Exploring the clinical and cellular mechanisms of LncRNA-KCNQ1OT1/miR-29a-3p/SOCS3 molecular axis in cases of unexplained recurrent spontaneous abortion.

OBJECTIVE: The objective of this study is to explore the functions and mechanisms of the LncRNA-KCNQ1OT1/miR-29a-3p/SOCS3 molecular pathway in the context of unexplained recurrent spontaneous abortion (URSA). METHODS: We conducted qRT-PCR to assess the levels of LncRNA-KCNQ1OT1, miR-29a-3p, and SOCS3 in both abortion tissues from women who experienced URSA and healthy early pregnant women. A dual-luciferase assay was employed to investigate whether miR-29a-3p targets SOCS3. Furthermore, RNA IP and RNA Pull-Down assays were employed to confirm the interaction between KCNQ1OT1 and SOCS3 with miR-29a-3p. RNA FISH was used to determine the cellular localization of KCNQ1OT1. Additionally, trophoblast cells (HTR8/SVneo) were cultured and the CCK-8 assay was utilized to assess cell proliferation, while flow cytometry was employed to analyze cell apoptosis. RESULTS: Compared to abortion tissues obtained from healthy early pregnant individuals, those from women who experienced URSA displayed a notable downregulation of KCNQ1OT1 and SOCS3, accompanied by an upregulation of miR-29a-3p. Suppression of KCNQ1OT1 resulted in the inhibition of cell proliferation and the facilitation of apoptosis in HTR8/SVneo cells. Our findings suggest that KCNQ1OT1 may exert a regulatory influence on SOCS3 through a competitive binding mechanism with miR-29a-3p. Notably, KCNQ1OT1 exhibited expression in both the cytoplasm and nucleus, with a predominant localization in the cytoplasm. Furthermore, we observed a negative regulatory relationship between miR-29a-3p and SOCS3, as the miR-29a-3p mimic group demonstrated significantly reduced cell proliferation and an increased rate of apoptosis when compared to the negative control (NC mimic) group. Additionally, the SOCS3 Vector group exhibited a substantial improvement in proliferation capability and a marked reduction in the apoptosis rate in comparison to the NC Vector group. The miR-29a-3p mimic + SOCS3 Vector group demonstrated a remarkable enhancement in proliferation and a reduction in apoptosis when compared to the miR-29a-3p mimic group. CONCLUSION: The competitive binding of miR-29a-3p to LncRNA-KCNQ1OT1 appears to result in the elevation of SOCS3 expression, consequently fostering the proliferation of trophoblast cells while concomitantly suppressing apoptosis.

Antioxidant, anticancer and apoptotic effects of the Bupleurum chinense root extract in HO-8910 ovarian cancer cells.

PURPOSE: The purpose of this study was to evaluate the anticancer, apoptotic and antioxidant properties of Bupleurum chinense (B.C) root extract against human epithelial ovarian cancer cells (HO-8910) in vitro. METHODS: MTT assay was used to evaluate the cell viability of HO-8910 cells after treatment with different B.C extract doses. Apoptotic and morphological effects induced by the extract were demonstrated by inverted phase contrast microscopy and fluorescence microscopy. The percentage of apoptotic cells was quantified by Annexin V/PI double staining assay. Flow cytometry using rhodamine-123 dye was used to measure disruption of mitochondrial membrane potential (Δψm). Gel electrophoresis was used to study the effects of the extract on DNA fragmentation. The antioxidant activity of the extract using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazolin-6-sulphonic acid) (ABTS) radical scavenging assays was also evaluated. RESULTS: The results showed that B.C extract could induce potent and dose-dependent cytotoxic effects on the HO-8910 cells as demonstrated by MTT assay. The extract also induced cell shrinkage, chromatin condensation and membrane blebbing which are the hallmark of apoptosis. The average proportion of Annexin V-staining positive cells (total apoptotic cells) significantly increased from 9.4% in control cells to 18.5, 28.2 and 50.5% in 20, 80 and 120 µg/ml B.C extract-treated cells respectively. Different doses of the extract (20, 80 and 120 µg/ml) after 48 hrs exposure led to a substantial increase in DNA fragmentation.The number of cells with disrupted Δψm increased from 6.6% in untreated (control cells) to 14.2, 42.1 and 73.4% in 20, 80 and 120 µg/ml in extract-treated cells, respectively CONCLUSION: The anticancer effects of Bupleurum chinense extract were mediated through the induction of apoptosis, DNA fragmentation and disruption of mitochondrial membrane potential.

[Determination of gold in copper matte and sintered copper material].

Ore sample, pretreated at 650 degrees C, was decomposed with aqua regia. Gold in the sample solution was then pre-concentrated by adsorbing with polyurethane foam plastic, released with thiourea solution, and determined by inductively coupled plasma-atomic emission spectrometry and flame atomic absorption spectrometry. Based on the characteristic of the copper matte and sinter containing copper, the effects of sample dissolving condition, matrix effect and interference of coexisting elements were investigated. The accuracy, precision and detection limit were discussed. The results of test show that both of the two methods were suitable for determining the contents of gold in copper matte and sintered copper material.

Expression of growth differentiation factor-9 and bone morphogenetic protein-15 in oocytes and cumulus granulosa cells of patients with polycystic ovary syndrome.

OBJECTIVE: To evaluate the effect of growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) on the development of follicles among patients with polycystic ovary syndrome (PCOS). DESIGN: Case-control study. SETTING: University Hospital. PATIENT(S): Twenty-two oocytes were obtained from 15 patients with PCOS and 67 oocytes from 58 controls. Cumulus granulosa cells (GC) were obtained from 16 patients with PCOS and controls treated with intracytoplasmic sperm injection. INTERVENTION(S): Immunofluorescence combined with laser scanning confocal microscopy and immunocytochemistry were used to analyze the expression of GDF-9 and BMP-15 in oocytes and cumulus GCs. MAIN OUTCOME MEASURE(S): To detect the protein expression levels. RESULT(S): No significant difference was found in the expression of GDF-9 and BMP-15 in the oocytes and BMP-15 in the cumulus GCs of patients with PCOS and controls. However, the expression of GDF-9 in cumulus GCs of patients with PCOS was decreased significantly compared with controls (8.88 +/- 1.52 vs. 5.01 +/- 0.83). CONCLUSION(S): The expression of GDF-9 and BMP-15 in the oocytes of patients with PCOS who received ovulation induction treatment was in the normal range, but the GDF-9 expression in cumulus GCs from patients with PCOS was significantly lower than the normal. Reduced GDF-9 expression in cumulus GCs of patients with PCOS appears to be associated with decreased long-term developmental potential of the oocytes of patients with PCOS.

[Preliminary study of protein expression profiling of PCOS on different state].

OBJECTIVE: To screen the serum protein expression profiles in patients having polycystic ovary syndrome (PCOS) with or without insulin resistance (IR) and search for discriminatory proteins. METHOD: Fasting serum samples of 30 PCOS patients with IR, 30 PCOS patients without IR, and 30 control individuals from Reproductive Center of Peking University Third Hospital were studied. RESULTS: There were 27 differential protein peaks between PCOS IR patients and controls, 17 between PCOS non-IR patients and controls, and 19 between PCOS IR patients and non-IR patients. Marker proteins from differentially expressed proteins were screened out using support vector machine (SVM), and were used to establish three diagnostic models for PCOS IR, PCOS non-IR, and IR, respectively. CONCLUSIONS: There were significantly different serum proteomic patterns in different types of PCOS. Using Protein Chip combined with SVM, computer diagnostic models for PCOS with and without IR were set up quickly and efficiently. These discriminatory proteins may help us understand the proteomic changes in serum and find out potential biomarkers of PCOS and IR.

[The measurement of proportion and function of regulatory T cells in unexplained recurrent spontaneous abortion].

OBJECTIVE: To investigate the proportion and function of CD(4)(+)CD(25)(+) regulatory T cells (CD(4)(+)CD(25)(+) Tr) in unexplained recurrent spontaneous abortion (URSA). METHODS: (1) Proportion measurement: the proportion of CD(4)(+)CD(25)(+) Tr cells in peripheral blood was measured by double-label flow cytometric analysis. The samples were taken from 15 URSA women, 15 normal non-pregnancy women and 13 normal pregnancy women. (2) Function measurement: CD(4)(+)CD(25)(+) Tr cells and CD(4)(+)CD(25)(-) T cells were extracted from peripheral blood lymphocytes by the microbeads separation. The purity of CD(4)(+)CD(25)(+) Tr cells and CD(4)(+)CD(25)(-) T cells was measured by flow cytometry. The growth inhibitory effect of CD(4)(+)CD(25)(+) Tr cells on CD(4)(+)CD(25)(-) T cells was assessed in vitro. RESULTS: The proportion of CD(4)(+)CD(25)(+) Tr cells was decreased significantly in URSA women (6.9 +/- 1.8)% than that in normal non-pregnancy women [(10.8 +/- 1.1)%] (P<0.05) and normal pregnancy women [(11.2 +/- 1.4)%] (P<0.01). Moreover, the suppressive rate of CD(4)(+)CD(25)(+) Tr cells was decreased significantly in URSA women (75 +/- 6)% than that in normal non-pregnancy women [(89 +/- 4)%] (P<0.05) and normal pregnancy women [(90 +/- 4)%] (P<0.01). However, with respect to the proportion and function of CD(4)(+)CD(25)(+) Tr cells, there was no significant difference between normal non-pregnancy women and normal pregnancy women (P > 0.05). CONCLUSION: The results suggest that decrease in proportion and function of CD(4)(+)CD(25)(+) Tr cells may be associated with URSA.

[Monocyte chemoattractant protein-1 and its correlation with lipoprotein in polycystic ovary syndrome].

OBJECTIVE: To measure serum monocyte chemoattractant protein-1 (MCP-1) levels and study its associations with lipoproteins in patients with polycystic ovary syndrome (PCOS). METHODS: Sixty-five PCOS women and 20 ovulating normal women with body mass index (BMI) < 25 kg/m2 as controls were recruited. PCOS women were divided to two groups: 27 BMI >or = 25 kg/m2 patients as obese group; 38 BMI < 25 kg/m2 as non-obese group. Serum MCP-1 was assayed by enzyme-linked immunosorbent assays (ELISA). Serum prolactin (PRL), follicle stimulating hormone (FSH), luteinizing (LH), estradiol (E2) and testosterone (T) were assayed by chemoluminescence method. Serum androstenedione (A) was assayed by radioimmunity method in patients. And triglycerides (TG), total cholesterol (TC), apoprotein A (ApoA), apoprotein B (ApoB) , lipoprotein (a) [LP(a)], high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol (HDL-C and LDL-C) were measured. RESULTS: MCP-1 (P = 0.001) and ApoB (P = 0.018) levels were found to be significantly increased in PCOS groups compared with that of controls, but the ratio of ApoA/ ApoB was significantly decreased in groups PCOS (P = 0.015). PCOS obese group had markedly higher MCP-1 serum levels than non-obese group (P = 0.012), and MCP-1 serum levels in PCOS non-obese group higher than controls (P = 0.03). Univariate analysis revealed that serum MCP-1 levels were significantly and positively correlated with BMI (r = 0.350, P = 0.001), LH(r = 0.262, P = 0.016), TG (r = 0.480, P = 0.000) and ApoB (r = 0.289, P =0.008); but significantly and negatively correlated with the ratio of ApoA/ ApoB (r = -0.282, P = 0.009). Partial correlation showed that serum MCP-1 levels were correlation with LH (r = 0.2577, P = 0.020) and TG (r = 0.4611,P = 0.000). Multiple regression analysis showed that MCP-1 levels was influenced by BMI and TG. Furthermore, TG showed more effect on MCP-1 levels. CONCLUSION: PCOS obese and non-obese patients had higher serum MCP-1 levels than controls. MCP-1 was correlated with BMI, LH ,TG, ApoB and the ratio of ApoA/ ApoB. BMI and TG were two major determining factors of MCP-1 in patients with PCOS. Furthermore,TG had more effect on MCP-1 levels. Based on the above findings, we presume that MCP-1 is likely to participate in the pathophysiology and long-term complication of PCOS.

[Protein expression profilings of polycystic ovary syndrome].

OBJECTIVE: To screen out serum protein profiling of polycystic ovary syndrome(PCOS) by surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) for discovering the discriminatory proteins. METHODS: Thirty one women with PCOS and thirty healthy women were detected by Weak Cation Exchange(WCX2)chip. ProteinChip reader and Biomarker Wizard software from Ciphergen Inc were combined with a bioinformatics method (support vector machines, SVM ) to analyze protein fingerprinting. RESULTS: There were 4 proteins which were obviously different between the PCOS group and the control. Three of them were up-regulated, and one down-regulated. To set up a analysis model by SVM using the 4 proteins could successfully distinguish between PCOS and the normal control. The corresponding sensitivity, specificity and positive predict value were 86.7%, 83.3%, 87.2%, respectively. CONCLUSION: Using ProteinChip technology can screen out serum discriminatory proteins quickly and efficiently. Combined with SVM, an optimal fingerprinting model has been set up, which can easily predict PCOS. In disease state of PCOS, there are significant variations which consist of four proteins. To investigate those four discriminatory proteins, especially the protein m/z 6 628 may be of benefit to pathogenic study and the development of biomarkers for PCOS.

[Determination of natural gas by gas chromatography with external standard-area normalization method].

A GC method for determination of natural gas by external standard-normalization has been established. O2, N2, CH4, CO2, and C2H4 were separated by 13X molecular sieves (1 m x 3 mm i.d., 50 degrees C) connected with PORAPAK T columns (2 m x 3 mm i.d.) in series and detected by TCD and the hydrocarbons of C3 and higher were separated by an SE-30 column (50 m x 0.32 mm i.d.) at 50 degrees C and detected by FID at first. Then O2 + N2 + CH4, CO2 and C2-C5 hydrocarbons were separated on a column (6 m x 3 mm i.d.) with mixed phases of beta, beta'-oxydipropionitrile and dibutyl phthalate at ambient temperature, and detected by TCD. Peak areas from SE-30 column and mixed phase column were correlated with C5, and area normalization was used for quantitative analysis; then normalization correction factors of each component in sample on 13X molecular sieves, PORAPAK T and SE-30 columns were calculated. In analysis, only the first step, area normalization was used for quantitative analysis. The method achieved calibration by SP-6000 Natural Gas Analyzer itself. It is economic, convenient, rapid and accurate.