P68 RNA helicase promotes invasion of glioma cells through negatively regulating DUSP5.

Gliomas are the most common central nervous system tumors. They show malignant characteristics indicating rapid proliferation and a high invasive capacity and are associated with a poor prognosis. In our previous study, p68 was overexpressed in glioma cells and correlated with both the degree of glioma differentiation and poor overall survival. Downregulating p68 significantly suppressed proliferation in glioma cells. Moreover, we found that the p68 gene promoted glioma cell growth by activating the nuclear factor-κB signaling pathway by a downstream molecular mechanism that remains incompletely understood. In this study, we found that dual specificity phosphatase 5 (DUSP5) is a downstream target of p68, using microarray analysis, and that p68 negatively regulates DUSP5. Upregulating DUSP5 in stably expressing cell lines (U87 and LN-229) suppressed proliferation, invasion, and migration in glioma cells in vitro, consistent with the downregulation of p68. Furthermore, upregulating DUSP5 inhibited ERK phosphorylation, whereas downregulating DUSP5 rescued the level of ERK phosphorylation, indicating that DUSP5 might negatively regulate ERK signaling. Additionally, we show that DUSP5 levels were lower in high-grade glioma than in low-grade glioma. These results suggest that the p68-induced negative regulation of DUSP5 promoted invasion by glioma cells and mediated the activation of the ERK signaling pathway.

Identifying survival-associated modules from the dysregulated triplet network in glioblastoma multiforme.

BACKGROUND: Long noncoding RNAs (lncRNAs) can act as competitive endogenous RNAs (ceRNAs) to compete with mRNAs for binding miroRNAs (miRNAs). The dysregulated triplets, composed by mRNAs, lncRNAs, and miRNAs, contributed to the development and progression of diseases, such as cancer. However, the roles played by triplet biomarkers are not fully understand in glioblastoma multiforme (GBM) patient survival. OBJECTIVES: Here, we constructed a differential triplet interaction network (TriNet) between GBM and normal tissues and identified GBM survival related triplets. METHODS: Four significantly dysregulated modules, enriched differentially expressed molecules, were identified by integrating affinity propagation method and hypergeometric method. Furthermore, knockdown of TP73-AS1 was implemented by siRNA and the expression of RFX1 was examined in U87 cells by qRT-PCR. The apoptosis of U87 cells was investigated using MTT assay and Acridine orange/Ethidium bromide (AO/EB) assay. RESULTS: We randomly split GBM samples into training and testing sets, and found that these four modules can robustly and significantly distinguish low- and high-survival patients in both two sets. By manually curated literatures for triplets mediated by core interactions, we found that members involved tumor invasion, proliferation, and migration. The dysregulated triplets may cause the poor survival of GBM patients. We finally experimentally verified that knockdown of TP73-AS1, an lncRNA of one triplet, could not only reduce the expression of RFX1, an mRNA of this triplet, but also induce apoptosis in U87 cells. CONCLUSIONS: These results can provide further insights to understand the functions of triplet biomarkers that associated with GBM prognosis.

Role of RbBP5 and H3K4me3 in the vicinity of Snail transcription start site during epithelial-mesenchymal transition in prostate cancer cell.

EMT (epithelial-mesenchymal transition) occurs in a wide range of tumor types, and has been shown to be crucial for metastasis. Epigenetic modifications of histones contribute to chromatin structure and result in the alterations in gene expression. Tri-methylation of histone H3 lysine 4 (H3K4me3) is associated with the promoters of actively transcribed genes and can serve as a transcriptional on/off switch. RbBP5 is a component of the COMPASS/ -like complex, which catalyzes H3K4me3 formation. In this study, we found that in the process of TGF-Beta1 induced EMT in the prostate cancer cell line DU145, H3K4me3 enrichment and RbBP5 binding increased in the vicinity of Snail (SNAI1) transcription start site. Knocking-down of RbBP5 notably decreased Snail expression and EMT. Recruitment of RbBP5 and formation of H3K4me3 at Snail TSS during EMT depend on binding of SMAD2/3 and CBP at Snail TSS. This study links the SMAD2/3 signal with Snail transcription via a histone modification - H3K4me3. Furthermore, our research also demonstrates that RbBP5 and even WRAD may be a promising therapeutic candidates in treating prostate cancer metastasis, and that DU145 cells maintain their incomplete mesenchymal state in an auto/ paracrine manner.

[Cloning of human uracil N-glycosylase and its detection in cancer tissues by quantitative RT-PCR].

The uracil in DNA comes from either the misincorporation of dUTP in place of dTTP or deamination of cytosine. In the latter case, it can result in a GC to AT transition mutation if the uracil is not removed before DNA replication. Base excision repair (BER) is a major pathway for removing DNA lesions arising from endogenous processes as well as those induced by exposure to exogenous chemicals or irradiation. BER is initiated by DNA glycosylases that excise aberrant bases from DNA by cleavage of the N-glycosidic bond linking to the base of its deoxyribose sugar. Uracil N-glycosylase (UNG) is the enzyme responsible for the first step in the BER pathway that specifically removes uracil from DNA. The UNG gene undergoes both temporal and spatial regulation mainly at the level of transcription. Normally cancer cells undergo over-proliferation and up-regulate their UNG during tumorigenesis. In this study we examine the correlation between UNG level and carcinogenesis, and explore the possibility of using UNG as a marker for cancer diagnosis. Human UNG gene was amplified from the total RNA of the human choriocarcinoma cell line, JEG-3, by RT-PCR. After purification, the 942bp full-length UNG cDNA coding sequence was digested with EcoR I and Sal I, and cloned into the digested pET-21 to construct a recombinant vector, pUNG. The UNG protein was expressed under the control of T7 promoter in E. coli BL21 (DE3) cells induced with IPTG. After ultrasonic treatment, the cell lysate and precipitate were analyzed by SDS-PAGE and a 39kD band was detected. The plasmid was serially diluted at appropriate concentrations and employed as standards in the subsequent quantification. Total RNAs were extracted from 18 pairs of clinical samples, each pair contains a sample of esophageal squamous cell carcinoma (ESCC) tissue and its surrounding normal esophageal epithelia. The copy numbers of UNG mRNA in these RNA samples were determined by real-time quantitative RT-PCR using a Lightcycler (Roche). UNG was present in 13 cases of ESCC (13/18, n = 18) but absent in all of the normal tissues. The results indicated that there was a correlation between high level of UNG expression and the carcinogenesis of ESCC.