lncRNA CASC2 suppresses the growth of hemangioma cells by regulating miR-18a-5p/FBXL3 axis.

Dysregulation of lncRNA cancer susceptibility candidate 2 (CASC2) is involved in the pathogenesis of multiple malignancies. However, the underlying mechanisms by which lncRNA CASC2 regulates the proliferation of hemangiomas (HAs) remain undocumented. Herein, the expression levels of lncRNA CASC2 and VEGF in proliferating or involuting phase HAs were assessed by qRT-PCR analysis, and the effects of lncRNA CASC2 on HAs cell growth were evaluated by MTT, colony formation assays and Western blot analysis. lncRNA CASC2 specific binding with miR-18a-5p was confirmed by luciferase report assay. Consequently, we found that the expression of lncRNA CASC2 was reduced in proliferating phase HAs as compared with the involuting phase HAs or normal tissues, and possessed a negative correlation with VEGF expression in proliferating phase HAs. Restored expression of lncRNA CASC2 repressed cell viability and colony formation and downregulated VEGF expression, while silencing lncRNA CASC2 showed the opposite effects. Moreover, lncRNA CASC2 was confirmed to bind with miR-18a-5p, which could reverse lncRNA CASC2-induced anti-proliferative effects by targeting FBXL3 in HAs cells. Altogether, our findings demonstrated that lncRNA CASC2 suppressed the growth of HAs cells by regulating miR-18a-5p/FBXL3 axis.

Knockdown of HMGB1 inhibits cell proliferation and induces apoptosis in hemangioma via downregulation of AKT pathway.

The high mobility group box 1 (HMGB1) as a conserved non-histone nuclear protein has been involved in a variety of biological processes of cancer, such as cell proliferation, apoptosis, angiogenesis and metastasis. Despite the increased expression of HMGB1 in many malignant tumors, the functions and molecular mechanisms by which HMGB1 contributes to the formation of hemangioma (HA) remain unclear. In the present study, immunohistochemistry was used to detect the expression levels of HMGB1 in different phases of human HAs. Cell function experiments, including MTT, cell colony formation and flow cytometry analysis were performed to evaluate the effects of HMGB1 knockdown on cell proliferation and apoptosis in HA CRL-2586 EOMA cells. As a consequence, we found that HMGB1 expression was significantly increased in proliferating phase HAs compared with the involuting phase HAs and normal skin tissues (P less than 0.01). Moreover, knockdown of HMGB1 gene in vitro suppressed EOMA cell proliferation and colony formation and induced cell apoptosis and cycle arrest at G0/G1 phase by downregulation of PCNA, CyclinD1, p-AKT and upregulation of p53 and cleaved PARP. Taken together, our findings demonstrate that HMGB1 may be implicated in the formation of HA through upregulation of AKT pathway, and represent a potential therapeutic target for treating HA.

Oxymatrine suppresses proliferation and induces apoptosis of hemangioma cells through inhibition of HIF-1a signaling.

Oxymatrine (OMT), a natural quinolizidine alkaloid, has been known to have anti-inflammation, anti-anaphylaxis, and chemopreventive effects on various cancer cells. To clarify the underlying role and molecular mechanisms of OMT in human hemangioma (HA), in the present study, we examined the expression of hypoxia-inducible factor-1a (HIF-1a) and vascular endothelial growth factor (VEGF) in different phases of human HA. After HA derived endothelial cells (HDEC) were pretreated with different concentrations of OMT, cell proliferation, apoptosis, and cycle distribution were evaluated by MTT assay and flow cytometry analysis, respectively. The effects of OMT on expression of HIF-1a signaling were determined by real-time PCR and western blot assays. Our results showed that, the expression of HIF-1a and VEGF was significantly increased in proliferating phase HA, but decreased in involuting phase HA. Moreover, OMT in vitro inhibited proliferative activities and induced cell apoptosis and cycle arrest in G0/G1 phase in HA cells with decreased expression of HIF-1a, VEGF, Bcl-2, and CyclinD1, and increased expression of p53. Taken together, our findings suggest that, the expression of HIF-1a and VEGF is increased in proliferating phase HA, and OMT suppresses cell proliferation and induces cell apoptosis and cycle arrest in proliferative phase HA through inhibition of the HIF-1a signaling pathway, suggesting OMT may provide a novel therapeutic strategy for the treatment of HA.

Knockdown of Livin inhibits growth and invasion of gastric cancer cells through blockade of the MAPK pathway in vitro and in vivo.

Livin, a novel member of the human inhibitors of apoptosis protein family, has been shown to be critical for tumor progression and poor prognosis for several types of malignancies. However, limited reports exist regarding the biological functions of Livin in human gastric cancer (GC). The present study investigated the clinical significance of Livin and caspase-3 (CAS-3) in human GC using immunohistochemistry assay, and explore the potential using RNA interference to knockdown Livin expression, including the subsequent effects on tumor growth and invasion in GC cells in vitro and in vivo. Our results showed that the rate of positive expression of Livin was significantly higher in GC tissues compared to that in adjacent non-cancer tissues (ANCT) (64.1 vs. 30.8%, P<0.001), while CAS-3 was lower in GC tissues than in ANCT (33.3 vs. 66.7%, P=0.001). Livin expression was positively correlated with tumor differentiation and lymph node metastases (P=0.009; P=0.007), while CAS-3 was negatively correlated with them (P=0.036; P=0.002) in patients with GC. Furthermore, knockdown of Livin inhibited cell proliferative activities and invasive potential, and induced cell in situ apoptosis in GC cells, accompanied with decreased expression of p38 MAPK, VEGF and MMP-2 and increased expression of CAS-3. In addition, the tumor volumes in the SGC7901 subcutaneous nude mouse model treated with Lv-shLivin was significantly smaller compared to those of the PBS group (P<0.01). Taken together, our findings indicate that the expression of Livin is increased in human GC and correlates with tumor differentiation and lymph node metastases, while knockdown of Livin inhibits cell growth and invasion through blockade of the MAPK pathway in GC cells, suggesting that Livin may be a potential therapeutic target for the treatment of GC.

Knockdown of VEGFR2 inhibits proliferation and induces apoptosis in hemangioma-derived endothelial cells.

Angiogenesis is a process of development and growth of new capillary blood vessels from pre-existing vessels. Angiogenic growth factors play important roles in the development and maintenance of some malignancies, of which vascular endothelial growth factor (VEGF)/VEGFR2 interactions are involved in proliferation, migration, and survival of many cancer cells. The aim of this study was to investigate the function of VEGFR2 in human hemangiomas (HAs). Using immunohistochemistry assay, we examined the expression levels of VEGF, VEGFR2, Ki-67, glucose transporter-1 (Glut-1), phosphorylated protein kinase B (p-AKT) and p-ERK in different phases of human HAs. Positive expression of VEGF, VEGFR2, Ki-67, Glut-1, p-AKT and p-ERK was significantly increased in proliferating phase HAs, while decreased in involuting phase HAs (P=0.001; P=0.003). In contrast, cell apoptotic indexes were decreased in proliferating phase HAs, but increased in involuting phase HAs (P<0.01). Furthermore, we used small hairpin RNA (shRNA)-mediated VEGFR2 knockdown in primary HA-derived endothelial cells (HemECs) to understand  the  role  of  VEGF/VEGFR2 signaling. Knockdown of VEGFR2 by Lv-shVEGFR2 inhibited cell viability and induced apoptosis in primary HemECs companied with decreased expression of p-AKT, p-ERK, p-p38MAPK and Ki-67 and increased expression of caspase-3 (CAS-3). Overexpression of VEGFR2 promoted cell viability and blocked apoptosis in Lv-VEGFR2-transfected HemECs. Taken together, our findings demonstrate that, increased expression of VEGFR2 is involved in the development of primary HemECs possibly through regulation of the AKT and ERK pathways, suggesting that VEGFR2 may be a potential therapeutic target for HAs. 

Knockdown of IGF2R suppresses proliferation and induces apoptosis in hemangioma cells in vitro and in vivo.

Insulin-like growth factor-II (IGF-II)/IGF2R signaling plays a pivotal role in cell growth, migration and differentiation in many malignancies. An individual with high IGF-II expression levels has a high risk of developing cancer, but IGF2R is often considered to be a tumor suppressor. To date, little has been reported about the role of IGF-II/IGF2R signaling in hemangiomas (HAs). Thus, uncovering the mechanisms of IGF-II/IGF2R signaling is very important to understanding the development of HAs. In the present study, the expression of IGF-II and IGF2R was investigated in 27 cases of HAs of different phases by immunohistochemistry. Through lentivirus-mediated IGF2R siRNA (Lv-siIGF2R) in HA-derived endothelial cells (HDECs), we observed the effects of IGF2R knockdown on the biological behavior of HA cells. We found that the expression of IGF-II and IGF2R was significantly increased in proliferating phase HAs, but decreased in involuting phase HAs. Furthermore, knockdown of IGF2R in vitro significantly diminished the proliferative activity and induced apoptosis and cycle arrest with decreased expression of PCNA, Ki-67, Bcl-2, Cyclin D1 and E and increased the expression of Bax in the proliferative phase HAs (HDEC and CRL-2586 EOMA cells). In addition, the tumor volumes in a subcutaneous HDEC nude mouse model treated with Lv-siIGF2R were significantly smaller than those of the control group. Taken together, our findings indicate that the expression of IGF-II and IGF2R is increased in proliferating phase HAs, and knockdown of IGF2R suppresses proliferation and induces apoptosis in HA cells in vitro and in vivo, suggesting that IGF2R may represent a novel therapeutic target for the treatment of human HAs.