特异性下调葡萄糖调节蛋白78表达对肝细胞癌侵袭和转移能力的影响

为了研究特异性下调葡萄糖调节蛋白(Grp)78对肝细胞癌侵袭和转移能力的影响。通过小干扰RNA(siRNA)技术特异性下调人肝细胞癌细胞株BEL7402中Grp78的表达,并应用Transwell法和划痕法对肝细胞癌侵袭、转移能力的改变进行分析,应用免疫沉淀技术和GST-pulldown技术分别对黏着斑激酶(FAK)的磷酸化水平和小GTPase RhoA的活性进行研究,应用免疫印迹技术检测E-钙黏着蛋白、N-钙黏着蛋白和波形蛋白的表达。结果发现,Transwell实验和划痕实验结果显示特异性下调Grp78表达可以抑制肝细胞癌的侵袭和转移,免疫沉淀结果显示特异性下调Grp78表达可以降低FAK的磷酸化水平,GST-pulldown实验结果表明特异性下调Grp78表达可以上调RhoA的活性。免疫印迹实验结果表明特异性下调Grp78可以下调N-钙黏着蛋白、波形蛋白的表达,上调E-钙黏着蛋白的表达。结果表明特异性下调Grp78在体外可以抑制肝细胞癌的侵袭和转移,这种抑制作用是通过FAK脱磷酸化和抑制肿瘤的上皮-间叶转化实现的。     

MXR7 facilitates liver cancer metastasis via epithelial-mesenchymal transition

MXR7 is a cell-surface protein and highly expressed in hepatocellular carcinoma(HCC). The aim of this study is to determine the expression profile of MXR7 in HCC and investigate the influence of MXR7 on invasion and metastasis of HCC cells. For this purpose, immunohistochemical assay was used to identify the differential expression of MXR7 in 94 HCC specimens. Expression of MXR7 in 4 pairs of HCC and portal vein tumor thrombus(PVTT) was also tested. The motility of HCC cells were characterized by transwell migration and matrigel invasion assays. In vivo metastasis potential was determined via tail vein injection assay.Moreover, compared with noninvasive HCC tumors or human HCC cell lines with low metastatic potential, invasive HCC samples and HCC cell lines with high metastatic potential exhibited higher MXR7 expression. Furthermore, forced expression of MXR7 in SMMC-7721 promoted cell proliferation, migration and invasion in vitro and accelerated tumor growth and metastasis in vivo. Conversely, knockdown of MXR7 expression in HuH7 cells inhibited proliferation and motility of cells. Mechanically,overexpression of MXR7 promoted epithelial-mesenchymal transition(EMT) progress, and MXR7 depletion repressed the EMT phenotype. In conclusion, MXR7 is a mediator of EMT and metastasis in HCC and may serve as a novel therapeutic target.     

Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.     

Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.     

SIRT7琥珀酰化修饰对肝癌生存、免疫浸润及预后的相关性分析

摘要 目的：探究SIRT7基因琥珀酰化修饰对肝癌患者的生存、免疫浸润及预后的相关性分析。方法：采用生物信息分析法对SIRT7在肝癌组织中的表达情况及其对肝癌患者预后的影响进行分析；采用蛋白免疫印迹法（Western blot）检测其转染效果。结果：（1）生物信息分析结果显示：SIRT7在多种肿瘤（包括肝癌）组织中呈高表达（P<0.05）；SIRT7的表达与肿瘤的生存曲线相关（P<0.05）；肝癌患者的SIRT7相对表达量与其预后相关，高表达组肝癌患者的总生存情况（P=0.017）和无进展生存情况较低表达组缩短（P=0.004）；免疫浸润和肿瘤微环境分析结果显示，SIRT7表达水平与多数免疫细胞浸润水平、肿瘤微环境(ESTIMATES core)均有明显负相关。（2）Western blot显示，SIRT7在肝癌细胞中表达高于正常细胞。因此，SIRT7 可作为肝癌的潜在预后标志物。结论：SIRT7表达水平与肝癌(HCC)患者的预后、免疫细胞浸润性、肿瘤微环境免疫细胞和基质细胞浸润等相关。     

Knockdown of MACC1 expression suppressed hepatocellular carcinoma cell migration and invasion and inhibited expression of MMP2 and MMP9

Expression of MACC1 (metastasis-associated in colon cancer-1) protein is associated with metastasis of various human cancers. This study analyzed MACC1 protein expression in hepatocellular carcinoma (HCC) tissue specimens and then investigated the effects of MACC1 knockdown on HCC cell migration and invasion, and gene expression levels. Sixty pairs of HCC and adjacent normal liver tissues from HCC patients were analyzed for MACC1 expression immunohistochemically. The HCC cell lines Hep3B, Huh7, MHCC97H, SMMC-7721, Bel-7402, and HepG2 and the normal liver cell line LO2 were used to assess expressions of MACC1 mRNA and MACC1 protein using qRT-PCR and western blot, respectively. MACC1 short hairpin RNA (shRNA) was used to knockdown MACC1 protein expression in Huh7 cells. Changes in the tumor phenotype of these cells were analyzed with wound healing assay and invasion assays, and differences in gene expression were evaluated via western blot. Immunofluorescence was used to locate MACC1 protein in the above cell lines. MACC1 was highly expressed in HCC tissues and the nuclear expression of MACC1 protein was associated with poor tumor differentiation and intrahepatic metastasis or portal invasion. Moreover, MACC1 mRNA and MACC1 protein was also expressed in HCC cell lines. Immunostaining showed that MACC1 protein was localized in both nuclei and cytoplasm of HCC cell lines and the nuclear localization of MACC1 protein was associated with increased aggressiveness of HCC in cell lines. Knockdown of MACC1 expression using MACC1-shRNA reduced Huh7 cell migration and invasion abilities, which was associated with downregulation of MMP2, MMP9, and c-Met proteins in Huh7 cells. Localization of MACC1 protein to the nucleus may predict HCC progression. Knockdown of MACC1 expression using MACC1 shRNA warrants further evaluation as a novel therapeutic strategy for control of HCC.     

LncRNA CRNDE promotes the epithelial-mesenchymal transition of hepatocellular carcinoma cells via enhancing the Wnt/β-catenin signaling pathway

Colorectal neoplasia differentially expressed (CRNDE) is a significantly upregulated long noncoding RNA in hepatocellular carcinoma (HCC). CRNDE could promote cell proliferation, migration, and invasion, while its molecular mechanisms were still largely unclear. In this study, we investigated the expression and function of CRNDE. CRNDE was significantly upregulated in tumor tissues compared with adjacent normal tissues. In vitro, we revealed that knockdown of CRNDE inhibited cell proliferation, migration, and cell invasion capacities in HCC. Animal studies indicated that CRNDE knockdown represses both growth and metastasis of HCC tumors in vivo. Moreover, knockdown of CRNDE suppressed the cell epithelial-mesenchymal transition (EMT) process by increasing the expression of E-cadherin and ZO-1, whereas, decreasing the expression of N-cadherin, slug, twist, and vimentin in HCC cells. We also revealed that knockdown of CRNDE suppressed the Wnt/β-catenin signaling in HCC. Thus, CRNDE could modulate EMT of HCC cells and knockdown of CRNDE impaired the mesenchymal properties. CRNDE increased invasion of HCC cells might be through activating the Wnt/β-catenin signaling pathway.     

Fibronectin promotes cervical cancer tumorigenesis through activating FAK signaling pathway

Cervical cancer is a cancer arising from the cervix, and it is the fourth most common cause of death in women. Overexpression of fibronectin 1 (FN1) was observed in many tumors and associated with the survival and metastasis of cancer cells. However, the mechanism by which FN1 promotes cervical cancer cell viability, migration, adhesion, and invasion, and inhibits cell apoptosis through focal adhesion kinase (FAK) signaling pathway remains to be investigated. Our results demonstrated that FN1 was upregulated in patients with cervical cancer and higher FN1 expression correlated with a poor prognosis for patients with cervical cancer. FN1 knockdown by small interfering RNA (siRNA) inhibited SiHa cell viability, migration, invasion, and adhesion, and promoted cell apoptosis. FN1 overexpression in CaSki cell promoted cell viability, migration, invasion, and adhesion, and inhibited cell apoptosis. Further, phosphorylation of FAK, a main downstream signaling molecule of FN1, and the protein expression of Bcl-2/Bax, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), and N-cadherin was upregulated in CaSki cells with FN1 overexpression, but caspase-3 protein expression was downregulated. The FAK phosphorylation inhibitor PF573228 inhibited FN1 overexpression-induced expression of those proteins in CaSki cells with FN1 overexpression. In vivo experiment demonstrated that FN1 knockdown significantly inhibited FN1 expression, phosphorylation of FAK, and tumor growth in xenograft from the nude mice. These results suggest that FN1 regulates the viability, apoptosis, migration, invasion, and adhesion of cervical cancer cells through the FAK signaling pathway and is a potential therapeutic target in the treatment of cervical cancer.     

Association of focal adhesion kinase with Grb7 and its role in cell migration.

Focal adhesion kinase (FAK) has been implicated to play a key role in integrin-mediated signal transduction in cell migration. Grb7 is an Src homology (SH) 2-containing and pleckstrin homology domain-containing molecule, which shares significant homology with the Caenorhabditis elegans gene for Mig-10 involved in cell migration during embryogenesis. Here, we report that the SH2 domain of Grb7 can directly interact with FAK through Tyr-397, a major autophosphorylation site in vitro and in vivo. This interaction is cell adhesion-dependent, suggesting that the FAK-Grb7 complex is involved in integrin signaling. Using tetracycline-regulated expression system, we showed that overexpression of Grb7 enhanced cell migration toward fibronectin, whereas overexpression of its SH2 domain alone inhibited cell migration. In addition, we found that phosphorylation of FAK or p130(cas) was not affected by the expression of either Grb7 or its SH2 domain alone, suggesting that Grb7 is downstream of FAK and does not compete with Src for binding to FAK in vivo. Taken together, these results suggest that the FAK-Grb7 complex plays a role in cell migration stimulated by integrin signaling through FAK.     

KMT6在肝癌细胞转移中的作用及对肝癌预后的影响

目的:探讨组蛋白赖氨酸甲基转移酶6(histone lysine methyltransferase 6,KMT6)对人肝癌细胞转移的调控作用及对肝癌患者预后的影响。方法:采用RNA干涉技术合成靶向KMT6的小干扰RNA(KMT6 siRNA)片段,瞬时转染至人肝癌细胞系Huh-7。随后,分别采用细胞黏附实验、细胞侵袭实验以及划痕迁移实验检测肝癌细胞的黏附、侵袭和迁移等肿瘤细胞转移能力。运用Oncomine数据库和cBioportal肝癌数据库分析KMT6的表达情况及对肝癌预后的影响。结果:靶向KMT6的小干扰RNA(KMT6 siRNA)片段瞬时转染至人肝癌细胞系Huh-7后,KMT6蛋白的表达显著下降,且黏附率、侵袭率和划痕修复率均显著降低(P<0.05)。Oncomine肝癌数据库分析表明肝癌组织KMT6的表达增加,cBioportal肝癌数据库分析表明KMT6基因改变与肝癌患者预后不良密切相关。结论:KMT6分子可促进肝癌细胞黏附、侵袭和迁移,其基因改变与肝癌患者预后不良显著相关。KMT6分子有望成为肝癌治疗的新靶点。     

Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.     

The expression and significance of NET-1 and Contactin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. In this study, the significance of NET and Contactin on the pathogenesis and prognosis of HCC was investigated, and further to explore their functions in vitro and in vivo by down regulation with siRNA. The expression of NET-1 and Contactin in HCC and in adjacent non-tumor tissues (ANT) were evaluated by immunohistochemistry, and the correlations of the expression of NET-1 and Contactin with the clinicopathological characteristics and survival of HCC patients were also analyzed. After inhibited by single-target siRNA or dual-target siRNA, the expressions of NET-1 and Contactin mRNA and protein in SMMC-7221 cells were determined by RT-PCR, Western blot and immunofluorescence stain. The cell proliferation and apoptosis were assessed by CCK-8 assays and flow cytometry (FCM). The ability of cell migration and invasion were evaluated by wound-healing migrating assay and transwell chamber assays, respectively. Subsequently, transmission system encapsulated cationic liposome was used to deliver dual-siRNA into HCC xenografts in mice. The expressions of NET-1, Cortactin, Ki67, Bax, Bcl2 and Survivin in xenograft tumor were detected by immunohistochemical staining, respectively. The positive rates of NET-1 and Cortactin in HCC tissues were significantly higher than those in ANT. In HCC, the expression of NET-1 was related to Edmondson''s grade (P<0.05), cirrhosis background (P<0.001) and TNM stage (P<0.05). The expression of Cortactin was related to tumor infiltration (P<0.05), vascular invasion (P <0.001) and TNM stage (P<0.001). The expressions of NET-1 and Cortactin were positively correlated (r=0.280, P=0.004). Significant differences in the 5-year survival rates were seen between the NET-1 negative group and the positive group (P<0.05), and between the Contactin negative group (50%) and Contactin positive group (28.0%, P<0.01). The 5-year overall survival rate (OS) in NET-1 and Contactin co-expression cases (27.78%) were remarkably lower than that in both NET-1 and Contactin negative cases (54.54%) and in NET-1 positive while Contactin negative cases (47.06%, P<0.05). Univariate and multivariate Cox regression analysis revealed that NET-1 and Contactin over-expression were independent indicators for OS in HCC patients (P<0.01). There were higher expressions of NET-1 and Contactin in SMMC-7721 cells than that in other HCC cells. Dual-siRNA was demonstrated to be more effective on inhibiting cancer cell proliferation, migration and inducing apoptosis than individual siRNAs used alone in vitro and in vivo (P<0.05). The results suggest that dual-siRNA may be a great potential in siRNA-based therapeutic applications.     

血小板反应蛋白4通过诱导肿瘤相关巨噬细胞M2型极化促进肝癌细胞转移

血小板反应蛋白4 (thrombospondin 4, THBS4)属于THBS家族成员,是细胞外基质分泌的蛋白质,参与调控细胞增殖、黏附及血管生成等多种生理过程。近来研究表明,机体在炎症刺激下加速产生THBS4并诱导巨噬细胞粘附与积累。我们的前期研究证实,THBS4在肝癌(hepatocellular carcinoma, HCC)中发挥促癌作用,但THBS4对肝癌免疫微环境的影响尚不明确。本文旨在分析THBS4通过诱导肿瘤相关巨噬细胞M2型极化,促进肝癌细胞转移的作用。通过肝癌条件培养基(HCC conditioned medium, HCM)模拟肿瘤微环境,发现在HCM作用下巨噬细胞中THBS4表达呈时间依赖性升高(P<0.05);下调THBS4促使M1型巨噬细胞标志物IL-1β、CD86的表达升高(P<0.01),而M2型标志物IL-10和CD206表达降低(P<0.01)。进一步通过Transwell共培养实验检测THBS4诱导的M2型巨噬细胞对肝癌转移的影响。将下调THBS4的M2型巨噬细胞(M2-TAMs)与HepG2肝癌细胞进行共培养。结果显示,下调T...     

Effects of stable knockdown of Aurora kinase A on proliferation, migration, chromosomal instability, and expression of focal adhesion kinase and matrix metalloproteinase-2 in HEp-2 cells

Overexpression of Aurora kinase A (AURKA) is frequently observed in various cancers, including laryngeal squamous cell carcinoma (LSCC). We investigated the effects of knockdown of AURKA on laryngeal cancer HEp-2 cells both in vitro and in vivo. A plasmid containing short hairpin (sh)RNA against AURKA was constructed and transfected into HEp-2. Measurements included the CCK-8 assay for viability and proliferation, flow cytometry for apoptosis and effects on the mitotic checkpoint, a trans-well assay for migration, immunofluorescence for assessment of genomic instability, and western blotting for protein expression. AURKA knockdown inhibited proliferation, migration, and colony formation in vitro and tumorigenicity in vivo. The knockdown induced the accumulation of cells in G2-M phase and eventual apoptosis. Knockdown of AURKA caused delayed entry into mitosis after treatment with nocodazole, reduced chromosomal instability, and decreased expression of focal adhesion kinase (FAK), phosphorylated FAK, and matrix metalloproteinase-2 (MMP-2), key regulators in cell adhesion and invasion. Knockdown of AURKA inhibits the growth and invasiveness of this LSCC cell line both in vitro and in vivo. These effects may partially result from the reduced expression of FAK and MMP-2. Knockdown of AURKA expression may represent a promising therapeutic strategy for the treatment of LSCC.     

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.     

HBO1 overexpression is important for hepatocellular carcinoma cell growth

Hepatocellular carcinoma (HCC) is a common primary liver malignancy lacking effective molecularly-targeted therapies. HBO1 (lysine acetyltransferase 7/KAT7) is a member of MYST histone acetyltransferase family. Its expression and potential function in HCC are studied. We show that HBO1 mRNA and protein expression is elevated in human HCC tissues and HCC cells. HBO1 expression is however low in cancer-surrounding normal liver tissues and hepatocytes. In HepG2 and primary human HCC cells, shRNA-induced HBO1 silencing or CRISPR/Cas9-induced HBO1 knockout potently inhibited cell viability, proliferation, migration, and invasion, while provoking mitochondrial depolarization and apoptosis induction. Conversely, ectopic overexpression of HBO1 by a lentiviral construct augmented HCC cell proliferation, migration and invasion. In vivo, xenografts-bearing HBO1-KO HCC cells grew significantly slower than xenografts with control HCC cells in severe combined immunodeficient mice. These results suggest HBO1 overexpression is important for HCC cell progression.Subject terms: Targeted therapies, Oncogenes     

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 p130Cas as a Mediator of Focal Adhesion Kinase–promoted Cell Migration

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130^Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130^Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130^Cas and its subsequent binding to several SH2 domains, which depended on both the p130^Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130^Cas further increased cell migration on FN and coexpression of the p130^Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130^Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130^Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.     

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。     

MicroRNA-550a Acts as a Pro-Metastatic Gene and Directly Targets Cytoplasmic Polyadenylation Element-Binding Protein 4 in Hepatocellular Carcinoma

MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that are often found at chromosomal breakpoints and play a vital role in human cancer. Our previous study found that miR-550a, a frequently amplified miRNA on 7p14.3, was upregulated in hepatocellular carcinoma (HCC). However, the possible functions and molecular mechanisms of miR-550a in HCC remain unknown. In this study, gain-of-function and loss-of-function assays revealed that miR-550a markedly promoted HCC cell migration and invasion. In addition, we discovered that cytoplasmic polyadenylation element binding protein 4 (CPEB4) was a potential target of miR-550a in HCC. Further analyses showed that knockdown of CPEB4 expression significantly facilitated HCC cell migration and invasion, which phenocopied the effects of miR-550a on HCC cells. Moreover, a decrease in CPEB4 expression mediated miR-550a-induced liver cancer cell migration and invasion. Interestingly, CPEB4 is frequently downregulated in HCC, and its expression levels correlate with the overall survival of HCC patients. Together, these results suggested that this newly identified miR-550a-CPEB4 axis may be involved in HCC cell metastasis. Moreover, the expression levels of CPEB4 could be used to predict outcomes in HCC patients. Our findings provide novel potential targets for HCC therapy and prognosis.