Valproic acid inhibits the proliferation of SHSY5Y neuroblastoma cancer cells by downregulating URG4/URGCP and CCND1 gene expression

Valproic acid (VPA), used for the treatment of epilepsy and bipolar disorder, regulates several signaling pathways in brain cells. The up-regulated gene 4 (URG4/URGCP) is a novel gene located on 7p13. URG4/URGCP stimulates cyclin D1 (CCND1) mRNA expression, and URG4/URGCP silencing diminishes CCND1 mRNA expression in HepG2 cells. This study was performed to investigate the anti-cancer mechanism of action of VPA by analyzing the expression of novel gene URG4/URGCP, CCND1, p21, p53, p65 (RelA), Bax, and Bcl-2 in SHSY5Y neuroblastoma (NB) cancer cells. Cytotoxic effects of VPA in SHSY5Y were noticed in time and dose dependent manner with the IC₅₀ doses within the range of 0.5–10 mM. IC₅₀ doses in the SHSY5Y were detected as 7.5 mM. Expression profiles were determined by semi quantitative RT-PCR and URG4/URGCP protein change by western blot analysis. Our results suggest that VPA induces cell cycle arrest in SHSY5Y due to the decrease in URG4/URGCP, CCND1 gene expression and the increase in p65. To conclude, VPA may be a prospective agent for the treatment of NB as a single agent or in combination with other drugs. Thus, more studies should be designed to find a safe dose with the best effects of VPA.     

Leukemogenesis as a new approach to investigate the correlation between up regulated gene 4/upregulator of cell proliferation (URG4/URGCP) and signal transduction genes in leukemia

The aim of the study is to the determine the profiles of cell cycle genes and a new candidate oncogene of URG4/URGCP which play role in leukemia, establishing the association between the early prognosis of cancer and the quantitation of genetic changes, and bringing a molecular approach to definite diagnosis. In this study, 36 newly diagnosed patients’ with ALL-AML in the range of 0–18 years and six control group patients’ bone marrow samples were included. Total RNA was isolated from samples and then complementary DNA synthesis was performed. The obtained cDNAs have been installed 96 well plates after prepared appropriate mixtures and assessed with LightCycler^® 480 Real-Time PCR quantitatively. CHEK1, URG4/URGCP, CCNG1, CCNC, CDC16, KRAS, CDKN2D genes in the T-ALL group; CCND2, ATM, CDK8, CHEK1, TP53, CHEK2, CCNG2, CDK4, CDKN2A, E2F4, CCNC, KRAS genes in the precursor B-ALL group and CCND2, CDK6 genes in the AML group have shown significant increase in mRNA expression level. In the featured role of acute leukemia the regulating signaling pathways of leukemogenesis partially defined, although identification of new genetic markers in acute leukemia subgroups, will allow the development of early diagnostic and new treatment protocols.     

上调基因4与癌症

上调基因4（up-regulated gene-4,URG4）是受乙肝病毒X蛋白（hepatitis B virus X protein,HBx）激活的下游基因之一,最初在转染HBx的HepG2细胞中被克隆,因其上调细胞增殖效应而得名。与未转染的细胞相比,URG4在HBx转染后的HepG2细胞中表达明显增加,证实了URG4与乙肝病毒相关的肝细胞癌发生相关。近年来,人们发现URG4不仅在肝癌细胞中高表达,还与胃癌、骨肉瘤、非小细胞肺癌、神经母细胞瘤等多种癌症相关。本文结合近年来对URG4的研究成果,包括URG4基因及其蛋白质的结构与功能,URG4在癌症发生发展中的作用,以及在癌症早期诊断和预后中的意义进行综述,可望为后续深入地开展URG4与癌症的研究提供参考。     

The hepatitis B x antigen anti-apoptotic effector URG7 is localized to the endoplasmic reticulum membrane

Hepatitis B x antigen up-regulates the liver expression of URG7 that contributes to sustain chronic virus infection and to increase the risk for hepatocellular carcinoma by its anti-apoptotic activity. We have investigated the subcellular localization of URG7 expressed in HepG2 cells and determined its membrane topology by glycosylation mapping in vitro. The results demonstrate that URG7 is N-glycosylated and located to the endoplasmic reticulum membrane with an N_lumen–C_cytosol orientation. The results imply that the anti-apoptotic effect of URG7 could arise from the C-terminal cytosolic tail binding a pro-apoptotic signaling factor and retaining it to the endoplasmic reticulum membrane.     

The RNA expression signature of the HepG2 cell line as determined by the integrated analysis of miRNA and mRNA expression profiles

Understanding miRNAs' regulatory networks and target genes could facilitate the development of therapies for human diseases such as cancer. Although much useful gene expression profiling data for tumor cell lines is available, microarray data for miRNAs and mRNAs in the human HepG2 cell line have only been compared with that of other cell lines separately. The relationship between miRNAs and mRNAs in integrated expression profiles for HepG2 cells is still unknown. To explore the miRNA–mRNA correlations in hepatocellular carcinoma (HCC) cells, we performed miRNA and mRNA expression profiling in HepG2 cells and normal liver HL-7702 cells at the genome scale using next-generation sequencing technology. We identified 193 miRNAs that are differentially expressed in these two cell lines. Of these, 89 miRNAs were down-regulated in HepG2 cells compared with HL-7702 cells, while 104 miRNAs were up-regulated. We also observed 3035 mRNAs that are significantly dys-regulated in HepG2 cells. We then performed an integrated analysis of the expression data for differentially expressed miRNAs and mRNAs and found several miRNA–mRNA pairs that are significantly correlated in HepG2 cells. Further analysis suggested that these differentially expressed genes were enriched in four tumorigenesis-related signaling pathways, namely, ErbB, JAK–STAT, mTOR, and WNT, which until now had not been fully reported. Our results could be helpful in understanding the mechanisms of HCC occurrence and development.     

LipoxinA4通过调节肿瘤相关巨噬细胞对肝癌细胞株HepG2microRNA表达谱的影响

肿瘤与炎症密切相关,作者以往已证实内源性促炎症缓解介质脂氧素A4（Lipoxin A4,LXA4）能在整体和细胞水平发挥抗肝癌细胞增殖和转移的作用。为进一步探讨LⅪ钆通过调节肿瘤相关巨噬细胞对肝癌细胞株HepG2microRNAs（miRNAs）表达谱的影响,该文首先提取经脂多糖（LPS）或LPS＋LXA4作用24h的人巨噬细胞株U937培养上清液,分别称为ACM或LCM,以模拟肿瘤的炎症微环境,并用此上清液刺激HepG2细胞,24h后提取细胞总RNA,采用microRNA芯片miRCURY^TM LNA Array（V16．0）检测,计算各样本中的miRNAs标准值及比值。以两组间Hy3荧光标记信号强度的比值≤0．5或≥2为标准判定差异表达miRNA。Real-time PCR检测Nhsa-miR-623的相对含量以验证基因芯片的结果。结果发现,与对照组细胞相比,经过ACM作用24h的HepG2细胞有35个miRNAs上调、130个miRNAs下调。LCM组与ACM组相比,HepG2细胞有185个miRNAs上调、71个miRNAs下调。Real-time PCR检测的结果证实,hsa-miR-623的变化与基因芯片趋势一致。综上所述,LXA4能通过肿瘤相关巨噬细胞而间接发挥其调节HepG2细胞miRNAs表达谱的作用。     

The expression of URGCP gene in prostate cancer cell lines: correlation with rapamycin

Molecular targets in prostate cancer are continually being explored, for which there are currently few therapeutic options. Rapamycin (RPM) is an antifungal macrolide antibiotic isolated from Streptomyces hygroscopicus which can inhibit the G1 to S transition. URGCP (upregulator of cell proliferation) is a novel gene located on chromosome 7p13. We aimed to investigate the role of URGCP gene expression changes in PC3, DU145, and LNCAP cell lines with/out RPM. Average cell viability and cytotoxic effect of rapamycin were investigated at 24?h intervals for three days by using Trypan blue dye exclusion test and XTT assay. Cytotoxic effects of rapamycin in DU145, PC3 and LNCAP cells were detected in time and dose dependent manner with the IC₅₀ doses within the range of 1–100?nM. As the results were evaluated, IC₅₀ doses in the DU145, PC3, and LNCaP cells were detected as 10, 25, and 50?nM, respectively. The mean relative ratios of URGCP gene expression in DU145, LNCAP and PC3 cells were found as ?1.48, 6.59 and ?13.00, respectively, when compared to rapamycin-free cells. The False Discovery Rate adjusted p value in DU145, LNCAP and PC3 were 1.25?×?10^?5, 2.20?×?10^?8 and 6.20?×?10^?9, respectively. When the URGCP gene expression level is compared between the dose and control group, we found that URGCP gene expression was significantly decreased in dose groups of DU145 and PC3 cells.     

miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs

miRDeepFinder is a software package developed to identify and functionally analyze plant microRNAs (miRNAs) and their targets from small RNA datasets obtained from deep sequencing. The functions available in miRDeepFinder include pre-processing of raw data, identifying conserved miRNAs, mining and classifying novel miRNAs, miRNA expression profiling, predicting miRNA targets, and gene pathway and gene network analysis involving miRNAs. The fundamental design of miRDeepFinder is based on miRNA biogenesis, miRNA-mediated gene regulation and target recognition, such as perfect or near perfect hairpin structures, different read abundances of miRNA and miRNA*, and targeting patterns of plant miRNAs. To test the accuracy and robustness of miRDeepFinder, we analyzed a small RNA deep sequencing dataset of Arabidopsis thaliana published in the GEO database of NCBI. Our test retrieved 128 of 131 (97.7%) known miRNAs that have a more than 3 read count in Arabidopsis. Because many known miRNAs are not associated with miRNA*s in small RNA datasets, miRDeepFinder was also designed to recover miRNA candidates without the presence of miRNA*. To mine as many miRNAs as possible, miRDeepFinder allows users to compare mature miRNAs and their miRNA*s with other small RNA datasets from the same species. Cleaveland software package was also incorporated into miRDeepFinder for miRNA target identification using degradome sequencing analysis. Using this new computational tool, we identified 13 novel miRNA candidates with miRNA*s from Arabidopsis and validated 12 of them experimentally. Interestingly, of the 12 verified novel miRNAs, a miRNA named AC1 spans the exons of two genes (UTG71C4 and UGT71C3). Both the mature AC1 miRNA and its miRNA* were also found in four other small RNA datasets. We also developed a tool, ??miRNA primer designer?? to design primers for any type of miRNAs. miRDeepFinder provides a powerful tool for analyzing small RNA datasets from all species, with or without the availability of genome information. miRDeepFinder and miRNA primer designer are freely available at http://www.leonxie.com/DeepFinder.php and at http://www.leonxie.com/miRNAprimerDesigner.php, respectively. A program (called RefFinder: http://www.leonxie.com/referencegene.php) was also developed for assessing the reliable reference genes for gene expression analysis, including miRNAs.     

Inhibition of mir-21, which is up-regulated during MYCN knockdown-mediated differentiation, does not prevent differentiation of neuroblastoma cells

Background

Neuroblastoma is a malignant childhood tumour arising from precursor cells of the sympathetic nervous system. Genomic amplification of the MYCN oncogene is associated with dismal prognosis. For this group of high-risk tumours, the induction of tumour cell differentiation is part of current treatment protocols. MicroRNAs (miRNAs) are small non-coding RNA molecules that effectively reduce the translation of target mRNAs. MiRNAs play an important role in cell proliferation, apoptosis, differentiation and cancer. In this study, we investigated the role of N-myc on miRNA expression in MYCN-amplified neuroblastoma. We performed a miRNA profiling study on SK-N-BE (2) cells, and determined differentially expressed miRNAs during differentiation initiated by MYCN knockdown, using anti-MYCN short-hairpin RNA (shRNA) technology.

Results

Microarray analyses revealed 23 miRNAs differentially expressed during the MYCN knockdown-mediated neuronal differentiation of MNA neuroblastoma cells. The expression changes were bidirectional, with 11 and 12 miRNAs being up- and down-regulated, respectively. Among the down-regulated miRNAs, we found several members of the mir-17 family of miRNAs. Mir-21, an established oncomir in a variety of cancer types, became strongly up-regulated upon MYCN knockdown and the subsequent differentiation.Neither overexpression of mir-21 in the high-MYCN neuroblastoma cells, nor repression of increased mir-21 levels during MYCN knockdown-mediated differentiation had any significant effects on cell differentiation or proliferation.

Conclusions

We describe a subset of miRNAs that were altered during the N-myc deprived differentiation of MYCN-amplified neuroblastoma cells. In this context, N-myc acts as both an activator and suppressor of miRNA expression. Mir-21 was up-regulated during cell differentiation, but inhibition of mir-21 did not prevent this process. We were unable to establish a role for this miRNA during differentiation and proliferation of the two neuroblastoma cell lines used in this study.     

Microarray-Based Approach Identifies Differentially Expressed MicroRNAs in Porcine Sexually Immature and Mature Testes

Background

MicroRNAs (miRNAs) are short non-coding RNA molecules which are proved to be involved in mammalian spermatogenesis. Their expression and function in the porcine germ cells are not fully understood.

Methodology

We employed a miRNA microarray containing 1260 unique miRNA probes to evaluate the miRNA expression patterns between sexually immature (60-day) and mature (180-day) pig testes. One hundred and twenty nine miRNAs representing 164 reporter miRNAs were expressed differently (p<0.1). Fifty one miRNAs were significantly up-regulated and 78 miRNAs were down-regulated in mature testes. Nine of these differentially expressed miRNAs were validated using quantitative RT-PCR assay. Totally 15919 putative miRNA-target sites were detected by using RNA22 method to align 445 NCBI pig cDNA sequences with these 129 differentially expressed miRNAs, and seven putative target genes involved in spermatogenesis including DAZL, RNF4 gene were simply confirmed by quantitative RT-PCR.

Conclusions

Overall, the results of this study indicated specific miRNAs expression in porcine testes and suggested that miRNAs had a role in regulating spermatogenesis.     

microRNA-1274a, a modulator of sorafenib induced a disintegrin and metalloproteinase 9 (ADAM9) down-regulation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a complex and heterogeneous tumor with poor prognosis. Sorafenib, a multikinase inhibitor, has been widely used to treat patients with advanced HCC in clinic. We postulated that microRNAs (miRNA) might be involved in HCC target-chemotherapy with sorafenib. MiRNA profile of HepG2 was evaluated after cells were treated with vehicle or sorafenib and alterations in miRNA expression occurred with 14 miRNAs. MiR-1274a, which is up-regulated by sorafenib, could significantly repress expression of ADAM9, a protease that is involved in sorafenib target-therapy of HCC, in HCC cells. Taken together, our data emphasizes a new miRNA-based mechanism of sorafenib antitumor therapy.     

Hepatitis B Virus HBx Activates Notch Signaling via Delta-Like 4/Notch1 in Hepatocellular Carcinoma

Hepatitis virus B (HBV) infection is one of the major causes of hepatocellular carcinomas (HCC). HBx protein encoded in HBV genome is one of the key viral factors leading to malignant transformation of infected cells. HBx functions by interfering with cellular functions, causing aberration in cellular behaviour and transformation. Notch signalling is a well-conserved pathway involved in cellular differentiation, cell survival and cell death operating in various types of cells. Aberration in the Notch signalling pathways is linked to various tumors, including HCC. The role of HBx on the Notch signalling in HCC, however, is still controversial. In this study, we reported that HBV genome-containing HCC cell line HepG2 (HepG2.2.15) expressed higher Notch1 and Delta-like 4 (Dll4), compared to the control HepG2 without HBV genome. This upregulation coincided with increased appearance of the cleavage of Notch1, indicating constitutively activated Notch signalling. Silencing of HBx specifically reduced the level of Dll4 and cleaved Notch1. The increase in Dll4 level was confirmed in clinical specimens of HCC lesion, in comparison with non-tumor lesions. Using specific signalling pathway inhibitors, we found that MEK1/2, PI3K/AKT and NF-κB pathways are critical for HBx-mediated Dll4 upregulation. Silencing of HBx clearly decreased the level of phosphorylation of Akt and Erk1/2. Upon silencing of Dll4 in HepG2.2.15, decreased cleaved Notch1, increased apoptosis and cell cycle arrest were observed, suggesting a critical role of HBx-Dll4-Notch1 axis in regulating cell survival in HCC. Furthermore, clonogenic assay confirmed the important role of Dll4 in regulating cell survival of HBV-genome containing HCC cell line. Taken together, we reported a link between HBx and the Notch signalling in HCC that affects cell survival of HCC, which can be a potential target for therapy.     

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.     

In silico analysis of RNA interference components and miRNAs-like RNAs in the seed-borne necrotrophic fungus Alternaria brassicicola

RNA interference is a mechanism of suppressing gene expression in plants, animals and fungi. This regulation mechanism involves three main enzymes, Dicers (Dcr), Argonautes (Ago) and RNA Dependent RNA Polymerases (Rdrp) allowing to produce smallRNAs. RNA interference and smallRNAs have a role in the plant–microorganisms interaction, either in a pathogenic or in a symbiotic relationships. Alternaria brassicicola is a pathogenic fungus of the Brassicaceae plants. During plant infection, it is able to transmit itself vertically and horizontally, giving advantages for new infection and dissemination. To investigate RNA interference and the presence of smallRNAs in A. brassicicola, an in silico analysis was achieved. Two DCR, 4 AGO and 3 RDRP genes were identified comforting the presence of smallRNAs in A. brassicicola. SmallRNA sequencing from wild-type strain and DCR deleted mutants allowed the identifcation of 17 miRNAs in A. brassicicola. The synthesis of these miRNAs is only weakly influenced by the inactivation of DCR genes suggesting the possible existence of an alternative Dicer-independent miRNA synthesis pathway. Target's prediction of A. brassicicola miRNAs identified genes in the fungus and in the plant model Arabidopsis thaliana. Some miRNAs were predicted to target A. thaliana genes involved in the methylation of histone and in the disease resistance.     

Alpha-Fetoprotein Promoter-Driven Cre/LoxP-Switched RNA Interference for Hepatocellular Carcinoma Tissue-Specific Target Therapy

Backgroud

RNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.

Methods

Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3) and non-HCC cell lines (L-02, Hela and SW1116) were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.

Results

The AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.

Conclusions

An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.     

Androgen receptor suppresses vasculogenic mimicry in hepatocellular carcinoma via circRNA7/miRNA7‐5p/VE‐cadherin/Notch4 signalling

Androgen receptor (AR) can suppress hepatocellular carcinoma (HCC) invasion and metastasis at an advanced stage. Vasculogenic mimicry (VM), a new vascularization pattern by which tumour tissues nourish themselves, is correlated with tumour progression and metastasis. Here, we investigated the effect of AR on the formation of VM and its mechanism in HCC. The results suggested that AR could down‐regulate circular RNA (circRNA) 7, up‐regulate micro RNA (miRNA) 7‐5p, and suppress the formation of VM in HCC Small hairpin circR7 (ShcircR7) could reverse the impact on VM and expression of VE‐cadherin and Notch4 increased by small interfering AR (shAR) in HCC, while inhibition of miR‐7‐5p blocked the formation of VM and expression of VE‐cadherin and Notch4 decreased by AR overexpression (oeAR) in HCC. Mechanism dissection demonstrated that AR could directly target the circR7 host gene promoter to suppress circR7, and miR‐7‐5p might directly target the VE‐cadherin and Notch4 3′UTR to suppress their expression in HCC. In addition, knockdown of Notch4 and/or VE‐cadherin revealed that shVE‐cadherin or shNotch4 alone could partially reverse the formation of HCC VM, while shVE‐cadherin and shNotch4 together could completely suppress the formation of HCC VM. Those results indicate that AR could suppress the formation of HCC VM by down‐regulating circRNA7/miRNA7‐5p/VE‐Cadherin/Notch4 signals in HCC, which will help in the design of novel therapies against HCC.