The association between polymorphisms of genes related to inflammation and recurrent pregnancy loss.

AIM: Recurrent pregnancy loss (RPL) occurs in 1-2% of pregnant women and about 50% of RPL cases are unexplained. Previous studies have shown that genetic variation in immune response genes can contribute to the risk in pregnancy maintenance during pregnancy. The aim of the present study was to evaluate the relationship between RPL and genes those have previously been associated with an inflammatory process on 107 RPL cases and 187 healthy controls. METHODS: In this work, the single-nucleotide polymorphisms was examined by utilizing the direct sequencing and the Sequenom MassARRAY system. RESULTS: The FAU rs769440 G allele had higher frequencies in patients with RPL (p = .019). No association was observed between other polymorphisms and RPL. CONCLUSION: The results showed an association between FAU rs769440 polymorphism and RPL in Chinese Han population.

Alarmin high mobility group box-1 in maternal serum as a potential biomarker of chorioamnionitis-associated preterm birth.

Chorioamnionitis is associated with an increased risk of spontaneous preterm birth. The aim of this study was to investigate the serum levels of high mobility group box-1 (HMGB1) in pregnancies with histological chorioamnionitis (HCA)-associated preterm labor (PTL) with intact membranes or preterm premature rupture of membranes (PPROM), and to access the role of serum HMGB1 in HCA and HCA-associated PTL. A total of 190 pregnant women were enrolled in this study: PLT patients with (n = 28) or without HCA (n = 36), PPROM patients with (n = 26) or without HCA (n = 65), and non-HCA PTL controls (n = 35). Maternal serum levels of HMGB1 were measured by enzyme-linked immunosorbent assay. Serum HMGB1 levels were significantly higher in PTL or PPROM patients than in control group (p < 0.01, respectively). The PPROM patients also exhibited higher serum HMGB1 levels compared to PTL patients (p = 0.015). HCA patients were characterized by significantly increased levels of serum HMGB1 when compared with non-HCA patients (p < 0.01). Therefore, maternal serum HMGB1 may become a potential biomarker of HCA and HCA-associated PTL.

MicroRNA-495 downregulates FOXC1 expression to suppress cell growth and migration in endometrial cancer.

MicroRNAs (miRNAs) are a class of noncoding RNAs and function as key regulators of gene expression at the post-transcriptional level. In this study, we found that miR-495 reduces cell growth, induces apoptosis and suppresses the migration of endometrial cancer by directly inhibiting FOXC1 expression. Further analysis revealed that FOXC1 promotes growth and migration and functions as an oncogene in vitro. FOXC1 overexpression reversed the cellular responses mediated by miR-495 in endometrial cancer cells. We also found that miR-495 suppresses the growth of endometrial cancer in vivo. Altogether, these results indicate that miR-495 acts as a tumour suppressor gene by targeting FOXC1 at the post-transcriptional level in endometrial cancer.

MicroRNA-182 promotes cell growth, invasion, and chemoresistance by targeting programmed cell death 4 (PDCD4) in human ovarian carcinomas.

As an important tumor suppressor, programmed cell death 4 (PDCD4) influences transcription and translation of multiple genes, and modulates different signal transduction pathways. However, the upstream regulation of this gene is largely unknown. In this study, we found that microRNA-182 (miRNA-182, miR-182) was upregulated, whereas PDCD4 was downregulated in ovarian cancer tissues and cell lines. Blocking or increase of miR-182 in ovarian cancer cell lines led to an opposite alteration of endogenous PDCD4 protein level. Using fluorescent reporter assay, we confirmed the direct and negative regulation of PDCD4 by miR-182, which was dependent on the predicted miR-182 binding site within PDCD4 3' untranslated region (3' UTR). MTT and colony formation assays suggested that miR-182 blockage suppressed, whereas miR-182 mimics enhanced viability and colony formation of ovarian cancer cells. These effects may partly be attributed to the cell cycle promotion activity of miR-182. miR-182 also contributed to migration and invasion activities of ovarian cancer cells. Furthermore, miR-182 reduced the chemosensitivity of ovarian cancer cells to CDDP and Taxol, possibly by its anti-apoptosis activity. Importantly, all the alterations of the above cellular phenotypes by blocking or enhancing of miR-182 could be alleviated by subsequent suppression or ectopic expression of its target PDCD4, respectively. We conclude that in ovarian cancer cells, miR-182 acts as an oncogenic miRNA by directly and negatively regulating PDCD4.

[Role of epidermal growth factor signaling system in the pathogenesis of endometriosis under estrogen deprivation conditions].

OBJECTIVE: To study the role of epidermal growth factor (EGF) , epidermal growth factor receptor(EGFR), extracellular signal-regulated kinase 1/2 (p-ERK1/2) in the pathogenesis of endometriosis under estrogen deprivation conditions. METHODS: The estrogen was quickly-stripped in medium and the female nude mice were castrated by bilateral oophorectomy to build estrogen deprivation in vitro and in vivo experimental models, respectively. (1) In vitro experiments:according to different treatments the estrogen deprived ectopic endometrial cells were classified into 4 groups: a. EGF group:the ectopic endometrial cells were cultured for 72 hours with different concentrations of EGF (0.01, 0.1, 1, 10, 50, 100 ng/ml), the results of EGF group were represented by the result of cells treated by 10 ng/ml EGF cultured for 72 hours; b. EGF+PD98059 group:the ectopic endometrial cells were cultured for 72 hours with 5×10(-2) mol/L PD98059 (inhibitor of ERK), followed by a cultivation for 72 hours treated by 10 ng/ml EGF+5×10(-2) mol/L PD98059; c. EGF+ ICI182780 group: the ectopic endometrial cells were cultured for 72 hours with 10(-6) mol/L ICI182780 [inhibitor of estrogen receptor(ER)], followed by a cultivation for 72 hours treated by 10 ng/ml EGF+10(-6) mol/L ICI182780; d. Blank control group:the ectopic endometrial cells were cultured with no treatment. The proliferation activity of ectopic endometrial cells in all groups after treatment were examined by methyl thiazolyl tetrazolium (MTT) method represented by absorbance value (A). The expression of p-ERK1/2 protein were detected by western blot. (2) In vivo experiments: 64 female nude mice were randomly divided into control and castration groups (both n=32) using random number chart. The mice in castration group were castrated by bilateral oophorectomy on 3 weeks after the endometriosis model was established. The levels of EGF, EGFR, p-ERK1/2 protein in ectopic lesions of both groups were measured on 4, 6, 8 and 10 weeks after the endometriosis model was established by western blot. RESULTS: (1) The proliferation activity of ectopic endometrial cells:the proliferation activity of ectopic endometrial cells treated by different concentrations of EGF (0.01, 0.1, 1, 10, 50, 100 ng/ml) for 72 hours were 0.310±0.010, 0.340±0.020, 0.670±0.010, 0.980±0.030, 1.360±0.020, 1.670±0.020, respectively, the proliferation activity was increased along with of EGF concentrations.The proliferation activity was 0.680±0.030 at EGF+ PD98059 group, the differences exhibited significant difference when compared with that at EGF group with 100 ng/ml for 72 hours (P<0.01) .The proliferation activity of EGF+ ICI182780 and blank control groups were 0.330±0.030 and 0.310±0.030, respectively, which did not reached statistical differences (P>0.05). (2) The expression of EGF, EGFR, pERK1/2 protein: a. In vitro experiments:the levels of p-ERK1/2 protein in EGF and blank control groups were 0.670±0.020 and 0.600±0.010, respectively, which reached statistical differences (P<0.05). The level of p-ERK1/2 protein in EGF+ PD98059 group was 0.610±0.020, which exhibited significant differences with that at blank control group (P>0.05). b. In vivo experiments:at 4, 6 and 8 weeks after the endometriosis models were established, the expression of EGF protein in the ectopic lesions of castration group and control group were (0.530±0.015 versus 0.610±0.015), (0.400±0.029 versus 0.620±0.018), (0.560±0.026 versus 0.630±0.021), respectively, the levels of EGFR protein were (0.500±0.030 versus 0.640±0.030), (0.470±0.020 versus 0.630±0.020), (0.510±0.030 versus 0.610±0.020) respectively, and the level of p-ERK1/2 protein were (0.500±0.020 versus 0.580±0.020), (0.490±0.020 versus 0.580±0.020), (0.570±0.020 versus 0.590±0.020), respectively. The difference of EGF, EGFR, p-ERK1/2 protein expression levels between two groups did not exhibited significant difference (P<0.01, P<0.01, P<0.05). At 10 weeks after the endometriosis models were established, the levels of EGF protein in castration group and control group were both 0.620±0.020, the levels of EGFR protein were both 0.610±0.020, and the level of p-ERK1/2 protein were 0.590±0.010 and 0.600±0.020. No statistical difference (P>0.05) was found between those two groups (P>0.05). CONCLUSIONS: EGF could stimulate the proliferation of ectopic endometrial cells by activating the ERK pathway under estrogen deprivation conditions. The inhibition of EGF signaling system in ectopic lesions was alleviated along with the prolongation of the period of estrogen deprivation.

[Inhibitory effects on Candida albicans of vagina cells transferred with antimicrobial peptide LL-37 and human defensin 5 recombinant plasmids].

OBJECTIVE: To evaluate the inhibitory effects on Candida albicans of vagina cells transferred with antimicrobial peptide LL-37 and human defensin 5 (HD5) recombinant plasmids and observe secretion of IL-8. METHODS: (1) The epithelial cells from female vagina were culture primarily. The pcDNA3.1(+)/HD5-EGFP and pcDNA3.1(+)/LL-37-EGFP eukaryotic recombinant plasmids were separately or jointly transferred into the fourth generation of vaginal epithelial cells. Two test groups were defined: one test group was no Candida albicans group including four subgroups which were untransferred group, HD5 group transferred with pcDNA3.1(+)/HD5-EGFP, LL-37 group transferred with pcDNA3.1(+)/LL-37-EGFP, combining transferring group transferred with pcDNA3.1(+)/HD5-EGFP and pcDNA3.1(+)/LL-37-EGFP; the other test group was with Candida albicans group which the Candida albicans were coincubated with the four subgroups described above. (2) For examination of cytokines and chemokines, at 6, 12, 24 and 48 hours, the supernatant of every group was collected. ELISA was applied to detect the levels of LL-37, HD5 and IL-8. At each time point, the growth inhibition of Candida albicans was calculated by glucose consumption testing. RESULTS: (1) The max level of LL-37, HD5 and IL-8 reached max level after being transferred for 24 hours, then showed decreasing trend. The secretion of LL-37, HD5 and IL-8 was significant higher in combining transferring group in Candida albicans group than other groups, and the secretion level of LL-37, HD5 and IL-8 was (100.16 ± 0.81) ng/ml, (58.50 ± 2.08) µg/ml and (101.03 ± 1.59) pg/ml (P < 0.01). (2) In different time point, the absorbance of each subgroup without Candida albicans declined slowly, and there were no statistically significant difference (P > 0.05), as while as in LL-37 subgroup and HD5 subgroup with Candida albicans. In group with Candida albicans, the absorbance of combining transferring subgroup were 3.210 ± 0.010, 3.150 ± 0.030, 3.099 ± 0.030 and 2.970 ± 0.040 at 6, 12, 24 and 48 hours, respectively, which was significantly higher than those in the other cells (P < 0.01), and the declined trend was the slowest. CONCLUSIONS: The antifungal ability of vaginal epithelial cell became stronger after being transferred with LL-37 and HD5 recombinant plasmids. LL-37 and HD5 could also possess immunomodulatory activity and induce chemokine IL-8 production.

[Inhibitory effects of soluble endoglin on invasive ability of cultured cytotrophoblasts of first trimester of pregnancy].

The present study aims to investigate the effects of soluble endoglin (sEng) on invasive ability of cultured cytotrophoblasts of first trimester of pregnancy. Cytotrophoblasts of normal 6 to 8-week pregnancy were cultured by trypsin digestion method, and were incubated with cell culture medium without (control group) and with 10 µg/L sEng (sEng group), respectively for 24 h. The invasive ability was determined by transwell invasion assay, and expressions of MMP-2, MMP-9 mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The results showed that the invasive ability of cytotrophoblasts in sEng group was lower than that in control group (P < 0.05). Compared with control group, the expressions of MMP-2 and MMP-9 mRNA and protein of cytotrophoblasts were significantly lower (P < 0.05). In conclusion, sEng may participate in the genesis of preeclampsia by affecting the invasive ability of cytotrophoblasts through regulation of the expression of MMP-2 and MMP-9.

[Human endostatin antiangiogenic gene therapy mediated by recombinant adeno-associated virus vector in nude mouse with endometriosis].

OBJECTIVE: To study the therapeutic effect of recombinant adeno-associated virus carrying human endostatin gene therapy on endometriosis in mice model. METHODS: Recombinant adeno-associated virus vector carrying human endostatin gene and enhanced green fluorescent proteins gene (rAAV2-endostatin-EGFP) was constructed. Endometrium was from 12 patients with leiomyoma undergoing hysterectomy in Second Hospital, Tianjin Medical University between November and December 2008. Endometriosis models of nude mice were established by transplanting human endometrial fragments intooperitoneal surface. After 1 week, those 60 mice were divided into 3 groups: treatment group including 20 mice injected with rAAV2-endostatin-EGFP to ectopic lesion, control group including 20 mice injected with rAAV2-EGFP to ectopic lesion and blank control group including 20 mice injected with phosphate buffered saline (PBS) to the ectopic lesion. At 1, 2 and 3 weeks after treatment, those mice underwent laparotomy to observe the location and size of ectopic lesion in abdominal cavity. The expression of endostain protein, number of gland, microvessel density (MVD) and vascular endothelial growth factor (VEGF) were measured in ectopic lesions. The serum level of estradiol and progesterone were detected in nude mice among every groups. RESULTS: (1) All endometriosis of nude mice models were established successfully through peritoneum transplanting. After 1 week's treatment, flat lesion nodes, decreased gland number and narrow and atrophy glandular cavity were observed by light microscope. (2) The endostatin gene was transferred into nude mice successfully and expressed effectively. It was observed that endostatin protein expression was shown with enhanced green fluorescent proteins in ectopic lesion. (3) Glands number of ectopic lesion in rAAV2-endostatin-EGFP group (7.8 ± 1.9, 7.0 ± 1.5 and 5.5 ± 1.7) were significantly less than 10.1 ± 1.7, 10.2 ± 2.0 and 9.8 ± 2.4 in rAAV2-EGFP control group and 10.2 ± 2.2, 10.0 ± 2.0 and 9.7 ± 2.2 in PBS control group at 1, 2 and 3 weeks after treatment (all P < 0.05). Glands number of ectopic lesion in rAAV2-endostatin-EGFP group at 3 weeks was significantly less than those at 1 and 2 weeks after treatment (P < 0.05). (4) MVD of ectopic lesion in rAAV2-endostatin-EGFP group (12.2 ± 1.5, 11.4 ± 2.1 and 9.0 ± 1.4) was significantly less than those at rAAV2-EGFP control group (16.5 ± 1.7, 16.5 ± 1.9 and 16.9 ± 1.9) and PBS control group (16.2 ± 1.6, 16.0 ± 1.6 and 16.3 ± 1.7) at 1, 2 and 3 weeks after treatment (all P < 0.05). MVD of ectopic lesion in rAAV2-endostatin-EGFP group at 3 weeks was significantly less than those at 1 and 2 weeks after treatment (P < 0.05). (5) The rate and density of VEGF expression at ectopic lesion in rAAV2-endostatin-EGFP group (35%, 30%, 25% and 1.60 ± 0.43, 1.33 ± 0.30, 1.03 ± 0.36) were significantly less than those at rAAV2-EGFP control group (80%, 75%, 85% and 2.43 ± 0.53, 2.43 ± 0.29, 2.66 ± 0.45) and PBS control group (85%, 90%, 90% and 2.36 ± 0.53, 2.64 ± 0.57, 2.53 ± 0.52) at one 1, 2 and 3 weeks after treatment (all P < 0.05). The expression of VEGF at ectopic lesion in rAAV2-endostatin-EGFP group at 3 weeks was significantly less than those at 1 and 2 weeks after treatment (P < 0.05). (6) The level of estradial and progesterone in serum of nude mice of rAAV2-endostatin-EGFP group [E(2): (48 ± 7) pmol/L, P: (61 ± 8) nmol/L] did not reach statistical difference when compared with those at rAAV2-EGFP control group [E(2): (50 ± 9) pmol/L, P: (60 ± 10) nmol/L] and PBS control group [E(2): (48 ± 7) pmol/L, P: (58 ± 10) nmol/L, P > 0.05]. CONCLUSIONS: The recombinant adeno-associated virus carrying human endostatin gene therapy could inhibit angiogenesis at endometriotic lesions and not influence steroid level. The antiangiogenic gene therapy might become a novel option for endometriosis.

SMYD2 promotes cervical cancer growth by stimulating cell proliferation.

BACKGROUND: Cervical cancer is the most common gynecological malignancy with low terminal cure rate, and therefore new therapeutic targets are urgently needed to combat this disease. SMYD2, as an oncogene, is abnormal highly expressed in multiple types of tumors and further affects the occurrence and development, but the potential correlations between SMYD2 expression and cervical cancer progression is still unclear. METHODS: We first used the bioinformatics website to screen the data of cervical cancer in (The Cancer Genome Atlas) TCGA and survival analysis was used to find the different survival rates in the SMYD2 high expression group and low expression group. Through immunohistochemistry, the association between SMYD2 expression and clinical-pathological features of cervical cancer patients was further evaluated. Quantitative PCR and Immunoblot were applied to investigate the relative mRNA and protein expression levels, respectively. In vivo and in vitro experiments were performed to explore the function of SMYD2 in cancer progression. RESULTS: We first found a high expression of SMYD2 in cervical cancer, and survival analysis found that the poorer survival rate in the SMYD2 high expression group than that in the low expression group. Herein, our study demonstrated that the expression of SMYD2 in patients with cervical cancer was associated with FIGO stage, tumor size and further correlated with poor prognosis. Our data further showed that the inhibition of SMYD2 expression in cervical cancer cell line Caski and Siha could dramatically block the proliferation of cervical cancer cells. Additionally, SMYD2-shRNA lentivirus infected remarkably inhibited tumorigenesis in mice compared with the scramble group. CONCLUSIONS: Taken together, this study provides strong evidence of the involvement of SMYD2 in cervical cancer growth and indicates that it could have high potential as a therapeutic target of cervical cancer.

Expression of phosphatase and tensin homolog deleted on chromosome ten in liver of athymic mice with hepatocellular carcinoma and the effect of Fuzheng Jiedu Decoction.

AIM: To explore the expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in liver of athymic mice with hepatocellular carcinoma (HCC) and the effect of Fuzheng Jiedu Decoction (FJD). METHODS: Forty eight male BALB/c athymic mice models were built by Bel-7402 with an indirect method. After 24 h of postoperation, the 48 athymic mice were distributed randomly into 4 groups: A, B, C, D, each group had 12 athymic mice. Group A were were treated by intragastric administration with FT207 (Tegafur) for 4 wk. Group B, C and D were treated by intragastric administration with FJD (complex prescription of Chinese crude drug) that had been delegated into 3 kinds of density as the low, middle, and high for 4 wk. At last, athymic mice were put to death, live time, volume of tumors, exponent of tumors and the tumor metastasis in livers were observed; and PTEN was detected in hepatic tissue, latero-cancer tissue and cancer tissue by immunohistochemistry. RESULTS: Four weeks later, the total survival rate in treatment group (A + B + C) was 50% and higher than the control group (0%) treated by FT207, (P < 0.01). The survival rate in group A, B, C was higher than in group D, and except group A with D, there was significant differences (Fisher's Exact Test P = 0.05 or 0.01). And no differences were observed between the treatment groups and the control group in volume of tumors and exponent of tumors (P > 0.05). Tumor metastasis in livers of the treatment group was less than the controls (Fisher's Exact Test, P = 0.021). The result of immunohistochemistry showed that the intensity of PTEN in latero-cancer tissue was the highest, and then the hepatic tissue, the lowest was cancer tissue (Kruskal-Wallis test, chi2 = 60.67, P = 0.000). It also showed that the intensity of PTEN in treatment groups (A, B, C) was higher than the control group (D) (F = 5.90, P = 0.002 in hepatic tissue and F = 15.99, P = 0.000 in latero-cancer tissue and chi2 = 26.08, P = 0.000 in cancer tissue), and group B is the highest in the treatment groups (P < 0.05, r = 0.01. respectively). However, there was no significant statistic difference between group A and group C (P > 0.05). CONCLUSION: FJD can prolong the survival time and decrease tumor metastasis in livers of these experimental mice. Mechanisms of FJD healing HCC may partially be explained by enhancing the expression of PTEN in liver.

[Expression of PTEN in athymic mice with HCC treated by complex prescription of Chinese crude drug].

OBJECTIVE: To research the treatment effect of complex prescription of Chinese crude drug in BALB/c athymic mice with human liver cancer, which were built by Bel-7402. METHOD: 48 male BALB/c athymic mouse models were built by Bel-7402 with an indirect method. After 24 hours of postoperation, the 48 athymic mice were distributed randomly into 4 groups which were treated by intragastric administration with complex prescription of Chinese crude drug that had been deliquated into 3 groups by the different density as the low, middle, and high and FT207 (Tegafur) for 4 weeks. At last, athymic mice were put to death and PTEN was detected in hepatic tissue, latero-cancer tissue and cancer tissue by immunohistochemistry (PowerVision Two-Step Histostaining Reagent). RESULT: All of the 48 athymic mice survived 12 to 28 days (Ms 24 days) and every mouse with liver cancer demonstrated by dissection. The result of immunohistochemistry represents that the intensity of PTEN in latero-cancer tissue is the highest, and then the hepatic tissue, the lowest is cancer tissue, P < 0.01. It also represents that the intensity of PTEN in treatment groups (A, B, C) is more higher than the control group (D), P < 0.05 or P < 0.01, and group B is the highest in the treatment groups, P < 0.05 or P < 0.01. However, there is no significant statistic difference between group A and group C. CONCLUSION: The higher expression of PTEN in the laterocancer tissue can represent the protective reaction of stress of the organism. And anticancer effect of this complex prescription of Chinese crude drug relates to an eligible density of it. Mechanisms of this complex prescription of Chinese crude drug healing HCC may partially be explained by enhancing the expression of PTEN in liver.

[Clinical study of method of strengthening body resistance and disintoxication disintoxication in patients with HCC of post-TACE].

OBJECTIVE: To research the effect of a complex prescription of Chinese crude drug with the function of strengthening body resistance and disintoxication disintoxication in patients with HCC of post-TACE. METHOD: 45 patients with HCC of post-TACE, as the treatment group, were treated by a complex prescription of Chinese crude drug with the function of strengthening body resistance and disintoxication disintoxication and routine methods of protecting liver. Other 37 patients, as the control group, with the same clinical feature were treated by routine methods of protecting liver only. In the later 1 month, accumulated points of clinical symptom, hepatic function and AFP were observed in all of the patients. And the clinical effect of the two groups was compared. RESULT: One week later, in the treatment group, there is no improvement in anorexia but nausea, abdominal distention and lassitude were improved more obviously than pretherapy in both a week and one month later (P < 0.01 or P <0.05). In the control group, anorexia were improved a week later (P <0.05), but there is no improvement in nausea, abdominal distention and lassitude at the same time, and one month later all of the indexes above improved (P <0.01 or P <0.05). Accumulated points of clinical symptom was decreased more obviously in the treatment group than in the control group in both a week and one month later (P <0.05). At the end of the therapy, in the both groups, ALT, TBIL and AFP all improved except ALB, (P <0.01 or P <0.05). And TBIL improved more obviously in the treatment group than in the control one month later (P <0.05). CONCLUSION: This complex prescription of Chinese crude drug can lighten the adverse reaction of post-TACE. And also it can promote the recovery of liver function and evaluate the quality of lives of such patients.

[Study of antitumous effect of the complex prescription of Chinese crude drug in athymic mice with HCC].

OBJECTIVE: To research the treatment effect of a complex prescription of Chinese crude drug in BALB/c athymic mice. METHODS: 48 male BALB/c athymic mouse models were built by Bel-7402 with an indirect method. After 24 hours of postoperation, they were distributed randomly into 4 groups which were treated by intragastric administration with complex prescription of Chinese crude drug of different density as the low, middle, high and FF207 (Tegafur) for 4 weeks. At last, athymic mice were put to death and survial time, volume of tumors, exponent of tumors and the tumor metastasis in livers were observed; and PTEN was detected in hepatic tissue, latero-cancer tissue and cancer tissue by immunohistochemistry. RESULTS: 4 weeks later, the total survival rate in treatment group (A + B + C) was 50% and more higher than the control group (0%) treated by FT207, P < 0.01. The survival rate in group A, B, C was higher than group D, except group A, there was significant differences, P < 0.05 or 0.01. And no differences were observed between the treatment groups and the control group in volume of tumors and exponent of tumors, P > 0.05. Tumor metastasis in livers of the treatment group is less than the control's, P < 0.05. Negative correlation between volume of tumors and survival time, positive correlation between tumors volume and spleen exponent were observed, P < 0.01. The result of immunohistochemistry represented that the intensity of PTEN in latero-cancer tissue was the highest, and then the hepatic tissue, the lowest was cancer tissue, P < 0.01. It also represented that the intensity of PTEN in treatment groups (A, B, C) was more higher than the control group (D), and group B was the highest in the treatment groups, P < 0.05 or P < 0.01. However, there was no significant statistic difference between group A and group C, P > 0.05. CONCLUSION: This complex prescription of Chinese crude drug can prolong the survival time and decrease tumor metastasis in livers of these experimental mice. Too large tumor volume is an main death cause of model. Mechanisms of this complex prescription of Chinese crude drug healing HCC may partially be explained by enhancing the expression of PTEN in liver.

Isolation, culture and identification of human adipose-derived stem cells.

The aim of the present study was to improve methods for the isolation and identification of adipose-derived stem cells (ASCs). Human subcutaneous adipose tissue was collected during liposuction surgery, without ultrasound-assisted liposuction and other assisted techniques, and digested with 0.075% collagenase I. First (P1) and second (P2) passage ASCs were applied to the subsequent experiments. ASCs were observed under a microscope, the growth curves of the cells were assessed using a cell counting kit-8 assay and the membrane expression of cell surface antigens, including cluster of differentiation (CD)44, CD105 and CD45, were detected by flow cytometry. In addition, ASCs were induced to differentiate into lipocytes and osteocytes. Oil red staining was applied to examine adipogenic induction, whereas alkaline phosphatase (ALP) staining was used to assess osteogenic induction. Primary ASCs adhered to the culture vessel wall after 72 h, were fusiform in appearance at 5 days and exhibited stable growth with active proliferation. In total, 1×105 stem cells were gained per 50 ml of lipo-aspirate. ASCs were plated in a 25 cm2 culture flask at a density of 5×104/ml; the cells underwent the first logarithmic growth period after 72 h and grew to 90% confluence within 3 days. Flow cytometry demonstrated that the cells were highly positive for CD105 and CD44, and weakly positive for CD45; 18.6% of P1 cells and 90.7% of P2 cells were CD44+CD45-CD105+. Oil red and ALP staining were positive. The results of the present study suggested that ASCs may be considered a promising cell type for tissue engineering. Furthermore, the present study established an effective method for the isolation and identification of ASCs, which reduced damage to the stem cells and simplified the identification procedure.

MiR-214 inhibits cell migration, invasion and promotes the drug sensitivity in human cervical cancer by targeting FOXM1.

OBJECT: MicroRNAs (miRNAs) play key roles in progression of cervical cancer. In the present study, we investigated the role of miR-214 in the process of migration, invasion and drug sensitivity to cisplatin in cervical cancer. METHODS: We detected the differential expression of miR-214 in 19 cases cervical cancer tissues and normal tissues as well as 4 cervical cancer cells and one normal cervical cells by Real-time PCR. Then, wound healing assay, transwell invasion assay and MTT were used to detect the effects of migration, invasion and sensitivity to cisplatin of cervical cancer when miR-214 was overexpressed. Western blot, immunofluorescence and Flow Cytometry were used to detect the mechanism of migration, invasion and sensitivity to cisplatin. Next, bioinformatics analysis was used to find the target of miR-214. Through the luciferase reporter assay, Real-time PCR and western blot, we confirmed the binding relationship of miR-214 and FOXM1. In cervical cancer tissues, the expression of FOXM1 was detected by western blot and Immunohistochemistry. We also knocked down FOXM1 in cervical cancer cells, wound healing assay, transwell invasion assay and MTT were performed to detect the migration, invasion and sensitivity to cisplatin abilities of FOXM1. Western blot and Flow Cytometry were used to detect the mechanism of migration, invasion and sensitivity to cisplatin by FOXM1. Finally, we performed rescue expriments to confirm the function relationship between miR-214 and FOXM1. RESULTS: 1. Our results showed that miR-214 was frequently downregulated in tumor tissues and cancer cells especially in CIN III and cervical cancer stages. 2. Overexpression of miR-214 significantly inhibited migration and invasion of cervical cancer cells and prompted the sensitivity to cisplatin. 3. FOXM1 was identified as a target of miR-214 and down-regulated by miR-214. 4. Knocking down FOXM1 could inhibited migration and invasion of cervical cancer cells and prompted the sensitivity to cisplatin. 5. FOXM1 was upregulated in tumor tissues. 6. The mechanism of migration, invasion and sensitivity to cisplatin were the resluts of changes of EMT and apoptosis. 7. The restoration of FOXM1 expression can counteract the effect of miR-214 on cell migration, invasion and sensitivity to cisplatin of cervical cancer cells. CONCLUSIONS: These findings indicate that miR-214 acts as a tumor suppressor during the process of migration, invasion and drug sensitivity through targeting FOXM1, suggesting miR-214 as a potential new diagnostic and therapeutic target for the treatment of cervical cancer.

[Expression of interleukin1-alpha, beta and interferon-gamma in macrophages from endometrium of women with endometriosis].

OBJECTIVE: To assess interleukin (IL)-1alpha, beta and interferon (IFN) gamma expression in macrophages in eutopic and ectopic endometrium of women with endometriosis. METHODS: In situ hybridization was used to examine the expression of IL-1alpha and beta and IFN-gamma in macrophages from eutopic and ectopic endometrium of 40 women with endometriosis and 15 control women. Eutopic endometrial samples were histologically classified into proliferative and secretary phases. Cervical polyp tissue was used as positive control. RESULTS: Expression of IL-1alpha and beta in macrophages from eutopic and ectopic endometrium of women with endometriosis were significantly higher than that in the control group (P < 0.05). Both gene expression in macrophages from ectopic endometrium was higher than that in eutopic endometrium of patients with endometriosis (P < 0.05). The expressions of the two genes were significantly increased in secretory phase of endometrium when compared to that in proliferative phase (P < 0.05). There was no difference in IFN-gamma expression in macrophages of endometium between patients with endometriosis and control (P > 0.05). No cycle dependent variation of the gene expression in the macrophages was found either in endometriosis group or in control group. CONCLUSION: There is a significant increase in the expression of IL-1alpha and beta in macrophages of endometriosis. IL-1 and activated macrophages may play an important role in the development and progression of endometriosis.

MicroRNA-503 suppresses proliferation and cell-cycle progression of endometrioid endometrial cancer by negatively regulating cyclin D1.

MicroRNAs (miRNAs) are post-transcriptional inhibitor regulators of gene expression that act by directly binding complementary mRNA and are key determinants of cancer initiation and progression. In this study, we revealed a role for the tumor-suppressor miRNA miR-503 in endometrioid endometrial cancer (EEC) cells. The miR-503 expression level gradually decreases across normal endometrial tissues, endometrial tissues with complex atypical hyperplasia, and EEC tissues. A relatively high level of miR-503 in EEC tissues indicates a longer survival time in EEC patients. The expression of a cell cycle-associated oncogene encoding cyclin D1 (CCND1) was inversely correlated with miR-503 expression in EEC tissues and cell lines. CCND1 has a binding sequence of miR-503 within its 3' untranslated region, and was confirmed to be a direct target of miR-503 by the fluorescent reporter assays. Increasing the miR-503 level in EEC cells suppressed cell viability, colon formation activity and cell-cycle progression, and the inhibited oncogenic phenotypes induced by miR-503 were alleviated by ectopic expression of CCND1 without the untranslated region sequence. Furthermore, in vivo studies also suggested a suppressive effect of miR-503 on EEC cell-derived xenografts. miR-503 increased in cell cycle-arrested EEC cells, and was restored to a normal level in EEC cells after cell cycle re-entry, while CCND1 displayed the opposite expression pattern. Collectively, this study suggested that miR-503 plays a tumor-suppressor role by targeting CCND1. Abnormal suppression of miR-503 leads to an increase in the CCND1 level, which may promote carcinogenesis and progression of EEC.