LncRNA NNT-AS1 contributes to the cisplatin resistance of cervical cancer through NNT-AS1/miR-186/HMGB1 axis.

BACKGROUND: Cisplatin (DDP) is a major chemotherapeutic drug which was widely used for cervical cancer (CC) patients with advanced or recurrent although its limitation in the development of resistance. LncRNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) has been reported to be involved in the DDP resistance. However, the role of NNT-AS1 in DDP resistance in CC remain unknown. METHODS: The mRNA expression of NNT-AS1, microRNA-186 (miR-186) and HMGB1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation and apoptosis abilities were measured via MTT assay or flow cytometry, respectively. Western blot was used to measure the expression level of HMGB1, Bax, Bcl-2, Cleaved-caspase 3, N-cadherin, Vimentin and E-cadherin. Cell migration and invasion abilities were analyzed using Transwell assay. The interaction among NNT-AS1, miR-186 and HMGB1 was confirmed by luciferase reporter assay and RNA pull-down assay. Murine xenograft model was established using stably transfected SiHa/DDP cells. RESULTS: NNT-AS1 level was significantly elevated in CC tissues and cells, especially in DDP-resistant tumors and cell lines. Subsequently, loss-of function assays indicated that NNT-AS1 silence could attenuate DDP resistance by inhibiting proliferation, metastasis and EMT but inducing apoptosis in DDP-resistant CC cells. Besides that, knockdown of NNT-AS1 also antagonized DDP resistance in vivo. Bioinformatics predication revealed NNT-AS1 directly bound to miR-186 and HMGB1 was a target of miR-186. Additionally, NNT-AS1 could regulate HMGB1 expression via targeting miR-186. Furthermore, restoration experiments showed NNT-AS1 knockdown might improve DDP-sensitivity of CC cells via blocking HMGB1 expression by competitive interaction with miR-186. CONCLUSION: NNT-AS1 improved chemoresistance of DDP-resistant CC cells via modulating miR-186/HMGB1 axis.

MicroRNA-1297 contributes to the progression of human cervical carcinoma through PTEN.

BACKGROUND: The human cervical carcinoma oncogenic mechanisms still remain elusive. Thus, we proposed to understand the biological role of a newly discovered therapeutic miRNA. METHODS: MiR-1297 related to human cervical carcinoma was selected for this study. TaqMan qRT- PCR assay was used to profile miRNA, phosphatase and tensin homolog (PTEN) expression in randomly chosen tumour with non-tumour tissues, and the apoptosis factors expression. Cell proliferation was monitored by CCK-8 assay and colony formation assay. Apoptosis was determined by flow cytometry. Protein level was determined by western blotting. 3'UTR was performed to validate the direct binding sites of miR-1297 on PTEN. SPSS was used for statistical analyses. RESULTS: MiR-1297 is repressed and PTEN activated in human cervical cancer tissues. After miR-1297 overexpression, HeLa cells had an increase in cell proliferation and decrease in apoptosis. PTEN expression is negatively correlation with miR-1297. PTEN silencing display the similar pattern as miRNA-1297 overexpression to inhibit HeLa cell growth and apoptosis in vitro. CONCLUSIONS: Our data indicate that miR-1297 contribute to the human cervical carcinoma through PTEN. miR-1297 could be a reasonable miRNA for future studies.

miR424-5p functions as an anti-oncogene in cervical cancer cell growth by targeting KDM5B via the Notch signaling pathway.

AIMS: Aberrant expression of miRNAs exert the critical roles in carcinogenesis, including cervical cancer. Recent study corroborated the down-regulation of miR424-5p in uterine cervix adenocarcinoma. This research aimed to investigate the function and underlying mechanisms of miR424-5p in cervical cancer cell growth. MAIN METHODS: Tissues samples were collected from patients with cervical cancer and healthy control. The expression levels of miR424-5p were determined by qRT-PCR. After transfection with miR424-5p mimics or inhibitor, cervical cancer cell proliferation and apoptosis were evaluated by WST-1 and flow cytometry assay, respectively. The underlying mechanism involved in aforementioned processes was also explored. KEY FINDINGS: Expression of miR424-5p was notably decreased in cervical cancer tissues and cells. Overexpression of miR424-5p restrained cell proliferation and promoted cell apoptosis, but with little function in miR424-5p inhibitor-treated groups. Furthermore, KDM5B was identified as a direct target of miR424-5p as the evidence that miR-424-5p inhibited KDM5B expression and luciferase activity of KDM5B 3'-UTR. Here, KDM5B elevation majorly reversed miR424-5p-triggered inhibition in cell proliferation and increase in cell apoptosis. Moreover, silencing KDM5B expression also restrained cell growth. Additionally, miR424-5p overexpression inhibited the expression of Notch1 and Notch2, which was obviously rescued after KDM5B up-regulation. Simultaneously, blocking KDM5B also attenuated the activation of Notch pathway. Importantly, treatment with Notch agonist Jagged1 antagonized miR424-5p-mediated suppression on cell growth. SIGNIFICANCE: This research suggests that miR424-5p may act as a novel anti-oncogene in cervical cancer by blocking cell growth through targeting KDM5B-Notch pathway. Accordingly, our study will support a promising therapeutic strategy against cervical carcinoma.

Effects of letrozole in combination with low-dose intramuscular injection of human menopausal gonadotropin on ovulation and pregnancy of 156 patients with polycystic ovary syndrome.

OBJECTIVE: To explore the effects of letrozole (LE) in combination with low-dose intramuscular injection of human menopausal gonadotropin (HMG) on the ovulation induction and pregnancy of patients with polycystic ovary syndrome (PCOS). METHODS: A total of 156 patients with PCOS infertility were randomly divided into an LE group, a clomiphene citrate (CC) group and an LE + HMG group (n= 52). LE and CC were orally taken according to the prescribed dosage on the 3rd-5th days of menstruation respectively, and 75 IU HMG was given through intramuscular injection. The ovulation induction parameters and pregnancy outcomes were observed. RESULTS: The number of ovulation cycle of LE + HMG group was significantly higher than that of LE group (χ2=8.451, P<0.001). After injection of human chorionic gonadotropin, both endometrial thickness and number of mature follicles of LE + HMG group were significantly higher than those of other two groups (P<0.001), and the daily estradiol (E2) level was also higher (q=4.531, P<0.05). The pregnancy rate of LE + HMG group was 55.7%, which exceeded those of other two groups (compared to LE group, χ2=4.012, P<0.05). In LE + HMG group, the average medication cycle of clinically pregnant patients was (2.9 ± 0.3) weeks, which was significantly shorter than those of CC and LE groups (F=17.241, P<0.001). CONCLUSION: The regimen using LE in combination with low-dose intramuscular injection of HMG has satisfactory therapeutic effects on ovulation induction, short medication cycle and high clinical pregnancy rate, which is promising for treating patients with PCOS infertility.

[Effects of plasma from patients with preeclampsia on the proliferation and apoptosis of human umbilical vein endothelial cells and its relationship with lysophosphatidic acid receptors].

OBJECTIVES: To investigate the effects of plasma from the patients with preeclampsia on proliferation and apoptosis of human umbilical vein endothelial cells (HUVEC), and to explore the relationship between cell damage and lysophosphatidic acid (LPA) receptors. METHODS: Sixty patients with preeclampsia were recruited from October 2011 to June 2012 in the First Affiliated Hospital of Zhengzhou University. Among them, thirty cases were defined as the mild preeclampsia group and thirty cases were defined as the severe preeclampsia group. The other thirty healthy pregnant women were recruited in the healthy pregnant women group. The levels of plasma LPA in the three groups were measured. The HUVEC were cultured in vitro with plasma from the three groups, and a blank control group was set up as well. Proliferation and apoptosis of HUVEC were measured by MTT assay and flow cytometry. Immunohistochemistry of biotin streptomyces protein peroxidase (SP) method was used to measure the protein expression level of Edg 2, 4, 7. RESULTS: (1) The plasma LPA levels in the healthy pregnant woman group, mild preeclampsia group and severe preeclampsia group were (3.38 ± 2.08) µmol/L, (6.12 ± 0.22) µmol/L, (9.10 ± 0.17) µmol/L, respectively. The plasma levels of LPA in patients with preeclampsia were significantly higher than that in the healthy pregnant women (P < 0.01). (2) The proliferation rate of HUVEC in the mild and severe preeclampsia groups [(65.2 ± 2.7)% and (51.9 ± 2.8)%] were significantly lower than that in the healthy pregnant women group and the control group [(84.3 ± 3.1)% and (100.0 ± 0.0)%, P < 0.01]. (3) The early apoptosis rate, middle-late apoptosis rate and total apoptosis rate of HUVEC in the mild and severe preeclampsia groups [total apoptosis rate were (30.4 ± 2.0)% and (43.4 ± 2.5)%] were significantly higher than those in the healthy pregnant women group and the control group [total apoptosis rate were (18.6 ± 1.6)% and (8.0 ± 1.5)%, P < 0.01]. (4) The expression positive rates of Edg 2, 4, 7 proteins in the four groups were as following: mild preeclampsia group 83%, 80% and 73%; severe preeclampsia group 97%, 93% and 90%; healthy pregnant women group 40%, 40% and 37%, and the control group 10%, 10% and 7% respectively. The positive rates of HUVEC in the mild and severe preeclampsia groups were significantly higher than those in the healthy pregnant women group and the control group (P < 0.01). CONCLUSIONS: The plasma of patients with preeclampsia could inhibit proliferation and promote apoptosis of HUVEC, and induce the expression of Edg 2, 4, 7 proteins. It suggested that the increase of lysophosphatidic acid in plasma could be one of the reasons of endothelial cell damage in patients with preeclampsia.

[Influence of pertussis toxin on GPER-mediated activation of phosphatidylinositol 3-kinase/protein kinase B signaling induced by 17ß-estradiol in endometrial carcinoma cells].

OBJECTIVE: To investigate the influence of pertussis toxin (PTX) on G protein-coupled estrogen receptor (GPER)-mediated activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling activated by 17ß-estradiol (17ß-E2) in endometrial carcinoma cells. METHODS: Expressions of GPER protein were detected by immunohistochemical SP method in Ishikawa and HEC-1A cells. Changes of levels of GPER, ERα and ERß protein and the activation of Akt protein were observed by western blot in the two cells after they were treated by PTX for 30 minutes at different concentrations (0, 0.1, 0.5, 1.0 µg/ml), and then co-stimulated with with 1×10(-6) mol/L 17ß-E2 respectively at different time (Ishikawa 30 minutes, HEC-1A 15 minutes). RESULTS: (1) Immunohistochemical SP method showed that GPER was positive stained in cell cytoplasm of Ishikawa and HEC-1A cell. (2) After co-treated with PTX at different concentrations (0, 0.1, 0.5, 1.0 µg/ml) and 10(-6) mol/L 17ß-E2, in Ishikawa cell, the ratio of p-Akt/Akt was 0.74 ± 0.54, 0.34 ± 0.06, 0.18 ± 0.03, 0.07 ± 0.15, the gray values of GPER was 0.872 ± 0.490, 0.395 ± 0.054, 0.145 ± 0.014, 0.034 ± 0.008, and with increasing concentration of PTX, the ratio of p-Akt/Akt and the expression of GPER decreased gradually (P < 0.05), which was most obviously when the concentration was 1.0 µg/ml (F = 63.729, P = 0.0001; F = 160.284, P = 0.0001); ERα and ERß protein had no significant change among different groups (P > 0.05). In HEC-1A cell, the ratio of p-Akt/Akt was 0.73 ± 0.09, 0.26 ± 0.14, 0.11 ± 0.03, 0, the Gray values of GPER is 0.927 ± 0.134, 0.485 ± 0.022, 0.194 ± 0.004, 0, and with increasing concentration of PTX, the ratio of p-Akt/Akt and the expression of GPER decreased gradually (P < 0.05), which were also completely inhibited when the concentration was 1 µg/ml (F = 1039.321, P = 0.0001; F = 109.646, P = 0.0001), ERα protein had no significant differences (P > 0.05) among different groups. ERß was negatively expressed. CONCLUSION: The results proposed that the activation of PI3K/Akt signaling in Ishikawa and HEC-1A cells could be inhibited after blocking the role of GPER by PTX.

The G protein-coupled receptor GPR30 mediates the nontranscriptional effect of estrogen on the activation of PI3K/Akt pathway in endometrial cancer cells.

OBJECTIVE: The goal of this study was to investigate the effect of G protein-coupled receptor 30 (GPR30) on the activation of PI3K/Akt pathway induced by E2 in endometrial cancer cells. METHODS AND MATERIALS: Immunohistochemistry was performed to determine the location and expression of GPR30, estrogen receptors (ERs), Akt, and phosphorylated Akt. We also investigated the expression of GPR30, ERs, and the level of phosphorylation of Akt induced by E2 in endometrial cancer cells, Ishikawa cells, and HEC-1A cells. We down-regulated the expression of GPR30 in endometrial cancer cell lines by transfection with shGPR30-pGFP-V-RS, a GPR30 antisense expression vector. The cells were then subjected to a proliferation assay. Immunoprecipitation assay was performed to determine whether GPR30 directly bind to PI3K. The stable transfected cells resuspension of 100 µL (5 × 10(6) cells) was injected subcutaneously into the right flank of athymic mice to perform xenograft tumor formation assays. RESULTS: E2 stimulated cell proliferation and induced GPR30 expression and PI3K/Akt pathway activation in endometrial cancer cells, Ishikawa cells, and HEC-1A cells, whereas the expression of ERs remained unchangeable. Down-regulation of GPR30 decreased the phosphorylation of Akt and reduced cell proliferation, and GPR30 did not bind to PI3K. Down-regulation of GPR30 significantly inhibited the tumor growth of HEC-1A cells in athymic nude mice. CONCLUSIONS: These findings suggest that GPR30 mediates the nontranscriptional effect of estrogen on the activation of PI3K/Akt pathway in endometrial cancer cells.

HDAC6 siRNA inhibits proliferation and induces apoptosis of HeLa cells and its related molecular mechanism.

OBJECTIVE: To investigate the effects of histone deacetylase 6 (HDAC6) siRNA on cell proliferation and cell apoptosis of the HeLa cervical carcinoma cell line and the molecular mechanisms involved. METHODS: Division was into three groups: A, the untreated group; B, the control siRNA group; and C, the HDAC6 siRNA group. Lipofectamine 2000 was used for siRNA transfection, and Western blot analysis was used to determine the protein levels. Cell proliferation and apoptosis were characterized using a CCK-8 assay and flow cytometry, respectively. RESULTS: HDAC6 protein expression in the HDAC6 siRNA-transfection group was significantly lower (P < 0.05) than in the untreated and control siRNA groups. The CCK-8 kit results demonstrated that the proliferation of HeLa cells was clearly inhibited in the HDAC6 siRNA transfection group (P < 0.05). In addition, flow cytometry revealed that the early apoptotic rate (26.0% ± 0.87%) was significantly elevated (P < 0.05) as compared with the untreated group (10.6% ± 1.19%) and control siRNA group (8.61% ± 0.98%). Furthermore, Western blot analysis indicated that bcl-2 protein expression in the HDAC6 siRNA-transfection group was down-regulated, whereas the expression of p21 and bax was up-regulated. CONCLUSION: HDAC6 plays an essential role in the occurrence and development of cervical carcinoma, and the down-regulation of HDAC6 expression may be useful molecular therapeutic method.

Effect of coincubation time of sperm-oocytes on fertilization, embryonic development, and subsequent pregnancy outcome.

Several studies have reported improved IVF by shortening the time of sperm-oocyte coincubation from 16-18 hours to 1-4 hours. The objective of this study was to examine the advantages and disadvantages of a shortened sperm-oocyte coincubation time in order to assess the effects of this insemination method for clinical IVF practice. Two insemination methods, the shortened method (4 hours) and the standard method (16-18 hours) of coincubation of sperm-oocytes for two groups of patients based on the quality of sperm were compared. Group I, was composed of couples without male factor; Group II, involved couples with mild male factor. Fertilization, good quality embryos, clinical pregnancy, and implantation rates were compared by two different insemination methods. In Group I, fertilization, clinical pregnancy, and implantation rates were not different between the two insemination methods. However, the polyspermy rate was significantly higher (P < 0.05) in the shortened (7.3%) than in the standard (4.1%) insemination method. In Group II, the fertilization rate was significantly lower (P < 0.05) using the shortened insemination method (62.6%) compared to the standard insemination method (68.7%). When fertilization failed with the shortened insemination method, the clinical pregnancy and implantation rates were 34.7% and 24.1%, respectively, from the rescue intracytoplasmic sperm injection (ICSI). The live birth rate from the rescue ICSI was 32.0% with normal infants. The duration of sperm-oocyte coincubation does not affect fertilization, embryo quality, clinical pregnancy, and implantation rates. However, fertilization rates will decrease with the shortened insemination method when the sperm parameters are poor. From the results of the present study we suggest that the combination of the shortened sperm-oocyte coincubation and rescue ICSI method may be an efficient method for IVF treatment in order to prevent fertilization failure when sperm parameters were poor as mild male factor.

[Effect of GPER on the activation of PI3K/Akt induced by 17ß-estradiol in endometrial carcinoma cells].

OBJECTIVE: To investigate the expression of G protein-coupled ER (GPER) and ER in the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) induced by 17ß-estradiol (17ß-E(2))in endometrial carcinoma cells, Ishikawa and HEC-1A. METHODS: Expressions of GPER, ERα and ERß protein in Ishikawa and HEC-1A cells were detected by immunohistochemical SP method. Levels of GPER, ERα and ERß were examined by western blot in Ishikawa and HEC-1A cells after treated with 1×10(-6) mol/L 17ß-E(2) at different time (0, 15, 30, 60, 120 minutes). RESULTS: GPER was positive expressed in Ishikawa and HEC-1A cells. ERα and ERß were both positive expressed in Ishikawa cells. While, ERα was weakly expressed and ERß was almost negatively expressed in HEC-1A cells. Western blot analysis showed that 1×10(-6) mol/L 17ß-E(2) treatment, the Ishikawa and HEC-1A cells GPER protein level for 15 minutes markedly increased (P < 0.05), which Ishikawa 30 minutes, when cells reached the highest level (0.192 ± 0.004), HEC-1A cells for 15 minutes and reached the highest level (0.184 ± 0.006); Ishikawa and HEC-1A cells, Akt, activation of 15 minutes from the treatment start was significantly increased (P < 0.05), which Ishikawa cells for 30 minutes and reached the highest level (0.666 ± 0.021), HEC-1A cells for 15 minutes and reached maximum (0.788 ± 0.035); Ishikawa and HEC-1A cells, ERα and ERß protein expression did not change significantly (P > 0.05). CONCLUSION: GPER likely involved in non-nuclear activation of PI3K/Akt signaling pathways in endometrial carcinoma cells, Ishikawa and HEC-1A.

[Effects of PD98059 and LY294002 on subcutaneous xenograft of human endometrial carcinoma in nude mice].

OBJECTIVE: To investigate the effects of signal pathway inhibitors PD98059 and LY294002 on cell proliferation, apoptosis, expressions of phosphorylated extracellular signal-regulared kinase (p-ERK) and phosphorylated protein kinase B (p-Akt) in endometrial carcinoma xenografts. METHODS: Human endometrial carcinoma Ishikawa cells were cultured in vitro. The effects of PD98059 and LY294002 on proliferation, apoptosis, and cell cycle distribution of endometrial cancer cells were detected by monotetrazolium (MTT) assay and fluorescence-activated cell sorting technique. The models of xenografted tumor were established by the subcutaneous inoculation in 24 nude mice, and then they were randomly divided into 4 groups (n = 6), normal saline group, PD98059 group (PD group), LY294002 group (LY group) or PD98059 + LY294002 group (PD + LY group) by intraperitoneal injections, respectively. The anti-tumor efficacy was evaluated by measuring tumor volume and tumor growth status. The histopathological change of tumor specimens was observed using HE staining and terminal deoxynucleotidyl transferase-mediated dUTP-digoxigen in nick and labeling method (TUNEL) testing and the expression levels of p-ERK and p-Akt were detected by immunohistochemistry method. RESULTS: (1) The proliferation of Ishikawa cells were suppressed after treated by PD98059 and(or) Y294002, in which A(570) values of cells decreased showing both time-dependent and concentration-dependent manner (LY294002: F(group) = 9.801, P = 0.002; F(time) = 10.398, P = 0.001. PD98059: F(group) = 8.213, P = 0.015; F(time) = 6.839, P = 0.036). Cell cycle distribution analysis revealed that percentage of Ishikawa cells at G(0)/G(1) phase (F(time) = 35.049, P = 0.004; F(group) = 32.024, P < 0.01) increased and percentage of S phase cells (F(time) = 7.789, P = 0.049; F(group) = 30.132, P < 0.01) decreased significantly. The percentage of apoptotic cells increased significantly among PD group, LY group and PD + LY group, in which there were significant difference [(63.3 ± 0.5)% vs (30.7 ± 20.1)% vs (40.8 ± 1.3)%; F = 621.059, P < 0.01]. (2) Compared with the control group, the increasing of transplanting tumor volume in the treated groups were obviously (F = 23.545, P < 0.01), and the inhibited rate of the tumor was higher in PD + LY group than that in PD group or LY group [(68 ± 9)% vs (32 ± 16)% or (38 ± 17)%; F = 10.283, P < 0.05]. (3) HE staining shown that there were different degrees of necrosis for endometrial carcinoma cell in different groups. The apoptosis of tumor cells were significantly increased in treated groups by TUNEL testing [(13.7 ± 1.5)%, (14.1 ± 1.2)%, (29.0 ± 1.8)%; F = 320.344, P < 0.01]. Immunohistochemistry results demonstrated that the expressions of p-ERK and p-Akt in treated groups were lower than that in control group, of which LY + PD group was the lowest one. CONCLUSION: The signal pathway inhibitors PD98059 and LY294002 could inhibit the growth of human endometrial carcinoma in vivo and in vitro, in which may induce cell apoptosis.

BMP-6 inhibits MMP-9 expression by regulating heme oxygenase-1 in MCF-7 breast cancer cells.

PURPOSE: BMP-6, which belongs to the TGF-ß superfamily, is a multifunctional molecule with distinct abilities in embryogenesis and organogenesis. Our recent research has implied that BMP-6 may suppress breast cancer metastasis. In the present study, we extended to elucidate the molecular mechanism by which BMP-6 exerts its anti-tumorigenic effect. METHODS: The Boyden chamber assay was used to examine the ability of BMP-6 and HO-1 in MCF-7 malignant progress. RT-PCR, western blot, luciferase assay, and quantitative CHIP were used to determine the potential mechanism and signaling pathways by which BMP-6 and HO-1 function as anti-metastatic factors in MCF-7 cells. RESULTS: The Boyden chamber assay showed that BMP-6 inhibited the migration and invasion of MCF-7 cells, which effect was significantly deprived by knockdown of HO-1. We further demonstrated that BMP-6 treatment resulted in an activation of HO-1 transcription through the recruitment of Smad1/5 to the Smad-responsive element on its promoter. In addition, BMP-6-induced up-regulation of HO-1 exhibited an inhibitory effect on MMP-9 secretion in a paracrine action in MCF-7 cells. Overexpression of BMP-6 and HO-1 synergistically suppressed MMP-9 transcription, which effect was specifically mediated via the MAPK/p38/AP-1 signaling. However, blockade of HO-1 using ZnPPIX totally abolished BMP-6-regulated MMP-9 activation in MCF-7 cells. CONCLUSIONS: These observations suggest a novel role of BMP-6/HO-1 cascade to relieve breast cancer metastasis by regulating the secretion of growth factors in tumor microenvironment.

Homo-binding character of LMO2 isoforms and their both synergic and antagonistic functions in regulating hematopoietic-related target genes.

BACKGROUND: The human lmo2 gene plays important roles in hematopoiesis and is associated with acute T lymphocyte leukemia. The gene encodes two protein isoforms, a longer form LMO2-L and a shorter form LMO2-S. Both isoforms function as bridge molecules to assemble their partners together to regulate their target genes. A typical LMO2 binding site consists of two elements, a GATA site and an E-box, with an interval of 9 approximately 12 bp. METHODS: In this study, the combination of MBP pulldown assay and mammalian two hybrid assay were used to confirm the homo-binding character of LMO2-L/-S isoforms. Luciferase reporter assay and Real-time PCR assay were used to detect expression levels and relative promoter activities of LMO2-L/-S isoforms. Co-transfection and Luciferase reporter assay were used to reveal the detailed regulatory pattern of LMO2-L/-S isoforms on their targets. RESULTS: Herein we report the homo-interaction character of LMO2-L and LMO2-S and their major difference in manner of regulating their target genes. Our results showed that LMO2-L and LMO2-S could only bind to themselves but not each other. It was also demonstrated that LMO2-L could either positively or negatively regulate the transcription of its different target genes, depending on the arrangement and strand location of the two elements GATA site and E-box, LMO2-S, however, performed constitutively transcriptional inhibiting function on all target genes. CONCLUSION: These results suggest that LMO2 isoforms have independent functions while there is no interaction between each other and they could play synergetic or antagonistic roles precisely in regulating their different genes involved in normal and aberrant hematopoiesis.

DeltaEF1 promotes breast cancer cell proliferation through down-regulating p21 expression.

Although the zinc finger-homeodomain transcription factor deltaEF1 is implied as a regulatory factor at the crossroad between proliferation and differentiation in carcinogenesis, its potential effect in the regulation of cell cycle progression has not been well elucidated. In our present study, we provide novel finding that, in breast cancer, the ectopic expression of deltaEF1 in MDA-MB-231 cells significantly promoted cell proliferation by increasing the cell number in S phase of the cell cycle. In contrast, deltaEF1 knockdown by RNA interference exhibited an opposite effect, highlighting a potent role of deltaEF1 to promote G1-S transition of breast cancer cells. Moreover, we demonstrated that deltaEF1 down-regulated p21 and concurrently up-regulated the expressions of CDK2 and CDK4 during this process. Further, deltaEF1 inhibited p21 transcription by recruiting to the E(2) box element on the p21 promoter. Depletion of endogenous deltaEF1 in MDA-MB-231 cells was sufficient to allow an inherent release of p21 expression, thus resulting in the cell cycle arrest. In addition, the stimulatory effect of deltaEF1 on cell proliferation through p21 regulation was supported by an inverse correlation of deltaEF1 and p21 expressions observed in both breast cancer cell lines and clinical tumor specimens. Taken together, these observations suggest a dual effect of deltaEF1 in promoting breast cancer cell proliferation, by differentially regulating the cell cycle regulatory proteins.

[Effect of DNA methyltransferase 1 gene silencing on proliferation and apoptosis of HeLa cells].

OBJECTIVE: To observe the effect of DNA methyltransferase 1 (DNMT1) gene silencing by RNA interfering technology on the proliferation and apoptosis of HeLa cells. METHODS: Recombinant plasmid pshRNA-DNMT1-A, B and C were respectively transfected into HeLa cells by lipofectamine 2000, while cells transfected plasmid vector pSilencer3. 1-H1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and western blotting was used to detected the mRNA and protein expression of DNMT1 in HeLa cells transfected for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8) assay was used to investigate the proliferation of the HeLa cells after transfection, while apoptosis was detected by flowcytometry (FCM) method. RESULTS: Three DNMT1-targeted short hairpin RNA (shRNA) A,B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragments. The results indicated that both recombinant plasmid pshRNA-DNMT1-A and B could effectively knock down the expression of DNMT1 gene in human cervical cancer cells, of which pshRNA-DNMT1-B was the better choice. While no effect of pshRNA-DNMT1-C was seen. RT-PCR results showed that the relative mRNA expression of DNMT1 gene in HeLa cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were 0.406 +/- 0.057, 0.191 +/- 0.036 and 0. 104 +/- 0.015, which were significantly lower than that in HeLa cells transfected by empty vector and non-transfected cells (0.520 +/- 0.020, 0.537 +/- 0.041, respectively, P < 0. 05). The western blotting analysis manifested that the relative expression of DNMT1 protein of HeLa cells transfected by pshRNA-DNMT1 for 24, 48 and 72 hours were 0.197 +/- 0.024, 0.075 +/- 0.015, 0.040 +/- 0. 013, which were significantly lower than that in transfected cells by empty vector and non-transfected cells (0.273 +/- 0.010, 0.283 +/- 0.016, respectively, P < 0.05). The CCK-8 results showed that the cell survival rates of HeLa cells transfected by pshRNA-DNMT1 for 24, 48, 72, 96 and 120 hours were 70.8%, 64.8%, 51.6%, 45.3% and 38.0%, there were statistically different compared with cells transfected by empty vector and non-transfected cells at different time-points (P < 0.01). The results of FCM indicated that the apoptosis rate of HeLa cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were (17.7 +/- 1.3)%, (35.3 +/- 1.3)%, (47.6 +/- 1.6)%, which were significantly higher than empty vector transfected cells and non-transfected cells [(4.9 +/- 0.5)%, (5.1 +/- 0.7)%, respectively, P < 0.05]. CONCLUSIONS: DNMT1 can be successfully silenced by RNA interfering in cervical HeLa cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.

[Reconstruction of adenovirus vector contained canstatin gene and hTERT gene core promoter and its anti-tumor effect in human ovarian cancer].

OBJECTIVE: To study anti-tumor effects on human ovarian cancer xenografted tumors in mice by constructing adenoviral expression vector containing canstatin gene and hTERT gene core promoter (AdhTERT-Can). METHODS: AdhTERT-Can vector was constructed and identified by means of enzyme cutting, electrophoresis and sequencing. Then, transfected into HO8910PM cells by means of lipofectamine and confirmed by green fluorescence protein (GFP) expression under laser confocus microscope. The mRNA expression of canstatin gene was tested by RT-PCR. Human ovarian cancer xenografted tumor models in nude mice were established and randomly divided into AdhTERT-Can group received viral supernatant solution of AdhTERT-Can by tail vein injection, Ad-Can groups received viral supernatant solution of Ad-Can by tail vein injection, and control groups received phosphate buffer solution (PBS). The volume of tumors were measured and compared in each group to evaluated the anti-tumor efficacy. RESULTS: All the constructed vectors of AdhTERT-Can were verified by enzymed digestion. There were green fluorescence from 60% of HO8910PM cells transfected by AdhTERT-Can under laser confocus microscope, and the mRNA expression of canstatin gene in HO8910PM cells were also verified by RT-PCR. The growth of tumor in AdhTERT-Can group was significantly inhibited compared with those in Ad-Can groups and control groups from the 8th day (P<0.01). However, there were not significant difference between Ad-Can group and PBS group (P>0.05). On the 30th day, the tumors showed liquefaction necrosis and cystic degeneration in each group, especially in AdhTERT-Can group. CONCLUSIONS: The recombinant adenovirus vector of AdhTERT-Can has been constructed successfully and could steady express in ovarian cancer cell lines HO8910PM. The results shown that it could inhibit significantly the growth of human ovarian cancer xenografted tumors in mice and shown to be the target of gene therapy.

Two novel serum biomarkers for endometriosis screened by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and their change after laparoscopic removal of endometriosis.

OBJECTIVE: To explore novel endometriosis serum biomarkers by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). DESIGN: First, we aimed to discover the potential biomarkers of endometriosis by SELDI-TOF-MS. Second, blinded test was performed to characterize the effectiveness of the model by examining the sensitivity and specificity. Third, 29 postoperative patients with endometriosis were recruited to monitor the change of potential biomarkers after laparoscopic surgery. SETTING: Collaborative investigation in an academic research environment. PATIENT(S): Fifty-nine patients with endometriosis, 31 patients without endometriosis, and 30 healthy volunteers. INTERVENTION(S): Blood serum of endometriosis and control group patients. MAIN OUTCOME MEASURE(S): Protein expression. RESULT(S): Two endometriosis-specific proteins were found in the preliminary screening study. Furthermore, the blinded test was performed and showed a sensitivity of 86.67% and a specificity of 96.77% of the markers for detecting endometriosis, which are significantly higher than those of CA-125 for distinguishing patients with endometriosis from patients without endometriosis. After surgery, the levels of these biomarker proteins decreased to levels comparable with those of patients without endometriosis. CONCLUSION(S): We discovered the potential biomarkers of endometriosis and set up a diagnostic model with a sensitivity of 86.67% and a specificity of 96.77%, which is significantly higher than that of CA-125 for detecting endometriosis, The levels of these proteins decreased to levels comparable with those of patients without endometriosis.

Increased staining for phosphorylated AKT and nuclear factor-kappaB p65 and their relationship with prognosis in epithelial ovarian cancer.

AKT plays an important role in malignant behavior of tumors. The purpose of the present study was to determine the expression of phosphorylated AKT (P-AKT) and nuclear factor-kappaB (NF-kappaB) p65 and their association with clinicopathological parameters and prognosis in epithelial ovarian tumor. On immunohistochemistry 115 samples of ovarian tissue that included 68 specimens of epithelial ovarian cancer, 12 of borderline tumor, 24 of epithelial benign tumor and 11 of normal ovary, were evaluated. Sixty-three patients with ovarian cancer were followed up from 7 to 68 months. The positive expression rate of P-AKT and NF-kappaB p65 were higher in epithelial ovarian cancer than in normal ovarian tissue (P<0.01). Elevated P-AKT or NF-kappaB p65 expression was significantly correlated with late clinical stage (P<0.05 and P<0.01) and poor histological differentiation (both P<0.01). P-AKT expression was significantly correlated with NF-kappaB p65 immunostaining (phi=0.272, P<0.05). Elevated expression of P-AKT was negatively correlated with the survival of ovarian cancer patients, but it was not an independent prognostic factor after multivariate analysis. Overexpression of P-AKT and NF-kappaB p65 were involved in the carcinogenesis and metastasis of ovarian cancer. P-AKT might contribute to the malignant transformation through NF-kappaBp65 upregulation.

[Promoter methylation of DAPK gene in cervical carcinoma].

BACKGROUND & OBJECTIVE: Aberrant DNA methylation plays important roles during multistage carcinogenesis in various human organs. This study was to explore the relationship between the promoter methylation and inactivation of DAPK gene in cervical cancer. METHODS: The promoter methylation of DAPK was investigated with methylation-specific polymerase chain reaction (MSP) in 52 specimens of cervical cancer, 60 specimens of cervical intraepithelial neoplasia (CIN) and 20 specimens of normal cervical squamous epithelial tissues. Its correlation to clinicopathologic features of cervical cancer was analyzed. The protein expression of DAPK was detected by immunohistochemistry. RESULTS: The methylation rate of DAPK gene promoter was significantly higher in cervical cancer tissues than in CIN (65.4% vs. 18.3%, P<0.05); while no methylation of DAPK gene was found in normal cervical tissues. The methylation rate of DAPK gene was significantly higher in cervical squamous cell carcinomas than in adenocarcinomas (80.0% vs. 16.7%, P<0.001). Promoter methylation of DAPK was negatively correlated to its protein expression (r=-0.849, P<0.001). CONCLUSION: The promoter methylation may lead to inactivation of DAPK gene, and may be related with tumorigenesis of cervical cancer.

[Soluble expression of recombinant human BMP6 in Escherichia coli and its purification and bioassay in vitro].

BMP6 is a potent protein for future treatment strategies of bone regeneration as it is a very important regulator of bone homeostasis. Active BMP6 is a dimer containing multidisulfide bonds and is a highly hydrophobic protein prone to aggregation. To obtain soluble and active BMP6 in Escherichia coli, we compared the effects of four N-terminal fusion tags (TRX, GST, MBP and CBD) and N-terminal His6-tag. The expression and solubility were tested under the different conditions (expression hosts, temperatures and inductor concentrations). A series of experiments leads to the finding that the placement of MBP before the BMP6 is best in availing the soluble expression of the protein. Our study alsodemonstrates that in E. coli BL21trxB(DE3) cytoplasm, which is a thioredoxin reductase mutant strain, soluble homodimeric BMP6 can be formed. The overexpressed MBP-BMP6 fusion protein is purified by chromatography, and shown to be functionally active.