IGF-II inhibitory DNAzymes inhibit the invasion and migration of hepatocarcinoma cells

Metastasis and recurrence are the biggest obstacles to enhance the efficacy of surgical resection as a cure for hepatocellular carcinoma which is the second most deadly cancer in China. Here, we had showed that two DNAzymes (DRz1 and DRz2) targeted to IGF-II could inhibit invasion, motility and migration of SMMC-7721 cells in vitro. DRz1 was transfected into SMMC-7721 cells and the results were shown that IGF-II expression level dramatically reduced. Meanwhile, DRz1 effectively inhibited adhesion between SMMC-7721 cells with the extracellular matrix and Fb cells comparing to those untreated or transfected with inactive DRz (P < 0.05, ANOVA). Furthermore, vascular endothelial growth factor and matrix metalloproteinases were down-regulated in DRz1 treated cells. These results may help to identify novel therapeutic molecules targeting hepatocellular carcinomas.     

Microfilament regulatory protein MENA increases activity of RhoA and promotes metastasis of hepatocellular carcinoma

Mammalian enabled (MENA), usually known as a direct regulator of microfilament polymerization and bundling, promotes metastasis in various cancers. Here we focus on the role of MENA in hepatocellular carcinoma (HCC) metastasis and the relevant mechanism from the view of RhoA activity regulation. By HCC tissue microarray analysis, we found that MENA expression was positively associated with satellite lesions (P<0.01) and vascular invasion (P<0.01). Cases with membrane reinforcement of MENA staining in HCC tissues had significantly higher rates of early recurrence in the intermediate MENA expression group. Knockdown of MENA significantly suppressed HCC cell migration and invasion in vitro, as well as their intrahepatic and distant metastasis in vivo. Knockdown of MENA also decreased filopodia and stress fibers in SMMC-7721 cells. Furthermore, a decrease of RhoA activity was detected by a pull-down assay in SMMC-7721-shMENA cells. The ROCK inhibitor, Y-27632, suppressed migration of both MENA knockdown SMMC-7721 cells and control cells, but diminished their difference. Thus, our findings suggest that MENA promotes HCC cell motility by activating RhoA.     

OIP5对肝癌细胞SMMC-7721增殖和侵袭迁移能力的影响

目的:探讨OIP5对肝癌细胞SMMC-7721增殖和侵袭迁移能力的影响。方法:采用RNA干扰技术沉默肝癌细胞中OIP5的表达后,通过qRT-PCR和Western-blot技术检测OIP5的下调效率,CCK-8和平板克隆法检测肝癌细胞的增殖能力,Transwell法检测肝癌细胞的侵袭和迁移能力。结果:转染OIP5-siRNA后,肝癌细胞SMMC-7721中OIP5 mRNA和蛋白的表达水平均明显降低(P0.05);同时,与对照组相比,OIP5-siRNA组肝癌细胞SMMC-7721的CCK-8实验的OD值、平板克隆法测得的克隆球个数、Transwell法测得的迁移细胞数与侵袭细胞数均明显低于对照组(P0.05)。结论:OIP5能够促进肝癌细胞的增殖和侵袭迁移,可能作为肝癌治疗的潜在靶点。     

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.     

Notch信号通路对肝癌细胞迁移能力及E-cadherin，COX-2表达的影响

目的:探讨Notch信号通路对肝癌细胞迁移能力及钙粘附蛋白E(E-cadherin)、环氧化酶-2(COX-2)表达的影响。方法:体外培养肝癌细胞系(SMMC-7721、MHCC97H)、正常非肿瘤肝细胞系(HL-7702),Transwell小室用于测定细胞的迁移侵袭能力,Western blot蛋白印迹法用于测定Notch1、E-cadherin、COX-2蛋白的表达水平,并采用DAPT阻断Notch信号通路,比较肝癌细胞系与正常非肿瘤肝细胞系的迁移侵袭能力及肝癌细胞中E-cadherin、COX-2蛋白的表达水平的改变。结果:SMMC-7721细胞、MHCC97H细胞的迁移能力强于HL-7702细胞,差异有统计学意义(P0.05);相比于HL-7702细胞,MHCC97H细胞、SMMC-7721细胞中的Notch1、COX-2表达水平均显著升高,E-cadherin的表达水平明显降低(P0.05);DAPT处理后,SMMC-7721细胞、MHCC97H细胞发生迁移的能力均弱于对照组,差异有统计意义(P0.05);DAPT处理后,SMMC-7721细胞、MHCC97H细胞内COX-2、Notch1的表达量明显降低,而E-cadherin的表达水平升高(P0.05)。结论:Notch信号通路参与肝癌细胞迁移过程,其机制可能与E-cadherin、COX-2的表达相关。     

Caveolin enhances hepatocellular carcinoma cell metabolism,migration, and invasion in vitro via a hexokinase 2-dependent mechanism

The development and progression of hepatocellular carcinoma (HCC) have been associated with abnormal cellular metabolism. Gene Expression Profiling Interactive Analysis RNA sequencing data revealed caveolin-1 (CAV-1) and hexokinase 2 (HK2) messenger RNA (mRNA) were significantly upregulated in human HCC compared with normal tissues, and high HK2 expression was associated with significantly poorer overall survival in HCC ( p < 0.05). CAV-1 and HK2 mRNA and protein expression were upregulated and positively correlated in 42 fresh human HCC tissues compared with tumor-adjacent normal tissues. Overexpression of CAV-1 or HK2 in SMMC-7721 and HepG2 HCC cells enhanced glucose and lactate metabolism and increased cell migration and invasion in transwell assays; knocking down CAV-1 or HK2 had the opposite effects. Overexpression of CAV-1 increased HK2 expression; overexpression of HK2 did not affect CAV-1 expression. Knocking down HK2 partially reversed the ability of CAV-1 to promote cellular metabolism, invasion, and migration in HCC, indicating CAV-1 enhances glycolysis, invasion, and metastasis in HCC cells via HK2-dependent mechanism. Further studies of the function and relationship between CAV-1 or HK2 expression are warranted to explore the potential of these proteins as metabolic targets for the treatment of HCC.     

Synergistic effect of EMS1-shRNA and sorafenib on proliferation,migration, invasion and endocytosis of SMMC-7721

To investigate the synergistic effect of EMS1-PSilencer4.1-shRNA (EMS1-shRNA) and sorafenib on biological behaviors of HCC cell line SMMC-7721. EMS1-shRNA was constructed and transfected into SMMC-7721 cells. Decreased levels of EMS1/cortactin were tested in RT-QPCR and Western blot assay. Proliferation, migration, invasion, and endocytosis of SMMC-7721 were tested through CCK8 assay, scratch test, transwell invasion assay and transferrin endocytosis assay, respectively. Raf-1 was detected by Western blot assay. HCC xenograft model was prepared to observe tumor growth. Animals were euthanized and their subcutaneous lesions were weighed. Then the tissues were fixed and paraffin sections were prepared. Cortactin and PCNA (a proliferation marker) were then detected by immunohistochemistry. As compared with untreated group, the levels of EMS1 gene and cortactin protein in EMS1-shRNA-transfected group were significantly reduced; Among EMS1-shRNA-transfected group, sorafenib-treated group and combined group, the levels of proliferation at 48 h were reduced to 83.69, 57.18, 41.94 %; the levels of migration were reduced to 49.69, 60.83, and 21. 67 %; the levels of invasion were reduced to 42.97, 53.65, 18.18 %; the levels of endocytosis were reduced to 37.15, 97.95 % (p > 0.05), 20.68 % (p < 0.05, respectively). Western blot assay showed levels of Raf-1 were reduced to 68.56, 59.09, 21.90 %. The tumor volume and weight of nude mice HCC xenograft tumors were reduced significantly either (p < 0.05, respectively). Immunohistochemistry showed levels of cortactin and PCNA were reduced to 35.69, 93.84, 23.68 and 87.69, 43.84, 33.68 % in each group, respectively. The biological behaviors of SMMC-7721 were inhibited in the presence of EMS1-shRNA and sorafenib both alone and in combination. The combination of the agents improved the curative effect over either single agent, showing synergetic effect.     

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.     

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.     

Regulation of human hepatocellular carcinoma cells by Spred2 and correlative studies on its mechanism

Members of the Spred gene family are negative regulators of the Ras/Raf-1/ERK pathway, which has been associated with several features of the tumor malignancy. However, the effect of Spred genes on hepatocellular carcinoma (HCC) remains uninvestigated. In the present work, we analyzed the in vitro and in vivo effects of Spred2 expression on the hepatic carcinoma cell line, SMMC-7721. In addition to attenuated ERK activation, which inhibited the proliferation and migration of unstimulated and HGF-stimulated SMMC-7721 cells. Adenovirus-mediated Spred2 overexpression induced the activation of caspase-3 and apoptosis, as well as reduced the expression level of Mcl-1. Most importantly, the knockdown of Spred2 markedly enhanced tumor growth in vivo. In conclusion, these results suggest that Spred2 could qualify as a potential therapeutic target in HCC.     

Overexpression of miR-623 suppresses progression of hepatocellular carcinoma via regulating the PI3K/Akt signaling pathway by targeting XRCC5

It has been reported that miR-623 is deregulated in lung adenocarcinoma and inhibits tumor growth and invasion. However, it is unclear whether miR-623 has a role in the progression of hepatocellular carcinoma (HCC). Herein, we found that miR-623 was significantly downregulated in HCC, and that its expression was related to poor clinical outcomes of patients with HCC. Upregulation of miR-623 decreased cell proliferation, viability, migration, and invasion and further promoted apoptosis in 7721, Huh7, and Bel-7402 cells. Moreover, we also observed that miR-623 regulated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), Wnt/β-catenin, and extracellular regulated protein kinases/c-Jun N-terminal kinase (ERK/JNK) signaling pathways as well as the expression level of related proteins. Further, X-ray repair cross complementing 5 (XRCC5) was a direct target for miR-623, and the suppression of PI3K/Akt, Wnt/β-catenin, and ERK/JNK signaling pathways and cell proliferation and invasion abilities caused by miR-623 in HCC cells was significantly reversed by the upregulation of XRCC5. Collectively, our data suggested that miR-623 suppressed the progression of HCC by regulating the PI3K/Akt, Wnt/β-catenin, and ERK/JNK pathways by targeting XRCC5 in HCC in vitro, indicating that miR-623 may be a target for the therapy of HCC.     

Effects of microRNA-513b on cell proliferation,apoptosis, invasion,and migration by targeting HMGB3 through regulation of mTOR signaling pathway in non-small-cell lung cancer

This study aimed to explore the underlying mechanism of miR-513b and HMGB3 in regulating non-small-cell lung cancer (NSCLC). NSCLC tumor, adjacent tissues, and cell lines were extracted, and the expression of miR-513b and HMGB3 were determined by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot analysis. Then, miR-513b was overexpressed in NSCLC cell, and the proliferation, migration, invasion, and apoptosis of cells were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), wound healing, transwell, and flow cytometry, respectively. Regulatory relationship between miR-513b and HMGB3 was determined using luciferase activity reporter assay. Lastly, HMGB3 and/or miR-513b were overexpressed in NSCLC cells, and the proliferation, migration, invasion, and apoptosis of cells were determined. Compared with the controls, the expression of miR-513b was significantly downregulated in the NSCLC tissues and cells lines by RT-qPCR ( p < 0.05). However, the expression of HMGB3 was significantly downregulated at both messenger RNA and protein levels ( p < 0.05). Overexpression of miR-513b could significantly inhibit the proliferation, invasion, migration, and promote apoptosis of NSCLC cells ( p < 0.05). HMGB3 was a target of miR-513b, and overexpression of HMGB3 could obviously reverse the effect of miR-513 on the proliferation, invasion, migration, and apoptosis of NSCLC cells ( p < 0.05). The present results could suggest miR-513b was downregulated in NSCLC and could regulate the proliferation, invasion, migration, and apoptosis of NSCLC cells via HMGB3.     

Selenium, iron, copper, and zinc levels and copper-to-zinc ratios in serum of patients at different stages of viral hepatic diseases

Viral hepatic diseases, especially those induced by the hepatitis B virus, can progress into more serious pathological outcomes and eventually to hepatocellular carcinoma. A growing body of evidence indicates that many trace elements play important roles in a number of carcinogenic processes that proceed through various mechanisms. To examine the status of trace elements during the development of hepatic carcinoma, we determined the selenium, iron, copper, and zinc levels and copper-to-zinc ratios in the serum of patients at different stages of viral hepatic disease. We observed significant changes in the selenium, iron copper, and zinc levels in the serum of patients having hepatocellular carcinoma, relative to those of healthy controls (p<0.05). The mean serum copper level in patients with hepatocellular carcinoma was significantly higher than that of the control group. In contrast, the mean selenium, iron, and zinc levels in patients having hepatocellular carcinoma were significantly lower than those of the control group. In addition, the mean zinc level in the serum of patients with hepatic cirrhosis was significantly lower than that of the control group (p<0.05). Moreover, we found markedly elevated Cu: Zn ratios (p<0.05) in patients having hepatic cirrhosis or hepatocellular carcinoma. Our findings imply that the levels of some trace elements, such as selenium, iron, copper, and zinc, and Cu:Zn ratios, might serve as biomarkers for the increased severity of viral hepatic damage.     

The predictive value of a preoperative systemic immune-inflammation index and prognostic nutritional index in patients with esophageal squamous cell carcinoma

Growing evidence indicates that systemic inflammation response and malnutrition status are correlated with survival in certain types of solid tumors. The aim of this study is to evaluate the association between the systemic immune-inflammation index (SII) and prognostic nutritional index (PNI) and overall survival (OS) in patients with esophageal squamous cell carcinoma (ESCC) after esophagectomy. A consecutive series of 655 patients with resected ESCC who underwent esophagectomy were enrolled in the retrospective study. The preoperative SII was defined as platelet × neutrophil/lymphocyte counts. The PNI was calculated as albumin concentration (g/L) + 5 × total lymphocyte count (10⁹/L). The optimal cut-off values of SII, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and PNI were determined by receiver operating characteristic analysis. Survival analysis was performed using the Kaplan–Meier method with a log-rank test, followed by a multivariate Cox proportional hazards model. A high SII was significantly related to tumor size, histological type, invasion depth, and TNM stage (p < 0.05). A low PNI was significantly associated with age, tumor size, invasion depth, lymph node metastasis, and TNM stage (p < 0.05). Univariate analysis revealed that age, smoking history, tumor size, invasion depth, lymph node metastasis, SII, NLR, PLR, and PNI were predictors of OS (p < 0.05). Multivariate analysis identified age (p = 0.041), tumor size (p = 0.016), invasion depth (p < 0.001), lymph node metastasis (p < 0.001), SII (p = 0.033), and PNI (p = 0.022) as independent prognostic factors correlated with OS. There was a significant inverse relationship between the SII and PNI (r = 0.309; p < 0.001). The predictive value increased when the SII and PNI were considered in combination. Our results demonstrate that the preoperative high SII and low PNI are powerful indicators of aggressive biology and poor prognosis for patients with ESCC. The combination of SII and PNI can enhance the accuracy of prognosis.     

MicroRNA-142-3p, a new regulator of RAC1, suppresses the migration and invasion of hepatocellular carcinoma cells

RAC1 regulates a diverse array of cellular events including migration and invasion. MicroRNAs (miRNAs) have a key role in the regulation of gene expression. In this study, we demonstrated that microRNA-142-3p (miR-142-3p) acted as a negative regulator of human RAC1. Overexpression of miR-142-3p decreased RAC1 mRNA and protein levels. Moreover, the overexpression of miR-142-3p suppressed, while blocking of miR-142-3p increased colony formation, migration and invasion in hepatocellular carcinoma (HCC) cell lines (QGY-7703 and SMMC-7721). RAC1 overexpression without the 3'untranslated region abolished the effect of miR-142-3p in the QGY-7703 and SMMC-7721 cells. These results demonstrated that miR-142-3p directly and negatively regulates RAC1 in HCC cells, which highlights the importance of miRNAs in tumorigenesis.     

盐霉素抑制人肝细胞癌转移与侵袭的体外与体内实验研究

目的：肝癌的转移与复发是肝癌治疗的一大难题,盐霉素是近年来新发现的具有抗肿瘤作用的抗生素,本文研究了盐霉素在体外及体内对人肝细胞癌转移与侵袭能力的作用及机制。方法：在体外对肝癌细胞株HepG2,SMMC-7721,BEL-7402给予盐霉素处理,体内建立裸鼠肝脏原位肿瘤模型,并给予腹腔注射盐霉素治疗。观察肿瘤细胞的转移侵袭能力以及肝内肿瘤转移灶的情况,进一步测定E．cadherin,Vimentin的表达,来研究盐霉素对肝癌转移及侵袭能力的影响及机制。结果：经盐霉素处理后,肝癌细胞株HepG2,SMMC．7721,BEL．7402的转移及侵袭能力明显下降,肝内转移灶的数目也减少。分子机制检测发现盐霉素处理后E．cadherin表达增高,Vimentin表达下降。结论：盐霉素在体内与体外都抑制了肝癌的转移与侵袭,其机制可能抑制了肿瘤细胞的上皮间质化（EMT）过程。这为控制肝癌的转移和复发提供了新的治疗思路。