Expression and Localization of Lung Surfactant Proteins in Human Testis

Background

Surfactant proteins (SPs) have been described in various tissues and fluids including tissues of the nasolacrimal apparatus, airways and digestive tract. Human testis have a glandular function as a part of the reproductive and the endocrine system, but no data are available on SPs in human testis and prostate under healthy and pathologic conditions.

Objective

The aim of the study was the detection and characterization of the surfactant proteins A, B, C and D (SP-A, SP-B, SP-C, SP-D) in human testis. Additionally tissue samples affected by testicular cancer were investigated.

Results

Surfactant proteins A, B, C and D were detected using RT-PCR in healthy testis. By means of Western blot analysis, these SPs were detected at the protein level in normal testis, seminoma and seminal fluid, but not in spermatozoa. Expression of SPs was weaker in seminoma compared to normal testicular tissue. SPs were localized in combination with vimentin immunohistochemically in cells of Sertoli and Leydig.

Conclusion

Surfactant proteins seem to be inherent part of the human testis. By means of physicochemical properties the proteins appear to play a role during immunological and rheological process of the testicular tissue. The presence of SP-B and SP-C in cells of Sertoli correlates with their function of fluid secretion and may support transportation of spermatozoa. In seminoma the expression of all SP''s was generally weaker compared to normal germ cells. This could lead to a reduction of immunomodulatory and rheology processes in the germ cell tumor.     

Histone Demethylase Retinoblastoma Binding Protein 2 is Overexpressed in Hepatocellular Carcinoma and Negatively Regulated by hsa-miR-212

Background

The H3K4 demethylase retinoblastoma binding protein 2 (RBP2) is involved in the pathogenesis of gastric cancer, but its role and regulation in hepatocellular carcinoma (HCC) is unknown. We determined the function of RBP2 and its regulation in HCC in vitro and in human tissues.

Methods

We analyzed gene expression in 20 specimens each of human HCC and normal liver tissue by quantitative real-time PCR and immunohistochemistry. Proliferation was analyzed by foci formation and senescence by β-galactosidase staining. Promoter activity was detected by luciferase reporter assay.

Results

The expression of RBP2 was stronger in cancerous than non-cancerous tissues, but that of its binding microRNA, Homo sapiens miR-212 (hsa-miR-212), showed an opposite pattern. SiRNA knockdown of RBP2 significantly upregulated cyclin-dependent kinase inhibitors (CDKIs), with suppression of HCC cell proliferation and induction of senescence. Overexpression of hsa-miR-212 suppressed RBP2 expression, with inhibited cell proliferation and induced cellular senescence, which coincided with upregulated CDKIs; with low hsa-miR-212 expression, CDKIs were downregulated in HCC tissue. Inhibition of hsa-miR-212 expression upregulated RBP2 expression. Luciferase reporter assay detected the direct binding of hsa-miR-212 to the RBP2 3′ UTR.

Conclusions

RBP2 is overexpressed in HCC and negatively regulated by hsa-miR-212. The hsa-miR-212–RBP2–CDKI pathway may be important in the pathogenesis of HCC.     

Activation of GPER-1 Estradiol Receptor Downregulates Production of Testosterone in Isolated Rat Leydig Cells and Adult Human Testis

Purpose

Estradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis.

Materials and Methods

Isolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay.

Results

GPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20–30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs.

Conclusions

Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.     

Correlation of MicroRNA-16, MicroRNA-21 and MicroRNA-101 Expression with Cyclooxygenase-2 Expression and Angiogenic Factors in Cirrhotic and Noncirrhotic Human Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression.

Methods

Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue.

Results

COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma.

Conclusions

In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.     

Amyloid precursor protein interaction network in human testis: sentinel proteins for male reproduction

Background

Amyloid precursor protein (APP) is widely recognized for playing a central role in Alzheimer''s disease pathogenesis. Although APP is expressed in several tissues outside the human central nervous system, the functions of APP and its family members in other tissues are still poorly understood. APP is involved in several biological functions which might be potentially important for male fertility, such as cell adhesion, cell motility, signaling, and apoptosis. Furthermore, APP superfamily members are known to be associated with fertility. Knowledge on the protein networks of APP in human testis and spermatozoa will shed light on the function of APP in the male reproductive system.

Results

We performed a Yeast Two-Hybrid screen and a database search to study the interaction network of APP in human testis and sperm. To gain insights into the role of APP superfamily members in fertility, the study was extended to APP-like protein 2 (APLP2). We analyzed several topological properties of the APP interaction network and the biological and physiological properties of the proteins in the APP interaction network were also specified by gene ontologyand pathways analyses. We classified significant features related to the human male reproduction for the APP interacting proteins and identified modules of proteins with similar functional roles which may show cooperative behavior for male fertility.

Conclusions

The present work provides the first report on the APP interactome in human testis. Our approach allowed the identification of novel interactions and recognition of key APP interacting proteins for male reproduction, particularly in sperm-oocyte interaction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0432-9) contains supplementary material, which is available to authorized users.     

SPARC Is a Key Regulator of Proliferation, Apoptosis and Invasion in Human Ovarian Cancer

Background

Secreted protein acidic and rich in cysteine (SPARC), a calcium-binding matricellular glycoprotein, is implicated in the progression of many cancers. In this study, we investigated the expression and function of SPARC in ovarian cancer.

Methods

cDNA microarray analysis was performed to compare gene expression profiles of the highly invasive and the low invasive subclones derived from the SKOV3 human ovarian cancer cell line. Immunohistochemistry (IHC) staining was performed to investigate SPARC expression in a total of 140 ovarian tissue specimens. In functional assays, effects of SPARC knockdown on the biological behavior of ovarian cancer cells were investigated. The mechanisms of SPARC in ovarian cancer proliferation, apoptosis and invasion were also researched.

Results

SPARC was overexpressed in the highly invasive subclone compared with the low invasive subclone. High SPARC expression was associated with high stage, low differentiation, lymph node metastasis and poor prognosis of ovarian cancer. Knockdown of SPARC expression significantly suppressed ovarian cancer cell proliferation, induced cell apoptosis and inhibited cell invasion and metastasis.

Conclusion

SPARC is overexpressed in highly invasive subclone and ovarian cancer tissues and plays an important role in ovarian cancer growth, apoptosis and metastasis.     

Reduced CTGF Expression Promotes Cell Growth,Migration, and Invasion in Nasopharyngeal Carcinoma

Background

The role of CTGF varies in different types of cancer. The purpose of this study is to investigate the involvement of CTGF in tumor progression and prognosis of human nasopharyngeal carcinoma (NPC).

Experimental design

CTGF expression levels were examined in NPC tissues and cells, nasopharynx (NP) tissues, and NP69 cells. The effects and molecular mechanisms of CTGF expression on cell proliferation, migration, invasion, and cell cycle were also explored.

Results

NPC cells exhibited decreased mRNA expression of CTGF compared to immortalized human nasopharyngeal epithelial cell line NP69. Similarly, CTGF was observed to be downregulated in NPC compared to normal tissues at mRNA and protein levels. Furthermore, reduced CTGF was negatively associated with the progression of NPC. Knocking down CTGF expression enhanced the colony formation, cell migration, invasion, and G1/S cell cycle transition. Mechanistic analysis revealed that CTGF suppression activated FAK/PI3K/AKT and its downstream signals regulating the cell cycle, epithelial-mesenchymal transition (EMT) and MMPs. Finally, DNA methylation microarray revealed a lack of hypermethylation at the CTGF promoter, suggesting other mechanisms are associated with suppression of CTGF in NPC.

Conclusion

Our study demonstrates that reduced expression of CTGF promoted cell proliferation, migration, invasion and cell cycle progression through FAK/PI3K/AKT, EMT and MMP pathways in NPC.     

Hypermethylation Leads to Bone Morphogenetic Protein 6 Downregulation in Hepatocellular Carcinoma

Background

In the liver, bone morphogenetic protein 6 (BMP-6) maintains balanced iron metabolism. However, the mechanism that underlies greater BMP-6 expression in hepatocellular carcinoma (HCC) tissue than adjacent non-cancerous tissue is unclear. This study sought to investigate the epigenetic mechanisms of BMP-6 expression by analysing the relationship between the DNA methylation status of BMP-6 and the expression of BMP-6.

Methods

Methylation-specific polymerase chain reaction (PCR), bisulphite sequencing PCR, the MethyLight assay, and quantitative real-time PCR were performed to examine BMP-6 methylation and mRNA expression levels. Immunohistochemistry (IHC) was performed on tissue arrays to evaluate the BMP-6 protein level.

Results

BMP-6 mRNA expression was approximately 84.09% lower in HCC tissues than in adjacent non-cancerous tissues, and this low level of expression was associated with a poor prognosis. Moreover, the hypermethylation observed in HCC cell lines and HCC tissues was correlated with the BMP-6 mRNA expression level, and this correlation was validated following treatment with 5-aza-CdR, a demethylation agent. In addition, BMP-6 DNA methylation was upregulated by 68.42% in 114 clinical HCC tissue samples compared to adjacent normal tissues, whereas the BMP-6 staining intensity was downregulated by 77.03% in 75 clinical HCC tissue samples in comparison to adjacent normal tissues. Furthermore, elevated expression of BMP-6 in HCC cell lines inhibited cell colony formation.

Conclusions

Our results suggest that BMP-6 CpG island hypermethylation leads to decreased BMP-6 expression in HCC tissues.     

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma

Background

The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.

Methods

The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.

Results

Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient''s survival, while there was no connection with pathological stage.

Conclusion

In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.     

Immunohistochemical Characterisation of Cell-Type Specific Expression of CK1δ in Various Tissues of Young Adult BALB/c Mice

Background

Casein kinase 1 delta (CK1δ) phosphorylates many key proteins playing important roles in such biological processes as cell growth, differentiation, apoptosis, circadian rhythm and vesicle transport. Furthermore, deregulation of CK1δ has been linked to neurodegenerative diseases and cancer. In this study, the cell specific distribution of CK1δ in various tissues and organs of young adult BALB/c mice was analysed by immunohistochemistry.

Methodology/Principal Findings

Immunohistochemical staining of CK1δ was performed using three different antibodies against CK1δ. A high expression of CK1δ was found in a variety of tissues and organ systems and in several cell types of endodermal, mesodermal and ectodermal origin.

Conclusions

These results give an overview of the cell-type specific expression of CK1δ in different organs under normal conditions. Thus, they provide evidence for possible cell-type specific functions of CK1δ, where CK1δ can interact with and modulate the activity of key regulator proteins by site directed phosphorylation. Furthermore, they provide the basis for future analyses of CK1δ in these tissues.     

MiR-101 Is Involved in Human Breast Carcinogenesis by Targeting Stathmin1

Background

MicroRNA-101 (miR-101) expression is negatively associated with tumor growth and blood vessel formation in several solid epithelial cancers. However, the role of miR-101 in human breast cancer remains elusive.

Results

MiR-101 was significantly decreased in different subtypes of human breast cancer tissues compared with that in adjacent normal breast tissues (P<0.01). Up-regulation of miR-101 inhibited cell proliferation, migration and invasion, and promoted cell apoptosis in ER alpha-positive and ER alpha-negative breast cancer cells and normal breast cells. Down-regulation of miR-101 displayed opposite effects on cell growth and metastasis. Further investigation revealed a significant inverse correlation between the expression of miR-101 and Stathmin1 (Stmn1), and miR-101 could bind to the 3′-untranslated region (UTR) of Stmn1 to inhibit Stmn1 translation. The inhibition of cell growth and metastasis induced by up-regulation of miR-101 was partially restored by overexpresson of Stmn1. Knockdown of Stmn1 attenuates the down-regulation of miR-101-mediated enhancement of cell growth and metastasis. More importantly, in vivo analysis found that Stmn1 mRNA and protein level in different subtypes of human breast cancer tissues, contrary to the down-regulation of miR-101, were significantly elevated.

Conclusions

This study demonstrates that down-regulation of miR-101 in different subtypes of human breast cancer tissues is linked to the increase of cellular proliferation and invasiveness via targeting Stmn1, which highlights novel regulatory mechanism in breast cancer and may provide valuable clues for the future clinical diagnosis of breast cancer.