长链非编码RNA SPRY4-IT1对髓母细胞瘤增殖及侵袭转移的影响

目的：探讨长链非编码RNA SPRY4-IT1（LncRNA）对髓母细胞瘤增殖及侵袭转移的影响。方法：髓母细胞瘤Daoy细胞分为对照组和si-SPRY4-IT1组,分别利用脂质体Lipofectamine 2000将阴性对照荧光序列和SPRY4-IT1-siRNA转入细胞中,采用Real-time PCR检测转染后各组细胞中SPRY4-IT1的表达情况,CCK-8实验及平板克隆形成实验检测细胞增殖能力的变化,以细胞体外侵袭、迁移实验分别检测细胞侵袭、迁移能力的变化,Western blot检测SPRY4-IT1对基质金属蛋白酶（MMP）-2和MMP-9蛋白的影响。结果：si-SPRY4-IT1组SPRY4-IT1 mRNA表达水平、细胞体外增殖能力、细胞侵袭、迁移能力、细胞MMP-2蛋白表达均较对照组明显降低（P<0.05）,而MMP-9蛋白表达未见明显变化。结论：干扰长链非编码RNA SPRY4-IT1在髓母细胞瘤Daoy细胞中的表达能显著抑制细胞的增殖、侵袭和迁移能力。     

Long noncoding RNA SPRY4-IT1 regulates intestinal epithelial barrier function by modulating the expression levels of tight junction proteins

Epithelial cells line the intestinal mucosa and form an important barrier to a wide array of noxious substances in the lumen. Disruption of the barrier integrity occurs commonly in various pathologies. Long noncoding RNAs (lncRNAs) control diverse biological processes, but little is known about the role of lncRNAs in regulation of the gut permeability. Here we show that the lncRNA SPRY4-IT1 regulates the intestinal epithelial barrier function by altering expression of tight junction (TJ) proteins. SPRY4-IT1 silencing led to dysfunction of the epithelial barrier in cultured cells by decreasing the stability of mRNAs encoding TJ proteins claudin-1, claudin-3, occludin, and JAM-1 and repressing their translation. In contrast, increasing the levels of SPRY4-IT1 in the intestinal mucosa protected the gut barrier in mice exposed to septic stress by increasing the abundance of TJ proteins. SPRY4-IT1 directly interacted with TJ mRNAs, and this process was enhanced through the association with the RNA-binding protein HuR. Of interest, the intestinal mucosa from patients with increased gut permeability exhibited a decrease in the levels of SPRY4-IT1. These findings highlight a novel role for SPRY4-IT1 in controlling the intestinal epithelial barrier and define a mechanism by which SPRY4-IT1 modulates TJ expression by altering the stability and translation of TJ mRNAs.     

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.     

Retracted: Upregulation of microRNA-140-3p inhibits epithelial-mesenchymal transition,invasion, and metastasis of hepatocellular carcinoma through inactivation of the MAPK signaling pathway by targeting GRN

Invasion and metastasis in hepatocellular carcinoma (HCC) results in poor prognosis. Human intervention in these pathological processes may benefit the treatment of HCC. The aim of the present study is to elucidate the mechanism of miR-140-3p affecting epithelial-mesenchymal transition (EMT), invasion, and metastasis in HCC. Microarray analysis was performed for differentially expressed genes screening. The target relationship between miR-140-3p and GRN was analyzed. Small interfering RNA (siRNA) against granulin (GRN) was synthesized. EMT markers were detected, and invasion and migration were evaluated in HCC cells introduced with a miR-140-3p inhibitor or mimic, or siRNA against GRN. A mechanistic investigation was conducted for the determination of mitogen-activated protein kinase (MAPK) signaling pathway-related genes and EMT markers (E-cadherin, N-cadherin, and Vimentin). GRN was highlighted as an upregulated gene in HCC. GRN was a target gene of miR-140-3p. Elevation of miR-140-3p or inhibition of GRN restrained the EMT process and suppressed the HCC cell migration and invasion. HCC cells treated with the miR-140-3p mimic or siRNA-GRN exhibited decreased GRN expression and downregulated the expressions of the MAPK signaling pathway-related genes, N-cadherin, and Vimentin but upregulated the expression of E-cadherin. GRN silencing can reverse the activation of the MAPK signaling pathway and induction of EMT mediated by miR-140-3p inhibition. Taken together, the results show that miR-140-3p confers suppression of the MAPK signaling pathway by targeting GRN, thus inhibiting EMT, invasion, and metastasis in HCC.     

Tumor-derived secretory clusterin induces epithelial–mesenchymal transition and facilitates hepatocellular carcinoma metastasis

Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality. Metastasis is the major concern that causes death in HCC. The goal of this study was to identify tumor-derived proteins in serum during HCC metastasis using an orthotopic xenograft tumor model and explore the role of key protein in HCC metastasis. Serum samples collected from HCCLM3-R metastatic HCC tumor model at specific stages of metastasis (1 wk, 3 wks and 6 wks) were subjected to iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. Twenty tumor-derived proteins were identified through human specific peptides. Secretory clusterin (sCLU), which was significantly upregulated during cancer progression and metastasis, was chosen for further study. The expression of sCLU was significantly higher in metastatic HCC cell lines and samples from metastatic HCC patients. ShRNA-mediated down-regulation of sCLU resulted in a reduced migratory capacity in HCC cell lines, as well as a reduction in pulmonary metastasis in vivo. Overexpression of sCLU in HepG2 cell line showed increased cell migratory ability. Further study found that sCLU contributed to HCC migration and epithelial–mesenchymal transition (EMT) in vitro, and metastasis in vivo. In addition, sCLU also plays an important role in the regulation of TGF-β1-smad3 signaling. These findings suggest that sCLU may promote HCC metastasis via the induction of EMT process and may be a candidate target for HCC therapy.     

Mesenchymal stem cells in inflammation microenvironment accelerates hepatocellular carcinoma metastasis by inducing epithelial-mesenchymal transition

In response to inflammation, mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodeling and wound healing. Tumors apply persistent mechanical and pathological stress to tissues and causes continual infiltration of MSCs. Here, we demonstrate that MSCs promote human hepatocellular carcinoma (HCC) metastasis under the influence of inflammation. The metastasis promoting effect could be imitated with the supernatant of MSCs pretreated with IFNγ and TNFα. Interestingly, treatment of HCC cells with the supernatant leads to epithelial-mesenchymal transition (EMT), an effect related to the production of TGFβ by cytokines stimulated MSCs. Importantly, the levels of MSCs expressing SSEA4 in clinical HCC samples significantly correlated with poor prognosis of HCC, and EMT of HCC was strongly associated with a shorter cancer-free interval (CFI) and a worse overall survival (OS). Therefore, our results suggest that MSCs in tumor inflammatory microenvironment could promote tumor metastasis through TGFβ-induced EMT.     

SPRY4-IT1 promotes survival of colorectal cancer cells through regulating PDK1-mediated glycolysis

ABSTRACT

Colorectal cancer (CRC) becomes the third leading cause of cancer-related deaths worldwide recently. The prognosis of CRC is still poor in decades, and targeted therapy is still a potential effective treatment. Long non-coding RNAs (lncRNAs) could regulate series of cellular functions and developmental processes. LncRNA-SPRY4-IT1 (GenBank ID AK024556) is derived from an intron of the SPRY4 gene, which was highly expressed in melanoma cells and affected the progression of multiple types of cancers. However, the mechanism of SPRY4-IT1 in CRC progression remains unclear. Herein, we found the high level of SPRY4-IT1 in human colorectal cancer (CRC) tissues and cells, and correlated with patients’ prognosis. We further noticed that SPRY4-IT1 regulated CRC cell growth and glycolysis, and promoting PDK1 expression. Our data further confirmed that SPRY4-IT1 regulated CRC progression targeting PDK1. We therefore thought SPRY4-IT1 could serve as a promising molecular target for the treatment of CRC.     

去泛素化酶在肿瘤上皮间质转化中的作用

肿瘤的侵袭和转移是加剧肿瘤恶化的主要原因,也是导致患者预后不良的根本原因。近年来大量研究发现,大部分肿瘤的转移都依赖于上皮间质转化(epithelial-mesenchymal transition, EMT)的发生,此外EMT也与肿瘤干性和肿瘤耐药等诸多肿瘤恶性行为密切相关,因此有效的抑制EMT的发生将可能极大的有利于肿瘤的治疗。去泛素化酶(deubiquitinating enzymes, DUBs)的主要功能之一就是通过移除底物蛋白质上泛素链,避免其通过泛素蛋白酶体途径降解,来维持细胞内蛋白质水平的动态平衡。去泛素化酶作为调节蛋白质泛素化修饰的一类重要酶类,其异常表达或酶活性的改变通常都会导致疾病的发生。众多研究发现,部分去泛素化酶在肿瘤侵袭和转移过程中表达失衡,在肿瘤转移的过程中扮演着重要的角色。EMT是指由上皮型细胞转变为间质型细胞的动态细胞生物学过程,在该过程中涉及到例如Snial1、Slug、ZEB1等EMT相关转录因子和细胞表面的例如E-钙黏着蛋白、N-钙黏着蛋白等分子标志物表达水平的变化。这些蛋白质通常具有不稳定性,易被降解等特征。EMT过程的发生,涉及到许多蛋白质稳定性的调节,而去泛素化酶作为一类维持蛋白质稳定的重要酶类,在调节这些蛋白质的稳定性方面发挥着重要的作用。EMT的发生也与TGF-β通路、Wnt通路等细胞内众多信号通路的异常活化密不可分,去泛素化酶通过介导这些信号通路的活化,从而间接的调节EMT发生发展。去泛素化酶通过调节EMT相关分子或EMT相关信号通路等多种方式直接或间接影响EMT进展,因此,通过靶向于去泛素化酶抑制肿瘤的侵袭和转移,将为肿瘤治疗提供新的治疗手段和方案,从而有效的推动肿瘤的治疗。本文主要就去泛素化酶在调节EMT相关分子以及信号通路等方面,阐述去泛素化酶在EMT过程中所发挥的重要作用及其作为肿瘤治疗靶点的可能性。     

SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

SPHK1 (sphingosine kinase 1), a regulator of sphingolipid metabolites, plays a causal role in the development of hepatocellular carcinoma (HCC) through augmenting HCC invasion and metastasis. However, the mechanism by which SPHK1 signaling promotes invasion and metastasis in HCC remains to be clarified. Here, we reported that SPHK1 induced the epithelial-mesenchymal transition (EMT) by accelerating CDH1/E-cadherin lysosomal degradation and facilitating the invasion and metastasis of HepG2 cells. Initially, we found that SPHK1 promoted cell migration and invasion and induced the EMT process through decreasing the expression of CDH1, which is an epithelial marker. Furthermore, SPHK1 accelerated the lysosomal degradation of CDH1 to induce EMT, which depended on TRAF2 (TNF receptor associated factor 2)-mediated macroautophagy/autophagy activation. In addition, the inhibition of autophagy recovered CDH1 expression and reduced cell migration and invasion through delaying the degradation of CDH1 in SPHK1-overexpressing cells. Moreover, the overexpression of SPHK1 produced intracellular sphingosine-1-phosphate (S1P). In response to S1P stimulation, TRAF2 bound to BECN1/Beclin 1 and catalyzed the lysine 63-linked ubiquitination of BECN1 for triggering autophagy. The deletion of the RING domain of TRAF2 inhibited autophagy and the interaction of BECN1 and TRAF2. Our findings define a novel mechanism responsible for the regulation of the EMT via SPHK1-TRAF2-BECN1-CDH1 signal cascades in HCC cells. Our work indicates that the blockage of SPHK1 activity to attenuate autophagy may be a promising strategy for the prevention and treatment of HCC.     

The SPRY domain-containing SOCS box protein 1 (SSB-1) interacts with MET and enhances the hepatocyte growth factor-induced Erk-Elk-1-serum response element pathway

The suppressor of cytokine signaling (SOCS) protein family includes a SPRY (repeats in splA and RyR) domain-containing SOCS box protein (SSB) subfamily, which consists of four members, SSB-1, SSB-2, SSB-3, and SSB-4. These proteins contain a central SPRY domain and a C-terminal SOCS box. Although some of the SOCS protein subfamilies function as adaptors for a large family of ubiquitin-protein isopeptide ligases to regulate certain signaling pathways, the function of the SSB subfamily remains to be determined. In our previous studies, we have found that two SPRY domain-containing proteins, RanBP9 and RanBP10, interact with MET through the SPRY domain. In the present study, we explored the function of SSB proteins in the regulation of the hepatocyte growth factor (HGF)-MET signaling. Our results showed that all four SSB proteins also interacted with the MET. The MET interaction with SSB-1 was further investigated. We demonstrated that SSB-1 bound to MET tyrosine kinase domain through its SPRY domain. MET interacted with SSB-1 in both the absence and the presence of HGF, but HGF treatment resulted in the recruitment of more SSB-1 by MET. We showed that overexpression of SSB-1 but not other SSB proteins enhanced the HGF-induced serum response element (SRE)-luciferase activity. Overexpression of SSB-1 exhibited no effect on the basal level or epidermal growth factor-induced SRE-luciferase activity. SSB-1 also enhanced HGF-induced Erk phosphorylation. Suppression of SSB-1 by the RNA interference method down-regulated HGF-induced SRE-luciferase activity and decreased Elk-1 activation. These results suggest that SSB-1 may play an important role in enhancing the HGF-induced Erk-Elk-1-SRE pathway. Furthermore, we demonstrated that in response to HGF stimulation, the SSB-1 protein became phosphorylated at tyrosine residue 31. The phosphorylated SSB-1 protein bound to p120Ras-GTPase-activating protein (GAP) but did not promote the degradation of p120RasGAP, indicating that enhanced HGF-MET signaling by overexpression of SSB-1 was not dependent on p120RasGAP degradation.     

Rce1 suppresses invasion and metastasis of hepatocellular carcinoma via epithelial-mesenchymal transition induced by the TGF-β1/H-Ras signaling pathway

Ras converting enzyme 1 (Rce1) plays an important role in invasion and metastasis of malignancy. However, the mechanism has not yet been fully explored in hepatocellular carcinoma (HCC). Primarily, we investigated the expression of Rce1 and H-Ras influence on patient prognosis through the clinical data. Further, we analyzed the regulatory effects of Rce1/H-Ras signal pathway on the epithelial–mesenchymal transition (EMT) in vitro and in vivo. Finally, we screened out the protein which bonds with Rce1 by CO-IP experiment to discuss the mechanism of Rce1 in EMT of HCC. This research revealed a significantly decreased expression of Rce1 in HCC compared with noncancerous tissues (p < .05). In contrast, H-Ras expression was increased in the tumor. The expression of them was a close association with the differentiation and tumor-node-metastasis (TNM) stage of the tumor (p < .001; p = .035, respectively) and Rce1 was an independent prognostic indicator (95%Cl: 0.193–0.821; p = .013). Through targeted regulation of Rce1 by cDNA or small interfering RNA, results show that the lower expression of Rce1 facilitated EMT and promoted the invasion and metastasis of HCC (p < .05). Furthermore, the CO-IP experiment unfolded that Rce1 could bond with farnesyltransferase-β (FNTB) which mediated the expression of H-Ras. Conclusions: Rce1 inhibits EMT via target regulation H-Ras and suppress the early invasion and metastasis of HCC. It may be a potential therapeutic target and prognostic indicator for HCC.     

Delphinidin inhibits epidermal growth factor-induced epithelial-to-mesenchymal transition in hepatocellular carcinoma cells

Epithelial-to-mesenchymal transition (EMT), important cellular process in metastasis of primary tumors, is characterized by loss of their cell polarity, disruption of cell-cell adhesion, and gain certain properties of mesenchymal phenotype that enable migration and invasion. Delphinidin is a member of anthocyanidin belong to flavonoid groups, known as having pharmacological and physiological effects including anti-tumorigenic, antioxidative, anti-inflammatory, and antiangiogenic effects. However, the effects of delphinidin on EMT is rarely investigated. Epidermal growth factor (EGF) is known as a crucial inducer of EMT in various cancer including hepatocellular carcinoma (HCC). To determine whether delphinidin inhibits EGF-induced EMT in HCC cells, antiproliferative effect of delphinidin on Huh7 and PLC/PRF/5 cells were measured by Cell Counting Kit-8 assay. As a result, delphinidin inhibited cell proliferation in a dose-dependent manner. Based on the result of proliferation, to measure the effects of delphinidin on EGF-induced EMT, we designated a proper concentration of delphinidin, which is not affected to cell proliferation. We found that delphinidin inhibits morphological changes from epithelial to mesenchymal phenotype by EGF. Moreover, delphinidin increased the messenger RNA and protein expression of E-cadherin and decreased those of Vimentin and Snail in EGF-induced HCC cells. Also, delphinidin prevented motility and invasiveness of EGF-induced HCC cells through suppressing activation of matrix metalloproteinase 2, EGF receptor (EGFR), AKT, and extracellular signal-regulated kinase (ERK). Taken together, our findings demonstrate that delphinidin inhibits EGF-induced EMT by inhibiting EGFR/AKT/ERK signaling pathway in HCC cells.     

Long noncoding RNA CPS1-IT1 suppresses melanoma cell metastasis through inhibiting Cyr61 via competitively binding to BRG1

Long noncoding RNA CPS1-IT1 is recently recognized as a tumor suppressor in several cancers. Here, we investigate the role of CPS1-IT1 in human melanoma. Presently, our study reveals the low expression of CPS1-IT1 in human melanoma tissues and cell lines, which is significantly associated with metastasis and tumor stage. Besides, the potential of CPS1-IT1 as a prognosis-predictor is strongly indicated. Functionally, CPS1-IT1 overexpression inhibits cell migration, invasion, epithelial–mesenchymal transition, and angiogenesis in melanoma cells. CYR61, an angiogenic factor that participates in tumor metastasis as well as a recognized oncogene in melanoma, is shown to be confined under CPS1-IT1 overexpression in melanoma cells. Furthermore, enforced expression of Cyr61 in CPS1-IT1-silenced melanoma cells dramatically normalized the protein level of Cyr61 and that of its downstream targets vascular endothelial growth factor and matrix metalloproteinase-9, as well as the repressive effect of CPS1-IT1 overexpression on melanoma cell metastasis. BRG1, a core component of SWI/SNF complex, is implied to interact with both CPS1-IT1 and Cyr61 in melanoma cells. Moreover, CPS1-IT1 negatively regulates Cyr61 expression by blocking the binding of BRG1 to Cyr61 promoter. Jointly, CPS1-IT1 controls melanoma metastasis through impairing Cyr61 expression via competitively binding with BRG1, uncovering a novel potential therapeutic and prognostic biomarker for patients with melanoma.     

PAK1 promotes proliferation,migration and invasion of hepatocellular carcinoma by facilitating EMT via directly up-regulating Snail

BackgroundHepatocellular carcinoma (HCC) is a primary cause of cancer mortality. PAK1 plays key roles in many types of cancers. However, the role of PAK1 in HCC is not clear.MethodsqRT-PCR and Western blotting were used to determine expressions of PAK1, Snail and epithelial mesenchymal transition (EMT)-related proteins. Luciferase reporter assay was used to measure the interaction between PAK1 and Snail. Wound healing, transwell, colony formation assays and flow cytometry were used to assess cell migration, invasion, proliferation and apoptosis. Mouse tumor xenograft model was used to determine the effect of PAK1 on tumor growth in vivo.ResultsPAK1 and Snail were up-regulated in HCC cells. PAK1 knockdown suppressed cell proliferation, migration and invasion, and increased apoptosis of HCC cells. PAK1 knockdown also inhibited tumor growth in vivo. Mechanistically, PAK1 promoted EMT by targeting Snail. Knockdown of PAK1 could up-regulate pro-apoptotic proteins but down-regulate proliferation-related proteins via suppressing β-catenin signaling pathway.ConclusionPAK1 promotes EMT process by increasing Snail, and facilitates progression of HCC by activating β-catenin pathway.