MicroRNA-224 is upregulated in HepG2 cells and involved in cellular migration and invasion

Background and Aim:  MicroRNAs are a class of small non-coding RNAs that negatively regulate the expression of their target genes. The aim of the present study was to explore the effects of microRNA on biological behaviors of HepG2 cells and further analyze its characteristics.
Methods:  We detected different expression profiles of miRNAs in HepG2 and L02 cell lines by microRNA microarray. Northern blot, quantitative real-time polymerase chain reaction, methylthiazolyl tetrazolium, fluorescence-activated cell sorting, scratch wound, transwell migration and Matrigel invasion assays and western blot were carried out to determine whether or not microRNA-224 ( miR-224 ) can influence the biological behaviors of HepG2 cells.
Results:  MiR-224 was significantly upregulated in HepG2 cells. Cell proliferation, migration and invasion, but not cell cycles, were altered after changing the expression of miR-224 . Taking invasion and migration as a breakthrough, a close relationship between the expression of miR-224 and its proteins such as PAK 4 and MMP9 , which were involved in the invasion of tumor, was found.
Conclusions:  Overexpression of miR-224 was involved in the malignant phenotype of HepG2 cells, and it may be an important factor in regulating the migration and invasion of HepG2 cells.     

Direct effects of statins on cells primarily involved in atherosclerosis.

Statins are lipid-lowering agents which act by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This enzyme is responsible for the conversion of HMG-CoA to mevalonate. Products of mevalonate metabolism are critical for several cellular processes of eukaryotic cells, and inhibition of the mevalonate pathway by statins has pleiotropic effects. It has been reported that statins inhibit the migration and proliferation of vascular smooth cells (VSMCs) and macrophages, decrease interleukin-6 and inducible nitric oxide synthase expression in VSMCs, improve endothelial function and up-regulate endothelial nitric oxide synthase expression. The above effects of statins are independent of plasma cholesterol levels, and are completely blocked by exogenous mevalonate and some isoprenoids. These findings suggest that, in addition to their effects on plasma lipids, statins exert direct antiatherosclerotic effects on the cells primarily involved in atherosclerosis.     

短发夹状RNA抑制RhoA基因在HepG2细胞中的表达

目的构建针对人RhoA基因的短发夹状双链RNA(shRNA)真核表达载体,观察其对人肝癌细胞株HepG2 RhoA表达的特异性抑制效应。方法设计合成针对RhoA mRNA编码序列两个不同靶点的核苷酸片断,并定向克隆到真核表达载体Pgenesil-2的U6启动子下,构建重组质粒phsRNA-RhoA1和pshRNA-RhoA2,不针对任何基因组序列的重组质粒pshRNA-HK作为阴性对照。脂质体法转染到HepG2细胞后,应用逆转录聚合酶链反应(RT-PCR)和蛋白免疫印迹法(Western-blot)分别检测RhoA基因表达的抑制效应。结果酶切鉴定和测序结果证实重组质粒均构建成功。转染HepG2后,pshRNA-RhoA2组RhoA基因mRNA和蛋白的表达水平均显著降低,与pshRNA-HK组和空白细胞组相比,差异有统计学意义(r=-19.28,t=-7.08,P<0.05)。而pshRNA-RhoA1组抑制效应不明显,差异无统计学意义(r=-0.16,t=-0.30,P>0.05)。结论构建的重组质粒pshRNA-RhoA2能特异有效地抑制RhoA基因在肝癌细胞HepG2中的表达,为进一步研究RhoA基因在肝癌中的作用提供了有效的分子工具。     

树突状细胞HBsAg疫苗抗乙型肝炎病毒免疫的体外研究

目的 研究人单核细胞来源的树突状细胞（DC）激活的HBsAg特异性细胞毒性T淋巴细胞（CTL）对表达HBsAg的HepG2/S靶细胞的杀伤效应，以探索DC-HBsAg疫苗在抗乙型肝炎病毒（HBV）中的作用。方法 从健康外周血中分离邮单核细胞，在粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素4（IL-4）的作用下培养7d诱导出DC，然后以DC为刺激细胞在体外诱导出HBsAg特异性CTL；将携有HBV-S基因的pLXSN/S重组质粒电击导入肝癌细胞系（HepG2)，建立表达HBsAg的靶细胞模型HepG2/S；用染色法检测HBsAg特异性CTL对HepG2/S靶细胞的杀伤效应。结果 DC激活的HBsAg特异性CTL对HepG2/S靶细胞具有较强的杀伤效应，不同浓度HBsAg(0μg/L，50μg/L和100μg/L）诱导的CTL的杀伤率分别为3.8%,69.5%和85.1%，而CTL对HepG2细胞无明显杀伤效应。结论 由DC激活的HBsAg特异性CTL具有较强制 特异性抗HBV作用。     

Wnt信号通路在HepG2细胞株及其克隆形成细胞中表达的异质性

目的:研究Wnt信号传导通路的关键因子β-catenin和COX-2在肝癌细胞株HepG2及其克隆形成细胞中的表达,探讨Wnt信号传导通路在不同增殖能力细胞中表达的异质性.方法:以HepG2细胞为研究对象,采用软琼脂克隆形成实验筛选克隆形成细胞,应用RTPCR、免疫化学和Western blot等技术,检测Wnt信号传...     

Estrogen receptor beta is involved in the anorectic action of estrogen

OBJECTIVE: Estrogen has been implicated in feeding behavior and adiposity. This study was undertaken to elucidate the mechanism underlying the anti-obesity and anorectic action of estrogen and the role of estrogen receptor (ER) in the central nervous system. METHODS AND RESULTS: Ovariectomy in 8-week-old female Wistar rats induced hyperphagia along with an increase in body weight and abdominal fat accumulation compared to control sham-operated rats. These changes were fully reversed by subcutaneous replacement of estradiol and were abrogated by pair-feeding. Then, the effects of intracerebroventricular infusion of estradiol, alone or in combination with antisense oligodeoxynucleotides (ODN), for ER in ovariectomized rats were examined. The estradiol group showed 10-20% lower daily food intake, and after the 2-week infusion period a 14% reduction in body weight with a similar reduction in abdominal fat compared to the vehicle group. The inhibitory effect of estradiol on food intake and body weight was blocked by co-administration of ER-beta antisense ODN, whereas ER-alpha antisense ODN did not show any influence. CONCLUSION: These results indicate that ER-beta in the central nervous system is involved in the anorectic action of estrogen.     

Putrescine is involved in the vitamin D action in chick intestine

We have reported that a single injection of 1 alpha,25-dihydroxyvitamin D3 into vitamin D-deficient chicks produces a marked increase of putrescine accumulation in the duodenum from two different sources, ornithine and spermidine. In the present study, the effects of putrescine depletion and its supplementation on duodenal villus length and calcium absorption were examined in newborn and 5-week-old chicks. Administering either alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, or N1,N4-bis(2,3-butadienyl)-1,4-butanediamine, a specific inhibitor of polyamine oxidase, to newborn chicks significantly decreased the duodenal content of putrescine and calcium transport activity. The putrescine depletion also induced shortening of the duodenal villus length. The inhibition of calcium absorption and villus length in the putrescine-depleted chicks was almost completely restored by administering putrescine to the birds. The effect of the putrescine depletion and its supplementation on the duodenal villus length and the calcium absorption was reproduced in 5-week-old vitamin D-deficient chicks given vitamin D3 or 1 alpha,25-dihydroxyvitamin D3. These results clearly indicate that putrescine is somehow involved in the vitamin D action in maintaining the morphological and functional development of the intestinal villus mucosa.     

Hint2 is expressed in the mitochondria of H295R cells and is involved in steroidogenesis

Hint2 belongs to the superfamily of histidine triad hydrolase enzymes. Recently, it has been shown to influence the mitochondria-dependent apoptosis occurring in hepatocytes, but its mechanism of action is still obscure. Here, we demonstrate that Hint2 is expressed in the mitochondria of H295R cells and in normal adrenals, and that this protein is involved in steroidogenesis. The presence of Hint2 in H295R cells was revealed by RT-PCR and by immunoblot analysis of subcellular fractions. The protein appeared associated with mitochondrial membranes, probably facing the interior of the organelle. Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by angiotensin II or K+, whereas protein silencing with specific small interfering RNA resulted in a marked reduction of the steroidogenic response. The duration of the mitochondrial calcium signal induced by angiotensin II was also reduced upon Hint2 down-regulation with small interfering RNA, but not affected after its overexpression, suggesting that under basal conditions, Hint2 is optimally expressed, and not rate limiting in steroidogenesis. Moreover, Hint2 also appeared involved in Ca2+-independent pathways leading to steroid formation. Indeed, pregnenolone formation in response to either forskolin or a hydroxyl analog of cholesterol was markedly reduced after Hint2 silencing. Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favorable mitochondrial potential. In conclusion, these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.     

Adhesion to the extracellular matrix is positively regulated by retinoic acid in HepG2 cells.

Aims: In this work, we aimed to investigate the possible modulation of cell-matrix interactions by retinoic acid (RA), in view of the well-known role of the extracellular matrix (ECM) and integrins in hepatocyte differentiation and proliferation. For this purpose, we analysed the adhesion ability of HepG2 cells on different substrates in the presence and absence of RA evaluating both the expression and cellular localisation of major proteins involved in focal contacts, using Western blot and confocal microscopy. RESULTS: A positive and substrate-dependent effect of RA on cell-matrix adhesion was observed after long-term culture. The increased adhesiveness in the treated cells was accompanied by an enhanced expression of beta1 and alpha3 integrin subunits, together with a redistribution of beta1 receptors clustered at the basal surface. In contrast, the levels of focal adhesion kinase (FAK), paxillin and alpha-actinin were unchanged, as was the phosphorylation state of FAK. Nonetheless, a stronger association between beta1 integrin and intracytoplasmatic proteins of focal contacts was observed in coimmunoprecipitation experiments after RA treatment, suggesting improved connection with the actin cytoskeleton. These results are consistent with previously described antiproliferative and differentiative effects of RA on transformed hepatocytes, and confirm the hypothesis of a direct influence of RA on specific adhesion molecules.     

HepG2细胞电转染条件的优化

目的优化HepG2细胞电转染条件,进一步提高HepG2细胞的转染效率。方法采用电穿孔方法将pcDNA3．1-EGFP导入HepG2细胞中,在500μL电转染体系中,在不同电场强度、细胞数目、脉冲频率、脉冲时间、电转质粒数目、电转缓冲液、培养基血清浓度条件下,分别将pcDNA3．1-EGFP质粒电转染HepG2细胞,检测不同条件下细胞存活率和转染率。结果电转前4℃孵育电转体系混合液10min,方波电转条件在1个电脉冲、电压270V、细胞数为2×10^6个、质粒20μg、脉冲时间20ms、电转缓冲液为优化缓冲液、电转后置于37℃、含15％FBS的DMEM高糖培养基中培养48h,可获得高转染率（60．68±1．87）％。结论优化电穿孔法的电转染条件能够有效提高HepG2细胞的电转染效率。本研究为外源基因电转染HepG2细胞提供了可靠的试验参数。     

HepG2与HepG2．2．15细胞中P53表达及活性的比较

目的探讨HBV对抑癌基因P53表达及活性的影响。方法选用HepG2及转染了HBV表达质粒的HepG2．2．15细胞，采用Western blotting法检测两种细胞P53的表达状况；磷酸钙法将报告基因质粒PG13-CAT和P21-LUC分别转染细胞，通过检测报告基因的表达观察各细胞中P53的活性。结果HepG2．2．15细胞中P53蛋白表达水平高于HepG2细胞，而且两种报告基因的表达在HepG2．2．15细胞中也较高。结论HBV在肝癌细胞内的复制对P53的表达及功能具有一定的增强作用。     

白细胞介素-37在人HepG2细胞中的表达

[目的]研究白细胞介素-37(interleukin-37,IL-37)在人肝肿瘤细胞系(HepG2)细胞中的表达,及在炎症因子刺激下,HepG2细胞中IL-37表达水平的改变。[方法]在37℃、5%CO2条件下培养人HepG2细胞,以脂多糖(lipopolysaccharide,LPS)刺激细胞,分别于刺激24h、48h、72h后提取细胞,分别提取细胞总RNA、总蛋白,利用逆转录酶聚合链反应技术(RT-PCR)检测细胞中IL-37-mRNA的表达,利用Western blot技术检测细胞中IL-37蛋白的表达水平。并且在LPS刺激细胞72h后,运用免疫荧光和激光共聚焦技术检测IL-37在细胞中的表达定位。[结果]RT-PCR结果证明HepG2中存在IL-37-mRNA的表达,且随着LPS刺激时间的延长IL-37-mRNA表达增多,0h(无刺激)、24h、48h、72h灰度值分别为0.10±0.008、0.14±0.014、0.22±0.010、0.28±0.014;Western blot结果显示细胞中存在IL-37蛋白的表达,且其表达趋势与IL-37mRNA表达趋势相同,即随着刺激时间延长,HepG2中IL-37蛋白表达增多,由24h、48h、72h分别为0.54±0.16、0.87±0.04、1.51±0.18;免疫荧光共聚焦结果显示IL-37在细胞核和细胞质中均有表达,以细胞核周围表达最为丰富。[结论]人HepG2细胞中存在IL-37表达,炎症因子刺激后,IL-37mRNA及IL-37蛋白表达水平均呈升高,趋势相同;IL-37在HepG2细胞质内和细胞核内均表达,以细胞核周围表达最为丰富。     

Urotensin Ⅱ-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in Hep G2 cells

AIM: To investigated the effects of urotensin Ⅱ(UII) on hepatic insulin resistance in Hep G2 cells and the potential mechanisms involved.METHODS: Human hepatoma Hep G2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucoseoxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species(ROS) levels were detected with a multimode reader using a 2′,7′-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase(JNK), insulin signal essential molecules such as insulin receptor substrate-1(IRS-1), protein kinase B(Akt), glycogen synthase kinase-3β(GSK-3β), and glucose transporter-2(Glut 2), and NADPH oxidase subunits such as gp91 phox, p67 phox, p47 phox, p40 phox, and p22 phox were evaluated by Western blot.RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption(P 0.05)and glycogen content(P 0.01) in Hep G2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression(P 0.01) and phosphorylation of IRS-1(P 0.05), associated with down-regulation of Akt(P 0.05) and GSK-3β(P 0.05) phosphorylation levels, and the expression of Glut 2(P 0.001), indicating an insulin-resistance state in Hep G2 cells. Furthermore, UII enhanced the phosphorylation of JNK(P 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1(P 0.001), phosphorylation of IRS-1(P 0.001) and GSK-3β(P 0.05), and glycogen synthesis(P 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation(P 0.05) and NADPH oxidase subunit expression(P 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production(P 0.05), JNK phosphorylation(P 0.05), and insulin resistance(P 0.05) in HepG 2 cells. CONCLUSION: UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG 2 cells.     

分泌型卷曲相关蛋白2对HepG2细胞生物学行为的影响

目的 了解分泌型卷曲相关蛋白2(sFRP2)对HepG2细胞增殖、侵袭和迁移等生物学行为的影响.方法 采用sFRP2重组腺病毒感染HepG2细胞,四甲基偶氮唑盐法检测HepG2细胞增殖,流式细胞术检测细胞周期分布,免疫组织化学法检测肿瘤转移相关因子的表达,Westernblot检测β连环素的表达,Tmnswell小室检测细胞迁移能力.结果 sFRP2明显抑制HepG2细胞增殖,限制细胞周期从Go/G<,1>期进入S期;sFRP2显著增强nepG2细胞CD44和CD82/KAI1等与肿瘤转移抑制相关蛋白的表达,而明显降低有助于肿瘤侵袭转移的细胞外基质金属蛋白酶诱导因子的表达; sFRP2可降低HepG2细胞的迁移能力.sFRP2感染前后,HepG2细胞均有β连环素的表达,且其表达差异无统计学意义.结论 sFRP2的重组腺病毒能成功感染HepG2细胞,并对H印G2细胞的增殖、侵袭和转移具有抑制效应.     

Ghrelin is involved in the decidualization of human endometrial stromal cells

Successful implantation involves a complex interaction between the endometrium and the embryo. It is well known that several neuropeptides are expressed in the endometrium and placenta during embryonal implantation, suggesting an important role as chemical mediators of the feto-maternal relationship. Ghrelin has recently been identified as the endogenous ligand for the GH secretagogue receptor. Ghrelin is a peptide hormone with many physiological functions, and its expression in the human placenta has been reported. To investigate the involvement of ghrelin in embryonal implantation, we assessed the spatio-temporal expression pattern of ghrelin and its receptor in the human endometrium and placenta through the normal menstrual cycle and in early pregnancy. We also examined the effect of ghrelin on the decidualization of endometrial stromal cells (ESC). Weak expression of ghrelin mRNA was detected in the nonpregnant endometrium, and it was dramatically increased in the decidualized endometrium. A GH secretagogue receptor mRNA was detected in the endometrium throughout the normal menstrual cycle and in early pregnancy, but not in the first trimester placenta. Immunohistochemical analysis using an antighrelin antibody revealed strong signals in decidual cells and extravillous trophoblast cells. Coculture with first trimester placenta up-regulated ghrelin mRNA expression by primary cultured ESC, although sex steroids and 8-bromo-cAMP had no effect. In addition, ghrelin enhanced the decidualization of ESC induced by 8-bromo-cAMP (8-Br-cAMP) in vitro. Thus, ghrelin is a novel paracrine/autocrine factor that is involved in cross-talk between the endometrium and embryo during embryonal implantation.     

辛伐他汀抑制HepG2细胞增殖作用的机制

目的 体外观察辛伐他汀对人肝癌细胞HepG2增殖、细胞周期及细胞周期蛋白依赖激酶抑制因子p21蛋白表达的影响.方法 采用四甲基偶氮唑盐(MTT)法观察辛伐他汀对HepG2细胞增殖的影响,用流式细胞仪检测辛伐他汀对细胞周期的作用,用免疫细胞化学法观察辛伐他汀对细胞周期蛋白依赖激酶抑制因子p2l蛋白表达的影响.对数据进行析因设计与单因素方差分析.结果 体外辛伐他汀可抑制HepG2细胞的增殖(F浓度=1264,P＜0.001 ;F时间=17.466,P＜0.001;F浓度*时间=35.053,P＜0.001).辛伐他汀处理组G0/G1期细胞增多,但细胞凋亡不明显;体外辛伐他汀可增强HepG2细胞周期蛋白依赖激酶抑制因子p21蛋白的表达(F=512.133,P＜0.001).结论 体外辛伐他汀对HepG2细胞增殖有抑制作用,该作用可能与其使细胞生长阻滞于G0/G1期及增强细胞周期蛋白依赖激酶抑制因子p21蛋白表达有关.