mRNA的表达水平与mimic control组相比明显下降,miR-142-5p inhibitor组TGF-β2蛋白和mRNA的表达水平与inhibitor control组相比明显升高(P0.01);miR-142-5p mimic组细胞凋亡率明显高于mimic control组,TGF-β2 siRNA组细胞凋亡率明显高于siRNA control组(P0.01)。结论 miR-142-5p在动脉粥样硬化中过度表达,miR-142-5p通过下调靶基因TGF-β2促进人巨噬细胞凋亡。     

聚合酶链反应扩增小分子干扰RNA表达框筛选抑制HBV DNA复制的小分子干扰RNA

一些短片段的双链RNA可以通过促使特定基因的mRNA降解来高效、特异地阻断体内特定基因表达，诱使细胞表现出特定基因缺失的表型，称为RNA干扰（RNAi）。小分子干扰RNA（siRNA）就是这种短片段双链RNA分子，能够以序列同源互补的mRNA为靶目标，降解特定的mRNA。本研究针对HBV DNA多聚酶区的siRNA，运用PCR扩增siRNA表达框的方法筛选出对HBV DNA抑制作用最强的siRNA，观察其对HBV DNA复制及HBsAg和HBeAg分泌的抑制作用。     

IL-29通过调控lncRNA DLEU1/miR-149-5p通路影响结肠癌细胞的增殖和凋亡

目的探讨白介素-29(IL-29)对结肠癌SW480细胞增殖和凋亡的影响及作用机制。方法 IL-29诱导SW480细胞,四甲基噻唑蓝染色法(MTT)检测细胞增殖,流式细胞仪检测细胞凋亡,qRT-PCR检测lncRNA DLEU1和miR-149-5p表达,Western blot法检测细胞周期蛋白D1(CyclinD1)、p21、B淋巴细胞瘤-2(Bcl-2)和B淋巴细胞瘤-2相关蛋白(Bax)蛋白表达。双荧光素酶实验验证lncRNA DLEU1和miR-149-5p靶向关系。转染DLEU1小干扰RNA或miR-149-5p模拟物至SW480细胞,上述相同方法观察抑制DLEU1表达或过表达miR-149-5p对SW480细胞增殖和凋亡的影响。结果 IL-29、抑制DLEU1表达或过表达miR-149-5p均可降低SW480细胞OD值、CyclinD1和Bcl-2蛋白表达(P 0. 05),提高细胞凋亡率、p21和Bax蛋白表达(P 0. 05)。IL-29可降低SW480细胞DLEU1表达(P 0. 05),促进miR-149-5p表达(P 0. 05)。DLEU1靶向负调控miR-149-5p表达。过表达DLEU1可逆转IL-29对SW480细胞增殖和凋亡的影响。结论 IL-29可能通过调控DLEU1/miR-149-5p通路抑制结肠癌细胞增殖,并诱导其凋亡。     

槐角黄酮通过调控miR-188-5p/PRMT5基因表达对肺癌细胞增殖、迁移和侵袭的影响及其机制

目的研究槐角黄酮对肺癌A549细胞增殖、迁移和侵袭的影响和潜在的分子机制。方法 qRT-PCR检测蛋白质精氨酸甲基转移酶(PRMT)5和miR-188-5p mRNA的表达,Western印迹检测PRMT5、增殖相关蛋白细胞周期蛋白(Cyclin)D1和p21、迁移侵袭相关蛋白基质金属蛋白酶(MMP)-2和MMP-9蛋白表达水平,四氮唑蓝(MTT)法测定A549细胞增殖活性,Transwell实验检测细胞迁移和侵袭能力,双荧光素酶报告系统验证miR-188-5p与PRMT5的调控关系,流式细胞术检测细胞凋亡。结果槐角黄酮可抑制肺癌细胞A549的侵袭、迁移和增殖,且呈显著的剂量依赖性;槐角黄酮可以促进A549细胞中miR-188-5p表达,抑制PRMT5表达;双荧光素酶报告系统实验结果表明,miR-188-5p靶向调控PRMT5的表达;过表达miR-188-5p和抑制PRMT5均可抑制A549细胞增殖、迁移和侵袭;抑制miR-188-5p逆转了槐角黄酮对A549细胞增殖、迁移和侵袭的抑制作用。结论槐角黄酮通过miR-188-5p靶向PRMT5基因抑制A549细胞的增殖、迁移和侵袭。槐角黄酮对肺癌具有潜在治疗作用。     

MiR-197-3p/STAMP2对ox-LDL诱导的人脐静脉内皮细胞凋亡及炎症应激的影响

目的 探究miR-197-3p/前列腺六次跨膜蛋白（Six transmembrane protein of prostate,STAMP）2对氧化型低密度脂蛋白（ox-LDL）诱导的人脐静脉内皮细胞（HUVECs）凋亡和炎症应答。 方法 用ox-LDL处理HUVEC复制内皮细胞损伤模型;RT-PCR检测miR-197-3p和STAMP2表达;ELISA检测细胞凋亡;Western blot用于检测STAMP2,Bax和Bcl-2的蛋白水平;ELISA检测HUVECs中细胞间黏附分子（ICAM）-1,血管内皮细胞黏附分子（VCAM）-1和E选择素（E-selectin）的表达水平;生物信息学预测miR-197-3p的靶基因;并采用双萤光素酶报告系统、qRT-PCR及Western blot验证miR-197-3p与STAMP2的靶向调控关系。 结果 ox-LDL能明显上调miR-197-3p的表达（P<0.05）,同时降低STAMP2的表达（P<0.05）,且呈时间依赖性;下调miR-197-3p能够显著抑制ox-LDL诱导的细胞凋亡（P<0.05）,同时降低促凋亡蛋白Bax并增加抑凋亡蛋白Bcl-2（P<0.05）;抑制miR-197-3p明显降低ox-LDL诱导的炎症因子的表达（P<0.05）;此外,生物信息学预测提示STAMP2是miR-197-3p的靶基因,而且qRT-PCR及Western blot结果显示抑制miR-197-3p明显上调STAMP2 mRNA和蛋白水平（P<0.05）,从而证实miR-197-3p能够靶向调控STAMP2。 结论 MiR-197-3p可以通过靶向调控STAMP2从而调节ox-LDL诱导的HUVECs凋亡及炎症应答,为动脉粥样硬化的靶向治疗提供理论基础。     

痉挛性截瘫蛋白21对乙型肝炎病毒复制的影响及其机制

目的 探讨人痉挛性截瘫蛋白21 (SPG21)对乙型肝炎病毒(HBV)复制的影响,并探讨其调节机制.方法 将HBV感染性克隆pHBVl.3及其启动子pHBV-Luc分别转染HepG2细胞,加入不同浓度的SPG21蛋白,采用酶联免疫吸附法检测细胞上清液中HBsAg和HBeAg含量;RTPCR和Western blot法检...     

Inhibition of hepatitis B virus replication in 2.2.15 cells by expressed shRNA

Summary.  Hepatitis B virus (HBV) infection is a worldwide health problem. To determine whether RNA interference (RNAi) could inhibit ongoing HBV replication in 2.2.15 cells, we constructed shRNA-producing vector pU6P based on the mouse U6 RNA promoter and cloned 12 targeted sequences against HBV into the vector, resulting in a series of pU6-siHBV vectors. The recombinant vectors were transfected into 2.2.15 cells, HBsAg and HBeAg in cultured media were assayed using enzyme-linked immunosorbent assay at various days after transfection. The amount of HBV DNA in the culture medium was quantitated by real-time polymerase chain reaction. HBsAg and HBeAg expression were inhibited by 72.8 ± 5.4% ( P  = 0.00003) and 55.8 ± 6.2% ( P  = 0.000026), respectively, 4 days after transfection with pU6-siHBV5. The greatest inhibition of HBV DNA was decreased by approximately 1.9-fold ( P  = 0.013) on day 6 post transfection with pU6-siHBV11 compared with that of empty vector. No change was found for HBV protein expression and DNA replication on pU6-siGFP (negative control) transfected cells. Our data demonstrate that the transfection of HBV-targeted shRNA-producing vector in 2.2.15 cells could inhibit the HBV protein expression and HBV DNA replication specifically. RNAi may be considered as a potential antiviral approach for human HBV infection.     

Liver histology and immunohistochemical findings in asymptomatic Indians with incidental detection of hepatitis B virus infection.

BACKGROUND AND OBJECTIVES: The relationship between hepatocyte expression of hepatitis B virus (HBV) antigens, liver histology and viral replication in asymptomatic subjects with incidental detection of hepatitis B surface antigen (HBsAg) remains unclear. We evaluated the histological activity index (HAI) and hepatocyte expression of viral antigens with replicative status in asymptomatic chronic HBV infection. METHODS: Asymptomatic subjects with incidental detection of HBsAg and ALT levels less than twice the upper limit of normal were grouped as follows: Group A - negative for HBeAg and HBV DNA (no HBV replication); B - HBeAg negative, HBV DNA positive (low HBV replication or pre-core mutant); C - positive HBeAg and HBV DNA (high viral replication). Liver biopsies were assessed for HAI (Ishak's scoring system). These were also subjected to immunohistochemistry for expression of HBsAg and hepatitis B core antigen (HBcAg); distribution, staining pattern and quantitative measurement of antigen expression were assessed. RESULTS: Median HAI was similar in the three groups (1.0, 2.0 and 2.0 in groups A, B and C, respectively). All subjects in Group C showed discrete cytoplasmic expression of HBsAg, whereas the other two groups showed heterogeneity in distribution and pattern of HBsAg staining. Quantitative measurement of cytoplasmic HBsAg revealed similar results in the three groups. Core antigen (nuclear) was detected in 4 of 5 subjects in Group C and none of those in Groups A and B. Ground-glass hepatocytes were seen in 20 and orcein-positive cells in 26 cases. HBsAg was detected by immunohistochemistry in 37 biopsies. CONCLUSIONS: Among asymptomatic subjects with chronic HBV infection, those with high rate of viral replication had discrete cytoplasmic HBsAg expression and nuclear expression of core antigen; these findings were uncommon in subjects with low or no viral replication.     

Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro

AIM： To explore the inhibitory effects of pokeweed antiviral protein seed （PAP-S） and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus （HBV） replication in vitro. METHODS： HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold overlength plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS： The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 μg/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 μg and 2.0 μg plasmid pXF3H-PAP, the levels of HBV nucleocapside- associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION： Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.     

特异性乙型肝炎病毒X基因反义核酸体外抗病毒作用

为了观察互补于乙型肝炎病毒（ＨＢＶ）Ｘ基因的三段硫代反义核酸（ＡＳＯＮ）体外抗病毒作用，采用ＥＬＩＳＡ和ＰＡＰ－ＥＬＩＳＡ法检测ＡＳＯＮ作用前后２，２，１５细胞上清中乙型肝炎病毒表面抗原、ｅ抗原及Ｘ抗原（ＨＢｓＡｇ、ＨＢｅＡｇ、ＨＢｘＡｇ）含量变化及细胞原位杂交检测细胞内ＨＢＶＤＮＡ含量变化。结果表明，三段ＡＳＯＮ均可抑制ＨＢｓＡｇ、ＨＢｅＡｇ和ＨＢｘＡｇ的表达，其抑制率分别为８０．６５％、６２．７６％和７８．０７％；细胞内ＨＢＶＤＮＡ也明显减少。据此认为，ＨＢｘＡｇ表达量下降可能系ＡＳＯＮ序列特异性封闭作用所致，而ＨＢ－ｓＡｇ和ＨＢｅＡｇ表达量以及ＨＢＶＤＮＡ含量降低，可能是通过ＨＢｘＡｇ对ＨＢＶＤＮＡ启动子的反式激活功能降低而实现的。     

针对HBV S基因发夹状siRNAs表达载体的构建及其在HepG2215细胞中对HBV基因表达的抑制作用

目的探讨通过载体在体外培养的细胞中表达发夹状siRNAs对HBV基因表达的抑制作用。方法构建表达针对HBV S基因mRNA的发夹状siRNAs的质粒pSilencer 2.1-U6-S,并与ayw亚型HBV全基因组表达质粒共转染体外培养的HepG2215细胞,用半定量RT-PCR分析目标mRNA表达丰度的变化,用ELISA法观察HBsAg表达的变化。结果siRNA处理组细胞上清中HbsAg和HBeAg含量分别比空白对照组降低了63.4%和68.0%,细胞中的2.1kb的信使RNA降低了75.2%。结论siRNA在体外培养的细胞中能有效地抑制HBV基因的表达。     

1．2倍基因组长度C基因型乙型肝炎病毒重组体构建及其在HepG2细胞中表达和复制

目的构建基于pBlueBac4．5质粒的1．2倍基因组长度C基因型乙型肝炎病毒（HBV）重组体，并研究其在HepG2细胞中的表达和复制。方法以重组质粒pWT上的1．2倍基因组长度C基因型HBVDNA序列和pBB4．5HBV1．3（D基因型）上的pBlueBac4．5载体序列为模板，构建重组质粒pBB4．5HBV1．2（C基因型）。用FuGENEHD瞬时转染法，将pBB4．5HBV1．2导人HepG2细胞。用化学发光免疫分析法、Southern印迹杂交法、荧光定量PCR法，分别检测转染后不同时间点HBsAg和HBeAg、HBV复制中间体及HBVDNA水平。此外，对转染时重组质粒用量进行优化。结果酶切和序列分析证实，pBB4．5HBV1．2重组质粒构建成功。初步转染实验证实，在转染细胞培养上清中可检测到HBsAg和HBeAg。优化后转染条件为：使用60mm细胞培养皿，8～11tLgpBB4．5HBV1．2，质粒与转染试剂5：9（μg：μl）。在此条件下，5d实验周期内可检测到HBsAg和HBeAg持续表达（峰值一般出现在转染后第3天）、HBVDNA持续复制（10^6～10^8拷贝／ml）及HBVDNA复制中间体形成。结论在HepG2细胞中，建立了以杆状病毒转移载体pBlueBac4．5为基础的1．2倍基因组长度C基因型HBV体外培养体系，有望为研究HBV耐药、筛选新抗病毒药物等提供新技术平台。