Jine Yang

Effects of MicroRNA‐29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma

Based on microarray data, we have previously shown a significant down‐regulation of miR‐29 in hepatocellular carcinoma (HCC) tissues. To date, the role of miR‐29 deregulation in hepatocarcinogenesis and the signaling pathways by which miR‐29 exerts its function and modulates the malignant phenotypes of HCC cells remain largely unknown. In this study, we confirmed that reduced expression of miR‐29 was a frequent event in HCC tissues using both Northern blot and real‐time quantitative reverse‐transcription polymerase chain reaction. More interestingly, we found that miR‐29 down‐regulation was significantly associated with worse disease‐free survival of HCC patients. Both gain‐ and loss‐of‐function studies revealed that miR‐29 could sensitize HCC cells to apoptosis that was triggered by either serum starvation and hypoxia or chemotherapeutic drugs, which mimicked the tumor growth environment in vivo and the clinical treatment. Moreover, introduction of miR‐29 dramatically repressed the ability of HCC cells to form tumor in nude mice. Subsequent investigation characterized two antiapoptotic molecules, Bcl‐2 and Mcl‐1, as direct targets of miR‐29. Furthermore, silencing of Bcl‐2 and Mcl‐1 phenocopied the proapoptotic effect of miR‐29, whereas overexpression of these proteins attenuated the effect of miR‐29. In addition, enhanced expression of miR‐29 resulted in the loss of mitochondrial potential and the release of cytochrome c to cytoplasm, suggesting that miR‐29 may promote apoptosis through a mitochondrial pathway that involves Mcl‐1 and Bcl‐2. Conclusion: Our data highlight an important role of miR‐29 in the regulation of apoptosis and in the molecular etiology of HCC, and implicate the potential application of miR‐29 in prognosis prediction and in cancer therapy. (HEPATOLOGY 2010.)

MicroRNA‐29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent intrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. We previously found that microRNA‐29b (miR‐29b) down‐regulation was significantly associated with poor recurrence‐free survival of HCC patients. Therefore, the role of miR‐29b in tumor angiogenesis, invasion, and metastasis was further investigated in this study using in vitro capillary tube formation and transwell assays, in vivo subcutaneous and orthotopic xenograft mouse models, and Matrigel plug assay, and human HCC samples. Both gain‐ and loss‐of‐function studies showed that miR‐29b dramatically suppressed the ability of HCC cells to promote capillary tube formation of endothelial cells and to invade extracellular matrix gel in vitro. Using mouse models, we revealed that tumors derived from miR‐29b‐expressed HCC cells displayed significant reduction in microvessel density and in intrahepatic metastatic capacity compared with those from the control group. Subsequent investigations revealed that matrix metalloproteinase‐2 (MMP‐2) was a direct target of miR‐29b. The blocking of MMP‐2 by neutralizing antibody or RNA interference phenocopied the antiangiogenesis and antiinvasion effects of miR‐29b, whereas introduction of MMP‐2 antagonized the function of miR‐29b. We further disclosed that miR‐29b exerted its antiangiogenesis function, at least partly, by suppressing MMP‐2 expression in tumor cells and, in turn, impairing vascular endothelial growth factor receptor 2‐signaling in endothelial cells. Consistently, in human HCC tissues and mouse xenograft tumors miR‐29b level was inversely correlated with MMP‐2 expression, as well as tumor angiogenesis, venous invasion, and metastasis. Conclusion: miR‐29b deregulation contributes to angiogenesis, invasion, and metastasis of HCC. Restoration of miR‐29b represents a promising new strategy in anti‐HCC therapy. (HEPATOLOGY 2011;)

MicroRNA‐195 Suppresses Angiogenesis and Metastasis of Hepatocellular Carcinoma by Inhibiting the Expression of VEGF, VAV2, and CDC42

Hepatocellular carcinoma (HCC) is characterized by active angiogenesis and metastasis, which account for rapid recurrence and poor survival. There is frequent down‐regulation of miR‐195 expression in HCC tissues. In this study, the role of miR‐195 in HCC angiogenesis and metastasis was investigated with in vitro capillary tube formation and transwell assays, in vivo orthotopic xenograft mouse models, and human HCC specimens. Reduction of miR‐195 in HCC tissues was significantly associated with increased angiogenesis, metastasis, and worse recurrence‐free survival. Both gain‐of‐function and loss‐of‐function studies of in vitro models revealed that miR‐195 not only suppressed the ability of HCC cells to promote the migration and capillary tube formation of endothelial cells but also directly repressed the abilities of HCC cells to migrate and invade extracellular matrix gel. Based on mouse models, we found that the induced expression of miR‐195 dramatically reduced microvessel densities in xenograft tumors and repressed both intrahepatic and pulmonary metastasis. Subsequent investigations disclosed that miR‐195 directly inhibited the expression of the proangiogenic factor vascular endothelial growth factor (VEGF) and the prometastatic factors VAV2 and CDC42. Knockdown of these target molecules of miR‐195 phenocopied the effects of miR‐195 restoration, whereas overexpression of these targets antagonized the function of miR‐195. Furthermore, we revealed that miR‐195 down‐regulation resulted in enhanced VEGF levels in the tumor microenvironment, which subsequently activated VEGF receptor 2 signaling in endothelial cells and thereby promoted angiogenesis. Additionally, miR‐195 down‐regulation led to increases in VAV2 and CDC42 expression, which stimulated VAV2/Rac1/CDC42 signaling and lamellipodia formation and thereby facilitated the metastasis of HCC cells. Conclusion: miR‐195 deregulation contributes to angiogenesis and metastasis in HCC. The restoration of miR‐195 expression may be a promising strategy for HCC therapy. (Hepatology 2013;58:642‐653)

Human Macrophages Promote the Motility and Invasiveness of Osteopontin-Knockdown Tumor Cells

Increasing evidence indicates that macrophages in tumor stroma can significantly modify the malignant phenotypes of tumors. Osteopontin (OPN) is frequently overexpressed in cancers with high metastatic capacity and, thus, has been considered as a potential therapeutic target. To find out whether macrophages can affect the outcome of OPN-knockdown tumor cells, we used RNA interference (RNAi) to stably silence the OPN expression in the highly invasive human hepatoma cell line SK-Hep-1. Silencing of OPN markedly decreased the motility and invasiveness of the SK-Hep-1 cells. Further studies using this cell model revealed that coculture with human macrophages or macrophage-conditioned medium largely restored the migration and invasion potential of OPN-knockdown tumor cells. Moreover, such macrophage-promoted motility can be effectively blocked either by the addition of OPN-neutralizing antibody to the cocultured medium or by silencing OPN expression in macrophages. These results indicate that macrophage-derived OPN can compensate for the decrease of OPN and thereby restore the metastatic potential of OPN-knockdown tumor cells. Further characterization of the underlying mechanisms disclosed that macrophage-derived OPN exerted its function independently of the actin cytoskeleton rearrangement or the activation of matrix metalloproteinase and Rho families. Our results suggest that there are fine-tuned complex interactions between cancer cells and stroma cells, which may modify the outcome of cancer therapy, and therefore should be considered for the rational design of anticancer strategy.

Cutting Edge: IRF8 Regulates Bax Transcription In Vivo in Primary Myeloid Cells

A prominent phenotype of IRF8 knockout (KO) mice is the uncontrolled expansion of immature myeloid cells. The molecular mechanism underlying this myeloproliferative syndrome is still elusive. In this study, we observed that Bax expression level is low in bone marrow preginitor cells and increases dramatically in primary myeloid cells in wt mice. In contrast, Bax expression level remained at a low level in primarymyeloid cells in IRF8 KO mice. However, in vitro IRF8 KO bone marrow-differentiated myeloid cells expressed Bax at a level as high as that in wild type myeloid cells. Furthermore, we demonstrated that IRF8 specifically binds to the Bax promoter region in primary myeloid cells. Functional analysis indicated that IRF8 deficiency results in increased resistance of the primary myeloid cells to Fas-mediated apoptosis. Our findings show that IRF8 directly regulates Bax transcription in vivo, but not in vitro during myeloid cell lineage differentiation.

A novel AP-1/miR-101 regulatory feedback loop and its implication in the migration and invasion of hepatoma cells

MicroRNA-101 (miR-101) is frequently downregulated in various cancers. To date, the regulatory networks of miR-101 remain obscure. In this study, we demonstrated that miR-101 was mainly transcribed from human miR-101-2 and mouse miR-101bgene loci. Subsequent analyses revealed that activator protein-1 (AP-1) directly binded to the −17.4 to −16.4 k region upstream of pre-miR-101-2 and activated the expression of miR-101. On the other hand, miR-101 could inhibit the expression of ERK2 and c-Fos, two key factors of the AP-1 pathway, by binding to their 3′-UTRs. Furthermore, reintroduction of miR-101 efficiently suppressed the AP-1 activity and pri-miR-101-2 transcription. These data thus suggest a novel AP-1/miR-101 regulatory circuitry, that is, AP-1 promotes the transcription of miR-101, whereas the expression of miR-101 reduces the level of ERK2 and c-Fos and thereby attenuates the AP-1 signaling. Further investigation disclosed that the AP-1 activator TPA-induced MMP9 activity and the TPA-promoted migration and invasion of hepatoma cells were significantly attenuated by miR-101 but were enhanced by miR-101 inhibitor. Our results suggest that the AP-1/miR-101 feedback loop may prevent the excessive activation of metastatic signals imposed by ERK2/AP-1 and highlight the biological significance of miR-101 downregulation in cancer metastasis.

Interaction with Ppil3 leads to the cytoplasmic localization of Apoptin in tumor cells

Apoptin, a small protein encoded by chicken anemia virus (CAV), induces cell death specifically in cancer cells. In normal cells, Apoptin remains in the cytoplasm; whereas in cancerous cells, it migrates into the nucleus and kills the cell. Cellular localization appears to be crucial. Through a yeast two-hybrid screen, we identified human Peptidyl-prolyl isomerase-like 3 (Ppil3) as one of the Apoptin-associated proteins. Ppil3 could bind Apoptin directly, and held Apoptin in cytoplasm even in tumor cells. We then demonstrated that the nuclearcytoplasmic distribution of Apoptin is related to the expression level of intrinsic Ppil3. Moreover, extrinsic modifying of Ppil3 levels also resulted in nuclearcytoplasmic shuffling of Apoptin. The Apoptin P109A mutant, located between the putative nuclear localization and export signals, could significantly impair the function of Ppil3. Our results suggest a new direction for the localization mechanism study of Apoptin in cells.

Expression of microRNA-195 is transactivated by Sp1 but inhibited by histone deacetylase 3 in hepatocellular carcinoma cells

MiR-195 expression is frequently reduced in various cancers, but its underlying mechanisms remain unknown. To explore whether abnormal transcription contributed to miR-195 downregulation in hepatocellular carcinoma (HCC), we characterized the -2165-bp site upstream of mature miR-195 as transcription start site and the -2.4 to -2.0-kb fragment as the promoter of miR-195 gene. Subsequent investigation showed that deletion of the predicted Sp1 binding site decreased the miR-195 promoter activity; Sp1 silencing significantly reduced the miR-195 promoter activity and the endogenous miR-195 level; Sp1 directly interacted with the miR-195 promoter in vitro and in vivo. These data suggest Sp1 as a transactivator for miR-195 transcription. Interestingly, miR-195 expression was also subjected to epigenetic regulation. Histone deacetylase 3 (HDAC3) could anchor to the miR-195 promoter via interacting with Sp1 and consequently repress the Sp1-mediated miR-195 transactivation by deacetylating histone in HCC cells. Consistently, substantial increase of HDAC3 protein was detected in human HCC tissues and HDAC3 upregulation was significantly correlated with miR-195 downregulation, suggesting that HDAC3 elevation may represent an important cause for miR-195 reduction in HCC. Our findings uncover the mechanisms underlying the transcriptional regulation and expression deregulation of miR-195 in HCC cells and provide new insight into microRNA biogenesis in cancer cells.

MDR1 polymorphisms associated with risk and survival in diffuse large B-cell lymphoma

Abstract MDR1 encodes an adenosine triphosphate (ATP)-dependent efflux transporter that protects the body from environmental xenobiotics to maintain optimal health. Five single nucleotide polymorphisms (SNPs) of MDR1, T-2410C, T-129C, C1236T, G2677T/A and C3435T, were identified in our previous study. To investigate further the biological significance of these SNPs, we genotyped the SNPs in 135 patients with diffuse large B-cell lymphoma (DLBCL) and 376 age- and sex-matched controls. Statistical analysis indicated that the MDR1-129TC genotype was associated with an increased risk of DLBCL (p = 0.040) compared with the TT genotype, and the increased risk was more pronounced in older patients (> 50 years, p = 0.011). Patients with MDR1 2677TT displayed worse survival rates compared with those carrying MDR1 2677G/A alleles (p = 0.036). Multivariate Cox analysis revealed that the G2677T/A polymorphism was an independent prognostic factor for overall survival (OS). Further, we found a combined effect of MDR1 G2677T/A and C3435T on OS of patients with DLBCL. These results suggest that the MDR1 T-129C, G2677T/A and C3435T polymorphisms are associated with risk of and survival in DLBCL, although the p-values are not as strong after Bonferroni correction. Further investigations with a relatively larger number of patients and longer follow-up periods should be undertaken to confirm our results.

Identification of Siah-interacting protein as a potential regulator of apoptosis and curcumin resistance

The mechanism underlying curcumin (diferuloylmethane) resistance is still largely unknown. Here we employed proteomic approach to identify the Siah-interacting protein (SIP) as a candidate for detailed study, because the spot intensity of SIP on a two-dimensional gel displayed 70–90% reduction in curcumin-sensitive cells, but remained unchanged in curcumin-resistant sublines, after curcumin treatment. Both gain- and loss-of-function studies revealed that SIP promoted curcumin-induced apoptosis. Moreover, SIP underwent phosphorylation and nuclear translocation in curcumin-sensitive but not resistant cells, upon curcumin exposure. The nuclear translocation of SIP was remarkably impaired when a putative nuclear localization sequence (NLS, amino acid (aa) 143–159) was deleted or the serine 141 was mutated into alanine, whereas truncation of the N-terminal region (aa 1–43) obviously increased the nuclear import of SIP. In accordance with their nuclear localization, N-terminal truncation significantly enhanced the proapoptotic effect of SIP, whereas NLS deletion or Ser141Ala mutation attenuated the apoptosis-promoting activity of both wild-type- and N-terminal truncated-SIP. These data suggest that SIP plays a role in apoptosis and curcumin resistance, and the function of SIP may be regulated by different motifs, such as the NLS, N-terminal region and serine 141. Our findings provide new insights into the biological significance of SIP and the mechanisms of drug resistance.