Identification of LINC01615 as potential metastasis-related long noncoding RNA in hepatocellular carcinoma

Hepatocellular carcinoma is one of the most prevalent and fatal cancers. Studying the long noncoding RNA (lncRNA) alterations in hepatocellular carcinoma may lead to new therapeutic strategies. We checked whether there were correlations between The Cancer Genome Atlas expression profiles of the differentially expressed lncRNAs and their DNA methylation status or the copy number variations for hepatocellular carcinoma. We obtained 41 lncRNAs that were differentially expressed between tumor and normal samples, and their DNA methylation status was negatively correlated with the expression levels. We identified five lncRNAs that were recurrently amplified or deleted in tumor samples, but none of them were associated with the messenger RNA (mRNA) expression levels. To obtain the biological function of these lncRNAs, the coexpressed mRNAs in the hepatocellular carcinoma were figured out. A total of 10 lncRNAs were highly correlated with at least one gene. Six out of the ten lncRNAs were already known to be related with cancer previously. LINC01615 had 72 coexpressed genes, and we carried out the gene ontology (GO) term enrichment for these protein-coding genes. The results suggested that these lncRNAs were associated with extracellular matrix organization. To summarize, we identified 41 potentially cancer-related lncRNAs. In particular, we proposed that LINC01615 potentially affected the extracellular matrix and had further impacts on the metastasis of hepatocellular carcinoma.     

Partial reversion of the phenotype of a poorly differentiated hepatocellular carcinoma in a three-dimensional culture

In vivo, normal tissues and organs have a three-dimensional structure and function in a three-dimensional environment. The standard two-dimensional cell culture conditions drastically differ from those in vivo. For this reason, three-dimensional cultures based on different variants of the extracellular matrix are more adequate for analyzing normal and tumor cell growth. Culturing a poorly differentiated hepatocellular carcinoma in a collagen gel yielded spheroids whose growth pattern shifted towards the epithelial phenotype. The shift was expressed in changes in the cytoskeleton, enhanced formation of extracellular matrix fibrils between cells, and formation of fibronectin fibrils on the outer surface of spheroids. Analysis of 25 genes reflecting the level of morphological and functional hepatocyte differentiation showed that the expression of the gene encoding the transforming growth factor TGFβ2 was suppressed the most significantly.     

Origin of laminin in the extracellular matrix of human tumor xenografts in nude mice

Monoclonal antibodies reacting exclusively with laminin of human origin and a polyclonal antibody reacting with both murine and human laminin were used to immunohistochemically study the extracellular matrix of four human tumors grown as xenografts in nude mice: a lung carcinoma and a yolk sac carcinoma because they produced cell associated laminin in vitro; and two hepatocellular carcinomas which did not produce cell associated laminin in vitro. The extracellular matrix of the xenografts of the lung carcinoma and the yolk sac carcinoma contained laminin of both human and murine origin. Xenografts of liver carcinoma contained only laminin of mouse origin. This shows that the malignant cells capable of laminin production in vitro contribute this glycoprotein to the extracellular matrix of the solid tumor formed by them in vivo.     

Silencing cathepsin S gene expression inhibits growth, invasion and angiogenesis of human hepatocellular carcinoma in vitro

Cathepsin S (Cat S) plays an important role in tumor invasion and metastasis by its ability to degrade extracellular matrix (ECM). Our previous study suggested there could be a potential association between Cat S and hepatocellular carcinoma (HCC) metastasis. The present study was designed to determine the role of Cat S in HCC cell growth, invasion and angiogenesis, using RNA interference technology. Small interfering RNA (siRNA) sequences for the Cat S gene were synthesized and transfected into human HCC cell line MHCC97-H. The Cat S gene targeted siRNA-mediated knockdown of Cat S expression, leading to potent suppression of MHCC97-H cell proliferation, invasion and angiogenesis. These data suggest that Cat S might be a potential target for HCC therapy.     

Expression and structure of human SPARC transcripts: SPARC mRNA is expressed by human cells involved in extracellular matrix production and some of these cells show an unusual expression pattern

A mouse SPARC cDNA clone was used to elucidate the expression of SPARC mRNA in normal diploid human cells as well as in tumor cells. Among 40 cell lines examined, 19 showed expression. The mRNA transcribed by the majority of the expressors are 2.1 kb with a trace amount of 3 kb. However, three cell types, undifferentiated basal keratinocytes, their differentiated derivatives, and breast adenocarcinoma cells, showed an expression pattern distinct from the typical one, having abundant 3-kb mRNA but no detectable 2.1-kb mRNA. The mRNA was translated and the product secreted. This expression pattern was not observed before in human cells and was not found in tumor cells of keratinocytes, squamous carcinoma cells, or many other adenocarcinoma cells. We showed by Northern hybridization that the SPARC-expressing melanocytic melanoma cell lines produced laminin, a component of extracellular matrix. Other cell types expressing the SPARC mRNA were also reported to synthesize extracellular matrix components. Thus, our results indicate an association between SPARC gene expression and production of extracellular matrix. However, the opposite is not true since non-SPARC-producers may or may not produce extracellular matrix. For example, A431 cell line, which does not express SPARC mRNA, is known to produce extracellular matrix components while the normal diploid melanocytes and undifferentiated embryonal carcinoma cells, which do not express SPARC mRNA, do not produce extracellular matrix component.     

The expression and regulation of DFNA5 in human hepatocellular carcinoma DFNA5 in hepatocellular carcinoma

Hepatocellular carcinoma is a primary malignancy of hepatocytes which accounts for 80 % of all primary liver cancers. DFNA5 has been identified as a tumor suppressor gene with an important role in several frequent forms of cancers, while little is known about its role in hepatocellular carcinoma. Through comparison of the DFNA5 protein expression in hepatocellular carcinoma cells (HepG2) with human fetal lung fibroblast cells (MRC5), we found that the DFNA5 protein expression in hepatocellular carcinoma cells was significantly lower than that in normal cells. The transfection of DFNA5 gene into HepG2 cells could increase DFNA5 protein expression, which subsequently led to inhibition of cell proliferation. Underlying mechanism study revealed that decreased proliferation was due to increased apoptosis and cell cycle arrest. In view of the important role of DFNA5 gene in carcinogenesis, these findings are expected to provide new understanding on development and treatment of human hepatocellular carcinoma.     

基于肝癌CDK2 基因敲减对CyclinE 表达及树舌多糖GF对其影响的研究

目的:探究树舌多糖GF(GAPS.GF)对rAAV-shRNA-CDK2抑制肝癌细胞Cyclin E基因表达的辅助作用。方法:将细胞培养后的人肝癌HepG2以皮下注射的方式接种于裸鼠前肢腋下,接种后的裸鼠随机分为5组:NC(非相关序列)对照组、肿瘤组、rAAV-shRNA-CDK2组、树舌多糖GF组以及树舌多糖GF+rAAV-shRNA-CDK2组,各实验组均采取尾静脉注射定量给药。采用实时定量PCR和Western blot技术研究肝癌细胞Cyclin E基因m RNA和蛋白水平的表达情况,同时观察GASP.GF对其作用的影响。结果:树舌多糖GF+rAAV-shRNA-CDK2联合应用组对肝癌HepG2细胞增殖的抑瘤率为75.6%,对Cyclin E基因mRNA表达抑制率为69%,对Cyclin E基因蛋白表达抑制率为67.5%,比rAAV-shRNA-CDK2组分别提高了3.42%、1%和2.7%。结论:树舌多糖GF与rAAV-shRNA-CDK2联合应用可以显著提高肝癌的治疗效果,说明树舌多糖GF可以辅助r AAV-shRNACDK2对肝癌细胞Cyclin E基因的表达的抑制作用。     

Increasing matrix stiffness upregulates vascular endothelial growth factor expression in hepatocellular carcinoma cells mediated by integrin β1

Matrix stiffness as a novel regulation factor involves in modulating the pathogenesis of hepatocellular carcinoma (HCC) invasion or metastasis. However, the mechanism by which matrix stiffness modulates HCC angiogenesis remains unknown. Here, using buffalo rat HCC models with different liver matrix stiffness backgrounds and an in vitro cell culture system of mechanically tunable Collagen1 (COL1)-coated polyacrylamide gel, we investigated the effects of different matrix stiffness levels on vascular endothelial growth factor (VEGF) expression in HCC cells and explored its regulatory mechanism for controlling HCC angiogenesis. Tissue microarray analysis showed that the expression levels of VEGF and CD31 were gradually upregulated in tumor tissues with increasing COL1 and lysyl oxidase (LOX) expression, indicating a positive correlation between tumor angiogenesis and matrix rigidity. The expression of VEGF and the phosphorylation levels of PI3K and Akt were all upregulated in HCC cells on high-stiffness gel than on low-stiffness gel. Meanwhile, alteration of intergrin β1 expression was found to be the most distinctive, implying that it might mediate the response of HCC cells to matrix stiffness simulation. After integrin β1 was blocked in HCC cells using specific monoclonal antibody, the expression of VEGF and the phosphorylation levels of PI3K and Akt at different culture times were accordingly suppressed and downregulated in the treatment group as compared with those in the control group. All data suggested that the extracellular matrix stiffness stimulation signal was transduced into HCC cells via integrin β1, and this signal activated the PI3K/Akt pathway and upregulated VEGF expression. This study unveils a new paradigm in which matrix stiffness as initiators to modulate HCC angiogenesis.     

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.     

Downregulating the expression of heparanase inhibits the invasion, angiogenesis and metastasis of human hepatocellular carcinoma

Invasion and metastasis are key features of human hepatocellular carcinoma (HCC). Heparanase is an endoglycosidase that can degrade extracellular matrix by cleaving heparan sulfate chains of heparan sulfate proteoglycan, thus playing important roles in the invasion and metastasis of human cancers. Heparanase has been detected in various human cancers and regarded as a prospective target in human cancer treatments. However, the effects of inhibiting the expression of heparanase on human HCC have not been fully evaluated. In this article we show that downregulating the expression of heparanase either by antisense oligodeoxynucleotide or by RNA interferencing can significantly reduce the expression of heparanase in SMMC7721 human HCC cells, leading to inhibition of the invasiveness, metastasis, and angiogenesis of HCC cells both in vitro and in vivo. Our results suggest that genetic downregulation of the expression of heparanase may serve as an efficient cancer therapeutic for human HCC.     

非复制型腺病毒携带miR-1的表达诱导肝癌细胞凋亡

miR-1在多种肝癌细胞中被甲基化沉默,导致其下游多个癌基因MET,FoxP1,HDAC4等大量表达,促进了肝癌的增殖.本研究利用非复制型腺病毒Ad-easy携带miR-1（Ad-miR-1）在肝癌细胞PLC/PRF/5和HepG2中过表达miR-1,探讨miR-1抑制肝癌细胞的作用机制.结果表明,肝癌细胞PLC/PRF/5和HepG2中miR-1表达水平极低,感染Ad-miR-1后miR-1表达水平显著上升.肝癌细胞PLC/PRF/5和HepG2感染Ad-miR-1后,MET和FoxP1的mRNA水平和蛋白表达水平被显著下调,细胞凋亡水平显著增加,肝癌细胞的增殖被抑制. 可见,miR-1在肝癌细胞中可能是一个重要的抑癌因子.同时,利用腺病毒载体携带抗癌的microRNA（如miR-1）,也为今后的基因治疗研究提供了一种新方法.