Search

The purpose of the present study was to study the characteristics of epidemic growth factor receptor (EGFR) gene distribution in patients with non-small cell lung cancer (NSCLC), and to detect the mutation rate of EGFR gene by Sanger sequencing and amplification refractory mutation system (ARMS)-PCR. Paraffin-embedded sections of NSCLC tissues from 399 NSCLC patients diagnosed in Renmin Hospital of Wuhan University were collected, 103 of them were detected for exons 18-21 mutation of EGFR by Sanger sequencing method, 296 cases were detected for exons 18-21 mutation by ARMS-PCR method. DNA extraction of both groups was performed with Qiagen QLAamp DNA FFPE Tissue KIT. Comparisons of detection rates between the two methods were conducted by row X list chi-square test. The total mutation rate of EGFR gene detected by Sanger sequencing was 21.4%, exons 18-21 and combined mutation rates were 1.0%, 9.7%, 1.0%, 7.8% and 2.0%, respectively. And the proportions were 4.7%, 45.2%, 4.7%, 36.3% and 9.4% respectively. The total mutation rate detected by ARMS-PCR was 51.4%, exons 18-21 and combined mutation rates were 2.7%, 27%, 1.7%, 18.2% and 1.7%, respectively. The proportions were 5.3%, 52.6%, 3.3%, 35.5% and 3.3% respectively. Further analysis of mutation rate showed that there was significant difference between the two methods in detecting total mutation of EGFR gene (P

Protein phosphatase 2A (PP2A) was reported to play an important role in cancer development; however, the relationship between PP2A and cervical cancer development has yet to be fully understood. The present study aimed to explore the role of PP2A in the development of cervical cancer. Serum levels of PP2A were detected by ELISA in 23 patients with cervical cancer and 30 patients with benign cervical lesions. Furthermore, the PP2A activities and the mRNA and protein levels of PP2A were measured in cervical cancer (n=8) and chronic cervicitis (n=10) tissues. The results showed that the serum levels of PP2A were significantly reduced in patients with cervical cancer. Further studies showed that not only the activities of PP2A but also the mRNA and protein levels of PP2A were significantly decreased in cervical cancer tissues. Wound healing and Transwell assays demonstrated that pharmacological and genetic upregulation of PP2A could inhibit the migration of HeLa cells, but the downregulation of PP2A promoted cellular migration. The activation of PP2A also inhibited the remodeling of actin and the activity of mitogen-activated protein kinases (MAPKs) including p-JNK, p-p38 and p-ERK. Meanwhile, the activation of PP2A was found to downregulate MMP-9 levels, which further inhibited the migration and invasion of HeLa cells. In conclusion, our data suggest that the activity and expression of PP2A are significantly reduced in cervical cancer tissues, and the activation of PP2A may inhibit the migration of cervical cancer cells by inhibiting the phosphorylation of p-JNK, p-p38 and the p-ERK/MAPK signaling pathway as well as by downregulating MMP-9, implying that PP2A plays an important role in cervical cancer development.

We aimed to determine the effect of YY1 expression on the expression profile of long noncoding RNAs (lncRNAs) in trophoblasts, and we studied the involvement of certain lncRNAs and YY1 in the pathogenesis of recurrent miscarriage (RM). RT2 lncRNA PCR arrays revealed that YY1 overexpression in trophoblasts significantly promoted the expression of the HOX transcript antisense RNA HOTAIR and demonstrated that HOTAIR expression was significantly lower in the RM trophoblasts than in control trophoblasts. Ectopic HOTAIR overexpression and knockdown experiments revealed that it was a novel target of YY1. Bioinformatics analysis identified two YY1-binding sites in the HOTAIR promoter region, and chromatin immunoprecipitation (ChIP) analysis verified that YY1 binds directly to its promoter region. Interestingly, HOTAIR overexpression enhanced trophoblast invasion in an ex vivo explant culture model, while its knockdown repressed these effects. Furthermore, liquid chromatography-tandem mass spectrometry (LC-MS/MS) label-free quantitative proteomics screening revealed that HOTAIR overexpression activated phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling in trophoblasts. In an ex vivo explant culture model, HOTAIR overexpression effectively elevated matrix metalloproteinase 2 (MMP2) expression via the PI3K-AKT signaling pathway, enhancing trophoblast migration and invasion. These findings reveal a new regulatory pathway in which YY1 activates PI3K-AKT signaling via HOTAIR, promoting MMP2 expression, suggesting that HOTAIR is a potential therapeutic target for RM.

Development of a culture system that supports self-renewal and proliferation of spermatogonial stem cells (SSCs) is enormously valuable for experimental research and potential treatment for male infertility. Although several research groups had reported their successes in SSC isolation and culture, the two current accepted culture systems are different in cell enrichment methods, serum and growth factors. Previous researches also indicated SSCs from different mouse strains required different culture conditions. Here we report for the first time that SSCs from BALB/c mice could be cultured in an improved culture system for 3 months. The modified culture system consisted of an improved enzymatic procedure, the enrichment of undifferentiated spermatogonia by differential adherence selection of isolated SSCs, mouse embryonic fibroblast feeder cells, StemPro-34 SFM medium supplemented with glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor and GDNF-family receptor α1 (GFRα1). The improved digestion method increased the viability and enrichment efficiency of isolated testis cells. Furthermore, basal culture medium with 10% fetal bovine serum as selected medium could increase the number of germ cell colonies in the initiation stage of culture. Cultured SSCs were characterized morphologically and formed typical colonies. Immunocytochemical staining and RT-PCR showed that cultured SSCs expressed Oct-4, GFRα1, Sox2 and several other special genes resembling undifferentiated spermatogonia. Spermatogonia transplantation further confirmed that cultured SSCs were functionally normal and could restore complete spermatogenesis. The culture methods described here could serve as a paradigm to establish conditions for the culture of SSCs from other species, allowing identification of universal factors necessary for proliferation of SSCs.

Several reports on the association between the BRCC5 gene polymorphism and ovarian cancer risk have been published recently, but the estimates of the risk vary widely. We thus performed a meta-analysis in an effort to determine the association. To identify the eligible studies, we searched the PubMed, Embase, and CNKI databases, and reviewed all original studies retrieved as well as their citations. The risk of ovarian cancer was estimated using odds ratio (OR) and its 95 % confidence interval (CI). Meta-analysis of seven comparisons revealed an obvious rise in the risk of ovarian cancer under the CC vs. GG contrast model (OR = 1.52, 95 % CI = 1.07–2.16, P OR = 0.020). A similar increase was also indicated in the CC vs. GC + GG model (OR = 2.10, 95 % CI = 1.51–2.93, P OR

The objective of this study was to evaluate the biocompatibility of vessel extracellular matrix (VECM) from rabbit and to discuss the feasibility of vessel extracellular matrix as a matrix for urethral reconstruction. Primary cultured bladder smooth muscle cells isolated from New Zealand rabbits were implanted on VECM.The effects of VECM on rabbit bladder smooth muscle cells (RBSMCs) metabolic activity, attachment, proliferation were monitored in vitro with the aid of an inverted light microscope and a scanning electron microscope. The cell viability was monitored by MTT(methythiazolye tetrazolium bromide) after 1, 3, 5 days seeding. The in vivo tissue response to VECM was investigated by implanting them into the subcutaneous of rabbits. VECM exhibited a nontoxic and bioactive effect on RBSMCs. RBSMCs could be attached to and proliferated on VECM and maintained their morphologies. MTT assay showed RBSMCs cultured with the extracts of VECM were not significantly different from those of negative controls. In vivo, VECM demonstrated a favorable tissue compatibility without tissue necrosis, fibrosis and other abnormal response. VECM exhibited nontoxic and bioactive effects on RBSMC. It is a suitable material for urethral reconstruction.

Recently, microRNAs (miRNAs) have been shown to be involved in multiple biological pathways that can influence tumor progression and metastasis and they can serve as prognostic biomarkers in many cancers. The present study examined the prognostic significance of miR-215 in cervical cancer. The paraffin-embedded paired cervical scrape samples and tumor tissue samples from 302 patients with stage II cervical cancer were detected for the expression of miR-215 by using qRT-PCR. A miR-215-based classifier was established by using the Cox regression model. The prognostic and predictive accuracy of this classifier was determined in both the internal testing group of 138 patients, and the external independent group of 280 patients. Moreover, cervical cancer HeLa cells overexpressing miR-215 (HeLa-miR-215) were constructed and subcutaneously injected into the nude mice to examine the effect of miR-215 on tumor growth and metastasis in vivo. The results showed that the expression level of miR-215 was significantly higher in cervical cancer tissues than in paired normal tissues (P

To determine the frequencies and the characteristics of Y chromosome microdeletions (pl) in infertile men from central China to perform appropriate therapeutic choices by updated multiplex-PCR.In this study, we established a novel universal primer-multiplex-PCR (U-M-PCR) method to overcome the disadvantages of traditional multiplex PCR (M-PCR). We chose 15 sequence-tagged sites (STS) for detection of Y chromosome microdeletions. 540 infertile male patients and 100 healthy male controls were selected in the study.Of the 540 male infertility patients, 48 Y-chromosome microdeletions were detected, with a total deletion rate of 8.9 %. Of these deletions, the rate of AZFa deletions (sY84) was 0.5 % (3/540), the rate of AZFb deletions (sY143) was 0.7 % (4/540) and the rate of AZFc deletions (sY242, sY254 and sY255) was 7.6 % (41/540). Compared with AZF deletion rates by M-PCR, we found U-M-PCR could detect AZFc deletion more specifically (1.0 %7.6 %). No Y-chromosome microdeletions were detected in the 100 males with normal semen (the control group).U-M-PCR method was more specific to detect AZFc microdeletions. It is necessary to use the U-M-PCR method to offer genetic screening and counseling to infertile men prior to intracytoplasmic sperm injection (ICSI) or in-vitro fertilization (IVF).

Objective. To evaluate the influence of cold ischemia time on spermatogenesis in a rabbit model of testicular ischemia-reperfusion (I/R) injury. Material and Methods. The testicular I/R model was established in 24 male white rabbits. The left testes were preserved using HC-A solution at 0°C to 4°C. Cold ischemia time was 1, 2, 4, and 6 hours. The right testes without vascular occlusion were used as autologous controls. Twenty-four hours after reperfusion, the animals were sacrificed, and samples were obtained at bilateral orchiectomy. Another 8 normal testes were used as normal controls. Testicular tissue Johnsen score, malondialdehyde concentration, and apoptosis index were used to evaluate spermatogenesis. Results. The Johnsen score decreased and the apoptosis index increased with the duration of cold ischemia time in the I/R groups. The malondialdehyde concentration in the I/R groups was significantly higher than the sham and normal groups, and was highest at 4 hours of cold ischemia time. Conclusion. Testicular I/R injury is highly related to cold ischemia time. In rabbit models, testis transplantation is best performed within 4 hours of cold ischemia with traditional hypothermic protection.

To establish a long-term culture system for mouse spermatogonial stem cells (SSCs) and to discuss the key factor that supports mouse SSC self-renewal and proliferation.Testis cells from 4-6 days postpartum male transgenic BALB/c mce were collected by a modified two-step enzymatic digestion method and plated on 0. 2% elatin-coated tissue culture plates. The germ cells were enriched by differential adherence selections after respectively incubated for 1, 5 and 24 h and then plated on the mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder layer. The basal culture medium was StemPro-34 SFM supplemented with other 15 nutrient factors. The 20 ng/ml Glial cell line-derived neurotrophic factor (GDNF), 10 ng/ml basic fibroblast growth factor (bFGF) and 200 ng/ml GDNF-family receptor alpha 1 (GFRalpha1) were added to the serum-free medium to promote SSC proliferation. Several important surface markers and special genes were examined by immunocytochemical staining and RT-PCR analysis.After 3-4 days culture on the MEF feeder, SSCs proliferated continuously and formed typical colonies. SSCs from the BALB/c mice could be cultured in a steady state for 3 months. Immunocytochemical staining showed that Oct4 was specifically expressed in the cultured SSC nucleus and GFRalpha1 strongly expressed on the surface of the membrane. RT-PCR confirmed that the cultured SSCs expressed Oct-4, GFRalpha1, Sox2 and several other special genes resembling undifferentiated spermatogonia.SSCs from BALB/c mice could be cultured in the improved culture system for 3 months. This culture system could help further understand the regulating mechanism of SSCs and might provide an opportunity for the treatment of male infertility by SSC transplantation.