microRNA-148a-3p enhances the effects of sevoflurane on hepatocellular carcinoma cell progression via ROCK1 repression

BackgroundSevoflurane (SEVO) inactivates the aggressiveness of hepatocellular carcinoma (HCC) cells by mediating microRNAs (miRNAs). Hence, we delved into the functional role of miR-148a-3p mediated by SEVO in HCC.MethodsLiver cells (L02) and HCC cells (HCCLM3 and Huh7) were exposed to SEVO to detect cell viability in HCC. HCCLM3 and Huh7 cells were treated with restored miR-148a-3p or depleted Rho-associated protein kinase 1 (ROCK1) to elucidate their roles in HCC cells' biological characteristics. HCCLM3 and Huh7 cells were treated with SEVO, and/or vectors that changed miR-148a-3p or ROCK1 expression to identify their combined functions in HCC cell progression. Tumor xenograft in nude mice was performed to determine growth ability of tumor. The target relationship between miR-148a-3p and ROCK1 was verified.ResultsSEVO inhibited proliferation, invasion and migration and enhanced apoptosis of HCCLM3 and Huh7 cells. MiR-148a-3p up-regulation or ROCK1 down-regulation inhibited HCCLM3 and Huh7 cell progression. ROCK1 was determined to be target gene of miR-148a-3p. Down-regulating miR-148a-3p or overexpressing ROCK1 mitigated cell aggressiveness inhibition caused by SEVO.ConclusionOur study elucidates that microRNA-148a-3p enhances the effects of sevoflurane on inhibiting proliferation, invasion and migration and enhancing apoptosis of HCC cells through suppression of ROCK1.     

LncRNA SNHG3 induces EMT and sorafenib resistance by modulating the miR-128/CD151 pathway in hepatocellular carcinoma

Dysregulation of long noncoding RNAs (lncRNAs) plays important roles in carcinogenesis and tumor progression, including hepatocellular carcinoma (HCC). Small nucleolar RNA host gene 3 (SNHG3) has been considered as an lncRNA to be associated with a poor prognosis in patients with HCC. Here, we reported that SNHG3 expression was significantly higher in the highly metastatic HCC (HCCLM3) cells compared with the lowly metastatic HCC cells (Hep3B and PLC/PRF/5). Furthermore, forced expression of SNHG3 promoted cell invasion, epithelial-mesenchymal transition (EMT), and sorafenib resistance in HCC. Moreover, SNHG3 overexpression induced HCC cells EMT via miR-128/CD151 cascade activation. Clinically, our data revealed that increased SNHG3 expression is correlated with poor HCC survival outcomes and sorafenib response. These data suggest that SNHG3 may be a novel therapeutic target and a biomarker for predicting response to sorafenib treatment of HCC.     

Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S

A new cysteine proteinase inhibitor, cystatin SN, was purified from human whole saliva by chromatography with DE32, Sephacryl S200, and CM-Sepharose CL6B. Cystatin SN is immunologically related to cystatin S and both inhibitors have a similar molecular mass of about 13 kDa. The new inhibitor, however, was clearly distinguished from cystatin S by its much higher pI value. These inhibitors showed similar inhibitory activity for ficin, but cystatin SN was a much better inhibitor for papain and dipeptidyl peptidase I. The amino acid sequence of cystatin SN deduced in the light of the known structure of cystatin S indicates that they have 10 different amino acid residues in the sequence comprising in total 113 residues.     

microRNA-489 Plays an Anti-Metastatic Role in Human Hepatocellular Carcinoma by Targeting Matrix Metalloproteinase-7

Dysregulation of microRNAs (miRNAs) is actively involved in the pathogenesis and tumorigenicity of hepatocellular carcinoma (HCC). miR-489 was found to play either oncogenic or tumor suppressive roles in human cancers. Recent study reported that the levels of miR-489 in late recurrent HCC patients were evidently higher than that in early recurrent cases, suggesting that miR-489 may function as a tumor suppressive miRNA in HCC. Yet, the clinical value and biological function of miR-489 remain rarely known in HCC. Here, we presented that miR-489 level in HCC tissues was notably reduced compared to matched non-cancerous specimens. Its decreased level was evidently correlated with adverse clinical parameters and poor prognosis of HCC patients. Accordingly, the levels of miR-489 were obviously down-regulated in HCC cells. Ectopic expression of miR-489 in HCCLM3 and MHCC97H cells prominently inhibits the migration and invasion of tumor cells and reduced lung metastases in vivo, while miR-489 knockdown increased these behaviors of HepG2 and MHCC97L cells. Mechanically, miR-489 negatively regulated matrix metalloproteinase-7 (MMP7) abundance in HCC cells. Herein, MMP7 was found to be a downstream molecule of miR-489 in HCC. An inversely correlation between miR-489 and MMP7 was confirmed in HCC specimens. MMP7 knockdown prohibited cell migration and invasion while MMP7 overexpression showed opposite effects on HCC cells. Furthermore, restoration of MMP7 expression could abrogate the anti-metastatic effects of miR-489 on HCCLM3 cells with enhanced cell migration and invasion. Altogether, miR-489 potentially acts as a prognostic predictor and a drug-target for HCC patients.     

Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.     

Characterization and amino acid sequence of a new acidic cysteine proteinase inhibitor (cystatin SA) structurally closely related to cystatin S, from human whole saliva

A cysteine proteinase inhibitor (designated as cystatin SA) was isolated from human whole saliva by procedures including chromatography on DE 32 and DEAE-Sepharose CL-6B. The amino acid sequence determined by conventional methods showed sequence homology of 90 and 87% as compared with the sequences of cystatin S and cystatin SN, respectively, both of which are salivary inhibitors characterized previously. The new inhibitor consisted of 117 residues and had a pI value of 4.3. Cystatin SA inhibited ficin and papain more strongly than cystatin S or cystatin SN did. It also exhibited inhibitory activity toward dipeptidyl peptidase I but the activity was much weaker than those toward ficin and papain.     

Cystatin C Is Downregulated in Prostate Cancer and Modulates Invasion of Prostate Cancer Cells via MAPK/Erk and Androgen Receptor Pathways

Cystatin C is believed to prevent tumor progression by inhibiting the activities of a family of lysosomal cysteine proteases. However, little is known about the precise mechanism of cystatin C function in prostate cancer. In the present study, we examined the expression of cystatin C and its association with matrix metalloproteinases 2 (MMP2) and androgen receptor (AR) in a tissue microarray comparing benign and malignant specimens from 448 patients who underwent radical prostatectomy for localized prostate cancer. Cystatin C expression was significantly lower in cancer specimens than in benign tissues (p<0.001) and there was a statistically significant inverse correlation between expression of cystatin C and MMP2 (r_s ² = −0.056, p = 0.05). There was a clear trend that patients with decreased level of cystatin C had lower overall survival. Targeted inhibition of cystatin C using specific siRNA resulted in an increased invasiveness of PC3 cells, whereas induction of cystatin C overexpression greatly reduced invasion rate of PC3 in vitro. The effect of cystatin C on modulating the PC3 cell invasion was provoked by Erk2 inhibitor that specifically inhibited MAPK/Erk2 activity. This suggests that cystatin C may mediate tumor cell invasion by modulating the activity of MAPK/Erk cascades. Consistent with our immunohistochemical findings that patients with low expression of cystatin C and high expression of androgen receptor (AR) tend to have worse overall survival than patients with high expression of cystatin C and high AR expression, induced overexpression of AR in PC3 cells expressing cystatin C siRNA greatly enhanced the invasiveness of PC3 cells. This suggests that there may be a crosstalk between cystatin C and AR-mediated pathways. Our study uncovers a novel role for cystatin C and its associated cellular pathways in prostate cancer invasion and metastasis.     

Profiling of the tetraspanin CD151 web and conspiracy of CD151/integrin β1 complex in the progression of hepatocellular carcinoma

Tetraspanin CD151 has been implicated in metastasis through forming complexes with different molecular partners. In this study, we mapped tetraspanin web proteins centered on CD151, in order to explore the role of CD151 complexes in the progression of hepatocellular carcinoma (HCC). Immunoprecipitation was used to isolate tetraspanin complexes from HCCLM3 cells using a CD151 antibody, and associated proteins were identified by mass spectrometry. The interaction of CD151 and its molecular partners, and their roles in invasiveness and metastasis of HCC cells were assayed through disruption of the CD151 network. Finally, the clinical implication of CD151 complexes in HCC patients was also examined. In this study, we identified 58 proteins, characterized the tetraspanin CD151 web, and chose integrin β1 as a main partner to further investigate. When the CD151/integrin β1 complex in HCC cells was disrupted, migration, invasiveness, secretion of matrix metalloproteinase 9, and metastasis were markedly influenced. However, both CD151 and integrin β1 expression were untouched. HCC patients with high expression of CD151/integrin β1 complex had the poorest prognosis of the whole cohort of patients. Together, our data show that CD151 acts as an important player in the progression of HCC in an integrin β1-dependent manner.     

Inhibitory properties of cystatin F and its localization in U937 promonocyte cells

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.     

Downregulation of CCR1 inhibits human hepatocellular carcinoma cell invasion

CC chemokine receptor 1 (CCR1) has an important role in the recruitment of leukocytes to the site of inflammation. The migration and metastasis of tumor cells shares many similarities with leukocyte trafficking, which is mainly regulated by chemokine receptor-ligand interactions. CCR1 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues with unknown functions. In this study, we silenced CCR1 expression in the human HCC cell line HCCLM3 using artificial microRNA (miRNA)-mediated RNA interference (RNAi) and examined the invasiveness and proliferation of CCR1-silenced HCCLM3 cells and the matrix metalloproteinase (MMP) activity. The miRNA-mediated knockdown expression of CCR1 significantly inhibited the invasive ability of HCCLM3 cells, but had only a minor effect on the cellular proliferation rate. Moreover, CCR1 knockdown significantly reduced the secretion of MMP-2. Together, these findings indicate that CCR1 has an important role in HCCLM3 invasion and that CCR1 might be a new target of HCC treatment.     

基于CRISPR转录激活系统构建内源性着丝粒蛋白F稳定过表达的肝细胞癌细胞模型

临床研究表明着丝粒蛋白F(centromere protein F,CENPF)与肝细胞癌(hepatocellular carcinoma,HCC)的发生相关。由于CENPF蛋白的分子量高达358 kDa,目前有关CENPF致癌机制研究使用的体内外模型主要基于CENPF表达敲低,尚缺乏将CENPF过表达以探究其与癌变因果关系的研究,CENPF过表达改变到底是“因”还是“果”仍不清楚。本研究旨在利用包括lentiMPHv2和lentiSAMv2两个载体的CRISPR/dCas9转录激活协同激活介体(synergistic activation mediator,SAM)系统构建内源性CENPF稳定过表达的细胞模型,为阐明CENPF过表达与HCC发生发展的因果关系提供有效工具。首先设计并合成能够特异性识别CENPF基因转录起始位点的单向导核糖核酸(single guide RNAs,sgRNA),并将其插入lentiSAMv2质粒中。利用慢病毒载体将lentiMPHv2质粒导入Huh-7和HCCLM3细胞株中,用潮霉素B筛选后再用慢病毒载体将连接好的携带sgRNA序列的lentiSAMv2质粒导入细胞中,并用杀稻瘟菌素S继续筛选。对2种抗生素筛选获得的Huh-7和HCCLM3细胞株样品进行CENPF mRNA和蛋白表达水平的检测,结果显示sgRNA1和sgRNA4序列均能够激活内源CENPF的表达,其中sgRNA4诱导转录效果更加明显。本研究利用CRISPR/dCas9系统成功构建内源性CENPF稳定过表达的HCC细胞模型,为研究CENPF过表达与肿瘤发生的因果关系奠定基础,并为构建大分子量蛋白过表达的细胞模型提供参考方案。     

Incomplete Radiofrequency Ablation Enhances Invasiveness and Metastasis of Residual Cancer of Hepatocellular Carcinoma Cell HCCLM3 via Activating β-Catenin Signaling

Background

Radiofrequency ablation (RFA) is one of the curative therapies for hepatocellular carcinoma (HCC), however, accelerated progression of residual HCC after incomplete RFA has been reported more frequently. The underlying molecular mechanism of this phenomenon remains to be elucidated. In this study, we used an incomplete RFA orthotopic HCC nude mouse model to study the invasive and metastatic potential of residual cancer as well as the correlated mechanism.

Methods

The incomplete RFA orthotopic nude mouse models were established using high metastatic potential HCC cell line HCCLM3 and low metastatic potential HCC cell line HepG2, respectively. The changes in cellular morphology, motility, metastasis and epithelial–mesenchymal transition (EMT), and HCC cell molecular markers after in vitro and in vivo incomplete RFA intervention were observed.

Results

Pulmonary and intraperitoneal metastasis were observed in an in vivo study. The underlying pro-invasive mechanism of incomplete RFA appeared to be associated with promoting EMT, including down-regulation of E-cadherin and up-regulation of N-cadherin and vimentin. These results were in accordance with the in vitro response of HCC cells to heat intervention. Further studies demonstrated that β-catenin was a pivotal factor during this course and blocking β-catenin reduced metastasis and EMT phenotype changes in heat-treated HCCLM3 cells in vitro.

Conclusion

Incomplete RFA enhanced the invasive and metastatic potential of residual cancer, accompanying with EMT-like phenotype changes by activating β-catenin signaling in HCCLM3 cells.     

Tissue distribution of bovine cystatin C analysed by in situ hybridisation

Cysteine proteinase inhibitors of the cystatin superfamily have been identified in many living organisms. However, knowledge of the tissue distribution of such inhibitors is limited. To elucidate this distribution in mammals, we have investigated the expression of the gene for cystatin C, belonging to cystatin family II, in several bovine tissues. In situ hybridisation with a digoxigenin-labelled cRNA probe demonstrated a high concentration of bovine cystatin C mRNA in the secretory epithelial cells of the choroid plexus, and also intense staining in cells of lymphoid tissue and in Sertoli cells. Cystatin C mRNA was also present in scattered neurons and glial cells throughout the cerebrum and the cerebellum. In the submandibular gland, specific mRNA was found mainly in striated intralobular ducts and interlobular ducts. The expression of cystatin C in brain tissue is of particular interest, as the inhibitor appears to be involved in certain neurological diseases. The main production of cystatin C within the brain is believed to be by astrocytes. However, this work shows that also neurons from young, normal individuals express cystatin C.     

Cystatin F is secreted, but artificial modification of its C-terminus can induce its endocytic targeting

Cystatins constitute a superfamily of cysteine protease inhibitors. A member of the type II secreted cystatin family, cystatin F, has been identified through different gene array experiments to be specifically expressed in hematopoietic cells as well as to be associated with several malignant tumors, suggesting a role in immunity or cancer progression. Cystatin F specificity as a protease inhibitor is still elusive, and understanding the cellular traffic of this molecule is therefore a major step in its characterization. Although the mannosylation-6 phosphate of cystatin F has been suggested, no conclusive evidences of its endosomal targeting have been reported. Here we show using U937 cells that cystatin F is secreted as a disulfide bridge-linked dimer and is not associated with endosomes intracellularly. Interestingly, although cystatin F targeting to endosomes or lysosomes is not observed in U937, modification of its C-terminal end by the addition of several amino acids promotes its accumulation in the lysosomes of transfected HeLa cells. This observation suggests that cystatin F can be targeted to the endocytic pathway under specific conditions and its C-terminal domain might contribute to this event.     

Regulated expression and intracellular localization of cystatin F in human U937 cells.

Cystatin F is a cysteine peptidase inhibitor recently discovered in haematopoietic cells by cDNA cloning. To further investigate the expression, distribution and properties of the native human inhibitor the promyeloid cell line U937 has been studied. The cells expressed relatively large quantities of cystatin F, which was found both secreted and intracellularly. The intracellular levels were unusually high for a secreted cystatin ( approximately 25% of the cystatin F in 2- or 4-day culture medium). By contrast, U937 cells contained only 3-4% of the related inhibitor, cystatin C. Cystatin F purified from lysates of U937 cells showed three major forms carrying two, one or no carbohydrate chains. Immunocytochemistry demonstrated a marked cytoplasmic cystatin F staining in a granular pattern. Double staining with a marker for endoplasmic reticulum revealed no colocalization for cystatin F. Analysis of the promoter region of the cystatin F gene (CST7) showed that it, like that of the cystatin C gene (CST3), is devoid of typical TATA- and CAAT-box elements. In contrast to the cystatin C promoter, it does not contain multiple Sp1 binding sites, but has a unique site for C/EBPalpha, possibly explaining the restricted expression of the cystatin F gene. Cells stimulated with all-trans retinoic acid to differentiate them towards a granulocytic pathway, showed a strong ( approximately 18-fold) down-regulation of intracellular cystatin F and almost abolished secreted levels of the inhibitor. Stimulation with tetradecanoyl phorbol acetate, causing monocytic differentiation, also resulted in down-regulation (two fold to threefold) of cystatin F expression, whereas the cystatin C expression was essentially unaltered in both experiments. The results suggest that cystatin F as an intracellular cysteine peptidase inhibitor with readily regulated expression, may be a candidate to control the cysteine peptidase activity known to be essential for antigen presentation in different blood cell lineages.     

Immunolocalization of human cystatins in neutrophils and lymphocytes

Summary The endogenous inhibitors of cysteine proteinases, cystatin A and cystatin B, have been localized in sections of human liver by indirect immunofluorescence microscopy. Cystatin A was localized in neutrophils and cystatin B was found in lymphocytes. Both types of cell were present in the blood vessels and within the sinusoids between the parenchymal cells. Cystatin A was also found in some of the parenchymal cells surrounding the blood vesscts but, in contrast to the uniform fluorescence of the leucocytes, the staining in the parenchymal cells was granular and rather irregular.     

Immunolocalization of human cystatins in neutrophils and lymphocytes

The endogenous inhibitors of cysteine proteinases, cystatin A and cystatin B, have been localized in sections of human liver by indirect immunofluorescence microscopy. Cystatin A was localized in neutrophils and cystatin B was found in lymphocytes. Both types of cell were present in the blood vessels and within the sinusoids between the parenchymal cells. Cystatin A was also found in some of the parenchymal cells surrounding the blood vessels but, in contrast to the uniform fluorescence of the leucocytes, the staining in the parenchymal cells was granular and rather irregular.