蛋白激酶PRKX对人肝癌细胞粘附和迁移能力的影响

目的观察蛋白激酶PRKX对人肝癌细胞SMMC-7721粘附和迁移能力的影响。方法采用脂质体转染的方法,将PRKX表达质粒转染到SMMC-7721细胞中,蛋白印迹方法鉴定转染前后PRKX蛋白的表达。细胞-基质粘附实验测定对照组和PRKX转染组SMMC-7721细胞的粘附能力。细胞迁移实验测定对照组和PRKX转染组SMMC-7721细胞的迁移能力。结果 SMMC-7721细胞转染组PRKX蛋白的表达增加,SMMC-7721细胞转染组的粘附能力和迁移能力均较对照组增加。结论 PRKX可增加人肝癌细胞SMMC-7721的粘附和迁移能力。     

Downregulation of NIN/RPN12 binding protein inhibit the growth of human hepatocellular carcinoma cells

The ribosome assembly factor NIN/RPN12 binding protein (Nob1) has been suggested to be essential for processing of the 20S pre-rRNA to the mature 18S rRNA, and is also reported to participate in proteasome biogenesis. However, it is unclear whether Nob1 is involved in tumor cells growth. The aim of this study was to determine whether the suppression of Nob1 by short hairpin RNA (shRNA) inhibits the growth of human hepatocellular carcinoma (HCC) cells. Recombinant lentiviral shRNA expression vector carrying Nob1 was constructed and then infected into human HCC cell line SMMC-7721. The growth properties of SMMC-7721/pGCSIL-GFP-shNC and pGCSIL-GFP-shNob1 cells were determined by MTT, BrdU incorporation assay, and flow cytometric analysis. In addition, the colony formation and tumor growth ability in nude mice were detected to define the function of Nob1 in cell transformation and tumorigenesis. Our data showed that the growth and proliferation of SMMC-7721/pGCSIL-GFP-shNob1 cells were significantly reduced compared with the SMMC-7721/pGCSIL-GFP-shNC. In addition, the colony formation was impaired after the suppression of Nob1 in SMMC-7721 cells. And in vivo, the tumor formation ability of the SMMC-7721/pGCSIL-GFP-shNob1 cells was significantly reduced compared with the control cells. Our data support that Nob1 is an important regulator of the tumorigenic properties of human HCC and could be used as a candidate therapeutic target in human HCC.     

乙型肝炎病毒X基因真核表达载体的构建及表达

目的:构建含有天然完整的乙型肝炎病毒(HBV)X基因序列的真核表达载体,观察其在肝癌细胞株中的表达。方法:设计并合成HBV X基因的引物,用PCR方法从含完整HBV全基因的HepG2细胞中扩增X基因序列,并将其连接到真核表达载体pVAX-1上,酶切、PCR鉴定;用Triton X-114去除质粒内毒素后,采用电穿孔法将重组质粒pVAX-HBV X和空质粒pVAX-1分别转染SMMC-7721细胞,RT-PCR法检测HBV X基因mRNA的表达,Western印迹鉴定HBV X蛋白(HBx)的表达。结果:酶切和PCR鉴定证实pVAX-HBV X载体中包含完整的HBVX基因片段,该重组质粒转染的SMMC-7721细胞中HBV X基因mRNA及HBx蛋白的表达稳定。结论:构建了HBV X基因的真核表达载体,为X基因及其编码蛋白的生物学功能的研究提供了可靠的基因材料。     

沉默EVL表达抑制肝癌细胞SMMC-7721的增殖和迁移能力

Ena/VASP 样蛋白（Ena/VASP like protein,EVL）是Ena/VASP家族成员之一,它参与肌动蛋白细胞骨架重组,以及细胞迁移、收缩环形成和细胞间附着.EVL在肝癌SMMC-7721细胞中高表达. 抑制EVL蛋白表达后,SMMC-7721细胞的增殖与迁移能力降低.为研究EVL在肝癌细胞的功能,构建了靶向shRNA干扰表达载体,稳定转染肝癌SMMC-7721细胞. MTT实验和细胞集落形成实验显示,与转染对照比较,沉默EVL蛋白表达可明显抑制SMMC-7721肝癌细胞的增殖、集落形成能力. Transwell实验证明,沉默EVL表达导致SMMC-7721细胞迁移能力降低. 进而,流式细胞术揭示,沉默EVL表达的SMMC-7721细胞G0/G1期细胞比例增多.研究结果提示,EVL蛋白可促进肝癌细胞的增殖与迁移;该结果可解释EVL在肝癌细胞中高表达的意义.     

Osteopontin promotes hepatocellular carcinoma invasion by up-regulating MMP-2 and uPA expression

Osteopontin (OPN) is over-expressed in a variety of cancers, but its role in hepatocellular carcinoma (HCC) progression has not been clarified. In this study, weakly tumorigenic, non-metastastic human HCC cell line SMMC-7721 cells were forced to over-express OPN via stable transfection. A series of functional assays were performed to assess the effects of OPN on tumor cell behaviors and cDNA microarray was used to identify the genes regulated by OPN. The results showed that OPN significantly enhanced the migration and invasion of SMMC-7721 cells in vitro. In addition, CD44v6 antibody could significantly inhibit the invasion of OPN over-expressing SMMC-7721 cells. Moreover, MMP-2 and uPA expressions were significantly up-regulated in OPN over-expressing SMMC-7721 cells. Together, these findings indicate that OPN enhanced HCC cells invasion through interaction with its receptor CD44v6 and increased MMP-2 and uPA expressions, providing at least one mechanism for OPN-mediated HCC progression and metastasis.     

FOXQ1在肝癌中的临床意义及其对SMMC-7721细胞体外血管形成的影响

探讨叉头框蛋白Q1(forkhead box Q1, FOXQ1)基因在肝癌中的临床意义及对肝癌细胞体外血管生成作用.利用 qRT-PCR法及Western印迹法,检测24例肝癌、癌旁组织、正常肝细胞L02及肝癌细胞SMMC-7721中FOXQ1的mRNA和蛋白质的表达;利用免疫组织化学法检测68例肝癌及癌旁组织中FOXQ1的蛋白质表达.合成shRNA-FOXQ1及shRNA-NC慢病毒,转染到SMMC-7721细胞.用体外血管生成实验检测转染shRNA-FOXQ1的肝癌细胞血管生成能力. 用qRT-PCR和Western印迹法检测细胞间FOXQ1、VEGF基因和蛋白质的表达.结果显示,癌组织和SMMC-7721细胞中FOXQ1 mRNA和蛋白质的表达均高于癌旁组织和正常肝细胞(P<0.05),FOXQ1蛋白的表达与TNM分期、肿瘤分化程度、肿瘤数目、肿瘤大小等参数差异显著(P<0.05).shRNA-FOXQ1组血管生成能力明显低于shRNA-NC组和空白组(P<0.05),FOXQ1、VEGF基因和蛋白质的表达也明显低于shRNA-NC组和空白组(P<0.05).研究结果证实,FOXQ1在肝癌中高表达,如果沉默FOXQ1的表达可抑制肝癌细胞血管生成,与肝癌的临床病理特征密切相关.     

Effects of Gekko sulfated polysaccharide-protein complex on the defective biorheological characters of dendritic cells under tumor microenvironment

We previously isolated a sulfated polysaccharide–protein complex from Gekko swinhonis Guenther, a traditional Chinese medicine, and have demonstrated its direct anti-cancer effect on human hepatocellular carcinoma cell line SMMC-7721. Here we investigated the effects of Gekko sulfated polysaccharide–protein complex (GSPP) on the defective biorheological characters of dendritic cells (DCs) under SMMC-7721 microenvironment. Our findings have shown that the biorheological properties of DCs were severely impaired by SMMC-7721 microenvironment, including decreased cell deformability, migration, and electrophoresis mobility, increased osmotic fragilities, and changed organizations of cytoskeletal proteins. We also found decreased secretion of interleukin (IL)-12 and increased secretion of IL-10 in DCs. However, supernatant collected from nonmalignant liver cells had no effect on these parameters. SMMC-7721 cells were treated with GSPP and the supernatant was used to culture DCs. We found that the defective biorheological parameters of DCs, except for osmotic fragility, were partially or completely improved. The secretion of IL-12 did not change as compared with that of DCs in SMMC-7721 microenvironment, but the secretion of IL-10 was resumed to the control level. Our results indicate that GSPP could partially restore the defective biorheological characteristics of DCs via modifying the tumor microenvironment and decreasing the secretion of IL-10 of DCs.     

The inhibitory effect of transthyretin gene on growth of human hepatoma cells

Transthyretin(TTR) gene was highly expressed in normal liver and it has been found to be deleted in part of DNA samples from human hepatic cancer.Its mRNA expression was suppressed in most hepatoma samples.In order to study the biological effect of TTR gene on the growth of hepatoma cells,a recombinant vector containing TTR cDNA was constructed by pCMV,then it was transfected into hepatoma cell lines SMMC-7721 and Q3.It has been demonstrated that the inhibition of growth rate of TTR cDNA transfected hepatoma cells was about 50% in strength compared with that of the control.This inhibition was further enhanced when the transfected hepatoma cells were treated with all-trans retinoic acid.Hepatoma cells of cell lines PLC/PRF/5,SMMC-7721 and Q3 as well as hepatoma cells SMMC-7721 transfected with pCMV or pCMV-TTR were analyzed for TTR expression by Northern hybridization.The low level of TTR expression was found in both hepatoma cell lines and in SMMC-7721 cells transfected with pCMV alone.However,a remarkable TTR mRNA expression was observed in hepatoma SMMV-7721 cells transfected with pCMV-TTR.It seems possible that TTR gene might be a candidate of cancer suppressor gene for human hepatic cancer.     

Chitooligosaccharides induce apoptosis of human hepatocellular carcinoma cells via up-regulation of Bax

Chitooligosaccharides (COS) have been shown to regulate various cellular and biological functions. However, the effect of COS on apoptosis of hepatocellular carcinoma cells remains unclear. In this study, the activity and mechanism of COS against human hepatocellular carcinoma cells (SMMC-7721 cells) were investigated in vitro. The experiments showed that COS notably induced the apoptosis of SMMC-7721 cells and increased the cleavage of poly(ADP-ribose) polymerase. It presented a dose-dependent manner, and the apoptotic rate amounted to about 38% after treatment with 0.8 mg/ml COS for 72 h. The mRNA and protein levels of Bax were up-regulated by COS. These results demonstrated that COS induced apoptosis of SMMC-7721 cells. The possible mechanism is that COS up-regulate pro-apoptotic protein Bax, and trigger the cells a start-up of the apoptosis program.     

Two specific inhibitors of the phosphatidylinositol 3-kinase LY294002 and wortmannin up-regulate β1,4-galactosyltransferase I and thus sensitize SMMC-7721 human hepatocarcinoma cells to cycloheximide-induced apoptosis

Previous study indicated that β1,4-galactosyltransferase I (β1,4GT1) was up-regulated by cycloheximide (CHX) and thus enhanced apoptosis induced by CHX in SMMC-7721 cells. In this study, we reported that constitutively active Akt protein (myr-Akt) inhibited CHX-induced apoptosis in SMMC-7721 cells through down-regulating β1,4GT1. However, the two PI3K inhibitors LY294002 and wortmannin treatment up-regulated β1,4GT1 through enhancing Sp1 protein expression and consequently increased CHX-induced SMMC-7721 cells apoptosis. Besides, our results suggested that β1,4GT1 and cell surface galactose residues synthesized by elevated β1,4GT1 played an important role in SMMC-7721 cells apoptosis treated with CHX and PI3K inhibitor together. Moreover, we found that CHX accentuated β1,4GT1 through down-regulating Akt expression to mediate SMMC-7721 cells apoptosis. Taken together, PI3K inhibitors LY294002 and wortmannin up-regulated β1,4GT1 and enhanced CHX-induced apoptosis in SMMC-7721 cells, which suggested that PI3K inhibitors might have therapeutic potential when combined with CHX in the treatment of hepatoma.     

Stealth siRNA against CD147 inhibits hepatocellular carcinoma cell metastatic properties

CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to secrete 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 research was to investigate the inhibitory effects of stealth small interfering RNA (siRNA) against CD147 on HCC cell line (SMMC-7721) metastatic properties including invasion, adhesion to ECM, gelatinase production, focal adhesion kinase (FAK) and vinculin expression. Flow cytometry (FCM) and western blot assays were employed to detect the transfection efficiency of the stealth siRNA against CD147. Invasion assays and gelatin zymography were also used to detect the effects of stealth siRNA against CD147 on SMMC-7721 cells’ invasion and gelatinase production. The effects of stealth siRNA against CD147 on FAK and vinculiln expression in SMMC-7721 cells were also detected by western blot. The results showed that stealth siRNA against CD147 inhibited SMMC-7721 invasion, adhesion to ECM proteins, MMP-2 production, and FAK and vinculin expression. These findings indicate that CD147 is required for tumor cell invasion and adhesion. Perturbation of CD147 expression may have potential therapeutic uses in the prevention of MMP-2-dependent tumor invasion.     

RNA interference targeting CITRON can significantly inhibit the proliferation of hepatocellular carcinoma cells

CITRON (rho-interacting, serine/threonine kinase 21), which is a key component of the midbody, is essential for cytokinesis. However, the role of CITRON in hepatocellular carcinoma (HCC) is poorly understood. Here we first measured the expression of CITRON in HCC specimens by quantitative real-time RT–PCR and immunohistochemical staining. The results showed CITRON to be frequently up-regulated in HCC as compared with adjacent non-tumour tissues. Then we employed adenovirus-mediated RNA interference against CITRON to assess its anti-proliferation effect on SMMC-7721 cells, a representative HCC cell line. The resulting data demonstrated that CITRON knockdown was capable of inhibiting the proliferation and colony formation of SMMC-7721 cells, with an obvious increase of multinucleated cells. Furthermore, we subcutaneously injected the SMMC-7721 cells with the CITRON knockdown into nude mice to evaluate the tumourigenicity. The data indicated that adenovirus-mediated RNA interference can suppress tumourigenicity in vivo of HCC cells. Our data suggest that CITRON may be a potential target for therapeutic intervention in HCC.     

SMMC-7721肝癌细胞67kD层粘连蛋白受体的分离纯化

分离纯化肝癌细胞的 6 7kD层粘连蛋白受体 (6 7LR) ,以便进一步研究 6 7LR的结构、功能及其在肝癌浸润、转移过程中的作用 .以SMMC 772 1肝癌细胞和L 0 2正常肝细胞为材料 ,采用13 1I标记的层粘连蛋白测定其与细胞的结合能力 ;亲和层析法分离纯化层粘连蛋白受体 ,用SDS PAGE、放射自显影及体外竞争结合实验进行鉴定 .在相同条件下SMMC 772 1肝癌细胞与层粘连蛋白特异结合量为 17 5 4± 0 4 9ng 10 5细胞 ,而L 0 2正常肝细胞与层粘连蛋白的特异结合量为 8 36± 0 4 8ng 10 5细胞 .经过亲和层析 ,从SMMC 772 1肝癌细胞和L 0 2正常肝细胞均可获得纯化受体 ,SDS PAGE显示为单一条带 ,分子量为 6 7kD ,放射自显影及体外竞争结合实验表明其具有较强的与层粘连蛋白结合的活性 .体外竞争结合实验表明 ,SMMC 772 1肝癌细胞层粘连蛋白受体 (772 1LnR)的抑制率可达到 96 2 7± 2 2 9% ,而L 0 2正常肝细胞层粘连蛋白受体 (L 0 2LnR)的抑制率为 4 8 71± 3 79% ,这说明 772 1LnR与层粘连蛋白的亲和力明显高于L 0 2LnR(P <0 0 0 1) .结果表明 ,与L 0 2肝细胞比较 ,SMMC 772 1肝癌细胞具有与层粘连蛋白较强结合能力的特异受体 ,并从肝癌细胞膜上分离纯化到与层粘连蛋白有较强亲和力的 6 7LR     

Redifferentiation of human hepatoma cells (SMMC-7721) induced by two new highly oxygenated bisabolane-type sesquiterpenes

Bisabolane-type sesquiterpenes are a class of biologically active compounds that has antitumour, antifungal, antibacterial, antioxidant and antivenom properties. We investigated the effect of two new highly oxygenated bisabolane-type sesquiterpenes (HOBS) isolated from Cremanthodium discoideum (C. discoideum) on tumour cells. Our results showed that HOBS induced morphological differentiation and reduced microvilli formation on the cell surface in SMMC-7721 cells. Flow cytometry analysis demonstrated that HOBS could induce cell-cycle arrest in the G1 phase. Moreover, HOBS was able to increase tyrosine-α-ketoglutarate transaminase activity, decrease α foetoprotein level and γ-glutamyl transferase activity. In addition, we found that HOBS inhibited the anchorageindependent growth of SMMC-7721 cells in a dose-dependent manner. Taken together, all the above observations indicate that HOBS might be able to normalize malignant SMMC-7721 cells by inhibiting cell proliferation and inducing redifferentiation.     

6-Shogaol induces apoptosis in human hepatocellular carcinoma cells and exhibits anti-tumor activity in vivo through endoplasmic reticulum stress

6-Shogaol is an active compound isolated from Ginger (Zingiber officinale Rosc). In this work, we demonstrated that 6-shogaol induces apoptosis in human hepatocellular carcinoma cells in relation to caspase activation and endoplasmic reticulum (ER) stress signaling. Proteomic analysis revealed that ER stress was accompanied by 6-shogaol-induced apoptosis in hepatocellular carcinoma cells. 6-shogaol affected the ER stress signaling by regulating unfolded protein response (UPR) sensor PERK and its downstream target eIF2α. However, the effect on the other two UPR sensors IRE1 and ATF6 was not obvious. In prolonged ER stress, 6-shogaol inhibited the phosphorylation of eIF2α and triggered apoptosis in SMMC-7721 cells. Salubrinal, an activator of the PERK/eIF2α pathway, strikingly enhanced the phosphorylation of eIF2α in SMMC-7721 cells with no toxicity. However, combined treatment with 6-shogaol and salubrinal resulted in significantly increase of apoptosis and dephosphorylation of eIF2α. Overexpression of eIF2α prevented 6-shogaol-mediated apoptosis in SMMC-7721 cells, whereas inhibition of eIF2α by small interfering RNA markedly enhanced 6-shogaol-mediated cell death. Furthermore, 6-shogaol-mediated inhibition of tumor growth of mouse SMMC-7721 xenograft was associated with induction of apoptosis, activation of caspase-3, and inactivation of eIF2α. Altogether our results indicate that the PERK/eIF2α pathway plays an important role in 6-shogaol-mediated ER stress and apoptosis in SMMC-7721 cells in vitro and in vivo.     

独角莲对肝癌细胞SMMC-7721细胞增殖抑制作用机理的研究

临床上独角莲对包括肝癌在内的多种肿瘤有一定的治疗作用。为揭示其抑癌机理,我们用独角莲根茎水提物(the aqueous extract from dried powdered rhizomes of Typhonium giganteum Engl.,AEoTGE)作用肝癌细胞株SMMC-7721,研究独角莲对SMMC-7721细胞增殖;细胞周期和细胞凋亡的影响。噻唑氮蓝(MTT)比色实验和流式细胞仪(flow cytometry,FCM)测定表明：AEoTGE能较强抑制SMMC-7721细胞的生长,SMMC-7721细胞被阻滞在S期,井诱导细胞凋亡。提示独角莲是一种在肝癌治疗上有前景的中草药。