Correction to: miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells.

An amendment to this paper has been published and can be accessed via the original article.

miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells.

BACKGROUND: miRNA expression profiles in ectopic endometrium (EC) serving as pathophysiologic genetic fingerprints contribute to determining endometriosis progression; however, the underlying molecular mechanisms remain unknown. METHODS: miRNA microarray analysis was used to determine the expression profiling of EC fresh tissues. qRT-PCR was performed to screen miR-205-5p expression in EC tissues. The roles of miR-205-5p and its candidate target gene, angiopoietin-2 (ANGPT2), in endometriosis progression were confirmed on the basis of both in vitro and in vivo systems. miR-205-5p and ANGPT2 expression were measured by in situ hybridization and immunochemistry, and their clinical significance was statistically analysed. RESULTS: miR-205-5p was screened as a novel suppressor of endometriosis through primary ectopic endometrial stromal cell migration, invasion, and apoptosis assay in vitro, along with endometrial-like xenograft growth and apoptosis in vivo. In addition, ANGPT2 was identified as a direct target of miR-205-5p through bioinformatic target prediction and luciferase reporter assay. Re-expression and knockdown of ANGPT2 could respectively rescue and simulate the effects induced by miR-205-5p. Importantly, the miR-205-5p-ANGPT2 axis was found to activate the ERK/AKT pathway in endometriosis. Finally, miR-205-5p and ANGPT2 expression were closely correlated with the endometriosis severity. CONCLUSION: The newly identified miR-205-5p-ANGPT2-AKT/ERK axis illustrates the molecular mechanism of endometriosis progression and may represent a novel diagnostic biomarker and therapeutic target for disease treatment.

Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries.

Ovarian injury because of chemotherapy can decrease the levels of sexual hormones and potentia generandi of patients, thereby greatly reducing quality of life. The goal of this study was to investigate which transplantation method for human umbilical cord mesenchymal stem cells (HUMSCs) can recover ovarian function that has been damaged by chemotherapy. A rat model of ovarian injury was established using an intraperitoneal injection of cyclophosphamide. Membrane-labelled HUMSCs were subsequently injected directly into ovary tissue or tail vein. The distribution of fluorescently labelled HUMSCs, estrous cycle, sexual hormone levels, and potentia generandi of treated and control rats were then examined. HUMSCs injected into the ovary only distributed to the ovary and uterus, while HUMSCs injected via tail vein were detected in the ovary, uterus, kidney, liver and lung. The estrous cycle, levels of sex hormones and potentia generandi of the treated rats were also recovered to a certain degree. Moreover, in some transplanted rats, fertility was restored and their offspring developed normally. While ovary injection could recover ovarian function faster, both methods produced similar results in the later stages of observation. Therefore, our results suggest that transplantation of HUMSCs by tail vein injection represents a minimally invasive and effective treatment method for ovarian injury.

Comparison of cell proliferation, apoptosis, cellular morphology and ultrastructure between human umbilical cord and placenta-derived mesenchymal stem cells.

Research in mesenchymal stem cells (MSCs) is mainly focused on applications for treatments of brain and spinal cord injury as well as mechanisms underlying effects of MSCs. However, due to numerous limitations, there is little information on selection of appropriate sources of MSCs for transplantation in clinical applications. Therefore, in this study we compared various properties of human umbilical cord-derived MSCs (HUCMSCs) with human placenta-derived MSCs (HPDMSCs), including cell proliferation, apoptosis, cellular morphology, ultrastructure, and their ability to secrete various growth factors (i.e. vascular endothelial growth factor, insulin-like growth factors-1, and hepatocyte growth factor), which will allow us to select appropriate MSC sources for cellular therapy. Cell culture, flow cytometry, transmission electron microscope (TEM) and atomic force microscope (AFM) were used for assessment of HUCMSCs and HPDMSCs. Results showed that the two types of cells appeared slightly different when they were observed under AFM. HUCMSCs appeared more fibroblast-like, whereas HPDMSCs appeared as large flat cells. HUCMSCs had higher proliferative rate and lower rate of apoptosis than HPDMSCs (p<0.05). However, HPDMSCs secreted more of the three growth factors than HUCMSCs (p<0.05). Results of TEM revealed that the two types of MSCs underwent active metabolism and had low degree of differentiation, especially HUCMSCs. Results of AFM showed that HUCMSCs had stronger ability of mass transport and cell migration than HPDMSCs. However, HPDMSCs displayed stronger adhesive properties than HUCMSCs. Our findings indicate that different sources of MSCs have different properties, and that care should be taken when choosing the appropriate sources of MSCs for stem cell transplantation.

Prognostic implications of microRNA-100 and its functional roles in human epithelial ovarian cancer.

Dysregulation of microRNAs (miRNAs) has been found to be associated with a variety of diseases, including epithelial ovarian cancer (EOC). Recently, miR-100 was reported to be downregulated in human ovarian carcinoma, however, the clinical significance and functional roles of miR-100 expression in human EOC are unclear. TaqMan real-time quantitative RT-PCR assay was performed to detect the expression of miR-100 in 98 EOC tissues and 15 adjacent normal epithelial tissues. The relationship between miR-100 expression and clinicopathological factors in 98 EOC patients was statistically analyzed. The effect of miR-100 expression on patient survival was determined. Finally, the role of miR-100 in EOC cell growth and its possible mechanisms were analyzed with miR-100 precursor or inhibitor-transfected cells. We showed that the level of miR-100 was significantly lower in EOC tissues compared to adjacent normal tissues. Low miR-100 expression was found to be closely correlated with advanced FIGO stage, higher serum CA125 expression level and lymph node involvement. Also, low miR-100 expression was correlated with shorter overall survival of EOC patients, and multivariate analysis showed that the status of miR-100 expression was an independent predictor of overall survival in EOC. Additionally, miR-100 could affect the growth of EOC cells by post-transcriptionally regulating polo-like kinase 1 (PLK1) expression. Together, these results suggest that low miR-100 expression may be an independent poor prognostic factor and miR-100 can function as a tumor suppressor by targeting PLK1 in human EOCs.

[Inhibitory effect of short hairpin RNA targeting survivin gene on human ectopic endometrial cells].

OBJECTIVE: To construct a lentiviral expression vector for short hairpin RNA (shRNA) of human survivin gene, and assess its gene silencing effect in human ectopic endometrial cells. METHODS: Human survivin gene shRNA sequence was designed using a software available on-line. The synthesized shRNA sequence was cloned into the pGCL-GFP vector to construct LV-survivin shRNA, which was confirmed by PCR and DNA sequence analysis. The packaging 293T cells were cotransfected with LV-survivin shRNA, pHelper 1.0 and pHelper 2.0, and the titer of the lentivirus was determined. The recombinant lentivirus was injected into human ectopic endometrial cells and the survivin mRNA expression was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in comparison with that in the non-transfected and blank vector-transfected human ectopic endometrial cells. RESULTS: PCR analysis and DNA sequencing confirmed correct insertion of the shRNA sequence into the lentiviral vector. The titer of virus after packaging was 8x10(8) U/ml. Survivin mRNA expression in human ectopic endometrial cells transfected by LV-survivin shRNA was significantly inhibited compared with those in the non-transfected and empty vector transfected human ectopic endometrial cells (P<0.01), and no significant difference was found between the latter two groups. CONCLUSION: The lentiviral shRNA vector of survivin gene constructed can effectively inhibit the expression of survivin gene in human ectopic endometrial cells in vitro. This vector provides a tool for investigating the role of survivin gene in the occurrence and progression of endometriosis and for searching new therapeutic targets.

[Expression of hypoxia-inducible factor-1alpha in endometriosis].

OBJECTIVE: To detect the expression of hypoxia-inducible factor-1alpha(HIF-1alpha) in endometriosis and explore the possible role of HIF-1alpha in the pathogenesis of endometriosis. METHODS: Immunohistochemistry was performed to examine the expression of HIF-1alpha in 20 normal endometrium, 20 ectopic endometrium and 68 eutopic endometrium specimens from 68 endometriosis patients, and the results were analyzed statistically. RESULTS: The expression of HIF-1alpha was significantly increased in ectopic endometrium than in normal endometrium (P<0.01), and the expression did not undergo changes with the normal menstrual cycle in the three types of endometrium. CONCLUSION: HIF-1alpha expression increases in ectopic endometrium, suggesting that HIF-1alpha plays an important role in the pathogenesis of endometriosis.

[Evaluation of tumor necrosis factor-alpha and tumor necrosis factor-beta in serum of patients with endometriosis].

OBJECTIVE: To evaluate the levels of tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta) before and after conservative laparoscopic surgery in patients with endometriosis. METHODS: The levels of TNF-alpha and TNF-beta in the serum of both 82 patients with EMS and 68 controls were determined by ELISA. RESULTS: The levels of TNF-alpha and TNF-beta in the serum of patients with EMS were significantly higher than those of the controls (P < 0.01), and they increased with the clinical terms ( P < 0.05). After clearance of endometrosis foci with laparoscopic conservative surgery, the TNF-alpha levels decreased significantly in EMS III - IV, and TNF-beta levels decreased significantly in EMS I - IV. CONCLUSION: Measuring TNF-alpha and TNF-beta levels in the serum of patients with EMS may have important value in postoperative follow-up, surveillance and evaluation of the effectiveness of the surgery.

[Expressions of nuclear factor-kappaB and intercellular adhesion molecule-1 in endometriosis].

OBJECTIVE: To investigate the association of nuclear factor-kappaB (NF-kappaB) and intercellular adhesion molecule-1 (ICAM-1) with endometriosis. METHODS: Immunohistochemistry was used to examine the NF-kappaB and VEGF protein expression in 35 specimens of normal endometrium, 48 eutopic endometrium of endometriosis and 76 ectopic endometrium, and the results were analyzed statistically. RESULTS: The expressions of NF-kappaB and ICAM-1 were found mainly in the glandular epithelial cells of the three endometrium specimens, with the ectopic endometriotic tissues showing the highest and normal endometrium the lowest expressions (P<0.001). NF-kappaB and ICAM-1 expressions were positively correlated to normal, eutopic and ectopic endometrium (r=0.688, 0.773 and 0.777, respectively, P<0.01). CONCLUSION: NF-kappaB and ICAM-1 may participate in the process of the pathogenesis in endometriosis.

[Cloning, expression, purification and characterization of human endostatin gene].

OBJECTIVE: To procure biologically active human endostatin. METHODS: Human endostatin gene was acquired by means of reverse transcriptase (RT)-PCR and cloned into PGEM-T vector with subsequent sequence identification. The gene fragment was inserted into the prokaryotic expression vector pBV220 and transformed into E.coli DH5alpha strain. Endostatin expression in the E.coli was identified and the inclusion body isolated, purified and its activity analyzed. RESULTS: The obtained gene fragment 552 bp in length was identified as the functional section of human endostatin gene by sequence analysis, and SDS-PAGE analysis showed that the expressed product was the target protein with biological activity. CONCLUSION: Human endostatin gene was expressed in E.coli and the protein obtained can inhibit the proliferation of ECV 304 cells.

[Changes of serum epithelial neutrophil-activating peptide-78 in patients with endometriosis].

OBJECTIVE: To investigate the role of serum epithelial neutrophil-activating peptide-78 (ENA-78) in the pathogenesis of endometriosis. METHODS: Serum concentrations of ENA-78 were measured by means of enzyme-linked immunosorbent assay (ELISA) in 42 patients with endometriosis (20 in stages I and II and 22 in stages III and IV) in comparison with 25 women without endometriosis (control group). RESULTS: Serum concentrations of ENA-78 were significantly higher in endometriosis group than in the control group (2.97+/-1.91 ng/ml vs 0.72+/-0.24 ng/ml, P>0.001), and ENA-78 levels in stages III and IV were significantly higher than those in stages I and II (4.48+/-1.25 ng/ml vs 1.30+/-0.74 ng/ml, P>0.001). Significant difference in ENA-78 levels between follicular phase and luteal phase was found in neither of the groups. CONCLUSION: Patients with endometriosis have elevated serum ENA-78 levels, which might be a pathogenic factor of endometriosis.

[Clinical analysis of radical hysterectomy plus pelvic lymphadenectomy for patients with malignant uterine tumors: laparoscopy versus laparotomy].

OBJECTIVE: To compare clinical efficacy of laparoscopy with laparotomy in radical hysterectomy plus pelvic lymphadenectomy for patients with malignant uterine tumors. METHODS: This retrospective study population included two groups: (1) 26 patients underwent laparoscopy-assisted radical hysterectomy plus pelvic lymphadenectomy (group laparoscopy) and (2) 27 patients were treated with radical hysterectomy plus pelvic lymphadenectomy through laparotomy (group laparotomy). The tumor stage in these two groups of patients was matched. Multiple clinical parameters were observed and analyzed statistically. RESULTS: Compared to the patients in group laparotomy, the patients in group laparoscopy had a longer operative time (310 vs 238 min), more pelvic lymph nodes removed (22 vs 16), lower volume of blood loss (756 vs 1129 ml), and transfusion (321 vs 746 ml), a shorter postoperative gastrointestinal function recovery time (37 vs 62 h), a quicker return to normal temperature (5 vs 8 d), and a shorter period to use antibiotics (6 vs 8 d) (P < 0.01) However, there were no significant differences in the volume of pelvic drainage (321 vs 216 ml), urination recovery time (13 vs 10 d), number of WBC found (11 x 10(9)/L vs 10 x 10(9)/L), hospital stay (26 vs 28 d) and cost (25 986 vs 22 672 Yuan) between the two groups (P > 0.05). CONCLUSION: The clinical efficacy of radical hysterectomy plus pelvic lymphadenectomy by laparoscopy for patients with malignant uterine tumor is equal to that through laparotomy.

[Laparoscopic cytoreductive surgery for ovarian carcinoma: report of 4 cases].

Four cases of ovarian carcinoma treated with cytoreductive surgery under laparoscopy from October 2002 to June 2003 were reported. According to our preliminary experience, laparoscopy played an important role in diagnosis, staging and follow-up of ovarian carcinoma, and in comparison with conventional laparotomy, laparoscopic cytoreductive surgery may produce similar effect in the treatment of early-stage carcinoma.

[Expression of vascular endothelial growth factor and endostatin in peritoneal fluid of patients with endometriosis].

OBJECTIVE: To investigate the relationship of vascular endothelial growth factor (VEGF) and endostatin with endometriosis. METHODS: The levels of vascular endothelial growth factor and endostatin were measured using ELISA in 50 women with endometriosis (stage I-II26 cases and stage III-IV 24 cases) in comparison with 42 women without endometriosis. RESULTS: The VEGF, endostatin concentrations and VEGF/endostatin ratio in endometriosis group were significantly higher than the mean value in the control group (P<0.01). The VEGF and endostatin levels in stages III and IV were both significantly higher than those in stages I and II(397+/-81 pg/ml vs 315+/-64 pg/ml, 125+/-62 ng/ml vs 66+/-40 ng/ml, P<0.01). However, the VEGF/endostatin ratio was lower in the advanced stages. There was no significant difference between follicular phase and luteal phase except that the VEGF level in follicular phase was higher than that in luteal phases. CONCLUSIONS: Both VEGF and endostatin may play a role in the regulation of angiogenesis of endometriosis, and the balance of angiogenic stimulators and inhibitors may be critical to the development of endometriosis.

[Association between gene mutation of cytochrome P450 1A1 in exon 7 A4889G locus and susceptibility to endometriosis].

OBJECTIVE: To assess the possible association between gene mutation of cytochrome P450 1A1(CYP1A1) in exon 7 A4889G locus and the susceptibility to endometriosis (EM). METHODS: Allele specific-polymerase chain reaction method was used to analyze gene mutation in exon 7 A4889G locus of CYP1A1 in 76 patients with endometriosis and 80 healthy controls. RESULTS: The frequency of allele G on A4889G locus of CYP1A1 gene showed a significant difference between the study cohort and the control group (Chi2=7.498, P<0.01), with an odds ratio of 1.957. Statistically significant difference in the frequencies of genotypes AA, AG and GG was observed between the two groups (Chi2=6.915, P<0.05). Individuals with homozygotes for G allele were at higher risk of suffering from EM when compared against those with homozygotes for A allele, the odds ratio being 3.437 (Chi2=5.430, P<0.05). CONCLUSION: The above results suggest that gene mutation of CYP1A1 in exon 7 A4889G locus might be a genetic susceptible factor of endometriosis. The mutation allele of CYP1A1 gene appears to increase the risk of endometriosis.

[Association between glutathione S-transferase M1 gene deletion and genetic susceptibility to endometriosis].

OBJECTIVE: To evaluate the possible association of the glutathione S-transferase M1 (GSTM1) gene polymorphism with the susceptibility to endometriosis in women of Han nationality in Guangdong Province. METHODS: Polymerase chain reaction was used to identify the GSTM1 genotypes in 76 patients with endometriosis and 80 controls (surgical patients for gynecological problems other than endometriosis). RESULTS: The frequencies of the GSTM1 null genotypes in patients with endometriosis and controls were 65.8% and 46.3%, respectively, showing a significant difference between the endometriotic cohort and the control group (X(2)=6.03, P < 0.05). Individuals with GSTM1 null genotype were exposed to risks for endometriosis 2.24 times that of subjects without these genotypes OR=2.24, 95% CI=1.17-4.27 . CONCLUSION: GSTM1 gene deletion might bea risk factor for endometriosis in women of Han nationality who are native residents in Guangdong Province.

[Preparation and identification of monoclonal antibodies against Penicillium marneffei].

OBJECTIVE: To prepare and identify monoclonal antibodies (mAbs) against Penicillium marneffei. METHODS: Recombinant mannoprotein1 (MP1) of Penicillium marneffei was used to immunize BALB/c mice, and anti-MP1 mAbs were obtained by means of hybridoma. Screening and identification of the mAbs were subsequently performed with indirect enzyme-linked immunosorbent assay and Western blotting, respectively. RESULTS: Four hybridomas producing antibodies against Penicillium marneffei were obtained, and the IgG isotypes of the 4 mAbs were identified as IgG1 with affinity constants (K) of 8.2x10(-9), 4.7x10(-9), 6.5x10(-9) and 2.7x10(-9), respectively. Western blotting demonstrated specific recognition of Aspergillus fumigatus MP1 by the obtained mAbs. CONCLUSION: The 4 hybridomas producing anti-MP1 mAbs with high specificity and affinity can be of significant value in the diagnosis of Penicilliosis marneffei infections.

Association of HLA-DPB1 gene with endometriosis in women of Guangdong Province in China.

OBJECTIVE: To analyze the hereditary susceptibility in patients with endometriosis by way of genotyping of HLA-DPB1 alleles. METHODS: The allelic types of HLA-DPB1 were detected by sequence-based typing (SBT) in 38 patients with endometriosis and 36 healthy women as the control. RESULT: Significant differences in the frequency of HLA-DRB1 allele was not observed between endometriotic patients and normal subjects. CONCLUSION: HLA-DPB1 allele may not be related to endometriosis.

Expression of metalloproteinase-9 in ectopic endometrium in women with endometriosis.

OBJECTIVE: To study the expression of matrix metalloproteinase-9 (MMP-9) in ectopic and eutopic endometrium from women with endometriosis, thereby to determine the role of matrix metalloproteinases in the pathogenesis of endometriosis. METHODS: Both ectopic and eutopic endometrium tissue specimens were obtained from 30 women with endometriosis, and gelatinase activity in the specimens was detected by zymography and quantitated by Gel Documentation Analysis System. RESULTS: MMP-9 was detected in both eutopic and ectopic endometrium, but the latter contained higher levels of MMP-9 than the former did, and as the endometriosis worsened, MMP-9 expression tended to increase further. CONCLUSION: Endometriotic tissue possesses higher level of gelatinase activity than eutopic endometrium from patients with endometriosis does, and MMP-9 might be of importance for the implantation and invasive growth of endometriotic tissue that may lead to endometriosis.

[Susceptibility to endometriosis in women of Han Nationality in Guangdong Province associated with Msp I polymorphisms of cytochrome P450 1A1 gene].

OBJECTIVE: To assess the possible association of the Msp I polymorphisms of cytochrome P4501A1(CYP1A1) gene with the susceptibility to endometriosis in women of Han Nationality in Guangdong Province. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was employed to analyze the 3 genotypes m1m1, m1m2 and m2m2 in 3'-flanking region of CYP1A1 in 76 patients with endometriosis and 80 healthy controls. RESULTS: The frequencies of genotypes m1m1, m1m2 and m2m2 were 30.3 %, 50.0 % and 19.7 % in patients with endometriosis while 42.5 %, 45.0 % and 12.5 % in the controls, respectively, showing no statistically significant difference in the frequencies of the three genotypes between the 2 groups. The frequencies of two alleles were of no significant difference between the patients and controls, either. CONCLUSION: Msp I polymorphisms of cytochrome P4501A1 in itself might not be associated with the susceptibility to endometriosis in women of Han Nationality in Guangdong Province.