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ABSTRACT: 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.
Oncology Reports 04/2012; 27(4):1238-44. · 1.84 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 04/2010; 30(4):859-62.
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ABSTRACT: To investigate the effect of small interfering RNA(siRNA) targeting NF-kappaB on migration ability of human umbilical venous endothelial cells.
EAhy926 cells (Human umbilical vein endothelial cell line) were cultured with stimulation of vascular endothelial growth factor (VEGF), and then were transfected with NF-kappaB -siRNA (40 pmol/L) by using catiotic liposome lipofectamine(TM); 2000 as the transfecting agent. Cells transfected with negative control-siRNA (40 pmol/L), empty Liposome and nothing as control. Forty-eight hours later, cells were collected and prepared for RT-PCR, Western blot analysis, and determination of the migration inhibition rate.
Compared with normal control group, both mRNA and protein levels of NF-kappaB were decreased obviously and at the same time cell migration inhibition rate was increased obviously in NF-kappaB-siRNA group. There were no significant differences between empty liposome group, negative control siRNA group and normal control group, respectively (P>0.05).
Synthetic siRNA can effectively inhibit endothelial cell migration ability by means of blocking the signaling pathway of NF-kappaB. And siRNA targeting NF-kappaB may be useful for antiangiogenesis treatment.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 05/2009; 25(5):428-30.
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ABSTRACT: To investigate the effect of small interference RNA (siRNA) targeting nuclear factor-kappaB (NF-kappaB) on endometriosis.
The eutopic endometrium of women with endometriosis were transplanted into the nonvascular region of 8-day-old chicken embryo chorioallantocic membrane (CAM), and the effects of NF-kappaB p65 siRNA on the vascularization and endometriotic lesion formation were tested with proper controls.
Transplantation of the endometrium onto the CAM resulted in a strong angiogenic response in the chicken tissue. The angiogenesis was significantly reduced and endometriotic lesion formation significantly suppressed with siRNA targeting NF-kappaB in comparison with the control group.
The NF-kappaB pathway is involved in the development of endometriotic lesions in vitro, and NF-kappaB gene silencing reduces endometriotic angiogenesis and promotes cell apoptosis in the endometriotic lesions, suggesting that NF-kappaB might be a good target for endometriosis treatment.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 05/2009; 29(4):757-9.
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ABSTRACT: To investigate the protective effect of estrogen against cyclophosphamide (CTX)-induced ovarian failure in female rats.
Sixty female Wistar rats (2-3 months old) were randomized into 4 groups to receive treatments with normal saline, CTX, estradiol (E2) or CTX+E2. Serum estradiol and follicle-stimulating hormone (FSH) concentrations were measured regularly in the 4 groups, and the weight of the ovary and uterus, follicle number and mean diameter of the follicles were compared.
Compared with CTX+EE2 group, the CTX group showed significantly increased FSH levels and decreased EE2 levels (P<0.05). The weight of the ovary and uterus and follicle number were significantly lower in CTX group than in CTX+EE2 group (P<0.05). No obvious differences in the indexes were found between the control and CTX+E2 groups.
Estrogen administration provides protection against CTX-induced ovarian damage in rats.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 05/2009; 29(5):872-5.
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ABSTRACT: To construct a lentiviral vector carrying human bcl-2 gene and investigate its expression in human ovarian granulosa cells (GCs).
Human bcl-2 gene was amplified from the plasmid pCMV-SPORT6 using PCR and subcloned into the lentiviral vector pGC-FU to construct the lentiviral expression vector pGC-FU- bcl-2. The bcl-2 gene insert was confirmed by restriction enzyme digestion and sequencing. The recombinant lentiviruses generated by 293T cells co-transfected with pGC-FU-bcl-2 and the packaging plasmids pHelper1.0 and pHelper2.0. The resulting recombinant lentivirus GC-FU-bcl-2 carrying bcl-2 and EGFP genes were then used to infect human ovarian granulosa cells. EGFP and bcl-2 protein expressions in 293T and human ovarian GCs were detected by fluorescent microscope and Western blotting.
The plasmid pGC-FU-bcl-2 carrying the correct bcl-2 gene sequence could be expressed in human ovarian GC cells, and the recombinant lentivirus GC-FU-bcl-2 was generated by the packaging 293T cells. Stable expression of EGFP and bcl-2 proteins were detected by fluorescent microscope and Western blotting in 293T and human ovarian GCs after the infection. The recombinant lentivirus efficiently delivered bcl-2 gene into human ovarian GCs, in which bcl-2 expression was expressed efficiently and stably.
The recombinant lentivirus GC-FU-bcl-2 has been successfully constructed, which is capable of delivering the target gene bcl-2 into human ovarian GCs for its stable expression.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 11/2008; 28(10):1856-9.
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ABSTRACT: To investigate the expression of vascular endothelial growth factor (VEGF) in eutopic and ectopic endometrium of in nude mice models of endometriosis and understand the role of VEGF in the development of endometriosis.
Nude mouse models of endometriosis were established by subcutaneous implantation and abdominal injection of human endometrium xenograft from patients with endometriosis. The morphology of the foci was observed microscopically, and VEGF expression was measured with immunohistochemistry.
Models of endometriosis were successfully established in 8 of the 10 mice receiving subcutaneous xenograft implantation and in 7 of the 10 mice with abdominal graft injection. Increased VEGF expression was observed in the ectopic endometrium in these models.
VEGF expression is increased in the ectopic endometrium of the nude mouse models, and this animal model may facilitate future studies of angiogenesis and antiangiogenesis therapy.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 06/2008; 28(5):816-9.
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ABSTRACT: To construct a recombinant adenovirus carrying human endostatin gene with AdEasy system.
Endostatin gene fragment was amplified from Pshuttle-Endostatin plasmid with PCR and subcloned into the pAdTrack-CMV shuttle vector. The resultant plasimid was cotransduced into E.coli BJ 5183 cells with pAdEasy-1 plasmid for homologous recombination. The linearized recombinant plasmid was subsequently transfected into AAV 293 cells, and the recombinant adenovirus was detected by GFP, PCR and restriction analysis.
The positive clones of the recombinants were verified by restriction analysis and the titer of the virus reached 2.06 x 10(10)pfu/ml, suggesting successful construction of recombinant adenovirus pAd-Endo.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 11/2007; 27(10):1514-6.
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ABSTRACT: To investigate the efficient transfection of green fluorescent protein gene (GFP) mediated by recombinant adenovirus vector(Ad-GFP) to rat bone marrow mesenchymal stem cells (MSCs) in vitro.
Wistar rat bone marrow-derived MSCs were separated and purified in vitro by Percoll density gradient centrifugation combined with adherent cell culture followed by identification with flow cytometry. MSCs infected by Ad-GFP were observed and the transfection efficiency was assessed by fluorescence microscope. The proliferative ability of these cells was tested by CCK-8.
The transfection efficiency was as high as 90.0%. Expression of GFP gene of infected MSCs was stable for 1 month after infection. There was no statistically difference in proliferative ability between the infected MSCs and non-infected ones (P>0.05).
The infected MSCs with Ad-GFP expressed GFP with high efficiency and retain the ability of proliferation as non-infected MSCs. Transgection with Ad-GFP is a highly effective method for labeling MSCs.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 10/2007; 27(10):1517-20.
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ABSTRACT: To determine the polymorphism in +252 site of tumor necrosis factor-beta(TNF-beta) gene in patients with or without endometriosis, to evaluate the levels of TNF-alpha and TNF-beta in the serum with or without endometriosis, to explore the relation between polymorphism of TNF-beta gene and the genetic susceptibility of endometriosis, and to explore the pathogenic mechanism of endometriosis at gene level.
By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, polymorphism on +252 site of TNF-beta gene was measured in 82 patients with endometriosis (the endometriosis group) and 80 patients without endometriosis (the control group). With the sandwich-enzyme-linked immunosorbent assay (ELISA), the levels of TNF-alpha and TNF-beta in the serum of the two groups were determined.
The TNF-beta level in the serum in the endometriosis group with TNF-beta gene +252 site AA genotype significantly increased, compared with GG genotype (t=2.029, P<0.05); while TNF-alpha and TNF-beta level in the serum had no statistical significance in patients with other genotypes in TNF-beta gene +252 site in the endometriosis group and the control group.
TNF-beta gene +252 site AA genotype might be enhance TNF-beta level in the serum of patients with endometriosis.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 09/2007; 32(4):656-9.
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ABSTRACT: 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.
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.
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.
HIF-1alpha expression increases in ectopic endometrium, suggesting that HIF-1alpha plays an important role in the pathogenesis of endometriosis.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 05/2007; 27(4):538-40.
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ABSTRACT: To construct the recombinant adenovirus vector expressing human endostatin.
Human endostatin gene extracted from pGEM-T Easy vector containing the target gene fragment was successfully amplified using PCR and cloned into pShuttle2 vector. The target gene was subcloned into an adenovirus vector and the resulted recombinant adenovirus (Ad-hEndo) was linearized before transfected into HEK 293 packaging cells. The Ad-hEndo recombinant adenovirus was efficiently amplified in 293T cells and purified by CsCl density centrifugation, and the titer of the virus was determined.
The amplified hEndostatin cDNA was verified by PCR and sequencing, and the resulted virus titer reached 5.2 x 10(9) pfu/ml.
The recombinant adenovirus containing human endostatin gene has been successfully constructed, which may provide important basis for gene therapy research for angiogenesis-dependent diseases.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 01/2007; 26(12):1769-71.
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ABSTRACT: To study the polymorphism of +252 site in intron 1 of tumor necrosis factor (TNF)-beta gene in relation to genetic susceptibility of endometriosis.
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was employed to detect the polymorphism of +252 site in intron 1 of TNF-beta gene in 82 Chinese Han patients with endometriosis in Guangdong Province and 80 Han patients without endometriosis (control group), and the relation between TNF gene polymorphism and the risk of endometriosis was analyzed.
The +252 site of TNF-beta allele and genotype distribution showed significant difference between endometriosis and control groups (Chi2=6.562, P<0.05; chi2=6.562, P<0.05), and relative risk of endometriosis in relation to allele A was increased by 1.793 fold. The risk of endometriosis was 3.33-fold higher in women of AA genotype than those of GG genotype (Chi2=6.562, P<0.05).
Allele A in TNF-beta gene +252 site can significantly increase the relative risk of endometriosis in women in Guangdong, among which TNF-beta AA genotype might be one of the genetic susceptible factors for endometriosis.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 08/2006; 26(8):1163-5.
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ABSTRACT: To investigate the effects of human endostatin on proliferation of human umbilical venous endothelial cells, the apoptosis of the endothelial cells treated with recombinant human endostatin was examined by fluorescence method and the cell proliferation activity analyzed by MTT assay. The results showed that human endostatin inhibited the proliferation of the endothelial cells with ED(50) of 550 ng/ml, and the inhibitory effect was enhanced with the increase of endostatin concentration, suggesting a dose-dependent inhibitory effect of human endostatin on endothelial cell proliferation. The mechanism of antiangiogenesis induced by human endostatin is related to the inhibition of cell proliferation and induction of apoptosis of the endothelial cells.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 08/2006; 26(7):978-80.
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ABSTRACT: 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.
The levels of TNF-alpha and TNF-beta in the serum of both 82 patients with EMS and 68 controls were determined by ELISA.
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.
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.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 06/2005; 30(3):304-6.
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ABSTRACT: To investigate the association of nuclear factor-kappaB (NF-kappaB) and intercellular adhesion molecule-1 (ICAM-1) with endometriosis.
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.
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).
NF-kappaB and ICAM-1 may participate in the process of the pathogenesis in endometriosis.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 06/2005; 25(6):703-5.
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ABSTRACT: To procure biologically active human endostatin.
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.
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.
Human endostatin gene was expressed in E.coli and the protein obtained can inhibit the proliferation of ECV 304 cells.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 05/2005; 25(4):416-8.
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ABSTRACT: To investigate the role of serum epithelial neutrophil-activating peptide-78 (ENA-78) in the pathogenesis of endometriosis.
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).
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.
Patients with endometriosis have elevated serum ENA-78 levels, which might be a pathogenic factor of endometriosis.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 04/2005; 25(4):464-5.
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ABSTRACT: 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.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 05/2004; 24(4):479-80.
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ABSTRACT: To review our experience with hysteroscopic electroresection in the treatment of uterine benign lesions.
We conducted a retrospective analysis of 775 cases of hysteroscopic surgery in view of the operation time, blood loss, complications and prognosis.
The average operation time was 26.5+/-12.3 min and average blood loss 40.1+/-10.5 ml. The outpatients (n=539) were discharged 2 h after operation while the inpatients stayed for 2 or 3 d. Hyponatremia occurred in 1 case, and no death, uterine perforation or electric injury was recorded. During the follow-up for 3 months to 6 years, bleeding relapsed in 17 cases after transcervical resection of the endometrium, and the amenorrhea rate was 88.9%. The recurrence rate of endometrial polyp was 5.5% after electroresection of polyps. Menstruation as normal in all the cases after transcervical resection of the myoma. The uterine cavity became morphologically normal after transcervical resection of the septum without adhesions in the cavities. Improvement of menstruation was achieved in 55 cases (96.5%) after transcervical resection of the adhesion. A patient undergoing transcervical resection of the cervical lesion developed uterine and cervical adhesion 4 months later.
For the patients with intrauterine benign diseases, hysteroscopic electroresection provides effective therapeutic options for lessening the invasive injury and decreasing the complications and blood loss.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 05/2004; 24(4):467-9.
