TPX2 Is a Prognostic Marker and Contributes to Growth and Metastasis of Human Hepatocellular Carcinoma

Targeting protein for Xenopus kinesin-like protein 2 (TPX2), a microtubule-associated protein, impacts spindle assembly in human cells. Several studies have demonstrated that TPX2 is overexpressed in different types of human cancers and promotes tumor growth and metastasis. In this study, we found that the expression level of TPX2 was obviously higher in hepatocellular carcinoma (HCC) tissues than in matched nontumor tissues. Elevated expressions of TPX2 mRNA were observed in all HCC cell lines (HepG2, Hep3B, SMMC-7721, Bel-7402 and Huh7) as compared with that in a non-transformed hepatic cell line (LO2). Clinical analysis indicated that the positive expression of TPX2 was significantly correlated with venous infiltration, high Edmondson-Steiner grading and advanced TNM tumor stage in HCC. Furthermore, TPX2 was a novel prognostic marker for predicting 5-year overall survival (OS) and disease-free survival (DFS) of HCC patients. In vitro studies found that TPX2 knockdown significantly inhibited cell proliferation and viability in both Hep3B and HepG2 cells. Moreover, TPX2 knockdown obviously slowed down tumor growth in a nude mouse xenograft model. Otherwise, TPX2 knockdown prominently suppressed HCC cell invasion and migration. In conclusion, these results indicate that TPX2 may serve as a prognostic marker and promotes tumorigenesis and metastasis of HCC.     

MicroRNA-130b Promotes Cell Aggressiveness by Inhibiting Peroxisome Proliferator-Activated Receptor Gamma in Human Hepatocellular Carcinoma

MircroRNA-130b (miR-130b) is proposed as a novel tumor-related miRNA and has been found to be significantly dysregulated in tumors. In this study, the expression level of miR-130b was found to be obviously higher in hepatocellular carcinoma (HCC) tissues than that in nontumor tissues. Further, miR-130b was expressed at significantly higher levels in aggressive and recurrent tumor tissues. Clinical analysis indicated that high-expression of miR-130b was prominently correlated with venous infiltration, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) tumor stage in HCC. Elevated miR-130b expression was observed in all HCC cell lines (HepG2, SMMC-7721, Huh7, Hep3B and MHCC97H) as compared with that in a nontransformed hepatic cell line (LO2). Furthermore, an inverse correlation between miR-130b and E-cadherin and a positive correlation between miR-130b and Vimentin were observed in HCC tissues. Down-regulation of miR-130b expression reduced invasion and migration in both Hep3B and MHCC97H cells. Peroxisome proliferator-activated receptor gamma (PPAR-γ) was inversely correlated with miR-130b expression in HCC tissues. In addition, down-regulation of miR-130b restored PPAR-γ expression and subsequently suppressed epithelial-mesenchymal transition (EMT) in HCC cells. We identified PPARγ as a direct target of miR-130b in HCC in vitro. Notably, PPAR-γ knockdown abolished down-regulation of miR-130b-inhibited EMT in MHCC97H cells. In conclusion, miR-130b may promote HCC cell migration and invasion by inhibiting PPAR-γ and subsequently inducing EMT.     

Novel Ferrocene Derivatives Induce G0/G1 Cell Cycle Arrest and Apoptosis through the Mitochondrial Pathway in Human Hepatocellular Carcinoma

In this study, detailed information on hepatocellular carcinoma (HCC) cells (HepG-2, SMMC-7721, and HuH-7) and normal human liver cell L02 treated by ferrocene derivatives (compounds 1, 2 and 3) is provided. The cell viability assay showed that compound 1 presented the most potent and selective anti-HCC activity. Further mechanism study indicated that the proliferation inhibition effect of compound 1 was associated with the cycle arrest at the G0/G1 phase and downregulation of cyclin D1/CDK4. Moreover, compound 1 could induce apoptosis in HCC cells by loss of mitochondrial membrane potential (ΔΨm), accumulation of reactive oxygen species (ROS), decrease in Bcl-2, increase in BAX and Bad, translocation of Cytochrome c, activation of Caspase-9, -3, and cleavage of PARP. These results indicated that compound 1 would be a promising candidate against HCC through G0/G1 cell cycle arrest-related proliferation inhibition and mitochondrial pathway-dependent apoptosis.     

靶向高迁移率族蛋白A1基因RNA干扰真核表达载体的构建

目的构建针对人高迁移率族蛋白A1(HMGA1)基因的RNA干扰真核表达载体,为研究HMGA1基因在肿瘤细胞中的作用奠定实验基础。方法设计合成特异性针对人HMGA1基因的寡核苷酸序列,梯度退火后,与pU6mRFP载体连接,构建重组载体,转染人肝癌细胞SMMC-7721。通过激光共聚焦显微镜观察红色荧光,估测转染效率,RT-PCR法检测转染细胞HMGA1mRNA水平,流式细胞仪检测细胞凋亡和细胞周期。结果DNA测序证实,成功构建了特异性HMGA1siRNA真核表达载体,转染后72h,转染效率为40%左右。所构建的载体能够特异性沉默转染细胞HMGA1基因的表达。转染细胞HMGA1基因沉默后,细胞凋亡率(27·86%±2·44%)明显高于空载体对照组和SMMC-7721对照组(分别为2·82%±2·39%和2·04%±0·70%),G0-G1期细胞百分数(77·73%±1·78%)明显高于空载体对照组和SMMC-7721对照组(分别为42·19%±3·28%和39·23%±3·63%),G2-S期细胞百分数(22·27%±1·78%)明显低于空载体对照组和SMMC-7721对照组(分别为57·81%±3·28%和60·77%±3·63%)。结论成功构建了HMGA1的RNA干扰真核表达载体。     

[VEGF]hTERT复合调控序列介导HSV-tk自杀基因诱导肿瘤细胞的凋亡

目的构建人端粒酶催化亚单位(hTERT)核心启动子及血管内皮生长因子(VEGF)增强子复合调控序列的真核表达载体,并检测在复合调控序列调控下HSV-tk自杀基因对肺癌细胞A549和肝癌细胞SMMC-7721凋亡的影响。方法构建复合调控序列[VEGF]hTERT和胸苷激酶(tk)基因的真核表达载体pcDNA3.1(+)-[VEGF]hTERT-tk和PCMV启动子的真核对照表达载体pcDNA3.1(+)-tk,转染肺癌细胞A549和肝癌细胞SMMC-7721,G418筛选抗性克隆,给入前药丙氧鸟苷(GCV),MTT比色法测定细胞生长抑制率,流式细胞仪检测肿瘤细胞凋亡率。结果质粒pcDNA3.1(+)-[VEGF]hTERT-tk和pcDNA3.1(+)-tk经双酶切及PCR鉴定,证明构建正确。转染了带有不同启动子的真核表达载体的肿瘤细胞,在给入前药GCV后均能表现出对细胞生长的抑制作用,GCV对A549细胞的抑制率大于SMMC-7721细胞,并能引起肿瘤细胞凋亡。[VEGF]hTERT在A549细胞中的活性较在SMMC-7721中高。结论HSV-tk自杀基因在肿瘤细胞内瞬时表达即能引起靶细胞凋亡,且复合调控序列在肿瘤细胞中的活性较通用真核细胞转录启动子PCMV高,为深入进行自杀基因治疗的研究提供了一定理论依据。     

Albumin Suppresses Human Hepatocellular Carcinoma Proliferation and the Cell Cycle

Many investigations have revealed that a low recurrence rate of hepatocellular carcinoma (HCC) is associated with high serum albumin levels in patients; therefore, high levels of serum albumin are a major indicator of a favorable prognosis. However, the mechanism inhibiting the proliferation of HCC has not yet been elucidated, so we investigated the effect of serum albumin on HCC cell proliferation. Hep3B was cultured in MEM with no serum or containing 5 g/dL human albumin. As control samples, Prionex was added to generate the same osmotic pressure as albumin. After 24-h incubation, the expressions of α-fetoprotein (AFP), p53, p21, and p57 were evaluated with real-time PCR using total RNA extracted from the liver. Protein expressions and the phosphorylation of Rb (retinoblastoma) were determined by Western blot analysis using total protein extracted from the liver. For flow cytometric analysis of the cell cycle, FACS analysis was performed. The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACScalibur (BD) with appropriate software (ModFit LT; BD). The cell proliferation assay was performed by counting cells with using a Scepter handy automated cell counter (Millipore). The mRNA levels of AFP relative to Alb(−): Alb(−), Alb(+), and Prionex, were 1, 0.7 ± 0.2 (p < 0.001 for Alb(−)), and 1 ± 0.3, respectively. The mRNA levels of p21 were 1, 1.58 ± 0.4 (p = 0.007 for Alb(−) and p = 0.004 for Prionex), and 0.8 ± 0.2, respectively. The mRNA levels of p57 were 1, 4.4 ± 1.4 (p = 0.002 for Alb(−) and Prionex), and 1.0 ± 0.1, respectively. The protein expression levels of Rb were similar in all culture media. The phosphorylation of P807/811 and P780 of Rb protein was reduced in Alb(+). More cells in the G0/G1 phase and fewer cells in S and G2/M phases were obtained in Alb(+) than in Alb(−) (G0/G1: 60.9%, 67.7%, 61.5%; G2/M: 16.5%, 13.1%, 15.6%; S: 22.6%, 19.2%, 23.0%, Alb(−), Alb(+), Prionex, respectively). The same results were obtained in HepG2. Cell proliferation was inhibited in 5 g/dL albumin medium in both HepG2 cells and Hep3B cells in 24 h culture by counting cell numbers. The presence of albumin in serum reduces the phosphorylation of Rb proteins and enhances the expression of p21 and p57, following an increase in the G0/G1 cell population, and suppresses cell proliferation. These results suggest that albumin itself suppresses the proliferation of hepatocellular carcinoma.     

MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA

Folate depletion causes chromosomal instability by increasing DNA strand breakage, uracil misincorporation, and defective repair. Folate mediated one-carbon metabolism has been suggested to play a key role in the carcinogenesis and progression of hepatocellular carcinoma (HCC) through influencing DNA integrity. Methylenetetrahydrofolate reductase (MTHFR) is the enzyme catalyzing the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate that can control folate cofactor distributions and modulate the partitioning of intracellular one-carbon moieties. The association between MTHFR polymorphisms and HCC risk is inconsistent and remains controversial in populational studies. We aimed to establish an in vitro cell model of liver origin to elucidate the interactions between MTHFR function, folate status, and chromosome stability. In the present study, we (1) examined MTHFR expression in HCC patients; (2) established cell models of liver origin with stabilized inhibition of MTHFR using small hairpin RNA delivered by a lentiviral vector, and (3) investigated the impacts of reduced MTHFR and folate status on cell cycle, methyl group homeostasis, nucleotide biosynthesis, and DNA stability, all of which are pathways involved in DNA integrity and repair and are critical in human tumorigenesis. By analyzing the TCGA/GTEx datasets available within GEPIA2, we discovered that HCC cancer patients with higher MTHFR had a worse survival rate. The shRNA of MTHFR (shMTHFR) resulted in decreased MTHFR gene expression, MTHFR protein, and enzymatic activity in human hepatoma cell HepG2. shMTHFR tended to decrease intracellular S-adenosylmethionine (SAM) contents but folate depletion similarly decreased SAM in wildtype (WT), negative control (Neg), and shMTHFR cells, indicating that in cells of liver origin, shMTHFR does not exacerbate the methyl group supply in folate depletion. shMTHFR caused cell accumulations in the G2/M, and cell population in the G2/M was inversely correlated with MTHFR gene level (r = −0.81, p < 0.0001), MTHFR protein expression (r = −0.8; p = 0.01), and MTHFR enzyme activity (r = −0.842; p = 0.005). Folate depletion resulted in G2/M cell cycle arrest in WT and Neg but not in shMTHFR cells, indicating that shMTHFR does not exacerbate folate depletion-induced G2/M cell cycle arrest. In addition, shMTHFR promoted the expression and translocation of nuclei thymidine synthetic enzyme complex SHMT1/DHFR/TYMS and assisted folate-dependent de novo nucleotide biosynthesis under folate restriction. Finally, shMTHFR promoted nuclear MLH1/p53 expression under folate deficiency and further reduced micronuclei formation and DNA uracil misincorporation under folate deficiency. In conclusion, shMTHFR in HepG2 induces cell cycle arrest in G2/M that may promote nucleotide supply and assist cell defense against folate depletion-induced chromosome segregation and uracil misincorporation in the DNA. This study provided insight into the significant impact of MTHFR function on chromosome stability of hepatic tissues. Data from the present study may shed light on the potential regulatory mechanism by which MTHFR modulates the risk for hepatic malignancies.     

Antitumor Effect of Regorafenib on MicroRNA Expression in Hepatocellular Carcinoma Cell Lines

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is one of the leading causes of cancer-related deaths worldwide. Regorafenib, a multi-kinase inhibitor, is used as a second-line treatment for advanced HCC. Here, we aimed to investigate the mechanism of the antitumor effect of regorafenib on HCC and evaluate altered microRNA (miRNA) expression. Cell proliferation was examined in six HCC cell lines (HuH-7, HepG2, HLF, PLC/PRF/5, Hep3B, and Li-7) using the Cell Counting Kit-8 assay. Xenografted mouse models were used to assess the effects of regorafenib in vivo. Cell cycle analysis, western blotting analysis, and miRNA expression analysis were performed to identify the antitumor inhibitory potential of regorafenib on HCC cells. Regorafenib suppressed proliferation in HuH-7 cell and induced G0/G1 cell cycle arrest and cyclin D1 downregulation in regorafenib-sensitive cells. During miRNA analysis, miRNA molecules associated with the antitumor effect of regorafenib were found. Regorafenib suppresses cell proliferation and tumor growth in HCC by decreasing cyclin D1 via alterations in intracellular and exosomal miRNAs in HCC.     

Inhibition of Cell Proliferation and Metastasis by Scutellarein Regulating PI3K/Akt/NF-κB Signaling through PTEN Activation in Hepatocellular Carcinoma

Scutellarein (SCU) is a well-known flavone with a broad range of biological activities against several cancers. Human hepatocellular carcinoma (HCC) is major cancer type due to its poor prognosis even after treatment with chemotherapeutic drugs, which causes a variety of side effects in patients. Therefore, efforts have been made to develop effective biomarkers in the treatment of HCC in order to improve therapeutic outcomes using natural based agents. The current study used SCU as a treatment approach against HCC using the HepG2 cell line. Based on the cell viability assessment up to a 200 μM concentration of SCU, three low-toxic concentrations of (25, 50, and 100) μM were adopted for further investigation. SCU induced cell cycle arrest at the G2/M phase and inhibited cell migration and proliferation in HepG2 cells in a dose-dependent manner. Furthermore, increased PTEN expression by SCU led to the subsequent downregulation of PI3K/Akt/NF-κB signaling pathway related proteins. In addition, SCU regulated the metastasis with EMT and migration-related proteins in HepG2 cells. In summary, SCU inhibits cell proliferation and metastasis in HepG2 cells through PI3K/Akt/NF-κB signaling by upregulation of PTEN, suggesting that SCU might be used as a potential agent for HCC therapy.     

表达HIV-1 gp160膜蛋白靶细胞的制备及鉴定

目的获得表达INV-1 gp160膜蛋白的靶细胞,用于HIV-1特异性细胞毒性淋巴细胞(CTL)和抗HIV-1重组毒素细胞杀伤活性检测。方法采用PCR方法,从质粒pJen 10中扩增HIV-1 gp160基因,插入pGEM-T载体,测序后克隆入pIRESl neo载体中构建成重组质粒pIRE160,酶切鉴定正确后,转染人肝癌细胞(SMMG-7721),CA18加压筛选至细胞不再死亡为止,并采用Western blot和间接免疫荧光法对制备的靶细胞进行检测。结果HIV-1 gp160在SMMC7721表面表达,并裂解为gp120和gp41蛋白。结论已成功得到稳定表达HIV-1 gp160的靶细胞,且表达的蛋白具有良好的特异性。     

hTERT核心启动子调控HSV-TK自杀基因系统诱导肿瘤细胞的凋亡

目的观察人端粒酶催化亚单位(hTERT)启动子调控逆转录病毒介导的单纯疱疹病毒-胸苷激酶基因/丙氧鸟苷(HSV-TK/GCV)自杀基因系统诱导肿瘤细胞凋亡的效果。方法采用PCR方法扩增胸苷激酶(TK)基因,并构建由hTERT核心启动子调控的胸苷激酶重组逆转录病毒载体pLNC-hTERTp-TK,转染包装细胞PT67,G418筛选阳性细胞克隆,检测病毒滴度,PCR法检测转染细胞中的TK基因。将获得的重组逆转录病毒感染人肺癌细胞株A549、肝癌细胞株SMMC-7721、宫颈癌细胞株HeLa和正常人成纤维细胞系WI-38,G418筛选阳性克隆,MTT法检测重组逆转录病毒对肿瘤细胞的生长抑制作用,流式细胞仪检测肿瘤细胞的凋亡。结果重组逆转录病毒载体pLNC-hTERTp-TK经双酶切,可见1402bp的目的基因片段,表明质粒构建正确。从转染的PT67细胞中扩增出1131bp的基因片段,与TK基因大小一致。在成功转染重组逆转录病毒的PT67细胞中,病毒滴度最高者可达7.8×105CFU/ml。重组逆转录病毒感染的3种肿瘤细胞的增殖均受到抑制,且生长抑制率随着GCV浓度的增加而升高,经较低浓度GCV(10μg/ml)处理的3种肿瘤细胞,凋亡率分别可达37.06%、34.88%和33.59%。结论由hTERT启动子调控的逆转录病毒介导的HSV-TK/GCV自杀基因系统可诱导肿瘤细胞凋亡,且具有较强的特异性和高效性。     

CXCL10 Decreases GP73 Expression in Hepatoma Cells at the Early Stage of Hepatitis C Virus (HCV) Infection

Golgi protein 73 (GP73), which is up-regulated in hepatocellular carcinoma (HCC), has recently been identified as a novel serum marker for HCC diagnosis. Several reports also noted the increased levels of GP73 expression in chronic liver disease in patients with acute hepatitis of various etiologies, chronic Hepatitis C virus (HCV) infection and alcoholic liver disease. The molecular mechanisms of GP73 expression in HCV related liver disease still need to be determined. In this study, we aimed to evaluate the effect of HCV infection on GP73 expression. GP73 was highly expressed in Huh7, Hep3B, 293T and HUVEC cells, and was low-expressed in HepG2 cells. HCV infection led to down-regulation of GP73 in Huh7 and HepG2/CD81 cells at the early stage of infection. CXCL10 decreased GP73 expression in Huh7 and HepG2 cells. Up-regulation of GP73 was noted in hepatocytes with cytopathic effect at advanced stage of HCV infection, and further research is needed to determine the unknown factors affecting GP73 expression. In conclusion, our study provided additional evidence for the roles of GP73 in liver disease.     

Ruthenium Complexes Induce HepG2 Human Hepatocellular Carcinoma Cell Apoptosis and Inhibit Cell Migration and Invasion through Regulation of the Nrf2 Pathway

Ruthenium (Ru) complexes are currently the focus of substantial interest because of their potential application as chemotherapeutic agents with broad anticancer activities. This study investigated the in vitro and in vivo anticancer activities and mechanisms of two Ru complexes—2,3,7,8,12,13,17,18-Octaethyl-21H,23H-porphine Ru(II) carbonyl (Ru1) and 5,10,15,20-Tetraphenyl-21H,23H-porphine Ru(II) carbonyl (Ru2)—against human hepatocellular carcinoma (HCC) cells. These Ru complexes effectively inhibited the cellular growth of three human hepatocellular carcinoma (HCC) cells, with IC₅₀ values ranging from 2.7–7.3 μM. In contrast, the complexes exhibited lower toxicity towards L02 human liver normal cells with IC₅₀ values of 20.4 and 24.8 μM, respectively. Moreover, Ru2 significantly inhibited HepG2 cell migration and invasion, and these effects were dose-dependent. The mechanistic studies demonstrated that Ru2 induced HCC cell apoptosis, as evidenced by DNA fragmentation and nuclear condensation, which was predominately triggered via caspase family member activation. Furthermore, HCC cell treatment significantly decreased the expression levels of Nrf2 and its downstream effectors, NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO1). Ru2 also exhibited potent in vivo anticancer efficacy in a tumor-bearing nude mouse model, as demonstrated by a time- and dose-dependent inhibition on tumor growth. The results demonstrate the therapeutic potential of Ru complexes against HCC via Nrf2 pathway regulation.     

环氧化酶-2基因shRNA重组腺病毒的构建及鉴定

目的构建携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)标签的环氧化酶-2(cyclooxyge-nase-2,COX-2)基因shRNA重组腺病毒,并观察其对肝癌细胞SMMC-7721增殖的影响。方法将前期构建的真核表达质粒pGenesil-1-COX-2-shRNA及阴性对照质粒pGenesil-1-HK的表达启动子U6及shRNA序列亚克隆至腺病毒穿梭质粒pAdTrack中,构建重组腺病毒穿梭质粒pAdTrack-U6-COX-2-shRNA-EGFP和pAdTrack-U6-HK-EGFP,酶切及测序鉴定正确后,经PmeⅠ线性化,转化感受态AdEasier,构建重组腺病毒质粒pAd-U6-COX-2-shRNA-EGFP和pAd-U6-HK-EGFP,经PacⅠ线性化,转染AD293细胞,包装重组腺病毒Ad-U6-COX-2-shRNA-EGFP和Ad-U6-HK-EGFP,经3轮扩增后,测定滴度。RT-PCR和Western blot法检测感染细胞中COX-2基因mRNA的转录及蛋白的表达,MTS法观察重组腺病毒对肝癌细胞SMMC-7721增殖的影响。结果重组腺病毒穿梭质粒pAdTrack-U6-COX-2-shRNA-EGFP和pAdTrack-U6-HK-EGFP及重组腺病毒质粒pAd-U6-COX-2-shRNA-EGFP和pAd-U6-HK-EGFP经酶切和测序鉴定均构建正确,并成功转染AD293细胞,经包装和3轮扩增后,重组腺病毒Ad-U6-COX-2-shRNA-EGFP和Ad-U6-HK-EGFP的滴度分别为1.4×1012和2.0×1012pfu/ml。重组腺病毒感染的SMMC-7721细胞中COX-2基因mRNA、蛋白相对表达量及细胞增殖能力均明显低于空白对照组及阴性对照组(P均<0.05)。结论成功构建了COX-2基因shRNA重组腺病毒Ad-U6-COX-2-shRNA-EGFP,且可显著抑制肝癌细胞SMMC-7721的增殖,为进一步研究COX-2作为肝癌基因治疗靶点及机制奠定了基础。