LncRNA CRNDE promotes cell proliferation, migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis.

Ovarian cancer seriously threatens the health of women. LncRNA CRNDE is known to be upregulated in ovarian cancer. However, the mechanism by which CRNDE regulates the progress of ovarian cancer is largely unknown. MTT assay was applied to measure the cell viability. Colony formation assay was used to measure the cell proliferation. Cell migration was tested by wound healing, and Transwell assay was performed to detect cell invasion. In addition, the expression of miR-423-5p, CRNDE and FSCN1 were detected by RT-qPCR and western blotting, respectively. Meanwhile, dual-luciferase reporter assay and RIP assay were performed to explore the correlation between miR-423-5p and CRNDE (or FSCN1). CRNDE and FSCN1 were upregulated in ovarian cancer cells (SKOV3, CAOV-3, IGROV1, A2780 and C13K), while miR-423-5p was downregulated. Moreover, silencing of FSCN1/CRNDE significantly decreased proliferation, migration and invasion of ovarian cancer cells (SKOV3 and CI3K) via suppressing MMP-2 and MMP-9. In addition, CRNDE could sponge miR-423-5p, and FSCN1 was confirmed to be the direct target of miR-423-5p. Furthermore, CRNDE knockdown-induced inhibition of FSCN1 was notably reversed by miR-423-5p downregulation. Knockdown of CRNDE inhibited cell proliferation, migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis. Thus, CRNDE may serve a new target for ovarian cancer.

Serum Golgi protein 73 is a prognostic biomarker of liver transplantation patients.

BACKGROUND: The death after liver transplantation (LT) was most commonly caused by HCC recurrence. Golgi protein 73 (GP73), a type II Golgi membrane protein, has been proved to be a better serum marker for HCC. OBJECTIVE: This study aims to clarify the relationship between serum GP73 levels and tumor recurrence as well as survival of HCC patients after LT. METHODS: Between November 2003 and July 2008, serum samples from 60 liver transplantation patients and 72 healthy individuals were collected. ELISA and microparticle enzyme immunoassay were used to measure serum GP73 and AFP levels. Patient survival was analyzed using log-rank test along with Kaplan-Meier method. Receiver operating characteristic (ROC) curve was utilized to analyze the diagnostic value of serum GP73 levels. Cox regression was utilized to analyze prognostic factors with multiple variables. RESULTS: Serum GP73 concentrations in HCC patients were much higher than that in healthy controls (P<0.001). Patients with lower serum GP73 levels at LT-6Month had better overall survival and recurrence-free survival than those with higher serum GP73 levels. ROC analyzing results showed that higher serum GP73 levels at 6 month post-LT could significantly predict mortality (P=0.020) as well as HCC recurrence (P=0.001) after liver transplantation. Multivariate analysis revealed that serum GP73 levels at LT-6Month was an independent predictor of good prognosis (P=0.002). CONCLUSION: Serum GP73 levels could be used to predict tumor recurrence and survival in HCC sufferers after LT.

An Engineered Arginase FC Protein Inhibits Tumor Growth In Vitro and In Vivo.

Arginine is a semiessential amino acid required for the growth of melanoma and hepatocellular carcinoma, and the enzymatic removal of arginine by pegylated arginine deiminase (ADI) or arginase is being tested clinically. Here, we report a genetically engineered arginase FC fusion protein exhibiting a prolonged half-life and enhanced efficacy. The use of this enzyme to treat different tumor lines both inhibited cell proliferation and impaired cellular migration in vitro and in vivo. Our data reinforce the hypothesis that nutritional depletion is a key strategy for cancer treatment.

[Construction of eukaryotic plasmid of human GPC3 and expression and purification of recombinant GPC3 protein].

AIM: To obtain enough human glypican-3 (GPC3) protein for structural and functional research. METHODS: The full-length cDNA coding for GPC3 was cloned by RT-PCR from human fetal hepatocytes. The open reading frame (ORF) of the cDNA consists of 1 700 bases, encoding a mature protein of 556 amino acids. The cDNA was inserted into the pPICZ A vector to construct a expression plasmid, named pPICZ A-GPC3. Then the plasmid was transformed into a Pichia pastoris strain, GS115 and the positive strains were screened on the YPD plates with Zeocin. The positive strains were further screened on cellulose acetate and nitrocellulose membrane with HRP labeled His-tag antibody. The selected strains were induced by methanol and the supernatants were analyzed by SDS-PAGE and Western blotting. RESULTS: SDS-PAGE analysis showed an anticipated band on the gel that could bind with goatanti-GPC3 antibody. Furthermore, the strain was fermented and the expression level was about 5 mg/L, and the recombinant GPC3 protein was purified by cation-exchange chromatography from the fermentation supernatant. CONCLUSION: Human GPC3 was expressed successfully in Pichia pastoris and purified to obtain the recombinant protein from fermentation supernatant, which made it possible for further structural and functional studies on GPC3.

A liquid chromatographic method for determination of the modification degree of proteins: PEGylated arginase as an example.

A liquid chromatographic method was developed to determine the modification degree of PEGylated proteins. This method effectively separated free polyethylene glycol (PEG) from other species in conjugation mixtures on a C4 reversed-phase column using water-acetonitrile gradient elution. Then the concentrations of free PEG were determined according to the integrated area under the curve of its evaporative light scattering detector (ELSD) signal, which was normalized by the PEG standard with similar molecular weights. The actual numbers of PEG attached to proteins, not those of lysines modified, were calculated. This method was performed with PEGylated arginase mixtures as an example and showed clear advantages over 2,4,6-trinitrobenzenesulfonic acid (TNBS) assays.

[Study on expression of estrogen receptor isoforms in eutopic and ectopic endometrium of ovarian endometriosis].

OBJECTIVE: To investigate the distribution of ER isoforms in endometriosis and eutopic endometrium. METHODS: Tissue samples of patients with ovarian endometriosis, treated in People's Liberation Army General Hospital from January 2004 to December 2006, were retrieved. A total of 60 cases of ovarian endometriotic cysts with their corresponding eutopic endometrium (30 cases of proliferation phase and 30 of secretary phase eutopic endometrium) and 30 cases of normal endometrium (15 proliferative and 15 secretary phase endometrial samples respectively) were included. Expressions of ERalpha and ERbeta were analyzed using immunohistochemistry and the expression ratio was statistically analyzed by using SPSS 12.0 software. RESULTS: Expressions of both ERalpha and ERbeta in epithelial cells were positively correlated with that of the stromal cells. The expression of ERalpha in eutopic endometrium (73.3% in epithelium and 76.7% in stroma) was significantly higher than that in ovarian endometriotic cysts (43.3% in epithelium and 46.7% in stroma), or normal control (56.7% in epithelium and 50.0% in stroma, respectively, each P < 0.05. However, the expression of ERbeta (90.0% in epithelium and 76.7% in stroma) was higher in ovarian endometriotic cysts than that in the eutopic endometrium (68.0% in epithelium and 63.3% in stroma respectively, P < 0.05), and ERbeta expression in eutopic endometrium was higher than that in the normal control endometrium (36.7% in epithelium and 26.7% in stroma, respectively, P < 0.05). The expressions of both ERalpha and ERbeta changed periodically in eutopic and normal endometrium, whereas ERalpha and ERbeta level were less variable in the ectopic endometrium. The expression of ERbeta was statistically higher than that of ERalpha (P < 0.05) in ectopic endometrium, whereas no significant difference was seen between the two isoforms in the eutopic or normal endometrium. CONCLUSIONS: Both ERalpha and ERbeta have higher expression levels in eutopic endometrium of patients with ovarian endometriotic cysts. ERbeta is predominantly expressed in endometriotic cysts, where the expression of ERalpha is limited. The different distribution of ERalpha and ERbeta may play an important role in the development of ovarian endometriosis.