Neural-like cells from adipose-derived stem cells for cavernous nerve injury in rats.

Although the remaining nerve tissue can regenerate and partly restore erectile function when the cavernous nerve is compressed/severed and function lost, the limited regenerative ability of these nerve tissues often fails to meet clinical needs. Adipose-derived stem cells are easy to obtain and culture, and can differentiate into neural cells. Their proliferation rate is easy to control and they may be used to help restore injured cavernous nerve function. Sprague-Dawley male rats (n = 45) were equally randomized into three groups: fifteen rats as a sham-operated group, fifteen rats as a bilateral nerve crush (BINC) group (with no further intervention), fifteen rats as a BINC with intracavernous injection of one million neural-like cells from adipose-derived stem cells (NAS) (BINC + NAS) group. After 4 weeks, erectile function was assessed by stimulating the cavernous body. The number of myelinated axons in the dorsal cavernous nerve was determined by toluidine blue staining. The area of neuronal nitric oxide synthase-positive fibers in the dorsal penile nerve was measured by immunohistochemical staining. Masson staining was used to analyze the ratio of smooth muscle to collagen in penile tissue. The results demonstrate that maximal intracavernous pressure, the ratio of maximal intracavernous pressure to mean arterial pressure, the numbers of myelinated axons and neuronal nitric oxide synthase-positive fibers in the dorsal penile nerve, and the ratio of smooth muscle to collagen could be increased after cell transplantation. These findings indicate that neural-like cells from adipose-derived stem cells can effectively alleviate cavernous nerve injury and improve erectile function. All animal experiments were approved by the Animal Ethics Committee of Huazhong University of Science and Technology, China (approval No. 2017-1925) on September 15, 2017.

MicroRNA-30e inhibits adhesion, migration, invasion and cell cycle progression of prostate cancer cells via inhibition of the activation of the MAPK signaling pathway by downregulating CHRM3.

Prostate cancer (PCa) testing is currently based on measurement of serum prostate­specific antigen levels and digital rectal examination, which are limited by a low predictive value and the adverse effects associated with overdiagnosis and overtreatment. Recent studies have reported that the abnormal expression of microRNAs (miRNAs) is associated with the mechanism underlying the development of PCa. Thus, the aim of the present study was to investigate the effects of miR­30e and its target gene, M3 muscarinic acetylcholine receptor (CHRM3), on the adhesion, migration, invasion and cell cycle distribution of PCa cells via the mitogen­activated protein kinase (MAPK) signaling pathway. The differentially expressed genes were screened in the Gene Expression Omnibus database from a gene expression microarray (GSE55945) of PCa. PCa tissues and adjacent tissues were collected from patients with PCa. The PC­3 and DU145 human PCa cell lines were treated with activator, inhibitor and siRNAs. The effects of miR­30e on cell adhesion, migration, invasion and cell cycle distribution with the involvement of CHRM3 and the MAPK signaling pathway were investigated. The bioinformatics results demonstrated that the CHRM3 gene and the MAKP signaling pathway were involved in the progression of PCa, and has­miR­30e was selected for further study. The levels of miR­30e were significantly downregulated, while the levels of CHRM3 were obviously upregulated in PCa. CHRM3 was verified as a target gene of miR­30e. Upregulation of miR­30e and downregulation of CHRM3 decreased the levels of p­P38, p­extracellular signal­regulated kinase, p­c­Jun N­terminal kinase, p­c­fos and p­c­JUN, cell adhesion, migration and invasion ability, and the number of cells in the S phase, while they increased the number of cells in the G0 and G1 phases. The findings of the present study suggest that miR­30e inhibited the adhesion, migration, invasion and cell cycle entry of PCa cells by suppressing the activation of the MAPK signaling pathway and inhibiting CHRM3 expression. Thus, miR­30e may serve as a candidate target for the treatment of PCa.

MiR-33b inhibits osteosarcoma cell proliferation through suppression of glycolysis by targeting Lactate Dehydrogenase A (LDHA).

Osteosarcoma (OS) is one of the most common types of malignant bone tumor in adolescent. MicroRNAs (miRNAs) are widely studied regulatory molecules which play important roles in tumor development, differentiation, growth, invasion, chemosensitivity and cellular metabolism. Recently, miR-33b has been reported to act as a tumor suppressor in osteosarcoma. However, the detailed mechanism of miR-33b in regulating osteosarcoma cell proliferation remains unclear. In this study, we detected miR-33b was significantly downregulated in osteosarcoma tissues compared to their matched adjacent nontumor tissues. The decreased expressions of miR-33b were also found in multiple osteosarcoma cell lines, including MG63, Saos-2, U2OS and SOSP-9607 when compared to normal osteoblast cell line hFOB. Overexpression of miR-33b suppressed U2OS cell proliferation and anaerobic glycolysis. We identified Lactate dehydrogenase-A (LDHA) as a direct target of miR-33b in osteosarcoma tumors and cells by Western blot and luciferase assay. Moreover, inhibition of LDHA significantly suppressed glycolysis and cell proliferation of osteosarcoma cells. Restoration of LDHA in miR-33b-overexpressing osteosarcoma cells reversed the suppressive effect of miR-33b on cell proliferation. In addition, we report a significantly negative correlation between LDHA mRNA and miR-33b expression in osteosarcoma tumors: miR-33b is downregulated in OS tumors with high levels of LDHA (92.9%). Meanwhile, high miR-33b expressions were found majorly in OS tumors with low LDHA mRNA levels (82.4%). This study reveals that miR-33b plays a suppressive role in the regulation of osteosarcoma cell proliferation through direct targeting LDHA. The miR-33b/glycolysis/LDHA axis may contribute to development of therapeutic anti-tumor agents for osteosarcoma.

Cavernous nerve reconstruction with autologous vein graft and platelet-derived growth factors.

In this study, we investigated the feasibility of using autologous vein graft and platelet-derived growth factors to bridge transected cavernous nerve in a rat model. A short defect in the bilateral cavernous nerve was created and repaired with vein graft from the right jugular vein or vein graft plus platelet-derived growth factors. The 32 rats were divided into four groups, namely Group 1 - no repair as a negative control, Group 2 - vein graft alone, Group 3 - vein graft plus platelet-derived growth factors, and Group 4 - sham operation as a positive control. We evaluated nerve regeneration and functional recovery using retrograde tracing study with FluoroGold, Toluidine blue staining of cavernous nerve, and the intracavernous pressure at 3 months. Three months after surgery, rich FluoroGold-positive cells were observed in the sham and vein graft plus platelet-derived growth factors group, but very few were found in the no repair group. The number of myelinated axons of regenerated cavernous nerve and intracavernous pressure were increased obviously in the two vein graft groups, especially in the vein graft plus platelet-derived growth factors group. These findings confirm the feasibility of using autologous vein as guides for cavernous nerve regeneration, and the regeneration can be further enhanced when the vein is filled with platelet-derived growth factors.

Maternal gestational smoking, diabetes, alcohol drinking, pre-pregnancy obesity and the risk of cryptorchidism: a systematic review and meta-analysis of observational studies.

BACKGROUND: Maternal gestational smoking, diabetes, alcohol drinking, and pre-pregnancy obesity are thought to increase the risk of cryptorchidism in newborn males, but the evidence is inconsistent. METHOD: We conducted a systematic review and meta-analysis of studies on the association between maternal gestational smoking, diabetes, alcohol drinking, and pre-pregnancy obesity and the risk of cryptorchidism. Articles were retrieved by searching PubMed and ScienceDirect, and the meta-analysis was conducted using Stata/SE 12.0 software. Sensitivity analysis was used to evaluate the influence of confounding variables. RESULTS: We selected 32 articles, including 12 case-control, five nested case-control, and 15 cohort studies. The meta-analysis showed that maternal smoking (OR = 1.17, 95% CI: 1.11-1.23) or diabetes (OR = 1.21, 95%CI: 1.00-1.46) during pregnancy were associated with increased risk of cryptorchidism. Overall, the association between maternal alcohol drinking (OR = 0.97, 95% CI: 0.87-1.07), pre-pregnancy body mass index (OR = 1.02, 95% CI: 0.95-1.09) and risk of cryptorchidism were not statistically significant. Additional analysis showed reduced risk (OR = 0.89, 95% CI: 0.82-0.96) of cryptorchidism with moderate alcohol drinking during pregnancy. No dose-response relationship was observed for increments in body mass index in the risk of cryptorchidism. Sensitivity analysis revealed an unstable result for the association between maternal diabetes, alcohol drinking and cryptorchidism. Moderate heterogeneity was detected in studies of the effect of maternal alcohol drinking and diabetes. No publication bias was detected. CONCLUSION: Maternal gestational smoking, but not maternal pre-pregnancy overweight or obesity, was associated with increased cryptorchidism risk in the offspring. Moderate alcohol drinking may reduce the risk of cryptorchidism while gestational diabetes may be a risk factor, but further studies are needed to verify this.

Role of oxidative stress in surgical cavernous nerve injury in a rat model.

This study investigates the role of oxidative stress in surgical cavernous nerve (CN) injury in a rat model. Eighty-four male Sprague-Dawley rats were randomly divided into three groups: group 1, sham-operated rats; group 2, bilateral CN-crushed rats; and group 3, bilateral CN-transection-and-sutured-immediately rats. Oxidative stress was evaluated by malondialdehyde levels, super oxide dismutase (SOD) activities, and glutathione peroxidase (GPX) activities in serum. Erectile function was assessed by CN electrostimulation at 3 months with mean maximal intracavernous pressure (ICP) and maximal ICP per mean arterial pressure. Nerve injury was assessed by toluidine blue staining of CNs and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining of penile tissue. GPX protein expression and nitrotyrosine-3 (NT-3) levels in penile tissue were measured. Erectile function and the number of myelinated axons of CNs and NADPH-diaphorase-positive nerve fibers were statistically decreased between groups, from sham to crush to transection. For markers, both nerve-injury groups showed increased oxidative stress markers at early time points, with the transection group showing greater oxidative stress than the crushed group and values normalizing to sham levels by week 12. GPX expression and NT-3 levels in penile tissue were in concordance with the results of SOD and GPX. These results show that oxidative stress plays an important role in injured CNs, and different methods of CN injury can lead to different degrees of oxidative stress in a rat model.

Focal adhesion kinase regulates the phosphorylation protein tyrosine phosphatase-α at Tyr789 in breast cancer cells.

Protein tyrosine phosphatase (PTP)­α regulates the phosphorylation of focal adhesion kinase (FAK), which is important in cellular signal transduction and integration of proteins. It has been demonstrated that a FAK­Del33 mutation (deletion of exon 33; KF437463) in breast cancer tissues regulates cell migration through FAK/Src signaling activation. However, the detailed pathway for Src activation with FAK­Del33 remains to be elucidated. The present study used a retroviral expression system to examine changes in PTPα phosphorylation affected by the FAK­Del33 protein in breast cancer cells. Small interfering (si)RNA targeting PTPα interfered with the phosphorylation of Src. Wound­healing and migration assays were performed to identify cell morphology and quantitative analysis was performed by examining band color depth in western blot analysis. Significant differences were observed in the phosphorylation level of PTPα at Tyr789 between the FAK­Del33 and the wild­type breast cancer cells, suggesting that FAK regulated the phosphorylation level of PTPα at Tyr789 in breast cancer mutant FAK­Del33 cells. The gene expression profile with FAK siRNA did not alter the levels of phosphorylation in other mutants, including autophosphorylation disability (Y397F), ATP kinase dominant negative (K454R) and protein 4.1, ezrin, radixin, moesin domain attenuate (Δ375). FAK RNAi inhibited the activity of the FAK­Del33 at the Src site and rescued the elevated cell migration and invasion. The present study demonstrated for the first time, to the best of our knowledge, an increase in the phosphorylation level of PTPα­Tyr789 by its upstream activator, FAK­Del33, leading to Src activation in certain breast cancer cells, which has significant implications for metastatic potential.

Somatic mutational analysis of FAK in breast cancer: a novel gain-of-function mutation due to deletion of exon 33.

Focal adhesion kinase (FAK) regulates cell adhesion, migration, proliferation, and survival. We identified a novel splicing mutant, FAK-Del33 (exon 33 deletion, KF437463), in both breast and thyroid cancers through colony sequencing. Considering the low proportion of mutant transcripts in samples, this mutation was detected by TaqMan-MGB probes based qPCR. In total, three in 21 paired breast tissues were identified with the FAK-Del33 mutation, and no mutations were found in the corresponding normal tissues. When introduced into a breast cell line through lentivirus infection, FAK-Del33 regulated cell motility and migration based on a wound healing assay. We demonstrated that the expression of Tyr397 (main auto-phosphorylation of FAK) was strongly increased in FAK-Del33 overexpressed breast tumor cells compared to wild-type following FAK/Src RTK signaling activation. These results suggest a novel and unique role of the FAK-Del33 mutation in FAK/Src signaling in breast cancer with significant implications for metastatic potential.

[Protective effect of grape seed proanthocyanidin on spermatogenesis following testicular torsion/detorsion in mice].

OBJECTIVE: To investigate the protective effect of grape seed proanthocyanidin (GSP) on spermatogenesis following testicular torsion/detorsion in mice. METHODS: Twenty-four healthy male Kunming mice, aged 8 weeks and weighing 25 - 27 g, were randomly divided into a control, a torsion and a treatment group, each containing 8 animals. The unilateral testicular torsion/detorsion model was established in the treatment and torsion groups. Thirty minutes before detorsion, the animals of the treatment group were injected intraperitoneally with 50 mg/kg GSP, and those of the torsion group with normal saline at the same dose, both for 3 days postoperatively. On the 4th day after surgery, ipsilateral orchiectomy were performed to detect histopathological changes, the levels of superoxide dismutase (SOD) and malondialdehyde (MDA), and the apoptotic index (AI) of germ cells in all the mice. RESULTS: Compared with the torsion group, the treated mice showed significantly increased Johnsen score (5.00 +/- 1.85 vs 7.38 +/- 0.92, P < 0.05), seminiferous tubule diameter ([176.50 +/- 1.60]microm vs [178.75 +/- 1.58] microm, P > 0.05), spermatogenic cell layers (3.75 +/- 1.03 vs 5.75 +/- 0.71, P < 0.05) and SOD activity ([29.04 +/- 4.46] U/mg prot vs [52.67 +/- 3.57] U/mg prot, P < 0.05), but remarkably reduced level of MDA ([4.63 +/- 0.05] nmol/mg prot vs [2.91 +/- 0.04] nmol/mg prot, P < 0.05) and AI of germ cells ([40.50 +/- 1.60]% vs [16.25 +/- 1.67] %, P < 0.05). CONCLUSION: Grape seed proanthocyanidin has a protective effect against spermatogenic injury in mice, the mechanisms of which may be related to its actions of scavenging oxygen free radicals, inhibiting lipid peroxidation and improving the antioxidant ability of the body.

Downregulation of cold-inducible RNA-binding protein activates mitogen-activated protein kinases and impairs spermatogenic function in mouse testes.

Cold-inducible RNA-binding protein (CIRP) is an RNA-binding protein that is expressed in normal testes and downregulated after heat stress caused by cryptorchidism, varicocele or environmental temperatures. The purpose of this study was to investigate the functions of CIRP in the testes. We employed RNAi technique to knock down the expression of CIRP in the testes, and performed haematoxylin and eosin staining to evaluate morphological changes following knockdown. Germ cell apoptosis was examined by terminal deoxynucleotidal transferase-mediated dUTP nick end labelling (TUNEL) assay, and mitogen-activated protein kinase (MAPK) signalling pathways were investigated by Western blotting to determine the possible mechanism of apoptosis. We found that using siRNA is a feasible and reliable method for knocking down gene expression in the testes. Compared to controls, the mean seminiferous tubule diameter (MSTD) and the thickness of the germ cell layers decreased following siRNA treatment, whereas the percentage of apoptotic seminiferous tubules increased. The p44/p42, p38 and SAPK/JNK MAPK pathways were activated after downregulation of CIRP. In conclusion, we discovered that downregulation of CIRP resulted in increased germ cell apoptosis, possibly via the activation of the p44/p42, p38 and SAPK/JNK MAPK pathways.

[Estradiol stimulates proliferation of prostatic smooth muscle cells via estrogen receptor alpha and IGF1].

OBJECTIVE: To investigate the effects of estrogen receptor alpha (ERa) and insulin-like growth factor 1 (IGF1) on the proliferation of prostatic smooth muscle cells (PSMCs) in vitro. METHODS: The ERalpha shRNA expression frame was subcloned to the pGSadeno adenovirus vector by homologous recombination technology to construct the pGSaaeno-ERalpha vector. After the mouse PSMCs were transfected in vitro by pGSaaeno-ERalpha, the mRNA and protein expression levels of ERalpha were detected by RT-PCR and Western blot respectively. The expression of IGF1 in the ERa-reduced cells was determined by Western blot 6 hours after treatment with 17beta-estradiol (E2) at 10(-8) mol/L. The post-transfection activity of estrogen or exogenous IGF1 in the proliferation of PSMCs was evaluated by MTT chlormetric analysis. RESULTS: After treatment with E2, the proliferation of PSMCs and the expression of the IGF1 gene were significantly increased in the normal control group (P <0.05), but not obviously changed in the ERalpha-siRNA group (P> 0.05). And exogenous IGF1 failed to induce the proliferation of the ERalpha-reduced PSMCs. CONCLUSION: E2 induces the expression of IGF1 via ERalpha, and IGFl, with the interaction of ERalpha, promotes the proliferation of PSMCs.

[Induction of cell cycle arrest in bladder cancer RT4 cells by capsaicin].

OBJECTIVE: To study the effects of capsaicin on the growth of bladder cancer RT4 cell and its potential mechanism. METHODS: Cell counting kit-8 (CCK-8) assay and flow cytometry were employed to observe the effects of capsaicin (50, 100, 150, 200, 250 micromol/L) on cell growth, cell cycle and apoptosis. Capsaicin (0 micromol/L) was used as a control. The effects of mRNA and protein of transient receptor potential cation channel subfamily V 1 (TRPV1) on RT4 cells were tested by RT-PCR and immunofluorescence respectively. And the expressions of cell cycle protein P53, P21, CDK2 were detected by Western blot after the treatment of capsaicin. RESULTS: 100 micromol/L capsaicin significantly decreased the viability of RT4 cell [82.0% +/- 6.2% vs 100.0% +/- 12.4% (control), P = 0.036] while the cell viability was 7.8% +/- 2.9% at 250 micromol/L (P = 0.000). It was in a dose-dependent manner. On the other hand, capsaicin induced the cell cycle arrest of bladder cancer RT4 cells G(0)/G(1) phase in a dose-dependent way. The cell proportion of G(0)/G(1) phase in the control was 37.4% +/- 5.6%, however, it was 72.4% +/- 5.3% at 250 micromol/L (P = 0.000). It was showed that TRPV1 mRNA and protein were expressed in RT4 cells. After a 48-hour treatment with capsaicin, the expressions of P53 and P21 were up-regulated in contrary to the expression of CDK2. CONCLUSION: Capsaicin induces the cell cycle arrest of bladder cancer RT4 cells G(0)/G(1) phase and growth inhibition via TRPV1 receptor by modulating the expression of P53, P21 and CDK2.

[Role of CXCL16/CXCR6 axis in the metastasis of human prostate cancer].

OBJECTIVE: To explore the roles of chemokine CXCL16 and its receptor CXCR6 in the directional invasion of human prostate cancer (PCa). METHODS: The expression of CXCL16/CXCR6 in PCa samples and osseous tissues was determined by immunohistochemistry. The expression of CXCR6 in PC3 and LNCap cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Then the effects of CXCL16 upon the migration and invasion of human PC3 and LNCap cells were examined by Matrigel invasion assay. RESULTS: The expression of CXCR6 protein was detected in all clinical PCa samples. But no CXCL16 protein was detected. Positive CXCL16 expression was observed in human osseous tissues. Both PC3 and LNCap cells expressed CXCR6 mRNA (0.38+/-0.054 vs 0.41+/-0.019 respectively) and protein. In addition, CXCL16 could promote the in vitro migration and invasion of PC3 and LNCap cell lines (invading cells 211.50+/-5.60 vs 89.25+/-3.31 respectively). Such a promoting effect of CXCL16 could not be blocked influenced by antiCXCL12 or antiCXCR4. CONCLUSION: CXCL16/CXCR6 axis may be another independent chemokine factor playing a significant role in the metastasis of prostate cancer.

Capsaicin mediates cell death in bladder cancer T24 cells through reactive oxygen species production and mitochondrial depolarization.

OBJECTIVES: To investigate the effects of capsaicin (CAP) on proliferation of bladder cancer T24 cells in vitro as well as on xenografts in nude mice in vivo. METHODS: T24 cells were assessed for cell viability and apoptosis by 3-(4, 5-dimethylthiazol-2-yl)-3, 5-diphenyltetrazolium bromide assay and flow cytometry analysis after incubation with different concentrations of CAP. To uncover the mechanism by which CAP affected the viability of T24 cells, intracellular production of reactive oxygen species (ROS) and mitochondrial membrane potential were assessed. To study the in vivo effects of CAP, T24 cells were grown as xenografts in nude mice and CAP (5 mg/kg by wt) was subcutaneously injected into nude mice with bladder tumors. RESULTS: CAP decreased the viability of T24 cells in a dose-dependent manner without marked apoptosis. CAP induced ROS production and mitochondrial membrane depolarization, thereby inducing cell death, not apoptosis, in T24 cells at a concentration of 100 microM or higher. Furthermore, these effects of CAP could be reversed by capsazepine, the antagonist of transient receptor potential vanilloid type 1 channel. In vivo experiment showed that CAP significantly slowed the growth of T24 bladder cancer xenografts as measured by size (661.80 +/- 62.03 vs 567.02 +/- 43.94 mm(3); P <.01). CONCLUSIONS: CAP mediates cell death in T24 cells through calcium entry-dependent ROS production and mitochondrial depolarization, and it may have a role in the management of bladder cancer.

Optimal dose of busulfan for depleting testicular germ cells of recipient mice before spermatogonial transplantation.

Successful spermatogonial transplantation requires depletion of the host germ cells to allow efficient colonization of the donor spermatogonial stem cells. Although a sterilizing drug, busulfan (Myleran), is commonly used for preparing a recipient mouse before transplantation, the optimal dose of this drug has not yet been defined. The present study investigated the effects of different doses of busulfan (10-50 mg per kg body weight) on survival rate, testicular mass and histomorphology, and on the haploid spermatids and spermatozoa of male BALB/c mice. The results suggest that a dosage of 30 mg kg(-1) is optimal for the ablative treatment with busulfan used to prepare the recipient mice. This dose results in an adequate depletion of the host germ cells for colonization of donor-derived spermatogonial stem cells and causes the lowest death rate of the animals.

[Protective effects of L-carnitine upon testicular ischemia-reperfusion damage in rats].

OBJECTIVE: To investigate the protective effects of L-carnitine upon testicular ischemia-reperfusion injury in rats. METHODS: Sprague-Dawley rats were divided into 3 groups (n = 10). In those animals undergoing unilateral testicular torsion, right testes were rotated 720 degrees for 2 h. Sham operated group served as a control group. Torsion group underwent 2 h torsion and saline was injected intraperitoneally at 30 min pre-detorsion. Treatment group underwent similar torsion but L-carnitine (500 mg/kg) was infused intraoperatively. The right testes of 5 animals in each group were excised after 4 h reperfusion for measuring the levels of malondialdehyde (MDA) and heat shock protein 70 (HSP70), evaluation of activities of antioxidant enzyme including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Histopathological changes and germ cell apoptosis indices (AI) were determined at 24 h post-detorsion in right testes of the remaining 5 animals in each group. RESULTS: The mean number of apoptotic nuclei per tubule cross section and the malondialdehyde level were significantly lower in treatment group as compared with torsion group [AI( 6.87 +/- 2.47) vs (17.13 +/- 3.56), MDA (160 +/- 15) vs (199 +/- 15) nmol/g]. Activities of antioxidant enzyme and the level of HSP70 were significantly higher in treatment group than those in torsion group [SOD (1638 +/- 153) vs (1078 158) U/g, CAT (317 +/- 28) vs (188 +/- 33) U/g, GPx (667 +/- 94) vs (311 +/- 65) U/g, HSP70 (0.87 +/- 0.13) vs (0.25 +/- 0.04)]. The pathological damage of testes in the treatment group was lighter than that in the torsion group (all P < 0.05). CONCLUSIONS: The administration of L-carnitine exerts a beneficial effect upon testicular ischemia-reperfusion injury. This effect may be achieved through an induced expression of HSP70.

[Establishment of PEG10 transgenic mouse and effects of PEG10 on growth, metastasis of transplanted tumor in mice].

OBJECTIVE: To establish PEG10 transgenic mice model and study the effect of PEG10 transgene on tumor growth and metastasis in mice. METHODS: The linearized expression element of pALB-PEG10, which contained mouse albumin promoter, structural gene of PEG10, and polyaenylation signal sequence, was microinjected into 3741 KM mouse fertilized ova. The manipulated embryos were then transplanted into the oviducts of 94 pseudopregnant recipient mice. All the newborn mice were screened by PCR to detect genomic DNA in tail tissue, then PEG10 mRNA and protein expression were detected by RT-PCR and western blot, respectively in the positive mice. Hepatoma cell H22 was subcutaneously inoculated into the right armpit of wild type mice and No.17, No.33 transgenic mice. Tumor size was measured every week. Mice were sacrificed on day 12 and then the tumors were exercised and weighted. Tumors and livers were fixed in formaldehyde and sectioned. The sections were stained with hematoxylin/eosin and examined under microscope. The expression of PEG10 protein was detected with immunohistochemistry method. RESULTS: Among the 43 off-springs, 3 were positive for tail tissue PEG10 gene examination, PEG10 was successfully expressed in the liver of the randomly selected transgenic mouse. H22 tumor grew faster in all the transgenic mice than in wild type mice. The average size and weight of tumors between the transgenic mice and wild type mice were significantly different (P < 0.05). Most tumors in the transgenic mice invaded the surrounding tissues and showed liver metastasis, PEG10 protein was expressed in liver. In contrast, nearly all the tumors in wild type mice were capsulized and PEG10 was not expressed in liver. CONCLUSION: Our results showed that the PEG10 gene could be expressed in the liver of the transgenic mice. PEG10 promotes growth, invasion, and metastasis of transplanted H22 tumors in mice.

[The effect of diethylstilbestrol on inducing abdominal cryptorchidism and relevant genetic expression in rats].

OBJECTIVE: To study the effect of diethylstilbestrol (DES) at different doses on transabdominal testicular descent in rats and the expression of INSL3 in the testis and HOXA10 in the gubernaculum. METHOD: Fifty E13.5 (embryonic day 13.5) pregnant female SD rats were randomly divided into five groups that received a subcutaneous injection of DMSO, 2.5, 5.0, 10.0 and 20.0 mg/kg DES (group A, B, C, D and E), respectively. Male offspring were killed at E19.5, and then fetal mortality, the degree of transabdominal testicular ascent (DTA) was determined by a stereomicroscope. The mRNA expressions of INSL3 in the testis and HOXA10 in the gubernaculum were determined by RT-PCR. The expression of INSL3 protein was determined by Western blotting. RESULTS: Male fetal mortality in group A, B, C, D, and E were 3.57%, 6.90%, 12.00%, 19.23% and 36.36%, respectively, which showed a dose-effect relationship between DES and the male fatal mortality (r=0.999, P<0.01). DTA in group B, C, D and E were (23.7+/-1.7) U, (38.8+/-1.9) U, (49.3+/-1.8) U and (58.6+/-2.1) U that were significantly larger than that in group A [(8.5+/-1.3) U] (q=46.12, 88.53, 120.44 and 141.37, respectively, P<0.01). There was also a dose-effect relationship between DES and DTA. In group B, C, D, and E, the expression of INSL3 mRNA were 0.9570+/-0.1490, 0.6760+/-0.1380, 0.0170+/-0.0040 and 0.0013+/-0.0003, respectively; the expressions of INSL3 protein were 0.8360+/-0.1520, 0.5310+/-0.1070, 0.0140+/-0.0020 and 0.0011+/-0.0003, respectively, which were significantly larger than the expression of INSL3 mRNA (1.801+/-0.126) and INSL3 protein (1.612+/-0.134) in group A (qmRNA=40.4840, 52.4402, 83.1585 and 82.0582, respectively, and qprotein=38.6151, 52.2747, 77.2756 and 76.1983, respectively, P<0.01). The expression of HOXA10 mRNA in group A, B, C, D, and E were 0.945+/-0.125, 0.940+/-0.119, 0.656+/-0.115, 0.544+/-0.118 and 0.463+/-0.114, respectively. Compared with the expression of HOXA10 mRNA in group A, the expression of group B was not significantly different (q=0.2213, P>0.05), those in other groups were down-regulated significantly (q=12.4304, 17.2477 and 20.2789, respectively, P<0.01). CONCLUSION: DES inhibited transabdominal testicular descent dose-dependently via down-regulating the expression of INSL3. HOXA10 may play no role in low-dosage DES induced intra-abdominal cryptorchidism, but down-regulated HOXA10 mRNA was involved in high-dosage DES induced ones.

Prenatal exposure to diaethylstilbestrol in the rat inhibits transabdominal testicular descent with involvement of the INSL3/LGR8 system and HOXA10.

BACKGROUND: Prenatal exposure to diaethylstilbestrol (DES) has been found to lead to intra-abdominal cryptorchidism, but the mechanism is still not completely clear. This study investigated the roles of the INSL3/LGR8 system and HOXA10 in DES-induced intra-abdominal cryptorchidism (DIIAC). The effect of DES on steroidogenic factor-1 (SF-1), that has been reported to control transcription of insulin-like factor 3 (INSL3), was also investigated. METHODS: Fifty pregnant female SD rats at embryonic day 13.5 (E13.5) were randomly assigned to five groups that received a subcutaneous injections of dimethyl sulfoxide (control), 2.5 mg/kg, 5 mg/kg, 10 mg/kg, or 20 mg/kg of DES. Male offspring were sacrificed at E19.5, and fetal mortality and the degree of transabdominal testicular ascent (DTA) were determined under a stereomicroscope. The mRNA expression of INSL3 and SF-1 in the testis and leucine rich repeat-containing G protein-coupled receptors 8 (LGR8) and homeobox-A10 (HOXA10) in the gubernaculum were determined by RT-PCR. The expression of INSL3 protein was determined by Western blotting. RESULTS: Higher fetal mortality and DTA were induced by DES in a dose-dependent manner (P < 0.01). Compared with the control group, the expression of INSL3 and SF-1 mRNA were down-regulated in a dose-dependent manner (P < 0.01), as was INSL3 protein; HOXA10 in the 2.5 mg/kg group and LGR8 mRNA in the 2.5 mg/kg and 5 mg/kg groups were not significantly different (P > 0.05); HOXA10 mRNA in groups C, D, and E decreased significantly and LGR8 mRNA levels in groups D and E increased significantly (P < 0.05, P < 0.01, respectively). CONCLUSIONS: DES can inhibit transabdominal testicular descent in a dose-dependent manner via down-regulating the expression of INSL3, which is induced by down-regulating the expression of SF-1. HOXA10 may not be involved in DES induced intra-abdominal cryptorchidism at 2.5 mg/kg, but is involved at 5, 10 and 20 mg/kg. LGR8 may not be responsible for DES-induced transabdominal testicular maldescent.

[Expression of estrogen receptor beta in benign prostatic hyperplasia complicated by chronic prostatitis].

OBJECTIVE: To observe the expression of estrogen receptor-beta (ERbeta) in benign prostatic hyperplasia (BPH) complicated by chronic prostatitis, and to evaluate the correlation of chronic prostatitis with ERbeta expression. METHODS: Histological sections of prostate tissues were obtained from 60 BPH patients complicated by chronic prostatitis and divided into Group 1 (Grade 1), 2 (Grade 2) and 3 (Grade 3) according to the scores on the inflammation of the prostate tissues using the four-point scale designed by Irani et al. The expression of ERbeta was determined by the immunohistochemical method. RESULTS: There were 24 cases (40%) in Group 1, 21 (35%) in Group 2 and 15 (25%) in Group 3, with no statistically significant differences in age and prostate volume among the three groups (P > 0.05). The expression of ERbeta was significantly decreased in Groups 2 and 3 as compared with Group 1 (P < 0.01). CONCLUSION: The expression of ERbeta is reduced with increased scores on the inflammation of the prostate tissues in BPH patients, and the decreased ERbeta expression may be associated with the inflammatory stimulation of prostatitis.