Exploring the frequency of a TP53 polyadenylation signal variant in tumor DNA from patients diagnosed with lung adenocarcinomas, sarcomas and uterine leiomyomas.

The TP53 3'UTR variant rs78378222 A>C has been detected in different tumor types as a somatic alteration that reduces p53 expression through modification of polyadenylation and miRNA regulation. Its prevalence is not yet known in all tumors. Herein, we examine tumor tissue prevalence of rs7837822 in Brazilian cohorts of patients from south and southeast regions diagnosed with lung adenocarcinoma (LUAD, n=586), sarcoma (SARC, n=188) and uterine leiomyoma (ULM, n=41). The minor allele (C) was identified in heterozygosity in 6/586 LUAD tumors (prevalence = 1.02 %) and none of the SARC and ULM samples. Additionally, next generation sequencing analysis revealed that all variant-positive tumors (n=4) with sample availability had additional pathogenic or likely pathogenic somatic variants in the TP53 coding regions. Among them, 3/4 (75 %) had the same pathogenic or likely pathogenic sequence variant (allele frequency <0.05 in tumor DNA) namely c.751A>C (p.Ile251Leu). Our results indicate a low somatic prevalence of rs78378222 in LUAD, ULM and SARC tumors from Brazilian patients, which suggests that no further analysis of this variant in the specific studied regions of Brazil is warranted. However, these findings should not exclude tumor molecular testing of this TP53 3'UTR functional variant for different populations.

The role of FASL, BCL-2 and BAX polymorphisms in brazilian patients with prostate cancer and benign prostatic hyperplasia.

BACKGROUND: Prostate cancer (PCA) is one of the leading causes of death among men, being related to several factors, including the aging male population, like benign prostatic hyperplasia (BPH), a histopathological and hyperplastic alteration associated to prostate aging. The FASL, BCL-2 and BAX genes are involved in cell apoptosis regulation and can be related to the development of both cancer and hyperplasia. This study aimed to investigate the association of FASL - 844 (rs763110), BCL-2 -938 (rs2279115) and BAX - 248 (rs4645878) polymorphic variants in Southern Brazilian PCA and BPH patients and healthy controls. METHODS AND RESULTS: 348 samples were analyzed, being 123 from PCA patients, 143 BPH patients and 82 healthy controls, using PCR-RFLP techniques. The results of genotyping analysis were adjusted by age, and compared with PSA levels and prostate volume. The analyzes of genotype frequencies according to PCA, HPB and controls, were performed by logistic regression corrected by age, and showed that the FASL CC genotype can be a risk factor for PCA patients, when compared to controls (p = 0.041). The clinical data investigation indicated higher PSA levels in PCA patients with FASL CC genotype, as compared to TC genotype carriers (p = 0.044), higher PSA levels for healthy individuals with BCL-2 AA genotype, comparing with CC genotype (p = 0.027) and higher PSA levels in BPH group with FASL CC genotype, as compared to TC genotype (p = 0.044). CONCLUSIONS: Our data indicate the FASLCC genotype as a risk factor for prostate pathologies, whileBCL-2 CC can act as a protective genotype.

Stanniocalcin-1 protein expression profile and mechanisms in proliferation and cell death pathways in prostate cancer.

Prostate cancer (PCa) is one of the most prevalent male tumours. Stanniocalcin-1 (STC1) is a glycoprotein and, although the role of STC1 in human cancer is poorly understood, it is suggested to be involved in the development and progression of different neoplasms. This study investigated the protein expression profile of STC1 in PCa and benign prostatic hyperplasia (BPH) samples and STC1 signalling during cell proliferation and cell death in vitro using cell lines. We found higher levels of STC1 in PCa when compared to BPH tissue and that STC1 inhibited forskolin stimulation of cAMP in PC-3 cells. A monoclonal antibody against STC1 was effective in reducing cell proliferation, in promoting cell cycle arrest, and in increasing apoptosis in the same cells. Since STC1 acts as a regulator of prostatic tissue signalling, we suggest that this protein is a novel candidate biomarker for prostate tumour clinical progression and a potential therapeutic target.

PSA secretion and cell proliferation are affected by NCoR1 silencing in prostate cancer cells

Introduction: The androgen receptor (AR) plays an important role in normal development of the prostate gland, as well as in prostatic neoplasms. Transcriptional regulation by AR is modulated by its interaction with co-activators or co-repressors, such as NCoR1 (nuclear receptor co-repressor 1), which is involved in reducing AR activity over the target gene transcription. Methods: To identify the role of NCoR1 in the prostate cancer androgen independence in a cell line model, we aimed to evaluate the effects of silencing NCoR1 on prostate-specific antigen (PSA) gene expression, the proliferative response and PSA secretion on the supernatant of C4-2B and LNCaP cells that were submitted to small interfering RNAs (siRNAs) transfection, and to treatments with different androgen dosages. Results: In LNCaP and C4-2B cells with no dihydrotestosterone (DHT) treatment, a decrease in PSA mRNA expression was observed 48 hours and 72 hours after gene silencing in the siNCoR group when compared to the control and siNC groups. The LNCaP and C4-2B cells showed a biphasic pattern in response to dihydrotestosterone treatment in transfected groups (siNCoR and siNC) as well as in the control condition (without transfection). The secretion of PSA in cell supernatant of LNCaP and C4-2B cells was higher in the siNCoR group, and, in relation to hormonal treatment, higher in the 10-8 M DHT group. Conclusions: A reduction in the NCoR1 levels seems to have a double influence on the activity of AR in PCa cells. These results suggest that NCoR may act as an AR co-repressor depending upon hormonal stimulation.(AU)

Lapatinib Inhibits Amphiregulin-induced BeWo Choriocarcinoma Cell Proliferation by Reducing ERK1/2 and AKT Signaling Pathways.

BACKGROUND: Human choriocarcinoma is the most aggressive type of gestational trophoblastic neoplasia. The expression of epidermal growth factor receptor (EGFR) in choriocarcinomas is significantly higher than those of trophoblastic cells in healthy placentas. Lapatinib is a potent EGFR and human epidermal growth factor receptor 2 (HER2) inhibitor that inhibits cell proliferation and induces apoptosis in various human cancer cells. Amphiregulin (AREG) is the most abundant EGFR ligand in amniotic fluid during human pregnancy. AIM: To explore the role of AREG in human choriocarcinoma cell proliferation. MATERIALS AND METHODS: The effect of lapatinib and AREG on cell proliferation was examined by the MTT assay. Western blots were used to investigate EGFR and HER2 expression, and the activation of caspase-3, extracellular signal-regulated kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase /protein kinase B (PI3K/AKT) signaling pathways. RESULTS: Treatment with lapatinib reduced BeWo cell proliferation by inducing apoptosis. Moreover, AREG treatment stimulated BeWo cell proliferation by activating ERK1/2 and PI3K/AKT signaling pathways, which was blocked by lapatinib. CONCLUSION: Targeting EGFR/HER2 might be a useful therapeutic strategy for human choriocarcinoma.

Androgenic modulation of AR-Vs.

PURPOSE: The importance of androgen receptor variants (AR-Vs) is recognized in prostate cancer. AR-Vs have been the focus of many studies. Expression of AR-Vs has been proposed as a biomarker for resistance to androgen deprivation therapy for metastatic disease. Herein, we show dynamic changes in AR-Vs expression in response to androgen modulation. METHODS: The C4-2B cell line was exposed to low (10-13 M) and high (10-8 M) androgen (dihydrotestosterone, DHT) levels, with or without flutamide. mRNA and protein expression levels were assessed by qPCR and immunohistochemistry, respectively. RESULTS: We demonstrated that high levels of DHT downregulate AR-FL and AR-Vs. Even though AR-Vs did not present ligand-binding domain, thus were not capable of binding to DHT, they present dynamic changes under androgen treatment. Treatment with flutamide alone or in association with low levels of DHT stimulates growth of prostatic cells. CONCLUSIONS: Importantly, we provide evidence that AR-Vs respond differently to androgenic modulation. These findings have implications for a better understanding of the role of AR-Vs in prostate carcinogenesis.

Androgen receptor isoforms expression in benign prostatic hyperplasia and primary prostate cancer.

The role of molecular changes in the androgen receptor (AR) as AR variants (AR-Vs) is not clear in the pathophysiology of benign prostatic hyperplasia (BPH) and hormone-naïve PCa. The aim of the current work was to identify the presence of AR isoforms in benign tissue and primary PCa, and to evaluate the possible association with tumor aggressiveness and biochemical recurrence in primary PCa. The mRNA levels of full length AR (AR-FL) and AR-Vs (AR-V1, AR-V4 and AR-V7) were measured using RT-qPCR. The protein expression of AR-FL (AR-CTD and AR-NTD) and AR-V7 were evaluated by the H-Score in immunohistochemistry (IHC). All investigated mRNA targets were expressed both in BPH and PCa. AR-FL mRNA levels were similar in both groups. AR-V4 mRNA expression showed higher levels in BPH, and AR-V1 and AR-V7 mRNA expression were higher in PCa. The AR-V7 protein showed a similar H-Score in both groups, while AR-CTD and AR-NTD were higher in nuclei of epithelial cells from BPH. These results support the assumption that these constitutively active isoforms of AR are involved in the pathophysiology of primary PCa and BPH. The role of AR-Vs and their possible modulation by steroid tissue levels in distinct types of prostate tumors needs to be elucidated to help guide the best clinical management of these diseases.

Selection of developmentally competent human oocytes aspirated during cesarean section.

PURPOSE: To evaluate the efficiency/safety of Brilliant Cresyl Blue (BCB) staining as a selection method of developmentally competent immature human oocytes. MATERIALS AND METHODS: Immature oocytes of 32 pregnant women were recovered during cesarean section (CS). After retrieval, 92 oocytes were randomly divided into two groups: control (directly disposed to in vitro maturation - IVM) and treated - exposed to BCB 26 µM during 60 min. After staining, the treated group was classified as cytoplasm coloration, BCB positive (blue) or negative (colorless), and then disposed to IVM. Nuclear status was checked after 24 and 48 h of IVM. Nuclear maturation (polar body extrusion), meiosis resumption (absence of germinal vesicle) and degeneration rates were evaluated among the three groups (control, BCB positive and BCB negative) using Generalized Estimating Equations, followed by Bonferroni's correction for multiple comparisons. RESULTS: Nuclear maturation was higher in BCB positive compared to BCB negative, after 24 and 48 h of IVM (p = .004 and p = .032). The control group was equal to BCB positive. There was no difference among groups analyzing meiosis resumption and degeneration rates. CONCLUSION: The BCB test can be a good marker in pre-selection procedures of developmentally competent human oocytes aspirated during CS.

Ovarian steroid hormones modulate the expression of progesterone receptors and histone acetylation patterns in uterine leiomyoma cells.

Uterine leiomyomas are the most common benign smooth muscle cell tumors in women. Estrogen (E2), progesterone (P4) and environmental factors play important roles in the development of these tumors. New treatments, such as mifepristone, have been proposed. We evaluated the gene expression of total (PRT) and B (PRB) progesterone receptors, and the histone acetyltransferase (HAT) and deacetylase (HDAC) activity after treatment with E2, P4 and mifepristone (RU486) in primary cell cultures from uterine leiomyoma and normal myometrium. Compared to myometrium, uterine leiomyoma cells showed an increase in PRT mRNA expression when treated with E2, and increase in PRB mRNA expression when treated with E2 and P4. Treatment with mifepristone had no significant impact on mRNA expression in these cells. The HDAC activity was higher in uterine leiomyoma compared to myometrial cells after treatment with E2 and E2 + P4 + mifepristone. HAT activity was barely detectable. Our results suggest that ovarian steroid hormones modulate PR, and mifepristone was unable to decrease PRT and PRB mRNA. The higher activity of HDAC leiomyoma cells could be involved in transcriptional repression of genes implicated in normal myometrium cell function, contributing to the maintenance and growth of uterine leiomyoma.

Effect of Helicobacter pylori on NFKB1, p38α and TNF-α mRNA expression levels in human gastric mucosa.

Helicobacter pylori infects ~50% of the world population, causing chronic gastritis and other forms of cellular damage. The present study assessed the influence of H. pylori on the mRNA expression levels of nuclear factor-κB1 (NFKB1), p38α and tumor necrosis factor-α (TNF-α) in human gastric mucosa in a southern Brazilian population. Human gastric tissue was collected by upper endoscopy and H. pylori diagnosis was performed using a rapid urease test and histological analysis. Total RNA was extracted and purified for subsequent cDNA synthesis and analysis by quantitative polymerase chain reaction (qPCR). The gastric tissue samples were divided into four groups as follows: Normal, inactive chronic gastritis, active chronic gastritis and intestinal metaplasia. The SDHA gene was classified as the most stable when compared with ACTB, GAPDH, B2M and HPRT1 genes, and was therefore selected as the reference gene for qPCR data normalization. TNF-α mRNA expression was significantly higher in samples that were positive for H. pylori and with active chronic gastritis. However, no difference was detected in the mRNA expression levels of NFKB1 and p38α between the groups. The present study concluded that the presence of H. pylori is associated with TNF-α upregulation in human gastric mucosa, but had no effect on NFKB1 and p38α mRNA expression levels.

Safety of brilliant cresyl blue staining protocols on human granulosa and cumulus cells.

The selection of human immature oocytes destined for in vitro maturation (IVM) is performed according to their cumulus-oocyte complex (COC) morphology. In animal models, oocyte pre-selection with brilliant cresyl blue (BCB) staining improves fertilization and blastocyst rates and even increases the number of calves born. As the granulosa cells and cumulus cells (GCs and CCs) have a close relationship with the oocyte and are available in in vitro fertilization (IVF) programs, applying BCB staining to these cells may help to elucidate whether BCB shows toxicity to human oocytes and to determine the safest protocol for this dye. GCs and CCs were isolated from 24 patients who underwent controlled ovarian stimulation. After 48 h, cells were exposed to: Dulbecco's Modified Eagle Medium (DMEM) with or without phenol red, DPBS and mDPBS for 60 min; 13, 20 and 26 µM BCB for 60 min; and 60, 90 or 120 min to 13 µM BCB. Cellular viability was tested using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. The 20 and 26 µM BCB exposures resulted in lower cell viability, similar to when cells were exposed to BCB for 90 or 120 min. GCs and CCs viabilities were equal among control group and 13 µM BCB group after 60 min. BCB staining was not toxic to GCs and CCs when the regime of 13 µM BCB for 60 min was used. Due to the close molecular/biochemical relationship between these cells and the gamete, we propose that it is unlikely that the use of BCB could interfere with the viability/health of human oocytes.

Progesterone Upregulates Gene Expression in Normal Human Thyroid Follicular Cells.

Thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. There are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium-iodide symporter NIS, thyroglobulin TG, thyroperoxidase TPO, and KI-67 genes expression, in normal thyroid follicular cells, derived from human tissue. NIS, TG, TPO, and KI-67 mRNA expression increased significantly after TSH 20 µUI/mL, respectively: 2.08 times, P < 0.0001; 2.39 times, P = 0.01; 1.58 times, P = 0.0003; and 1.87 times, P < 0.0001. In thyroid cells treated with 20 µUI/mL TSH plus 10 nM progesterone, RNA expression of NIS, TG, and KI-67 genes increased, respectively: 1.78 times, P < 0.0001; 1.75 times, P = 0.037; and 1.95 times, P < 0.0001, and TPO mRNA expression also increased, though not significantly (1.77 times, P = 0.069). These effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. This work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth.

Decreased Expression of GPER1 Gene and Protein in Goiter.

Goiter is more common in women, suggesting that estrogen could be involved in its physiopathology. The presence of classical estrogen receptors (ERα and ERß) has been described in thyroid tissue, suggesting a direct effect of estrogen on the gland. A nonclassic estrogen receptor, the G-protein-coupled estrogen receptor (GPER1), has been described recently in several tissues. However, in goiter, the presence of this receptor has not been studied yet. We investigated GPER1 gene and protein expressions in normal thyroid and goiter using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. In normal thyroid (n = 16) and goiter (n = 19), GPER1 gene was expressed in all samples, while GPER1 protein was expressed in all samples of normal thyroid (n = 15) but in only 72% of goiter samples (n = 13). When comparing GPER1 gene and protein levels in both conditions, gene expression and protein levels were higher in normal thyroid than in goiter, suggesting a role of this receptor in this condition. Further studies are needed to elucidate the role of GPER1 in normal thyroid and goiter.

Modulation of apoptosis by sulforaphane is associated with PGC-1α stimulation and decreased oxidative stress in cardiac myoblasts.

Sulforaphane is a naturally occurring isothiocyanate capable of stimulating cellular antioxidant defenses and inducing phase 2 detoxifying enzymes, which can protect cells against oxidative damage. Oxidative stress and apoptosis are intimately involved in the pathophysiology of cardiac diseases. Although sulforaphane is known for its anticancer benefits, its role in cardiac cells is just emerging. The aim of the present study was to investigate whether sulforaphane can modulate oxidative stress, apoptosis, and correlate with PGC-1α, a transcriptional cofactor involved in energy metabolism. H9c2 cardiac myoblasts were incubated with R-sulforaphane 5 µmol/L for 24 h. Cell viability, ANP gene expression, oxidative stress and apoptosis markers, and protein expression of PGC-1α were studied. In cells treated with sulforaphane, cellular viability increased (12 %) and ANP gene expression decreased (46 %) compared to control cells. Moreover, sulforaphane induced a significant increase in superoxide dismutase (103 %), catalase (101 %), and glutathione S-transferase (72 %) activity, reduced reactive oxygen species levels (15 %) and lipid peroxidation (65 %), as well as stimulated the expression of the cytoprotective enzyme heme oxygenase-1 (4-fold). Sulforaphane also promoted an increase in the expression of the anti-apoptotic protein Bcl-2 (60 %), decreasing the Bax/Bcl-2 ratio. Active Caspase 3\7 and p-JNK/JNK were also reduced by sulforaphane, suggesting a reduction in apoptotic signaling. This was associated with an increased protein expression of PGC-1α (42 %). These results suggest that sulforaphane offers cytoprotection to cardiac cells by activating PGC1-α, reducing oxidative stress, and decreasing apoptosis signaling.

Are growth factor receptors modulated by metformin in human endometrial stromal cells after stimulation with androgen and insulin?

OBJECTIVE: To assess the effect of metformin on gene and protein expression of insulin receptor (IR) and IGF-1 (IGF-1R) receptor in human endometrial stromal cells after stimulation with androgen and insulin. STUDY DESIGN: Primary culture of endometrial stromal cells stimulated with estrogen, progesterone with or without androgen or insulin, and treated with metformin for 24 and 48 h, followed by RNA (qRT-PCR) and protein (Western blot) extraction and analysis. RESULTS: IR gene expression was increased after treatment with insulin (2.9-fold change, p = 0.027) and further after metformin treatment (4.7-fold change, p < 0.001), and in IGF-1R, the group treated with insulin (1.83-fold change) and metformin (1.78-fold change) showed more expression, than control group (p < 0.001). Similarly, IR protein expression was increased after addition of metformin and insulin (249,869 ± 15,878) in relation to the other groups (p < 0.001). Furthermore, cells treated with insulin (153,634 ± 29,123) and androgen plus insulin (162,854 ± 86,258) had a higher IR protein expression compared to control (104,654 ± 5,634) and androgen group (71,595 ± 3,439, (p = 0.045 and 0.021). In groups treated with insulin (127,711 ± 4,591) and androgen plus insulin (151,098 ± 5,194) the protein IGF-1R was increased compared to control (79,355 ± 3,470) and the androgen-only group (79,326 ± 3,114) (p < 0.001). CONCLUSION: Metformin in combination with insulin increased IR protein and gene expressions, while it had no influence on the protein expression of IGF-1R in endometrial stromal cells.

Human granulosa cells: insulin and insulin-like growth factor-1 receptors and aromatase expression modulation by metformin.

BACKGROUND/AIM: Granulosa cells are the source of the most important ovarian steroids. Even in patients without significant improvement in metabolic parameters, metformin has apparently an important effect on the ovary. The aim of this study was to evaluate gene and protein expression of an insulin receptor (IR), insulin-like growth factor-1 (IGF1) receptor (IGF1R) and aromatase in granulosa cells treated with metformin and insulin. METHODS: Luteinized granulosa cells were collected from 27 patients during in vitro fertilization procedures. Cells were isolated, stored in culture for 24 h and divided into four groups: control; metformin for 30 min, and metformin for 30 min plus insulin for 30 or 60 min. RESULTS: IR and IGF1R mRNA expression was significantly enhanced by metformin but was not affected by insulin. Aromatase mRNA expression was significantly reduced in metformin-incubated cells following stimulation with insulin for 30 min. No statistical differences were found in IGF1R and aromatase protein expression, and IR expression was not detected. CONCLUSION: A direct effect of metformin on the gene expression of IGF1R, IR and aromatase was observed. Further studies should investigate the role of IGF1R, IR and aromatase in ovarian physiology for a better understanding of the effect of metformin.

Mechanical processing of hyperviscous semen specimens can negatively affect sperm DNA fragmentation.

INTRODUCTION: The present study compared the DNA fragmentation in human sperm samples with reduced, physiological, and increased viscosity in order to evaluate whether the process used to reduce viscosity (expulsion of semen through a needle and syringe) alters significantly sperm DNA fragmentation. METHODS: The seminal parameters of semen samples from 123 patients were evaluated and classified according to their viscosity. Samples with increased viscosity were submitted to a process of expulsion of semen through a 10-mL syringe and an 18-gauge (18G) needle to reduce the seminal viscosity. The DNA fragmentation of all samples was analysed using TUNEL assay (Terminal deoxynucleotidyl transferase mediated dUTP Nick-end labelling assay); in samples with increased viscosity, the fragmentation was assessed before and after the process of expulsion with syringe and needle. RESULTS: There was no difference in DNA fragmentation between groups with different viscosity (P = 0.857). A significantly increase in sperm DNA fragmentation after expulsion of hyperviscous semen through the syringe was observed (P = 0.035). CONCLUSION: There was no difference in DNA fragmentation rate between samples with reduced, increased and physiological viscosities; however, the physical process of expulsion of semen through a syringe and needle increased sperm DNA fragmentation.

CCR2 and CCR5 genes polymorphisms in benign prostatic hyperplasia and prostate cancer.

Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are two chronic conditions, very common in aged men, that have been associated to inflammatory process. Chemokines and their receptors are recognized as critical mediators of inflammatory responses, they regulate immune cell migration and are implicated in tumor pathogenesis. The impact of two chemokine receptor gene polymorphisms, CCR2-64I (rs1799864) and CCR5-Δ32 (rs333), was evaluated in BPH and PCa. 385 DNA samples (130 BPH, 136 PCa, 119 healthy control) were genotyped. The allele frequencies were similar among control, BPH and PCa groups. Median of serum PSA levels was different between groups: 0.79, 1.45 and 6.91 ng/mL in control, BPH and PCa groups, respectively (all p<0.001). The prostate volume median was 20.00 cm(3) in the control group, thus, lower than BPH (35.35 cm(3)) and PCa (35.80 cm(3)) (both p<0.001), nevertheless no statistical significant difference was observed between BPH and PCa patients (p=0.172). Remarkably, CCR2-64I was a protective factor to PCa when compared with BPH (OR=0.550; 95%CI=0.311-0.975), although the statistically significant difference was lost after correction for multiple comparisons. No significant associations of CCR5-Δ32 variant were observed with BPH, PCa or PCa clinicopathologic status. Our data suggest the influence of CCR2-64I variant in the development of prostate cancer.

Reference gene for primary culture of prostate cancer cells.

Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

Androgen receptor GGC polymorphism and testosterone levels associated with high risk of prostate cancer and benign prostatic hyperplasia.

Polymorphic GGC repeats in the androgen receptor (AR) gene can alter transactivation of androgen-responsive genes and increase the risk of benign prostatic hyperplasia (BPH) and prostate cancer (PCa). We investigated the association between GGC repeat length, testosterone levels and the risk of developing PCa and BPH in a population from southern Brazil. A sample comprising 130 PCa, 126 BPH and 88 control patients was evaluated. DNA was extracted from leukocytes and the AR gene was analyzed by fragment analysis. The hazard ratio (HR) was estimated. GGC mean length was not different between the three study groups. The risk of developing PCa in individuals with GGC > 19 was 3.300 (95 %CI 1.385-7.874) higher when compared to the GGC ≤ 19 group (p = 0.007). The risk of developing PCa and BPH in individuals with total testosterone levels <4 ng/mL was 2.799 (95 % CI 1.362-5.754). (p = 0.005) and 2.786 (95 % CI 1.470-5.280) (p = 0.002), respectively. Total testosterone levels in patients with GGC > 19 were significantly lower when compared to patients in the GGC ≤ 19 group. Our data suggest that the presence of a high number of polymorphic GGC repeats in the AR gene is associated with an increased risk of developing PCa and BPH, and that lower testosterone levels also increase the risk of developing these diseases.