Carcinoembryonic antigen directed herpes viral oncolysis improves selectivity and activity in colorectal cancer

BACKGROUND: G207 is an oncolytic herpes virus whose replicative cycle requires cellular ribonucleotide reductase (RR) for viral DNA synthesis. We attempt to enhance viral cytotoxicity in carcinoembryonic antigen (CEA)-producing colorectal cancer (CRC) cells through CEA-driven RR production. METHODS: CEA enzyme-linked immunosorbent assay was performed on LS174T and HCT-8 human CRC cells. The CEA enhancer-promoter (CEA E-P) was functionally assessed by luciferase assay. CEA E-P was cloned upstream of UL39, the gene encoding the large subunit of RR. Cells were transfected with CEA E-P/UL39 and infected with G207 for cytotoxicity assays. LS174T, with or without CEA E-P/UL39, were implanted into athymic mouse flanks (n = 28) and treated with G207. RESULTS: CEA levels were 7-fold higher in LS174T versus HCT-8 ( P <.00001). CEA E-P increased luciferase expression 7.5-fold in LS174T ( P <.01), with no increase in HCT-8. G207 cytotoxicity of'CEA E-P/UL39-transfected LS174T cells increased 69% by day 10 versus nontransfected cells ( P <.001), with no significant increase in HCT-8. Combining CEA E-P/UL39 with G207 in LS174T flank tumors resulted in a 65% decrease in tumor volume versus G207, phosphate-buffered saline, or'CEA E-P/UL39 alone ( P <.0001). CONCLUSIONS: CEA-driven RR production by CEA-secreting CRC cells significantly improves oncolytic viral cytotoxicity and specificity in vitro, and reduces tumor burden in vivo.     

Utilizing tumor hypoxia to enhance oncolytic viral therapy in colorectal metastases

OBJECTIVE: To determine the effects of hypoxia-induced ribonucleotide reductase (RR) production on herpes oncolytic viral therapy. SUMMARY BACKGROUND DATA: Hypoxia is a common tumor condition correlated with therapeutic resistance and metastases. Attenuated viruses offer a unique cancer treatment by specifically infecting and lysing tumor cells. G207 is an oncolytic herpes virus deficient in RR, a rate-limiting enzyme for viral replication. METHODS: A multimerized hypoxia-responsive enhancer was constructed (10xHRE) and functionally tested by luciferase assay. 10xHRE was cloned upstream of UL39, the gene encoding the large subunit of RR (10xHRE-UL39). CT26 murine colorectal cancer cells were transfected with 10xHRE-UL39, incubated in hypoxia (1% O2) or normoxia (21% O2), and infected with G207 for cytotoxicity assays. CT26 liver metastases, with or without 10xHRE-UL39, were created in syngeneic Balb/C mice (n = 40). Livers were treated with G207 or saline. Tumors were assessed and stained immunohistochemically for G207. RESULTS: 10xHRE increased luciferase expression 33-fold in hypoxia versus controls (P < 0.001). In normoxia, 10xHRE-UL39 transfection did not improve G207 cytotoxicity. In hypoxia, G207 cytotoxicity increased 87% with 10xHRE-UL39 transfection versus nontransfected cells (P < 0.001). CT26 were resistant to G207 alone. Combining 10xHRE-UL39 with G207 resulted in a 66% decrease in tumor weights (P < 0.0001) and a 65% reduction in tumor nodules (P < 0.0001) versus G207 monotherapy. 10xHRE-UL39-transfected tumors demonstrated greater viral staining. CONCLUSIONS: Hypoxia-driven RR production significantly enhances viral cytotoxicity in vitro and reduces tumor burden in vivo. G207 combined with RR under hypoxic control is a promising treatment for colorectal cancer, which would otherwise be resistant to oncolytic herpes virus alone.     

AIBP在肝癌细胞中的表达及意义与含AIBP基因及AFP启动子驱动的双自杀基因表达载体构建

目的：探讨载脂蛋白A-I结合蛋白（AIBP）在肝癌细胞中的表达及意义。方法：用RT-PCR与Western blot法分别检测正常肝细胞株L02,AFP阳性人肝癌细胞株HepG2和Hep3B,及AFP阴性人肝癌细胞株SMMC7721中AIBP基因与蛋白的表达;构建AFP启动子驱动的双自杀基因（CD,TK）+AIBP基因过表达载体pcDNA3.1-AFP-AIBP-yCD/TK,并转染Hep3B和SMMC7721细胞,用MTT法检测转染后细胞的增殖能力,用RT-PCR和Western blot法检测细胞AIBP,血管内皮生长因子（VEGF）,VEGF受体2（VEGFR-2）,基质金属蛋白酶9（MMP-9）基因和蛋白的表达。结果：AIBP mRNA和蛋白在正常肝细胞中高表达,而在各肝癌细胞系中均表达下调,且Hep3B和SMMC7721细胞中下调明显。成功构建pcDNA3.1-AFP-AIBP-yCD/TK真核表达质粒并转染入Hep3B和SMMC7721细胞。转染后AFP阳性Hep3B细胞生长到明显抑制,但AFP阴性SMMC7721细胞增殖不受影响;两种细胞的AIBP基因与蛋白表达均明显上调,而VEGFR-2,VEGF和MMP-9基因与蛋白表达明显表达下调。定量指标间的差异均有统计学意义（均P<0.05）。结论：AIBP在肝癌细胞中表达下调,AIBP与肝癌细胞的侵袭转移能力有关,而与细胞增殖能力无关;成功构建了联合基因载体pcDNA3.1-AFP-AIBP-yCD/TK。

     

Mda-7/IL-24基因转染对肝癌细胞凋亡影响的研究

目的 为了研究Mda-7／IL-24基因对肝癌细胞系Hep3B凋亡的影响。方法 构建了Mda-7／IL-24基因的真核表达载体pcDNA3．1／Mda-7／IL-24，用脂质体介导的基因转染法分别将真核重组体pcDNA3．1／Mda-7／IL-24和pcDNA3．1空载体质粒导人人肝癌细胞系Hep3B细胞中，经G418筛选获得细胞克隆。通过聚合酶链式反应（PCR）、免疫组织化学等方法检测基因和蛋白的表达，同时检测了细胞的生长和凋亡情况。结果 pcDNA3．1／Mda-7／IL-24转染的Hep3B细胞能够检测到Mda-7／IL-24的表达，可以抑制细胞生长，DNA琼脂糖凝胶电泳显示凋亡细胞特有的“梯状”条带，转染空载体和未转染的Hep3B细胞未见凋亡表现。结论 将外源性Mda-7／IL-24基因导人Hep3B细胞后，可以促进凋亡，Mda-7／IL-24作为一种肝癌的治疗基因，具有广泛的应用前景。     

Oncolytic herpes viral therapy is effective in the treatment of hepatocellular carcinoma cell lines

The rising incidence of hepatocellular carcinoma (HCC) in western countries, along with the poor prognosis offered by present-day treatment modalities, makes novel therapies for this disease necessary. Oncolytic herpes simplex viruses (HSV) are replication-competent viruses that are highly effective in the treatment of a wide variety of experimental models of human malignancies. This study seeks to investigate the effectiveness of oncolytic herpes viruses in the treatment of primary HCC cell lines. Sixteen commercially available human HCC cell lines were studied. G207 is an attenuated, replication-competent, oncolytic HSV engineered to selectively replicate within cancer cells. Cell lines were tested for viral sensitivity to G207 and their ability to support viral replication using standard cytotoxicity and viral replication assays. Eleven of 16 cell lines were moderately to highly sensitive to G207 viral oncolysis. HCC cell lines additionally demonstrated the ability to support viral replication in vitro with as high as 800-fold amplification of the administered viral dose observed. G207 is cytotoxic to, and efficiently replicates within, HCC cell lines in vitro. From these data, we suggest that oncolytic HSV therapy may have a role in the treatment of HCC, and in vivo studies are warranted. Presented in part at the 2005 American Hepato-Pancreato-Biliary Association Congress, Hollywood, Florida, April 14–17, 2005. Supported by grants R01CA75461 and R01CA72632 from the National Institutes of Health, and by grant MBC-99366 from the American Cancer Society (Yuman Fong).     

Activation of human Kupffer cells by thymostimulin (TP-1) to produce cytotoxicity against human hepatocellular cancer

Background: In a small pilot study, thymostimulin (TP-1) produced tumor regression in almost 50% of patients with hepatocellular cancer (HCC) who were treated with TP-1 alone. However, the mechanism of the TP-1-mediated antitumor effect against HCC is unknown. Methods: Human hepatocytes and Kupffer cells were isolated from liver biopsy specimens by collagenase infusion and counterflow elutriation. Hepatocytes and Kupffer cells were incubated in vitro with clinically relevant doses of TP-1. Cell-free supernatants were collected at various time points after incubation. Hepatocyte and Kupffer cell supernatant levels for a panel of growth factors and monokines were determined by enzyme-linked immunosorbent assay. The cytotoxic activity of TP-1 alone and of TP-1-stimulated hepatocyte and Kupffer cell supernatants against Hep G2 and Hep 3B human HCC cells in vitro was measured by MTT assay. Results: Doses of TP-1 up to 100 µg/ml produced no cytotoxicity against Hep G2 or Hep 3B cells. Furthermore, supernatants from TP-1-treated hepatocytes produced no cytotoxicity against Hep G2 or Hep 3B cells, and TP-1 did not stimulate the release of transforming growth factor (TGF)-, TGF-, or hepatocyte growth factor. TP-1-treated Kupffer cell supernatants produced significant cytotoxicity against Hep G2 cells but produced no cytotoxicity against Hep 3B cells. Kupffer cells stimulated by TP-1 released significant amounts of tumor necrosis factor-alpha (TNF-), interleukin (IL)-1, and IL-6 compared with control Kupffer cells (p<0.01). The activity of TP-1-treated Kupffer cell supernatants against Hep G2 cells was blocked by anti-TNF- antibodies, whereas neither anti-IL-1 nor anti-IL-6 antibodies blocked cytotoxicity. Conclusions: These results demonstrate that TP-1 cytotoxicity against human HCC cells is not mediated directly or through hepatocytes, but occurs through activation of Kupffer cells and release of TNF-. Understanding the mechanism of TP-1 cytotoxicity against human HCC has been used to plan a phase I trial of TP-1 combined with regional infusion of doxorubicin to treat unresectable HCC.Presented at the 49th Annual Cancer Symposium of The Society of Surgical Oncology, Atlanta, Georgia, March 21–24, 1996.     

RNA干扰对肝癌细胞巨噬细胞移动抑制因子基因表达的抑制作用

目的 观察特异性小干扰RNA(siRNA)对肝癌细胞巨噬细胞移动抑制因子(MIF)基因表达的抑制作用.方法 脂质体方法将siRNA转染肝癌细胞PLC、Hep3B.定量RT-PCR、Western blot检测MIF mRNA和蛋白、MAPK信号分子的表达.噻唑蓝(MTY)比色法、体外细胞侵袭实验检测细胞增殖和对重组基底膜(matrigel)穿透能力.结果 100 nmol/L的MIF siRNA使MIF mR-NA表达下调81.3%和89.1%,蛋白水平降低69.50%、72.31%,与对照组比较其差异有统计学意义(P均＜0.01).细胞增殖率下降16.79%和47.14%(P均＜0.05).穿透matrigel的细胞数为51.00±11.27和18.56±4.72,与对照组比较差异有统计学意义(P均＜0.05).磷酸化MAPK下调.结论 MIF siRNA有效抑制MIF表达及肝癌细胞增殖和迁移,可能部分通过抑制MAPK磷酸化起作用.     

Ionizing radiation potentiates the antitumor efficacy of oncolytic herpes simplex virus G207 by upregulating ribonucleotide reductase

BACKGROUND: Replication-competent herpes simplex virus-1 (HSV-1) mutants have an oncolytic effect on human and animal cancers. The aim of this study was to determine whether G207, an HSV-1 mutant, can be combined with ionizing radiation (IR) to increase antitumor activity while decreasing treatment-associated toxicity. METHODS: This study was performed by using G207, a replication-competent HSV-1 mutant deficient in viral ribonucleotide reductase (RR) and the gamma(1)34.5 neurovirulence protein. The antitumor activity of G207 or IR was tested against HCT-8 human colorectal cancer cells in vitro and in an in vivo mouse subcutaneous tumor model. RESULTS: We demonstrated that G207 has significant oncolytic effect on HCT-8 cells in vitro in a cytotoxicity assay and in vivo in a mouse flank tumor model and that these effects are improved with low-dose IR. We further illustrated that the increased tumoricidal effect is dependent on the up-regulation of cellular RR by IR measured by a functional bioassay for RR activity. Chemical inhibition of RR by hydroxyurea abrogates the enhanced effect. In contrast to G207, R3616, the parent virus of G207 that expresses functional RR, does not exhibit enhanced oncolysis when combined with IR. CONCLUSIONS: These data encourage clinical investigation of combination radiation therapy and HSV oncolytic therapy.     

上调miR-449a表达对肝癌细胞生长和侵袭能力的影响

目的:探讨miR-449a在肝癌(HCC)细胞中的表达及其对HCC细胞生物学行为的影响。方法:用qRT-PCR检测miR-449a在正常肝细胞L02和4种HCC细胞株(HepG2、Hep3B、SMMC-7721和Bel-7402)的表达。用脂质体法将miR-449a模拟物或阴性对照序列转染HCC细胞后,分别用CCK-8法、流式细胞术、Transwell小室实验检测细胞增殖、细胞周期、侵袭能力的变化,并观察以上两种不同转染的HCC细胞在裸鼠体内的成瘤情况。结果:miR-449a在4种HCC细胞株的表达水平均明显低于L02细胞(均P0.05),其中在Bel-7402细胞表达的水平最低。与转染阴性对照序列的Bel-7402细胞比较,转染miR-449a模拟物的Bel-7402细胞增殖活性明显降低、G_1/S期阻滞明显增加、穿室细胞数明显减少(均P0.05);与转染阴性对照序列的Bel-7402细胞比较,转染miR-449a模拟物的Bel-7402细胞在裸鼠体内成瘤后的移植瘤质量与体积均明显减小(0.748 g vs.1.234 g;33.667 mm~3 vs.1 400.500 mm~3,均P0.05)。结论:HCC细胞中miR-449a表达降低,上调miR-449a表达可以抑制HCC细胞在体内外的生长。     

G207, modified herpes simplex virus type 1, kills human pancreatic cancer cells in vitro

Pancreatic cancer is often fetal, and farther effective therapeutic options are needed. This study was designed to assess whether the replication-restricted herpes simplex virus, G207, was effective in killing human pancreatic cancer cells in vitro. G207, a multimutated strain of herpes simplex virus type 1 carrying lacZ reporter gene, is capable of efficient cytolytic growth in many dividing cells, including certain tumor cells, but not in nondividing cells. Three human pancreatic cell lines, AsPC-1, MIA PaCa-2, and BxPC-3, were infected with G207 at different multiplicities of infection. After 24 hours, expression of the lacZ reporter gene was tested using a histochemical X-gal assay. In addition, cell lines were infected with G207 for 24 to 48 hours; then the virus obtained from cell pellets and media supernatant was used to infect Vero cells to obtain G207 titers by plaque assay. To assess whether increasing viral immediate early gene expression would improve cytolysis and virus production, similar experiments were performed with the addition of 0.5 mmol/L of hexamethylene bisacetamide (HMBA) 1 hour after viral infection. Finally, MTS cell viability assays were performed to measure viable cells at 24 to 96 hours post infection. The X-gal assay data revealed a viral dose-dependent β-galactosidase expression, indicating G207 infectivity and expression of the lacZ reporter gene. Plaque assays demonstrated a viral dose-dependent increase in plaque formation, indicating viral production from all three cell lines. In addition, HMBA data indicated a modest increase in viral production. The MTS assay data indicated a dose-dependent cytotoxicity for G207 in the cell lines tested. G207 infects, replicates in, and is cytotoxic to the above-listed human pancreatic cell lines in vitro and warrants therapeutic evaluation in models of pancreatic cancer. Supported by the Wilmot Cancer Research Fellowship Program funded by the James P. Wilmot Foundation. Presented at the Thirty-Ninth Annual Meeting of The Society for Surgery of the Alimentary Tract, New Orleans, La., May 17–20,1998.     

Effects of human hepatocyte growth factor on the proliferation of human hepatocytes and hepatocellular carcinoma cell lines

BACKGROUND: Hepatocyte growth factor (HGF) has been suggested to initiate both hepatocyte and tumor cell proliferation after partial hepatectomy, thereby supporting local tumor recurrence. The aim of this study was to clarify the role of HGF in the regeneration of human hepatocyte and the growth of residual hepatocellular carcinoma cells after liver resection. PATIENTS/METHODS: 36 patients who underwent partial hepatectomy for hepatocellular carcinoma (HCC) or living liver donation have been analyzed for HGF serum levels at day -1 through day 5 following surgery using an enzyme-linked immunosorbent assay. Isolated human hepatocytes and HCC cell lines (Hep 3B, Hep G2) were treated either with recombinant human (rh)-HGF, or sera from the 36 patients in the presence or absence of anti-HGF in order to measure their proliferative capacity using (3)H-thymidine incorporation. RESULTS: Basal HGF levels were significantly higher in HCC than in healthy patients (1,573 +/- 131 vs. 778 +/- 64 pg/ml; p < 0.001), however, the postoperative rise of HGF in healthy patients was higher (9,608 +/- 3111 vs. 2,060 +/- 148 pg/ml) than in HCC patients. Incubation of human hepatocytes and Hep 3B cells with rh-HGF revealed a dose-dependent increase in DNA synthesis, while anti-HGF partially abolished this effect. Sera from normal and resected HCC patients stimulated DNA synthesis only in human hepatocytes, whereas it was inhibited in HCC cell lines. CONCLUSION: HGF plays an important role in hepatocyte proliferation but contrary to in vitro results, HGF does not play a major role for the progression of hepatocarcinoma cells in vivo.     

缺氧诱导的lncRNA AC114803在肝细胞癌中的表达及其作用

背景与目的:缺氧是肝细胞癌(HCC)微环境的关键特征之一.缺氧可诱导编码基因和非编码RNA的表达,从而影响HCC的进展.既往研究发现缺氧反应性长链非编码RNA (lncRNA) AC114803与肾细胞癌预后相关,但其在HCC中尚未见研究.因此,本研究探讨HCC中lncRNA-AC114803表达与缺氧的关系及其功能....     

SiRNA干扰LI-cadherin对荷人肝癌裸鼠移植瘤的影响

目的 研究肝肠粘连蛋白(liver intestine cadherin,LI-cadherin)在荷瘤裸鼠体内对肝癌细胞Hep3B侵袭转移能力的作用.方法 体外培养肝癌细胞株Hep3B,并进行针对LI-cadherin的SiRNA转染.将Hep3B细胞及转染后的肿瘤细胞接种入裸鼠脾脏,建立裸鼠荷瘤模型.观察成瘤及转移效果,免疫组化检测转移灶组织结构,Western blot方法检测转染前后荷瘤裸鼠体内肿瘤转移灶中LI-cadherin的表达.结果 (1)SiRNA质粒转染入Hep3B肝癌细胞,转染率达到80％.(2)建立裸鼠荷人肝癌模型,30只裸鼠移植手术后均成活,荷瘤率达到60％以上.SiRNA转染组荷瘤率为80％,转移灶个数为26个,明显高于空白对照组和空质粒转染组.(3)裸鼠体内转移灶中Ll-cadherin 的含量SiRNA转染组明显低于空白对照组和空质粒转染组.结论 LI-cadherin对肝癌细胞的黏附能力有重要作用,对LI-cadherin进行SiRNA干扰可增强肝细胞癌在裸鼠体内的侵袭转移能力.     

趋化因子VCC-1在肝细胞癌中的表达及其意义

目的 研究趋化因子VCC-1在肝细胞癌中的表达及其临床意义.方法 采用RT-PCR法检测8种肝细胞癌细胞株、10例正常肝脏组织标本、42例肝癌组织及癌旁组织中的VCC-1的mRNA表达情况,并结合临床资料探讨VCC-1的临床意义.结果 肝细胞癌细胞株中,SUN398的VCC-1表达量最高,Hep3B和Huh7几乎不表达,SUN387、SUN449、SUN423、HepG2、PLC5的表达介于两者之间.42例肝癌患者标本中,癌组织及癌旁组织同时存在表达,癌组织高于癌旁组织26例(61％,14.9±7.6倍),癌旁组织高于癌组织16例(39％,6.9±5.4倍).癌组织表达上调(P＜0.01).VCC-1的表达水平与肿瘤分化程度、直径有关(P＜0.05).在10例正常肝脏标本中,8例无表达,2例微表达,其表达水平低于癌细胞株、肝癌组织及癌旁组织(P＜0.01),而复发的3例标本均有癌组织高表达(20.1±2.3倍).有癌栓的8例标本中,5例出现癌组织VCC-1高表达(17.3±4.5倍),3例低表达.结论 VCC-1表达升高可能与肝细胞癌的发生发展有关,并有可能成为肝细胞癌的治疗靶点.     

巨噬细胞移动抑制因子小干扰RNA对肝癌细胞表达血管内皮生长因子的抑制效应

目的 观察巨噬细胞移动抑制因子(MIF)小干扰RNA(siRNA)对肝癌细胞表达血管内皮生长因子(VEGF)的影响.方法 Western blot检测MIF和VEGF在肝癌和癌旁组织的表达情况;人工化学合成MIF siRNA和对照siRNA,将其转染PLC、HepG2肝癌细胞株,定量聚合酶链反应(PCR)和Western blot检测MIF和VEGF mRNA及其蛋白的表达.结果 MIF、VEGF mRNA和蛋白在肝癌组织中高表达.MIF siRNA(50、100 nmol/L)转染肝癌细胞株后,PLC细胞MIF和VEGFmRNA水平分别下调(78.8±7.2)%、(90.4±2.9)%和(60.6±2.6)%、(79.8±1.2)%;HepG2细胞MIF和VEGF mRNA水平分别下调(74.3±8.9)%、(88.4±4.6)%和(65.6±4.6)%、(80.7 ±2.2)%.MIF蛋白分别下调(57.3±3.4)%、(78.7±1.2)%和(54.8±6.8)%、(77.9±2.3)%,并呈剂量依赖关系;伴随MIF mRNA和蛋白的表达下调VEGF蛋白分别下调(52.6±12.9)%、(87.4±2.3)%和(52.4±11.1)%、(68.5±2.8)%,亦呈剂量依赖关系,与对照组比较差异有统计学意义(P<0.01),两干预组间的差异也有统计学意义(P<0.05).结论 MIF siRNA能特异性阻断PLC及HepG2细胞MIF mRNA和MIF蛋白的表达,并且同时有效下调VEGF mRNA及其蛋白的表达,MIF可能通过调控VEGF基因和蛋白的表达,参与肿瘤血管的生成和促进肿瘤细胞的转移.     

Stearoyl-CoA desaturase plays an important role in proliferation and chemoresistance in human hepatocellular carcinoma

Background

Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage, when it is not amenable for aggressive therapies such as surgical resection or liver transplantation. Current therapeutic options achieve clinical responses in only a small percentage of cases. As a consequence, effective approaches for prevention and treatment are greatly needed. Altered lipid metabolism has been recently linked to HCC pathogenesis. The aims of this study were to define the cellular and molecular mechanisms linking stearoyl-CoA desaturase (SCD), the rate-limiting enzyme and an essential regulator of lipid homeostasis in liver cells, to carcinogenesis in HCC.

Material and methods

HCC and normal liver specimens were collected. Human HCC cell lines: HepG2, Hep3B, and PLC/PLF/5 were used for immunoblot, cell viability, proliferation, and apoptosis assays. Small interfering RNAs were used for genetic inhibition, and 10, 12 conjugated linoleic acid was used for pharmacologic SCD inhibition.

Results

SCD was strongly expressed in surgically resected HCC (n = 64) and various human HCC cell lines (HepG2, Hep3B, and PLC/PLF/5). The levels of SCD negatively correlated with degree of tumor differentiation (P < 0.01). Treatment of these HCC cell lines with a panel of chemotherapeutic drugs resulted in a time-dependent, phosphatidylinositol 3 kinase- and c-Jun N-terminal kinases1/2–mediated upregulation of SCD expression, which paralleled the degree of resistance to drug-induced apoptosis. Specific genetic or pharmacologic SCD suppression resulted in inhibition of cell proliferation (P < 0.001) and significantly increased sensitivity to chemotherapy-induced apoptosis.

Conclusions

Our data suggest that increased SCD expression plays an important role in HCC development and resistance to chemotherapy-induced apoptosis, and this is in part mediated by phosphatidylinositol 3 kinase/c-Jun N-terminal kinases activation. Specific targeted interruption of this pathway in HCC could be a desirable approach in designing novel therapeutic strategies.     

miR-542-3p对肝癌细胞恶性生物学行为的影响及机制探讨

目的 探讨microRNA-542-3p(miR-542-3p)在人肝癌细胞系及组织中的表达水平及其对肝癌细胞增殖、侵袭转移能力的影响.方法 采用RT-PCR检测miR-542-3p在肝癌细胞系(HCCLM3、Hep3B、Huh7、SMMC-7721、MHCC-97H、MHCC-97L)以及人正常肝细胞LO2中的表达;...     

Hepatitis B virus gene expression in two cell lines, one derived from a natural human infection, the other experimentally infected in vitro

Hepatitis B virus (HBV) gene expression was investigated in the PLC/PRF/5 human hepatoma cell line and the mouse L-cell line transfected with HBV DNA by modulating the in vitro growth conditions. Both cell lines contain the viral genome integrated into cell chromosomes and both have been shown to produce hepatitis B surface antigen only. Treatment with 5-azacytidine (5-azaC) did not enhance hepatitis B core antigen (HBcAg) synthesis in experimentally transfected mouse L cells. Low levels of hepatitis B e antigen (HBeAg) were detected from the L cells regardless of 5-azaC treatment, and synthesis of HBeAg was dependent on the growth state of the cultures. No HBcAg or HBeAg was detected in PLC/PRF/5 cells with or without 5-azaC treatment. This observation suggests that there is a distinct difference between experimentally transfected cells with HBV and the naturally derived material.