miR-218 suppresses the growth of hepatocellular carcinoma by inhibiting the expression of proto-oncogene Bmi-1.

PURPOSE: To identify microRNAs (miRNAs) directly regulating the proto-oncogene Bmi-1 expression in the development of hepatocellular carcinoma (HCC) and to explore the underlying molecular mechanisms. METHODS: Four HCC cell lines, including HepG2, Bel7404, Huh7, and PLC5, the normal hepatocellular cell line MIHA, and 30 HCC biopsies were included in this study. Potential miRNAs, which interact with Bmi-1 and are involved in the development of HCC were identified through bioinformatic analyses. The expression of miRNA and Bmi-1 in HCC cell lines and HCC tissues was analyzed using fluorescence protein analysis, real-time quantitative PCR (RT-qPCR), and Western blotting. RESULTS: Bioinformatic analysis suggested that miR-218 is a potential miRNA regulating Bmi-1 expression. Fluorescence protein analysis, RT-qPCR, and Western blotting confirmed the direct interaction between miR-218 and Bmi-1. In addition, increased expression of Bmi-1 was detected in HCC cell lines and HCC tissues. In most HCC tissues, the expression of miR-218 was decreased and was associated with increased expression of Bmi-1. CONCLUSION: miR-p218 downregulates the expression of the proto-oncogene Bmi-1 in HCC, and it may be an effective target for the treatment of this disease.

Non-Genomic Action of Androgens is Mediated by Rapid Phosphorylation and Regulation of Androgen Receptor Trafficking.

BACKGROUND: Testosterone is critical for maintaining spermatogenesis and male fertility. The accomplishment of these processes requires the synergistic actions of the classical and non-classical signaling pathways of androgens. METHODS: A murine testicular Sertoli cell line, TM4 cell was used to examine androgen actions in Sertoli cells. Western blot analysis and immunofluorescence assay were employed to study the testosterone-induced Androgen receptor (AR) translocation. Protein phosphorylation antibody array was applied to identify the phosphorylation sites under testosterone treatment, and these findings were verified by Western blot analysis. RESULTS: We found that a physiological dose of testosterone induced fast membrane association of AR. By using a phosphorylation antibody array, several phosphorylation sites, such as MEK1/2 (Ser217/221), Akt (Ser473), and Erk1/2 (Thr202/Tyr204) were rapidly phosphorylated within 5 min of testosterone treatment. Inhibition of the MEK and Akt signaling pathways prevented AR trafficking. Blocking of AR by flutamide eliminated the stimulation effect of testosterone on kinase phosphorylation. Testosterone induced kinase Src phosphorylation, and inhibition of Src restricted AR translocation to the membrane and the nucleus. CONCLUSION: Findings suggested that the membrane association of AR was mediated by the MEK and Akt phosphorylation signaling pathways, which resulted in Src activation and was initiated by testosterone binding to the membrane-localized AR. This study provides new insights into the testosterone signaling pathway in Sertoli cells, which mediate spermatogenesis. In addition, the study can be used in the diagnosis and treatment of male infertility caused by disorders in spermatogenesis.

The Wilms tumor gene, Wt1, is critical for mouse spermatogenesis via regulation of sertoli cell polarity and is associated with non-obstructive azoospermia in humans.

Azoospermia is one of the major reproductive disorders which cause male infertility in humans; however, the etiology of this disease is largely unknown. In the present study, six missense mutations of WT1 gene were detected in 529 human patients with non-obstructive azoospermia (NOA), indicating a strong association between WT1 mutation and NOA. The Wilms tumor gene, Wt1, is specifically expressed in Sertoli cells (SCs) which support spermatogenesis. To examine the functions of this gene in spermatogenesis, Wt1 was deleted in adult testis using Wt1(flox) and Cre-ER(TM) mice strains. We found that inactivation of Wt1 resulted in massive germ cell death and only SCs were present in most of the seminiferous tubules which was very similar to NOA in humans. In investigating the potential mechanism for this, histological studies revealed that the blood-testis barrier (BTB) was disrupted in Wt1 deficient testes. In vitro studies demonstrated that Wt1 was essential for cell polarity maintenance in SCs. Further studies found that the expression of cell polarity associated genes (Par6b and E-cadherin) and Wnt signaling genes (Wnt4, Wnt11) were downregulated in Wt1 deficient SCs, and that the expression of Par6b and E-cadherin was regulated by Wnt4. Our findings suggest that Wt1 is important in spermatogenesis by regulating the polarity of SCs via Wnt signaling pathway and that WT1 mutation is one of the genetic causes of NOA in humans.

Overexpression of cyclooxygenase-1 correlates with poor prognosis in renal cell carcinoma.

The aim of this study was to evaluate expression of COX-1 in renal cell carcinoma (RCC) and its prognostic value. mRNA of COX-1 was detected in 42 paired RCC and adjacent normal tissues with quantitative real- time polymerase chain reaction (qRT-PCR). Expression of COX-1 was also evaluated in 196 RCC sections and 91 adjacent normal tissues with immunohistochemistry. Statistical analysis was performed to assess COX-1 expression in RCC and its prognostic significance. The results of qRT-PCR showed mRNA levels of COX-1 in RCC tissues to be significantly higher than that in adjacent normal tissues (p < 0.001). Immunohistochemical assays also revealed COX-1 to be overexpressed in RCC tissues (p < 0.001). Statistical analysis demonstrated high expression of COX-1 was correlated with tumour size (p = 0.002), pathological stage (p = 0.003), TNM stage (p = 0.003, 0.007, 0.027, respectively), and tumour recurrence (p < 0.001). Survival analysis indicated patients with high expression of COX-1 had shorter survival time (p < 0.001), and COX-1 was an independent predictor. This is the first study to reveal overexpression of COX-1 in RRC and point to use as a prognostic marker in affected patients.

RALY RNA binding protein-like reduced expression is associated with poor prognosis in clear cell renal cell carcinoma.

The molecular mechanisms involved in the progression of clear cell renal cell carcinomas (ccRCCs) are still unclear. The aim of this study was to analyse the relationships between expression of RALYL and clinical characteristics. In 41 paired samples of ccRCCs and adjacent normal tissues, we used real-time qPCR to evaluate the expression of RALYL mRNA. RALYL protein levels were determined in 146 samples of ccRCC and 37 adjacent normal tissues by immunohistochemistry. Statistical analysis was used to explore the relationships between expression of RALYL and the clinical characteristics (gender, age, tumor size, T stage, N stage, M stage, survival times and survival outcome) in ccRCC. In addition, these patients were follow-up period 64 months (range: 4~116 months) to investigate the influence on prognosis. We found significantly differences between ccRCC tissues and normal tissues (p<0.001, paired-sample t test) in mRNA levels of RALYL. Immunohistochemistry analyses in 146 ccRCC samples and 37 adjacent normal tissues showed significantly lower RALYL protein levels in ccRCC samples (χ2-test, p<0.001), inversely correlating with tumour size (p=0.024), T stage (0.005), N stage (p<0.001) as well as M stage (p=0.019), but not age (p=0.357) and gender (p=0.348). Kaplan-Meier survival analysis demonstrated that people with lower level of RALYL expression had a poorer survival rate than those with a higher level of RALYL expression, significantly different by the log-rank test (p=0.011). Cox regression analysis indicated that RALYL expression (p=0.039), N stage (p=0.008) and distant metastasis (p<0.001) were independent prognosis factors for the overall survival of ccRCC patients. We demonstrated that the expression of RALYL was significantly low in ccRCC and correlated with a poor prognosis in a large number of clinical samples. Our findings showed that RALYL may be a potential therapeutic target as well as a poor prognostic factor.