CD80 transfected human hepatocellular carcinoma cells activate cytotoxic T lymphocytes to target HCC cells with shared tumor antigens

Human HCC cell lines (BEL-7402, SMMC-7721 and QGY-7703) do not express CD80 molecules, although they express MHC class I molecules and ICAM-1. HCC's poor immunogenicity may therefore be due to lack of CD80 molecules. This study first investigated whether CD80 molecules could provide minimal co-stimulatory signal for establishing an efficient anti-tumor immunity in HCC and second, whether the transfection of CD80 into the BEL-7402 cell line could induce T cell activation for targeting other HCC cell lines expressing shared common antigens. The transfection of cDNA encoding CD80 into ICAM-1+ HCC BEL-7402 cells was confirmed by flow cytometrical analysis. The CD80-transfected cells could enhance the immunogenicity of BEL-7402 cells as detected by T cell proliferation assay, and also activated the T cells at a higher proliferation rate comparing with the BEL-7402 cells transfected with vector only. The CD80-transfected cell line was also found able to activate T cells which subsequently induced cell lysis of SMMC-7721, QGY-7703 and parent BEL-7402 cell lines as detected by cytotoxicity assay. It can be concluded that the cytotoxicity was due to MHC class I restricted CD8+ cytotoxic T lymphocytes, but not natural killer (NK) cells, since this cytotoxic effect could be blocked by anti-MHC class I antibody and the cytotoxicity was shown very low in NK-cell-sensitive K562 cell line. Electroporation of CD80 cDNA into human HCC cells could increase the expression of the functional CD80 molecules and enhance the immunogenicity of the genetically-modified HCC cells to activate T cells for targeting 3 HCC cell lines in an HLA-restricted manner.     

人肝癌相关抗原表达与细胞周期的关系

The expression of human hepatoma associated antigen A25 in relation to the different stages of cell cycle is reported as studied by monoclonal antibody and flow cytometry methods. A25 antigen was found to express all the four human hepatoma cell lines we tested. Hepatoma cell line PUMC-H1 expressed the antigen at higher levels than SMMC-7721 and QGY-7703 whereas BEL-7402 expressed at lower levels. Further studies showed that the cell lines PUMC-H1, SMMC-7721 and QGY-7703 were found to be at the similar stages of cell cycle while QGY-7703 was different from these three cell lines by having more cells in S state but fewer cells in G0-G1 stages. In the same cell line, A25 was expressed during all cell cycle stages though at different levels. It was expressed at the lowest level in G0-G1 stages, a slightly higher level in S stage and the highest in G2-M stage. The results in this paper suggest that the expression of A25 is related to the stage of cell cycle in the same cell line but not related to the stage of cell cycle in different cell lines with similar tissue origin.     

野生型RASSF1A基因抑制肝癌细胞增殖的分子机制

背景与目的:RASSF1A(ras association domain family 1A)基因抑制肿瘤增殖的分子机制目前尚未完全明确,本文将探讨野生型RASSF1A基因抑制肝癌细胞增殖的分子机制.方法:利用已构建成功的稳定表达野生型和突变型RASSF1A基因的肝癌细胞株QGY-7703,使用流式细胞仪对其进行细胞周期的测定,Western blot分析其cyclin D1、cyclin E及P21蛋白的表达水平,通过检测荧光素酶报告基因活性及RT-PCR检测野生型RASSFIA基因的表达来分析RASSF1A基因对cyclin D1启动子和mRNA表达水平的影响.结果:野生型RASSF1A基因的表达可使QGY-7703细胞的细胞周期发生G1/S期阻滞,使其cyclin D1蛋白的表达水平下调,但不影响cyclin E和P21蛋白的表达水平.外源性cyclin D1的表达可使野生型RASSF1A基因引起的QGY-7703细胞G1/S期阻滞消失.野生型RASSF1A基因表达不影响QGY-7703细胞cyclin D1启动子的活性和cyclin D1 mRNA的表达水平.结论:野生型RASSF1A基因通过转录后机制负性调控cyclin D1蛋白的表达,使肝癌细胞株QGY-7703细胞周期阻滞在G1/S期.     

Chemokine Expression Profiles of Human Hepatoma Cell Lines Mediated by Hepatitis B Virus X Protein

The hepatitis B virus X protein (HBx), which is encoded by hepatitis B virus (HBV), plays crucial roles in the tumorigenesis of HBV associated hepatocellular carcinoma (HCC). Recent studies suggest that the HBx is involved in regulation of host immune cytokines and chemokines in HBV-associated HCC patients. However, effects of the HBx on autocrine chemokine expression profiles of hepatoma cells, which were shown in modulation of tumor-immune cell interactions, have not been investigated comprehensively. In the present study, human hepatoma cell lines SMMC-7721 and HepG2 were transfected with HBx-expressing plasmid. Human chemokine antibody array 1 (RayBio®), which simultaneously detects 38 chemokine factors, was used to determine chemokine expression profiles. Real-time polymerase chain reaction (real-time PCR) was used to further confirm the differential expression of chemokines. Chemokine antibody array revealed that all 38 chomekines were found to be expressed by SMMC-7721 and HepG2 cell lines. Interleukin-8 (IL-8) was obviously up-regulated, and epithelial neutrophil-activating protein 78 (ENA78), eosinophil chemotactic protein-1 (Eotaxin-1), monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3 and macrophage inflammatory protein-3β (MIP-3β) were significantly declined in both cell lines following transfection of HBx-expressing plasmid. Other chemokines showed little or no significant changes. HBx-induced differential chemokine expression levels were validated by real-time PCR. Hierarchical cluster analysis identified a distinction of chomekine expression profiles between HBX-expressing hepatoma cell lines and controls. Our findings provide new evidence that HBx is able to selectively regulate chomekines in hepatoma cells that may be involved in the regulation of tumor-immune cell interactions.     

ARHI, as a novel suppressor of cell growth and downregulated in human hepatocellular carcinoma, could contribute to hepatocarcinogenesis

The identification of cancer genes differentially expressed in hepatocellular carcinoma (HCC) plays an important role in understanding the molecular mechanisms of hepatocarcinogenesis. Here, ARHI gene expression was analyzed by real-time RT-PCR and it was significantly downregulated in 33 of the 42 (78.6%, more than two folds) HCC specimens compared with adjacent noncancerous livers (P < 0.01). In addition, ARHI expression was reduced in some HCC samples at protein level confirmed by immunohistochemistry. Furthermore, our data suggested that the overexpression of ARHI can significantly inhibit cell growth and colony formation of Hep3B cells (P < 0.01), whilst silencing endogenous ARHI gene by RNAi could promote cell growth of Huh-7 and Focus. LOH of microsatellite markers D1S2806 and D1S2803 was only found in 2.4% (1 of 42 HCCs) of HCC cases. The expression of ARHI was obviously re-expressed in some HCC cells, Bel-7402, Bel-7405, QGY-7703 and Hep3B, by a demethylation agent, 5-aza-2'-deoxycytidine (DAC). DNA hypermethylation within ARHI promoter was identified in 47.1% of HCC specimens without ARHI expression. Our current observations provide evidences that ARHI downregulated in HCCs could play a role in liver cancer via acting as a tumor suppressor gene, which mainly was triggered by the epigenetic events in HCC specimens.     

TPX2 knockdown suppressed hepatocellular carcinoma cell invasion via inactivating AKT signaling and inhibiting MMP2 and MMP9 expression

Objective： Targeting protein for Xenopus kinesin-like protein 2 （TPX2） is a nuclear proliferation-related protein that plays a critical role in the formation of mitotic spindle. High expression of TPX2 has been observed in several types of tumors. However, the role of TPX2 in hepatocellular carcinoma （HCC） remains unclear. Our study aimed to investigate the effect of TPX2 on HCC cell invasion. Methods： The immortalized normal human liver cell line L02 and six HCC cell lines including SMMC- 7721, BEL-7402, Huh-7, HepG2, Hep3B and SKHepl were subjected to qRT-PCR and western blot for TPX2 mRNA and protein, respectively. Furthermore, TPX2 small interfering RNA （siRNA） was used to knock down TPX2 expression in SMMC-7721 and HepG2 cells. Cell proliferation and invasion were determined by MTT and transwell assays. Otherwise, expression of p-AKT, MMP2 and MMP9 were evaluated by western blot in SMMC-7721 cells. Results： The expression of TPX2 in HCC cell lines was markedly higher than that in normal human liver cell line. TPX2 knockdown using a specific TPX2-siP, NA reduced the number of invaded cells and inhibited cell proliferation in SMMC-7721 and HepG2 cells. Furthermore, TPX2 knockdown resulted in inactivation of AKT signaling and down-regulation of MMP2 and MMP9 expression in SMMC-7721 cells. Conclusions： Our study identified that TPX2 might contribute to tumor cell invasion through activating AKT signaling and subsequently increasing MMP2 and MMP9 in HCC.     

核因子蛋白90敲减对肝癌细胞增殖影响及机制的研究

目的探究核因子蛋白90(NF90)敲减对肝癌细胞增殖影响并探讨其可能机制。方法将靶向NF90的寡核苷酸链克隆至PLKO质粒后,包装出靶向敲减NF90的慢病毒shRNA(带有绿色荧光标签及G418抗性)。将肝癌细胞QGY-7703、SMMC-7721分为对照组及干扰组,分别感染包含随机序列shRNA和靶向NF90 shRNA的病毒液。48 h后流式分选出带有绿色荧光的阳性细胞,G418筛选阳性单克隆细胞株,Western blotting鉴定NF90的蛋白表达水平,最终在对照组中选取一株细胞命名shRNA-ns,在干扰组中选取NF90敲减效果良好且稳定的两株细胞命名为shRNA-1、shRNA-2。采用CCK-8法检测各株细胞的增殖情况,克隆形成试验分析各株细胞的克隆形成能力。质谱分析预测NF90的相关结合蛋白,内、外源免疫共沉淀确定NF90与多聚ADP核糖合成酶(PARP1)的关系,荧光定位分析NF90与PARP1在细胞内的定位情况。结果 shRNA-1、shRNA-2细胞中的NF90表达水平显著低于shRNA-ns细胞,表明包装的慢病毒敲减NF90效果良好。与shRNA-ns细胞比较,shRNA-1、shRNA-2细胞的生长缓慢,克隆形成数减少,差异均有统计学意义(P<0.01)。NF90与PARP1在细胞内相互结合并且共定位于细胞核。结论敲减NF90可抑制肝癌细胞增殖,其机制可能与PARP1相互作用有关。     

MiR 142 5p通过靶向IGF2BP3调控多柔比星诱导的肝癌细胞凋亡

目的:探讨miR-142-5p对多柔比星诱导的原发性肝细胞癌(hepatocellular carcinoma,HCC)细胞凋亡的影响及其作用机制.方法:收集广西医科大学附属肿瘤医院88例HCC患者手术切除的癌组织及癌旁组织(距癌灶组织边缘2～5cm)标本.采用实时荧光定量PCR检测人HCC组织和癌旁组织、人正常肝细胞以及HCC细胞系中miR-142-5p的表达量.向HCC细胞SMMC-7721中转染miR-142-5p mimics,流式细胞术检测过表达miR-142-5p后SMMC-7721细胞在多柔比星(doxorubicin)(1 μg/ml)诱导下凋亡的变化;生物信息学方法预测miR-142-5p可靶向结合胰岛素样生长因子2 mRNA结合蛋白3(in-sulin-like growth factor 2 mRNA-binding protein 3,IGF2BP3)基因,并采用荧光素酶报告基因实验进行验证.采用实时荧光定量PCR及Western blotting检测过表达miR-142-5p的SMMC-7721细胞中IGF2BP3的mRNA及蛋白表达情况.结果:与癌旁组织和正常肝细胞相比,HCC组织(-6.91±2.61vs-11.59±2.59,P＜0.01)和多种HCC细胞系中miR-142-5p呈明显低表达(均P＜0.01);过表达miR-142-5p可显著促进多柔比星诱导的HCC细胞SMMC-7721的凋亡[(49.40±3.47)％ vs (19.50±1.74)％,P＜0.01];过表达miR-142-5p可明显降低HCC细胞中IGF2BP3的mRNA及蛋白表达水平(P＜0.01),敲减IGF2BP3表达可进一步促进多柔比星诱导的SMMC-7721细胞的凋亡(P＜0.01).荧光素酶报告基因实验结果显示,miR-142-5p能够抑制IGF2BP3的3'UTR荧光素酶报告基因的活性.结论:miR-142-5p在HCC组织标本和体外培养细胞系中的表达水平均显著降低,转染miR-142-5p mimics后能够促进多柔比星诱导的HCC细胞的凋亡,其机制可能与miR-142-5p靶向作用IGF2 BP3从而促进HCC细胞凋亡有关.     

Downregulation of CD147 expression alters cytoskeleton architecture and inhibits gelatinase production and SAPK pathway in human hepatocellular carcinoma cells

Background

CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA) against CD147 (si-CD147) on hepatocellular carcinoma cells' (SMMC-7721) architecture and functions.

Methods

Flow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK), vinculiln and mitogen-activated protein kinase (MAPK) expression in SMMC-7721 cells.

Results

Downregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression.

Conclusion

CD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.     

肝癌细胞 67kDa层粘连蛋白受体的表达

背景与目的:本实验室最近发现人肝癌细胞 SMMC-7721表达的 67kDa层粘连蛋白受体( 67kDa Laminin receptor,67LR)与层粘连蛋白的结合能力明显高于正常肝细胞 L-02表达的 67LR,而 67LR在肝癌细胞和正常肝细胞中的表达水平尚不清楚.本研究旨在探讨 67LR在肝癌细胞中的表达,及其与层粘连蛋白结合能力的关系.方法:以人肝癌细胞 SMMC-7721、 HepG2和正常肝细胞 L-02为材料,采用 131I标记的层粘连蛋白测定其与细胞的结合能力;采用流式细胞术和 RT-PCR分析 67LR蛋白和 mRNA的表达水平. 结果: (1)相同条件下 SMMC-7721、 HepG2 细胞与层粘连蛋白特异结合量分别为 (17.54± 0.49) ng/105 cell、 (11.18± 0.53) ng/105 cell,而 L-02细胞的特异结合量为 (8.36± 0.48) ng/105 cell,表明肝癌细胞与层粘连蛋白的结合能力明显高于正常肝细胞( P< 0.01) ;(2)流式细胞术分析, SMMC-7721细胞的 67LR阳性表达率为 34.7%, L-02细胞的 67LR阳性表达率为 55.3%,而 HepG2 细胞则几乎不表达 67LR; (3)RT-PCR产物进行半定量分析发现,两株肝癌细胞的 67LR mRNA表达水平明显高于 L-02细胞.结论:肝癌细胞与层粘连蛋白的结合能力明显高于正常肝细胞 L-02,而细胞膜表面的 67LR水平却低于 L-02细胞,提示 SMMC-7721细胞 67LR的亲和力可能高于 L-02细胞,而且还涉及其它层粘连蛋白受体.     

miR-223-3p 通过靶向RAC1 调控肝细胞癌SMMC-7721 细胞的增殖和凋亡

目的：探讨miR-223-3p 通过调控Ras 相关C3 肉毒素底物1（Ras-related C3 botulinum toxin substrate 1，RAC1）对肝细胞癌（hepatocellular carcinoma，HCC）细胞增殖和凋亡的影响及其可能的作用机制。方法：选用2016 年8 月至2018 年8 月吉林市中心医院手术切除的30 例HCC 组织及其癌旁组织标本和人HCC 细胞系SMMC-7721、Bel-7402、HepG2 及人正常肝细胞QSG-7701，用qPCR检测HCC组织和细胞系中miR-223-3p的表达水平。分别将miR-223-3p mimics、miR-223-3p inhibitor 和siRAC1转染至SMMC-7721 细胞，通过CCK-8、克隆形成实验和Annexin V-FITC/PI 染色流式细胞术检测SMMC-7721 细胞的增殖、克隆形成和凋亡水平。用双荧光素酶报告基因实验检测miR-223-3p 与RAC1 的靶向关系，Western blotting 检测细胞中RAC1 蛋白的表达水平。结果：miR-223-3p 在HCC组织的表达水平显著低于癌旁组织（P<0.01），其表达水平与肿瘤大小、TNM分期及肿瘤分化病理特征相关（P<0.05 或P<0.01）；miR-223-3p 在HCC细胞系表达水平显著低于QSG-7701 细胞（均P<0.01），以在SMMC-7721细胞中表达水平最低。双荧光素酶报告基因实验证实RAC1 是miR-223-3p 靶基因，miR-223-3p 靶向负调控RAC1 的表达。转染miR-223-3p mimics 显著抑制SMMC-7721 细胞的增殖和克隆形成能力（P<0.05 或P<0.01），并促进细胞凋亡（P<0.01）；转染miR-223-3p inhibtor 则逆转miR-223-3p mimics 对细胞的抑制作用。结论：过表达miR-223-3p 抑制HCC细胞增殖和克隆形成能力并促进细胞凋亡，其机制可能与靶向下调RAC1表达有关。     

Chemosensitivity of human hepatocellular carcinoma cell line QGY-7703 is related to bcl-2 protein levels.

Bcl-2 protein is one of the major apoptosis regulators. The study examines the effect of Bcl-2 protein on the chemosensitivity of a human hepatocellular carcinoma cell line, QGY-7703. Western blot analysis showed that Bcl-2 and Bax proteins were expressed in QGY-7703 cells. Characteristic features of Taxol- and doxorubicin-induced apoptosis were evidenced by the Annexin-V binding assay, TUNEL and DAPI staining. At constant Bax protein levels, stable sense and antisense gene-transfected QGY-7703 cells showed that constitutive expression of Bcl-2 could render the cells more resistant to Taxol and doxorubicin. Contrarily, decreased Bcl-2 levels caused the cells to be more sensitive to the drugs. As Bcl-2 levels are directly proportional to the resistance of QGY-7703 cells to Taxol and doxorubicin, manipulation of Bcl-2 could be performed to enhance the sensitivity of liver cancer to chemotherapeutic agents. Copyright Copyright 1999 S. Karger AG, Basel     

RASSF1A基因对肝癌细胞化疗药物敏感性的影响

目的探讨RASSF1A基因的表达对肝细胞肝癌化疗药物敏感性的影响。方法利用已构建成功的稳定表达野生型和突变型RASSF1A基因的肝癌细胞株QGY-7703,化疗药物Mitomycin、Adriamycin、Etoposide、5-Fluorouracilur、Cisplatin分别作用各细胞株后,比较生长抑制率、细胞周期及P53、P21、Bax、Caspase-3蛋白的表达水平。结果野生型RASSF1A的表达可提高Mitomycin诱导的肝癌细胞生长抑制率、凋亡发生率（P<0.05）和Caspase-3的活性,对P53、P21、Bax基因的蛋白表达水平没有影响。结论野生型RASSF1A基因可提高肝癌细胞对Mitomycin的敏感性。     

双向电泳分析肝癌细胞线粒体的差异表达蛋白

背景与目的:线粒体在细胞凋亡中扮演关键的角色,对线粒体蛋白质组的研究可能有助于解释癌症的发生、早期诊断以及抗癌药物的研发。本研究对正常肝细胞L02线粒体、表阿霉素干预前后肝癌细胞QGY-7703线粒体进行二维电泳图谱的比较分析,筛选差异表达蛋白斑点。方法:运用蔗糖密度梯度的超离心方法分离纯化线粒体,双向凝胶电泳分离线粒体蛋白质,ImageMaster2D软件对所得图谱进行图像分析。结果:L02细胞线粒体、表阿霉素干预前QGY-7703细胞线粒体、表阿霉素干预后QGY-7703细胞线粒体三种标本超离心后,其纯化线粒体琥珀酸脱氢酶比活力分别提高13.8、11.8、10.2倍。L02细胞线粒体和表阿霉素干预前后QGY-7703细胞线粒体经双向凝胶电泳分辨出206、217、214个蛋白斑点,L02细胞与QGY-7703细胞比较筛选出49个差异表达蛋白斑点;表阿霉素干预前后的QGY-7703细胞进行比较,筛选到29个差异表达蛋白斑点。结论:运用超离心和双向电泳的方法成功筛选出一批在QGY-7703细胞线粒体中差异表达的蛋白斑点。     

人剪切修复基因XPD对肝癌细胞生长的抑制作用

目的探讨野生型人剪切修复基因着色性干皮病基因D(xeroderma pigmentosum D,XPD)对肝癌细胞SMMC-7721增殖的影响,并探讨其机制。方法用脂质体转染法瞬时转染SMMC-7721细胞,转染重组质粒XPD-N2和空载质粒N2,并用未转染的与XPD-N2、N2具有相同遗传背景和代数的SMMC-721细胞作为空白对照。荧光显微镜观察绿色荧光蛋白报告基因表达情况,流式细胞仪检测细胞周期,逆转录-聚合酶链反应（RT-PCR）、Western blot法检测细胞中XPD、c-myc、cdc25A、cdK2表达量变化,MTT法观察细胞增殖的活力。结果提取出的重组质粒pEGFP-N2-XPD用酶切鉴定,与Genebank上的相符。在荧光显微镜下,可以在SMMC-7721-pEGFP-N2、SMMC-7721-pEGFP-N2-XPD细胞中观察到绿色荧光蛋白的表达,质粒的转染效率为30%左右。流式细胞仪结果显示,pEGFP-N2-XPD重组质粒转染入细胞后,肝癌细胞进入S期发生阻滞,停滞在G1期。RT-PCR、Western blot检测发现SMMC-7721-pEGFP-N2-XPD细胞与SMMC-7721-pEGFP-N2和SMMC-7721两对照组相比,其XPD 表达明显增高（P<0.05）。c-myc、cdc25A、cdK2相对表达量明显减少,差异有统计学意义（P<0.05）。与两对照组相比,SMMC-7721-pEGFP-N2-XPD细胞增殖率明显减弱（P<0.05）。结论野生型XPD基因可以在转录和翻译水平抑制SMMC-7721细胞内c-myc、cdc25A、cdK2的表达。而且野生型XPD基因通过抑制cdK2的表达作用于S期DNA损伤检控点,从而抑制SMMC-7721细胞增殖。     

CSIG promotes hepatocellular carcinoma proliferation by activating c-MYC expression

Cellular senescence-inhibited gene (CSIG) protein significantly prolongs the progression of replicative senescence, but its role in tumorigenesis is unclear. To reveal the role of CSIG in HCC, we determined its expression in HCC tissues and surrounding tissues and its functions in tumor cell proliferation in vitro and in vivo. CSIG protein was overexpressed in 86.4% of the human HCC cancerous tissues as compared with matched surrounding tissues, and its protein expression was greater in HCC cells than the non-transformed hepatic cell line L02. Furthermore, upregulation of CSIG significantly increased the colony formation of SMMC7721 and HepG2 cells, and silencing CSIG could induce cell cycle arrest and cell apoptosis. The tumorigenic ability of CSIG was confirmed in vivo in a mouse xenograft model. Our results showed that CSIG promoted the proliferation of HepG2 and SMMC7721 cells in vivo. Finally, CSIG protein directly interacted with c-MYC protein and increased c-MYC protein levels; the ubiquitination and degradation of c-MYC protein was increased with knockdown of CSIG. CSIG could also increase the expression of c-MYC protein in SMMC7721 cells in vivo, and it was noted that the level of c-MYC protein was also elevated in most human cancerous tissues with high level of CSIG.     

转录水平RNA干扰肝素酶基因对肝癌细胞侵袭能力的影响

目的 探讨转录水平基因沉默（TGS）技术和转录后水平基因沉默（PTGS）技术对肝素酶（HPA）基因干扰效果及其对肝癌SMCC-7721细胞侵袭能力的影响。方法 从HPA基因启动子区和编码区分别设计并合成TGS和PTGS的小干扰RNA（siRNA）,并转染肝癌细胞SMMC-7721;实时半定量PCR和Western blotting检测TGS和PTGS siRNA转染后48、72和96h HPA的表达,并设空白组作为对照;Transwell小室实验检测干扰后SMMC-7721细胞的侵袭能力。结果 TGS和PTGS两种技术在转染48h后,从mRNA和蛋白水平上均能成功干扰HPA表达;转染后72h,PTGS组HPA恢复表达,而TGS组仍保持沉默;转染后96h,两组HPA均恢复表达。Transwell小室实验表明,TGS和PTGS组HPA基因均能使SMCC-7721的穿膜细胞数减少,TGS组效果更明显。结论 TGS技术沉默肝癌SMMC-7721细胞中HPA基因的能力优于PTGS技术,并使肝癌细胞的侵袭能力显著降低。     

肿瘤转移相关基因原肌球蛋白2的克隆及功能初步研究

目的构建携带肿瘤转移相关基因原肌球蛋白2(TM2)的绿色荧光蛋白(GFP)真核表达载体,初步探讨其在肿瘤细胞稳定表达后的生物学功能。方法采用逆转录多聚酶链反应(RT- PCR)从人胃癌细胞株AGS中扩增全长的TM2-cDNA序列,采用基因重组技术构建带TM2的绿色荧光蛋白真核表达载体pIRES2-EGFP;通过脂质体转染方法将重组基因转入TM2低表达肝癌细胞株QGY-7703中。荧光显微镜观察及流式细胞仪检测重组pIRES2-EGFP-TM2的荧光表达;Western blot检测细胞转染后TM2的蛋白表达;研究TM2基因与肝癌细胞株QGY-7703体外侵袭、移动及裸鼠成瘤等生物学特性的关系。结果成功构建了携带TM2的绿色荧光蛋白真核表达载体;荧光显微镜观察显示,在转染重组质粒的QGY-7703细胞中均有绿色荧光的表达;流式细胞仪检测显示,QGY-7703- EGFP-TM2的荧光表达率为90%以上;Western blot检测证实,TM2在转染的QGY-7703细胞系中表达明显升高;转染了TM2的细胞株侵袭能力(45.6±9.9)显著高于其亲代细胞(21.6±3.3,P<0.05);转染TM2的细胞株迁移能力(41.4±11.8)显著强于其亲代细胞(16.7±3.7,P<0.05);实验组、空白对照组和载体对照组肿瘤平均体积分别为(241.5±95.1)mm3、(123.7±92.3)mm3和(100.6±85.4)mm3,实验组与空白对照组比较,差异有统计学意义(P<0.01),表明转染TM2能明显促进肿瘤的生长。结论TM2基因对促进肝癌细胞的生长和转移发挥重要作用。     

The effect of NET-1 on the proliferation, migration and endocytosis of the SMMC-7721 HCC cell line

To explore the effect of NET-1 on the proliferation, migration and endocytosis in the hepatocellular carcinoma (HCC) cell line SMMC-7721, we constructed the pU6H1-NET-1-siRNA (NET-1siRNA) and pcDNA3.1/myc-NET-1 (myc-NET-1) vectors and transfected them into SMMC-7721 cells. The expression levels of NET-1 mRNA and protein were detected using real-time quantitative RT-QPCR and western blotting. The proliferation rates of SMMC-7721 cells were determined by CCK-8 assays, flow cytometry (FCM) and immunohistochemistry staining. The migration in two or three dimensional space of SMMC-7721 cells were determined by wound-healing assay and in vitro invasion assay. The extent of endocytosis in SMMC-7721 cells was estimated by observing the amount of transferrin (Tfn) absorbed with capture ELISA assays, and Tfn endocytosis was observed under confocal immunofluorescence microscopy. The results show that: i) after transfecting NET-1 siRNA, the expression of NET-1 mRNA and protein in SMMC-7721 cells decreased significantly, the growth of cells was suppressed, which induced cell cycle arrest, the proliferation rates were dramatically reduced and the expression of Ki67 declined, and migration and endocytosis in cells were inhibited, compared with untreated cells (every P<0.01); ii) Following transfection with myc-NET-1, the expression of NET-1 mRNA and protein in SMMC-7721 cells increased, and both the proliferation of cells and the cell cycle were promoted (P<0.01, respectively). However, the abilities of cell migration and endocytosis were not affected compared with untreated cells. These data suggest that: i) the NET-1 gene may play an important role in proliferation, migration and endocytosis of cells; ii) siRNA technology may efficiently suppress the expression and function of NET-1 in HCC, suggesting that NET-1 may be a therapeutic target for HCC.