MiR-22重组腺病毒的构建及对HepG2细胞葡萄糖摄取的影响

文中构建了miR-22重组腺病毒Ad-miR-22,分析了其对HepG2细胞胰岛素信号通路及葡萄糖摄取的抑制作用。通过PCR方法,扩增了miR-22的前体及侧翼序列,酶切后克隆至腺病毒穿梭载体pAdTrack-CMV中,构建穿梭质粒pAdT-22,经PCR及测序鉴定。穿梭质粒经PmeⅠ线性化后,直接转化含有腺病毒骨架载体的感受态细胞BJ5183,产生重组腺病毒质粒Ad-miR-22,最后经PacⅠ线性化后转染包装细胞系293A。重组腺病毒经过3轮扩增后感染HepG2细胞,通过荧光定量PCR检测miR-22表达水平。通过葡萄糖摄取实验观察Ad-miR-22对HepG2细胞葡萄糖摄取的影响。采用Western blotting检测Ad-miR-22对HepG2细胞SIRT1在蛋白质水平的表达及GSK-3β磷酸化水平的影响。采用荧光定量PCR检测miR-22对PEPCK及G6Pase等基因在mRNA水平表达的影响。结果表明,重组腺病毒Ad-miR-22感染显著增加HepG2细胞miR-22表达水平。此外,Ad-miR-22显著抑制胰岛素诱导的HepG2葡萄糖摄取,并通过下调GSK-3β磷酸化抑制胰岛素信号通路的激活。Ad-miR-22反转胰岛素对糖异生关键酶表达的抑制作用,并下调SIRT1基因在蛋白质水平的表达。综上所述,构建了miR-22的重组腺病毒,发现其显著增加糖异生,抑制HepG2细胞葡萄糖摄取,该作用可能与miR-22调节SIRT1在蛋白质水平的表达有关。     

miR-29b2c重组腺病毒的构建及对胃癌细胞增殖和迁移的影响

构建了miR-29b2c重组腺病毒Ad-miR-29b2c,考察了其对HGC-27、MGC-803胃癌细胞增殖及迁移的抑制作用。采用PCR从基因组扩增miR-29b2c片段,并克隆至腺病毒穿梭载体pAdTrack-CMV中,构建穿梭质粒pAdT-29b2c,经酶切及测序鉴定。穿梭质粒经PmeⅠ线性化后与腺病毒骨架载体共转化BJ5183感受态,产生重组腺病毒质粒Ad-miR-29b2c,再经PacⅠ线性化后转染293A细胞进行包装。重组腺病毒扩增后感染HGC-27细胞,通过MTT及细胞迁移实验观察Ad-miR-29b2c对HGC-27、MGC-803细胞增殖及迁移的影响。采用Western blotting检测Ad-miR-29b2c对HGC-27、MGC-803细胞δ-catenin蛋白表达的影响。酶切、测序及荧光定量PCR结果表明重组腺病毒构建成功,miR-29b及miR-29c在HGC-27细胞过表达。MTT实验表明Ad-miR-29b2c能显著抑制HGC-27、MGC-803细胞增殖。细胞迁移实验表明Ad-miR-29b2c能显著抑制HGC-27、MGC-803细胞迁移。此外,Ad-miR-29b2c能显著降低HGC-27、MGC-803细胞δ-catenin蛋白表达水平。综上所述,构建了miR-29b2c的重组腺病毒,并发现其可以抑制胃癌细胞HGC-27和MGC-803的增殖及迁移,该作用可能与miR-29抑制HGC-27、MGC-803细胞δ-catenin蛋白表达有关。     

构建MicroRNA-29a和microRNA-29c腺病毒并探讨其对膀胱癌细胞增殖能力的影响

构建miRNA-29a/c的重组腺病毒并观察其对膀胱癌T24细胞增殖能力的调控。以人全基因组DNA为模板,PCR扩增miR-29a、miR-29c,克隆至腺病毒穿梭载体pAdtrace-TO4-CMV。重组穿梭载体经pme I线性化后与腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌BJ5183,通过同源重组获得重组腺病毒质粒pAdEasy-1-miR-29a、pAdEasy-1-miR-29c,pac I线性化后转染HEK-293细胞,进行包装和扩增。实时荧光定量PCR检测感染腺病毒的膀胱癌T24细胞中miR-29a、miR-29c的表达水平,并利用CCK-8实验检测细胞增殖能力。经DNA测序和限制性内切酶分析显示,重组腺病毒质粒pAdEasy-1-miR-29a、pAdEasy-1-miR-29c构建成功;感染腺病毒Ad-miR-29a和Ad-miR-29c后,经实时荧光定量PCR检测,膀胱癌细胞中miR-29a、miR-29c表达显著增高(P0.01);过表达miR-29a/c后的CCK-8实验显示,细胞增殖能力明显低于对照组(P0.05)。以上说明已成功构建miR-29a、miR-29c腺病毒,过表达miR-29a/c可抑制膀胱癌细胞的增殖。     

miR-34a-5p通过靶向醛缩酶A抑制肝癌细胞的增殖和迁移

[目的]证明醛缩酶A(ALDOA)在肝癌细胞的增殖和迁移作用并探索miR-34a-5p靶向调控ALDOA的分子机制,为肝癌治疗提供潜在的分子靶标。[方法]分子生物学技术构建了ALDOA表达质粒,蛋白质印迹(Western Blotting)和实时荧光定量PCR(RT-PCR)检测ALDOA的过表达和敲降效果,CCK-8验证细胞的增殖,划痕实验验证细胞的迁移。[结果]在肝癌细胞中过表达ALDOA能促进肝癌细胞的增殖与迁移(P 0.05),敲降ALDOA后肝癌细胞的增殖与迁移受到抑制(P 0.05),miR-34a-5p是通过靶向结合ALDOA的3'UTR抑制其表达从而抑制肝癌细胞的增殖与迁移(P 0.05)。[结论]miR-34a-5p通过靶向ALDOA抑制肝癌细胞的增殖和迁移。     

白藜芦醇体外对肝癌HepG2细胞分化及P21WAF1/CIP1表达的影响

目的:探讨白藜芦醇(Resvratrol,Res)在体外对肝癌细胞分化及相关周期蛋白依赖激酶抑制因子P21WAF1/CIP1的影响.方法:体外培养肝癌HepG2细胞.用MTT法检白藜芦醇对HepG2细胞的生长抑制作用,用倒置显微镜观察肝癌细胞的形态改变,用放射免疫法检测其AFP分泌.以RT-PCR方法检测HepG2细胞中P21WAF1、CIP1mRNA的表达,用免疫细胞化学检测其P21WAF1、CIP1蛋白的表迭.结果:白藜芦醇呈时间剂量性抑制HepG2细胞株的增殖,使其亚细胞结构趋于正常,AFP分泌量下降,并显著上调HepG2细胞中P21WAF1/CIP1 mRNA和蛋白的表达.结论:白藜芦醇能诱导HepG2细胞在体外向正常肝细胞分化,并上调其P21WAF1/CIP1的表达.     

白藜芦醇体外对肝癌HepG2细胞分化及P21^WAF1/CIP1表达的影响

目的：探讨白藜芦醇（Resvratrol,Res）在体外对肝癌细胞分化及相关周期蛋白依赖激酶抑制因子P21^WAF1/CIP1的影响。方法：体外培养肝癌HepG2细胞,用MTT法检白藜芦醇对HepG2细胞的生长抑制作用,用倒置显微镜观察肝癌细胞的形态改变,用放射免疫法检测其AFP分泌,以RT-PCR方法检测HepG2细胞中P21^WAF1/CIP1mRNA的表达,用免疫细胞化学检测其P21^WAF1/CIP1蛋白的表达。结果：白藜芦醇呈时间剂量性抑制HepG2细胞株的增殖,使其亚细胞结构趋于正常,AFP分泌量下降,并显著上调HepG2细胞中P21^WAF1/CIP1mRNA和蛋白的表达。结论：白藜芦醇能诱导HepG2细胞在体外向正常肝细胞分化,并上调其P21^WAF1/CIP1的表达。     

双香豆素在HBx介导的肝癌细胞生长增殖中的作用及其机制研究

HCC是世界范围内最常见的恶性肿瘤之一,其中50%以上与乙型肝炎病毒(HBV)感染相关,且乙肝相关性肝癌患者术后复发率明显高于未合并乙肝患者。而病毒蛋白HBx是促进乙肝相关性肝癌进展的主要因素。因此,寻找能够有效抑制HBx促癌作用的小分子化合物,减缓乙肝相关性肝癌的疾病进展,对于延长患者生存期具有重要意义。该研究初步探讨了小分子化合物双香豆素在HBx介导的肝癌细胞生长增殖中的作用及机制。首先,利用MTT实验确定了双香豆素的使用浓度。其次,采用生长曲线、CCK-8和EdU实验检测了双香豆素对HBV稳定表达细胞(HepG2.2.15)、HBV感染细胞(HepG2-NTCP)、HBx过表达细胞(HepG2-HBx)、HBV瞬时表达细胞(HepG2 HBV-WT)及肝癌细胞(HepG2)生长增殖能力的影响;同时利用平板集落形成实验检测了细胞集落形成能力。然后,利用实时荧光定量PCR和Western blot分别检测了细胞生长增殖相关分子β-连环蛋白(β-catenin)及下游靶标c-Myc和细胞周期蛋白A2(Cyclin A2)的mRNA和蛋白水平。最后,检测了双香豆素发挥抑癌作用时对HBx的依赖性。结果显示,双香豆素呈浓度依赖地抑制HepG2.2.15细胞及HBV感染的HepG2-NTCP细胞的生长增殖及集落形成;同时,双香豆素显著抑制HBx过表达细胞(HepG2-HBx)及HBV瞬时表达细胞(HepG2 HBV-WT)的生长增殖。进一步机制解析发现,双香豆素显著下调β-catenin、c-Myc和Cyclin A2的水平进而抑制细胞生长增殖。最后,该研究证实了双香豆素对乙肝相关性肝癌生长增殖的抑制作用依赖于HBx。总之,该文发现双香豆素可有效抑制乙肝相关性肝癌生长增殖且此作用依赖于HBx。     

维甲酸对肝癌细胞HepG2糖基化磷脂酰肌醇特异性磷酯酶D表达及细胞生物学特性的影响

应用MTT、流式细胞仪、免疫印迹法检测全反式维甲酸(ATRA)单独或联合糖基化磷脂酰肌醇特异性磷酯酶D(GPI-PLD)特异性抑制剂1,10-二氮杂菲对肝癌细胞HepG2生物学特性的改变.ATRA使肝癌细胞HepG2 GPI-PLD基因表达及酶活性上调,并呈现剂量和时间依赖性.ATRA可抑制肝癌细胞HepG2增殖,使肝癌细胞Caspase-3表达水平显著增加,Bcl-2表达水平下调,促进肝癌细胞凋亡(P<0.05).ATRA联合1,10-二氮杂菲诱导组细胞,Bcl-2、细胞增殖活性较ATRA单独诱导组显著增强,Caspase-3、凋亡率显著下降.维甲酸可促进肝癌细胞HepG2 GPI-PLD基因表达上调,高活性的GPI-PLD有助于维甲酸抑制肝癌细胞增殖,促进肝癌细胞凋亡.     

MiR-33a抑制肝癌细胞的增殖、侵袭和迁移北大核心CSCD

miR-33a参与许多肿瘤发展的调控。然而,其对肝癌的作用还未完全清楚。本研究探讨了miR-33a在肝癌细胞中的表达及其功能。实时荧光定量PCR显示,与永生化的正常肝细胞系LO2相比,miR-33a在SMMC-7721、Bel-7405、Hep3B细胞中表达量较高,在SK-Hep-1、HepG2、Huh7细胞中表达量较低。其中,在SMMC-7721中表达量最高,在Huh7中表达量最低。分别用miR-33a模拟物和抑制物转染表达量最高和最低的细胞系SMMC-7721和Huh7,实时荧光定量PCR显示,模拟物转染细胞后,36 h转染效率最高;cy3染色法显示,抑制物转染细胞后,各时间点被标记的细胞均大于60%;CCK-8法和Transwell法显示,miR-33a抑制SMMC-7721和Huh7细胞增殖、迁移和侵袭;Western印迹显示,miR-33a抑制SMMC-7721、Huh7细胞claudin-1表达,促进E-钙粘蛋白表达;balb/c裸鼠成瘤实验表明,皮下注射miR-33a拮抗剂能促进肿瘤生长。上述结果证明,miR-33a在不同的肝癌细胞系中表达水平高低不一,在SMMC-7721中最高,Huh7中最低;miR-33a可以抑制肝癌细胞增殖,并可能通过抑制claudin-1及促进E-钙粘蛋白表达,抑制上皮间质转化进程抑制肝癌细胞侵袭和迁移。     

构建MicroRNA．29a和microRNA-29c腺病毒并探讨其对膀胱癌细胞增殖能力的影响

构建miRNA．29a／c的重组腺病毒并观察其对膀胱癌T24细胞增殖能力的调控。以人全基因组DNA为模板,PcR扩增miR-29a、miR-29c,克隆至腺病毒穿梭载体pAdtrace．TO4-CMV。重组穿梭载体经pmeI线性化后与腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌BJ5183,通过同源重组获得重组腺病毒质粒pAdEasy—1—miR-29a、pAdEasy—1．miR-29c,pacI线性化后转染HEK-293细胞,进行包装和扩增。实时荧光定量PcR检测感染腺病毒的膀胱癌T24细胞miR-29a、miR．29c的表达水平,并利用CCK．8实验检测细胞增殖能力。经DNA测序和限制性内切酶分析显示,重组腺病毒质粒pAdEasy—1—miR．29a、pAdEasy．1-miR-29c构建成功：感染腺病毒Ad—miR-29a和Ad—miR．29c后,经实时荧光定量PCR检测,膀胱癌细胞中miR．29a、miR．29c表达显著增高（P〈0．01）;过表达miR．29a／c后的CCK．8实验显示,细胞增殖能力明显低于对照组伊〈0．05）。以上说明已成功构建miR．29a、miR．29c腺病毒,过表达miR．29a／c可抑制膀胱癌细胞的增殖。     

Increased antitumor capability of fiber-modified adenoviral vector armed with TRAIL against bladder cancers

Adenoviral vectors are widely used for cancer therapy and show a tumor-suppressing effect. However, bladder cancers are found to be resistant against infection of Ad5-derived adenoviral vector, limiting the application of the existing strategy of gene therapy. Therefore, efforts to develop novel types of adenoviral vector aimed for improving the viral infection and enhancing expression level of tumor-inhibiting transgene is urgently required. We constructed a 5/35 fiber-modified E1A-deleted adenoviral vector armed with TRAIL gene. Its ability to express this gene for inhibition of bladder cancer cell growth was investigated in our work. The results showed that this modification in fiber region facilitates adenoviral infection to bladder cancer, perhaps due to high expression of CD46 on target cell surface. Subsequently, we found an enhanced expression level of TRAIL mediated by 5/35 fiber-modified adenoviral vectors in bladder cancer cells, leading to an increased tumor-inhibiting capability of 5/35 adenoviral vector against bladder cancer cells. Consistently, growth of xenograft tumors in mice was also effectively inhibited by 5/35 fiber-modified vector-mediated gene therapy strategy. The 5/35 fiber-modified adenoviral vector-based gene transfer shows an improved efficacy against bladder cancers. The application of this novel gene therapy vector may benefit the patients in clinical bladder cancer treatment.     

In vivo antitumor effect of herpes simplex virus thymidine kinase gene therapy in rat hepatocellular carcinoma: feasibility of adenovirus-mediated intra-arterial gene delivery

Transfer of the herpes simplex virus-thymidine kinase gene, followed by the administration of ganciclovir (HSV-tk/GCV), has been a major approach for cancer gene therapy. We investigated the antitumor effect of the HSV-tk/GCV strategy with the rat orthotopic hepatocellular carcinoma (HCC) model and the tumor-selective gene delivery by an adenovirus-mediated gene transfer through the hepatic artery. The complete antitumor effect was demonstrated, after the treatment with GCV in rat HCC established by the implantation of HSV-tk transferred rat HCC cells. The in vivo bystander effect was also observed. The marked infiltration of CD4+ and CD8+ T lymphocytes, macrophages and NK cells were found in the tumor area. After the injection of adenovirus carrying the LacZ gene into the hepatic artery, the selective expression of transgene in the tumor cell was achieved. These findings indicate that the HSV-tk/GCV strategy, using an adenoviral vector, could be a promising avenue for the treatment of hepatocellular carcinoma.     

A new oncolytic adenoviral vector carrying dual tumour suppressor genes shows potent anti-tumour effect

Cancer Targeting Gene-Viro-Therapy (CTGVT) is a promising cancer therapeutical strategy that strengthens the anti-tumour effect of oncolytic virus by expressing inserted foreign anti-tumour genes. In this work, we constructed a novel adenoviral vector controlled by the tumour-specific survivin promoter on the basis of the ZD55 vector, which is an E1B55KD gene deleted vector we previously constructed. Compared with the original ZD55 vector, this new adenoviral vector (ZD55SP/E1A) showed much better ability of replication and reporter gene expression. We then combined anti-tumour gene interleukine-24 (IL-24) with an RNA polymerase III-dependent U6 promoter driving short hairpin RNA (shRNA) that targets M-phase phosphoprotein 1 (MPHOSPH1, a newly identified oncogene) by inserting the IL-24 and the shRNA of MPHOSPH1 (shMPP1) expression cassettes into the new ZD55SP/E1A vector. Our results demonstrated excellent anti-tumour effect of ZD55SP/E1A-IL-24-shMPP1 in vitro on multiple cancer cell lines such as lung cancer, liver cancer and ovarian caner. At high multiplicity-of-infection (MOI), ZD55SP/E1A-IL-24-shMPP1 triggered post-mitotic apoptosis in cancer cells by inducing prolonged mitotic arrest; while at low MOI, senescence was induced. More importantly, ZD55SP/E1A-IL-24-shMPP1 also showed excellent anti-tumour effects in vivo on SW620 xenograft nude mice. In conclusion, our strategy of constructing an IL-24 and shMPP1 dual gene expressing oncolytic adenoviral vector, which is regulated by the survivin promoter and E1B55KD deletion, could be a promising method of cancer gene therapy.     

MicroRNA-122 Triggers Mesenchymal-Epithelial Transition and Suppresses Hepatocellular Carcinoma Cell Motility and Invasion by Targeting RhoA

The loss of microRNA-122 (miR-122) expression is strongly associated with increased invasion and metastasis, and poor prognosis of hepatocellular carcinoma (HCC), however, the underlying mechanisms remain poorly understood. In the present study, we observed that miR-122 over-expression in HCC cell lines Sk-hep-1 and Bel-7402 triggered the mesenchymal-epithelial transition (MET), as demonstrated by epithelial-like morphological changes, up-regulated epithelial proteins (E-cadherin, ZO-1, α-catenin, occludin, BVES, and MST4), and down-regulated mesenchymal proteins (vimentin and fibronectin). The over-expression of miRNA-122 also caused cytoskeleton disruption, RhoA/Rock pathway inactivation, enhanced cell adhesion, and suppression of migration and invasion of Sk-hep-1 and Bel-7402 cells, whereas, these effects could be reversed through miR-122 inhibition. Additional studies demonstrated that the inhibition of wild-type RhoA function induced MET and inhibited cell migration and invasion, while RhoA over-expression reversed miR-122-induced MET and inhibition of migration and invasion of HCC cells, suggesting that miR-122 induced MET and suppressed the migration and invasion of HCC cells by targeting RhoA. Moreover, our results demonstrated that HNF4α up-regulated its target gene miR-122 that subsequently induced MET and inhibited cell migration and invasion, whereas miR-122 inhibition reversed these HNF4α-induced phenotypes. These results revealed functional and mechanistic links among the tumor suppressors HNF4α, miR-122, and RhoA in EMT and invasive and metastatic phenotypes of HCC. Taken together, our study provides the first evidence that the HNF4α/miR-122/RhoA axis negatively regulates EMT and the migration and invasion of HCC cells.     

MicroRNA-147 Induces a Mesenchymal-To-Epithelial Transition (MET) and Reverses EGFR Inhibitor Resistance

Background

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression and may promote resistance to therapy. An analysis of patients (n = 71) profiled with both gene expression and a global microRNA assessment (∼415 miRs) identified miR-147 as highly anti-correlated with an EMT gene expression signature score and postulated to reverse EMT (MET).

Methods and Findings

miR-147 was transfected into colon cancer cells (HCT116, SW480) as well as lung cancer cells (A-549). The cells were assessed for morphological changes, and evaluated for effects on invasion, motility, and the expression of key EMT markers. Resistance to chemotherapy was evaluated by treating cells with gefitinib, an EGFR inhibitor. The downstream genes regulated by miR-147 were assayed using the Affymetrix GeneChip U133 Plus2.0 platform. miR-147 was identified to: 1. cause MET primarily by increasing the expression of CDH1 and decreasing that of ZEB1; 2. inhibit the invasion and motility of cells; 3. cause G1 arrest by up-regulating p27 and down-regulating cyclin D1. miR-147 also dramatically reversed the native drug resistance of the colon cancer cell line HCT116 to gefitinib. miR-147 significantly repressed Akt phosphorylation, and knockdown of Akt with siRNA induced MET. The morphologic effects of miR-147 on cells appear to be attenuated by TGF-B1, promoting a plastic and reversible transition between MET and EMT.

Conclusion

miR-147 induced cancer cells to undergo MET and induced cell cycle arrest, suggesting a potential tumor suppressor role. miR-147 strikingly increased the sensitivity to EGFR inhibitor, gefitinib in cell with native resistance. We conclude that miR-147 might have therapeutic potential given its ability to inhibit proliferation, induce MET, as well as reverse drug sensitivity.