The distribution and possible role of ERK8 in mouse oocyte meiotic maturation and early embryo cleavage.

It is well known that extracellular signal-regulated kinase 8 (ERK8) plays pivotal roles in various mitotic events. But its physiological roles in oocyte meiotic maturation remain unclear. In this study, we found that although no specific ERK8 signal was detected in oocyte at the germinal vesicle stage, ERK8 began to migrate to the periphery of chromosomes shortly after germinal vesicle breakdown. At prometaphase I, metaphase I (MI), anaphase I, telophase I, and metaphase II (MII) stages, ERK8 was stably detected at the spindles. By taxol treatment, we clarified that the ERK8 signal was stained on the spindle fibers as well as microtubule asters in MI and MII oocytes. In fertilized eggs, the ERK8 signal was not observed in the two pronuclei stages. At prometaphase, metaphase, and anaphase of the first mitosis, ERK8 was detected on the mitotic spindle. ERK8 knock down by antibody microinjection and specific siRNA caused abnormal spindles, failed chromosome congression, and decreased first polar body extrusion. Taken together, our results suggest that ERK8 plays an important role in spindle organization during mouse oocyte meiotic maturation and early embryo cleavage.

Uncoupling protein 2 expression affects androgen synthesis in polycystic ovary syndrome.

The roles of uncoupling protein-2 (UCP2) on the androgen synthesis of granulosa cells derived from patients with polycystic ovary syndrome (PCOS) and normal subjects were explored. Primary human granulosa cells from 18 patients who received in vitro fertilization (IVF) were examined; nine patients had PCOS with hyperandrogenism. Primary cultures were treated with genipin, a proton leak inhibitor, guanosine diphosphate (GDP), an UCP inhibitor, and triiodothyronine (T3), an inducer of UCP gene expression. Mitochondrial membrane potential was determined using the JC-1 assay. T3 induced P450scc and UCP2 expressions and testosterone synthesis in both normal and PCOS granulosa cells. Their expressions in response to T3 treatments were correlated in the PCOS group. Differences in testosterone synthesis were observed between normal and PCOS cells in response to genipin. Increased mitochondrial membrane potential was observed in response to genipin and GDP; while T3 decreased it. Increased ovarian UCP2 expression in response to T3 treatment in PCOS may alter pregnenolone synthesis by influencing P450scc expression, thus altering testosterone production. Further in vivo studies are necessary to fully elucidate the role of UCP2 in the hyperandrogenism commonly observed in PCOS.

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis.

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.

[Study on the relationship between altered expression of annexin A4 and endometrial receptivity during the implantation window in infertile patients with endometriosis].

OBJECTIVE: To identify the differential expressed proteins, and to investigate the relationship between altered expression of annexin A4 during window of implantation [WOI (at day-6 after ovulatory day)] in infertile patients with endometriosis and endometrial receptivity. METHODS: Two-dimensional fluorescence differential in-gel electrophoresis (2D-DIGE) and matrix-assist laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) were used to detect protein expression in endometrial WOI in 10 infertile cases with endometriosis as endometriosis group and 10 infertile cases with tubal factors as control group. The semi-quantitative validation of annexin A4 in the eutopic endometrial tissue during WOI was analyzed by western blot. RESULTS: By comparing protein profiles, there were 7 meaningful differential proteins during WOI in infertile patients with endometriosis. One protein with an isoelectric point of 5.84 and relative molecular weight of 36 100 were down regulated 348% in samples of endometriosis group. It was identified as annexin A4 by mass spectrometry. By western blot, relative intensity of annexin A4 in endometriosis group was 7.2 ± 0.9, which was lower than 17.8 ± 2.6 in control group significantly (t = 7.654, P = 0.002). CONCLUSION: Lower expression of annexin A4 during WOI in infertile patients with endometriosis might be associated with the decrease of endometrial receptivity.

Synaptotagmin1 is required for spindle stability and metaphase-to-anaphase transition in mouse oocytes.

Synaptotagmin1, a calcium sensor for exocytosis, forms the 7S complex, or so-called SNARE protein complex, together with SNAP -25, syntaxin and synaptobrevin to mediate docking and fusion of synaptic vesicles to the plasma membrane of the nerve terminal. Here, we identified the unique localization, expression and function of Syt1 during mouse oocyte meiotic maturation by using confocal microscopy, western blotting, Morpholino-based knockdown and time-lapse live cell imaging. We showed that Syt1 expression was gradually increased during oocyte maturation. Syt1 was localized at the oocyte cortex from GV to MII stages and at the spindle poles in MI and MII phases, with one third of a signal-free zone at the oocyte cortex, where the chromosomes are located, which is similar to the distribution pattern of CGs from the pro-MI to MII stages. Knockdown of Syt1 resulted in pro-MI/MI arrest and PB1 extrusion decrease, with severely disrupted spindles and misaligned chromosomes. Knockdown of Syt1 also caused abnormal localization of γ-tubulin, which became redistributed into the cytoplasm. Chromosome spreading showed failure of homologous chromosome segregation. The spindle assembly checkpoint protein Bub3 was detected at the kinetochores even after 10 h of oocyte culture. Live cell imaging analysis revealed that knockdown of Syt1 resulted in abnormal spindles with various morphologies and chromosomes arrested at the pro-MI/MI stage. Defective spindles failed to support chromosome alignment along microtubules, which led to repetitive unsuccessful metaphase-anaphase transitions and failure of PB1 extrusion after extended culture. Taken together, we suggest that Syt1 may act as a MTOC-associated protein to play important roles in mouse oocyte spindle organization/stability, and that it is indispensable for the metaphase-anaphase transition to promote mouse oocyte meiotic maturation.

[Relation between insulin resistance and insulin receptor gene methylation in the endometrium of patients with polycystic ovary syndrome].

OBJECTIVE: To study the relationship between insulin resistance and methylation of insulin receptor (INSR) gene in the endometrium of women with polycystic ovary syndrome (PCOS). METHODS: Based on the HOMA index, 35 patients with PCOS were divided into insulin resistant group (IR group, n=18) and non-resistant group (NIR group, n=18). The patients age, serum estriol, testosterone, FSH and LH, fasting insulin and fasting blood glucose were compared between the two groups. The endometrial samples were obtained from the patients to examine DNA methylation status of INSR gene in the endometrial cells using methylation-specific PCR. RESULTS: The BMI, WHR, fasting glucose, fasting insulin, and HOMA index differed significantly between the two groups (P<0.05). PCR analysis showed partial methylation in the promoter region of INSR gene in 13 samples in IR group and 11 samples in NIR group, without detection of full methylation of the INSR gene in either group. The methylation status showed no significant difference between the two groups (P=0.328). CONCLUSION: Partial methylation of the INSR gene occurs in the endometria of PCOS patients, but this study does not provide a strong evidence supporting the relationship between insulin resistance and INSR gene methylation in women with PCOS.

[Preliminary study on the value of ratio of serum luteinizing hormone/follicle-stimulating hormone in diagnosis of polycystic ovarian syndrome among women with polycystic ovary].

OBJECTIVE: To investigate the value of ratio of luteinizing hormone (LH) to follicle-stimulating hormone (FSH) in diagnosis of polycystic ovarian syndrome (PCOS) among women with polycystic ovary (PCO) and to compare the difference of the diagnostic criteria between the Rotterdam Consensus and the Committee for Reproductive and Endocrine in Japan Society of Obstetrics and Gynecology. METHODS: By means of transvaginal Doppler ultrasound, 195 women with PCO were diagnosed in Nanfang Hospital of Reproductive Medicine Center and compare difference of multiple clinical indexes according to Rotterdam consensus and Japan consensus respectively. In the mean time, the ratio of LH/FSH, the level of LH, testosterone (T) and recevier operating characteristic (ROC) curve were explored to on the value of diagnosis of PCOS. RESULTS: By Rotterdam consensus, 144 women were diagnosed with PCOS and 51 women were non-PCOS, while 111 were identified as PCOS and 84 were non-PCOS according to Japan consensus. LH/FSH in PCOS and non-PCOS were 1.59 ± 0.84 and 0.85 ± 0.47 respectively when based on Rotterdam consensus, and this ratio were 1.87 ± 0.76 in PCOS and 0.78 ± 0.39 in non-PCOS based on Japan consensus. When using LH/FSH to diagnosis PCOS by Rotterdam consensus and Japan consensus, areas under ROC curve are 0.786 and 0.942, respectively. CONCLUSIONS: The ratio of LH/FSH ≥ 1 provide the significant value in the diagnosis of PCOS. The criteria of the Committee for Reproductive and Endocrine in Japan Society of Obstetrics and Gynecology is more suitable for Chinese patients.

Abnormal expression of uncoupling protein-2 correlates with CYP11A1 expression in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) may result from hypersensitivity to insulin, which is negatively regulated by uncoupling protein (UCP)-2. Because cholesterol side-chain cleavage enzyme (CYP11A1) is closely linked to PCOS, the expression of UCP-2 and CYP11A1 in ovarian tissues from PCOS patients was examined in the present study. Twelve PCOS patients with hyperandrogenaemia who underwent laparoscopic ovarian wedge resection and 12 age-matched control patients who underwent contralateral ovarian biopsy were enrolled in the study. UCP-2 expression in early stage (primordial, primary and secondary) and late stage (sinus and mature) follicles was examined using immunohistochemistry, whereas UCP-2 and CYP11A1 mRNA and protein levels in ovarian tissue were determined using quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively. UCP-2 expression increased significantly with follicular development in both control and PCOS tissue, with expression in early stage follicles from PCOS patients significantly greater than that in controls. In addition, both UCP-2 and CYP11A1mRNA and protein levels, mean fasting blood glucose concentrations and fasting serum insulin levels were significantly higher in PCOS patients compared with the control group. Finally, a significant correlation between UCP-2 and CYP11A1 expression was found in PCOS but not control patients. In conclusion, in PCOS patients, there was a correlation between UCP-2 and CYP11A1 expression, which was significantly higher than in the control group. These changes in UCP-2 and CYP11A1 expression may mediate follicle development in PCOS.

GM130, a cis-Golgi protein, regulates meiotic spindle assembly and asymmetric division in mouse oocyte.

GM130, a cis-Golgi protein, plays key roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that GM130 was localized to the spindle poles at both metaphase I and metaphase II stages and associated with the midbody at telophase I stage. The association of GM130 with spindle poles was further confirmed by its colocalization with the centrosome-associated proteins, MEK1/2. By nocodazole treatment, we clarified that GM130 localization was consistently dependent on spindle assembly. Then we investigated the possible function of GM130 by specific morpholino microinjection. This treatment caused abnormal spindle formation, and decreased first polar body extrusion. Our results showed that knockdown of GM130 impaired the localization of MTOCs proteins γ-tubulin and Plk1. Using live cell imaging we observed that depletion of GM130 affected spindle migration and resulted in elongated spindle and large polar body extrusion. We further found that depletion of GM130 blocked p-MEK1/2 accumulation at the spindle poles. And, it was shown that GM130 detached from the spindle poles in oocytes treated with MEK specific inhibitor U0126. Taken together, our results suggested that GM130 regulates microtubule organization and might cooperate with the MAPK pathway to play roles in spindle organization, migration and asymmetric division during mouse oocyte maturation.

[Frozen embryo transfer combined with intrauterine administration of autologous peripheral blood mononuclear cells for repeated implantation failure: report of 3 cases].

OBJECTIVE: To explore the efficacy of frozen-thawed embryo transfer combined with intrauterine administration of autologous peripheral blood mononuclear cells (PBMCs) in the treatment of repeated implantation failure (RIF). METHODS: PBMCs obtained from 3 patients with RIF on the day of follicle rupture (natural cycle) or when the endometrial thickness reached 8 mm (hormone replacement cycle) were cultured in the presence of HCG for 48 h. The cultured PBMCs, along with freshly isolated PBMCs, were administered into the uterine cavity of the patients. Vitrified cleavage-stage embryos or blastocysts transfer was performed on day 3 or 5, respectively. RESULTS: Vitrified embryo or blastocyst transfer resulted in pregnancy and healthy live births in all the 3 patients. CONCLUSION: Frozen-thawed embryo transfer combined with intrauterine administration of autologous PBMCs may be an effective and safe approach to the treatment of RIF and may improve the outcomes of assisted reproduction.

p38α MAPK is a MTOC-associated protein regulating spindle assembly, spindle length and accurate chromosome segregation during mouse oocyte meiotic maturation.

P38αMAPK (p38α) is usually activated in response to various stresses and plays a role in the inhibition of cell proliferation and tumor progression, but little is known about its roles in meiotic spindle assembly. In this study, we characterized the dynamic localization of p38α and explored its function in mouse oocyte meiotic maturation. P38α specifically colocalized with γ-tubulin and Plk1 at the center of MTOCs and spindle poles. Depletion of p38α by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes probably via MK2 dephosphorylation. Notably, depletion of p38α led to significant spindle pole defects, spindle elongation, non-tethered kinetochore microtubules and increased microtubule tension. The disruption of spindle stability was coupled with decreased γ-tubulin and Plk1 at MTOCs. Overexpression of Eg5, a conserved motor protein, also caused spindle elongation and its morpholino injection almost completely rescued spindle elongation caused by p38α depletion. In addition, p38α-depletion decreased BubR1 and interfered with spindle assembly checkpoint (SAC), which resulted in aneuploid oocytes. Together, these data indicate that p38α is an important component of MTOCs, which regulates spindle assembly and spindle length, as well as stabilizes the spindle and spindle poles. Perturbed SAC and abnormal microtubule tension may be responsible for the misaligned chromosomes and high aneuploidy in p38α-depleted mouse oocytes.

[CA 125 expression in cervical and vaginal secretions in women in normal reproductive period].

OBJECTIVE: To investigate the tumor-associated antigen CA125 expression in the serum and cervical and vaginal secretions in women during normal reproductive period, and explore the clinical value of detecting tumor markers in the cervical and vaginal secretions. METHODS: A total of 145 women in reproductive period were divided into 3 age groups (20-29 years, 30-39 years, and over 40 years), and their CA125 levels in cervical secretion, vaginal secretion and serum were detected by automatic electro-chemiluminescent immunoassay. RESULTS: CA125 levels in the cervical secretion, vaginal secretion and serum showed no significant difference between the 3 age groups (P>0.05). In each group, CA125 levels differed significantly between the cervical secretion, vaginal secretion and serum (P<0.001). In the 145 women, the average CA125 level was 497.82 - or + 75.29 U/ml in the cervical secretion, 114.66 - or + 26.40 U/ml in vaginal secretion and 18.06 - or + 3.35 U/ml in serum, showing significant differences between them (P<0.001). CONCLUSION: CA125 expression level is significantly higher in the cervical and vaginal secretions than in the serum in women in normal reproductive period, and its levels in cervical and vaginal secretions can be more sensitive and convenient for early detection of related diseases.

Demethylation of LHR in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome.

The cause of polycystic ovary syndrome (PCOS), a complex endocrine disorder, is unknown, but its familial aggregation implies underlying genetic influences. Hyperandrogenemia is regarded as a major endocrine character of the PCOS. In this study, we employed bisulfite sequencing and bisulfite restriction analysis to investigate the DNA methylation status of LHR, AR, FSHR and H19 in dehydroepiandrosterone (DHEA)-induced mouse PCOS model. The result showed that methylation of LHR was lost in ovary from induced PCOS mouse. However, AR, FSHR and H19 had similar methylation pattern in DHEA-treated group and control groups. These data provide evidence for close linkage between DNA demethylation of LHR and PCOS.

[Inhibitory effect of RNA interference targeting BaxBak on apoptosis of human granulosa cells].

OBJECTIVE: To investigate the inhibitory effect of small interfering RNA (siRNA) targeting Bax-Bak on the apoptosis of human granulosa cells. METHODS: Human granulosa cells were transfected with Bax-siRNA and Bak-siRNA either alone or in comibnation, and the cell morphological changes were obsered and the cell apoptosis was detected with flow cytometry. Western blotting was performed to examine the changes in Bax and Bak expressions in the transfected cells. RESULTS: Western blotting demonstrated significantly weakened expressions of Bax and Bak in the transfected cells. The cell morphology of the cells tranfected with Bak siRNA and with both Bak and Bax siRNA remained normal; the cells with exclusive Bax siRNA transfection presented with basically normal cell morphology, but black spots were noted in the cytoplasm. In the positive and negative control groups, the cells became rounded and shrank with expanded intercellular spaces and numerous black spots in the cytoplasm. Flow cytometry showed apoptotic indexes of 3.44% and 3.97% in cells transfected with Bak siRNA and Bax-Bak siRNA, respectively, significantly lower than that in the negative group. Bax siRNA transfection resulted in an apoptotic index of 19.98%, similar to that in the negative group. CONCLUSION: Interference of the expression of Bak gene inhibits the apoptosis of human granulosa cells, and the inhibitory effect can be enhanced by simultaneous Bax interference, which, when used alone, does not obviosuly inhibit the apoptosis of human granulosa cells.

[Clinical application of prefilled pen and conventional syringe during controlled ovarian stimulation for in vitro fertilization].

OBJECTIVE: To investigate the efficacy, convenience and costs of recombinant follitropin alpha administered by a prefilled pen device and conventional syringe in Chinese women undergoing controlled ovarian stimulation for in vitro fertilization (IVF). METHODS: A total of 184 patients undergoing IVF treatment were enrolled in this study. According to a long-term recombinant follicle-stimulating hormone (rFSH) protocol, ovarian stimulation was performed with the prefilled pen and conventional syringe at random in these subjects, and the dose of follitropin, number of oocytes and embryo parameters and IVF-ET outcome were compared between the two groups. RESULTS: The total rFSH dose, cost, and frequency of hospital visits were significantly lower in the pen protocol group, but the residual rFSH amount was higher. Compared with conventional injections, the prefilled pen was associated with significantly lowered rate of local redness, high rate of local bruise, more frequent follitropin dose modulation and lower serum oestradiol levels on HCG day. No significant difference was found in the endometrial thickness, numbers of oocytes retrieved, MII oocytes, transferred embryo, or the clinical pregnancy rates between the two groups. The ratio of MII oocytes, good quality embryo rates and implantation rates was significantly higher in the pen group with lower incidences of moderate and severe ovarian hyperstimulation syndrome. CONCLUSION: The prefilled pen provides an easy, safe, effective and more patient-friendly means for controlled ovarian stimulation procedure in Chinese women, but more attention should be given to protocol optimization and patient education.

[Relationship between protein oxidation levels in the follicular fluid and the outcome parameters of in vitro fertilization-embryo transplantation].

OBJECTIVE: To explore the relationship between protein oxidation levels in the follicular fluid and the outcome parameters of in vitro fertilization-embryo transplantation (IVF-ET). METHODS: The levels of advanced oxidation protein products (AOPP) in the follicular fluid were measured in 64 women with tubal infertility undergoing IVF-ET. The relationship between the AOPP levels and IVF-ET outcome parameters was analyzed. RESULTS: AOPP levels showed significant inverse correlations between the proportion of mature oocytes (r=-0.401, P=0.001), fertilization rate (r=-0.257, P=0.045), cleavage rate (r=-0.290, P=0.024) and good embryo rate (r=-0.520, P=0.000). AOPP levels differed significantly between the groups with different retrieved oocyte numbers (F=3.851, P=0.027), being the lowest in women with 8 to 15 retrieved oocytes and the highest in those with retrieved oocytes below 8. The AOPP level in the non-pregnant women was significantly higher than that in the pregnant women (t=3.665, P=0.001). The AOPP levels also differed significantly with age (F=15.919, P=0.000), and the women >35 years of age had the highest level and those below 30 years had the lowest level. CONCLUSION: Protein oxidative stress is present in the follicular fluid of women on IVF-ET cycles. High level of AOPP may have adverse effects on the oocytes and early embryonic development and may affect the outcome of IVF-ET.

[Effect of two different doses of letrozole in promoting ovulation in infertile women with polycystic ovarian syndrome].

OBJECTIVE: To investigate the effect of two different doses of letrozole in promoting ovulation in infertile women with polycystic ovarian syndrome (PCOS). METHODS: Seventy-six PCOS infertile women undergoing intrauterine insemination (IUI) or with anovulation were divided into two groups and received oral letrozole at the daily dose of 2.5 (n=36) or 5.0 mg (n=40) from the 3rd to the 7th day of the menstrual cycle. Three days after discontinuation of the medication (the 10th day of the menstrual cycle ), ultrasound scanning was performed to monitor the follicle development. When the diameter of the biggest follicle reached 14 mm, the presence of luteinizing hormone (LH) was monitored; when LH positivity was detected, blood samples were drawn to test follicle-stimulating hormone (FSH), LH, estradiol (E2) and testosterone (T) levels. Human chorionic gonadotropin (hCG, 10 000 U) was then injected to induce ovulation. RESULTS: The ovulation rate, stimulation follicle days, diameter of the biggest follicle on the day of LH positivity and the thickness of endometrium were all similar between the two groups (P>0.05). But in women receiving 5.0 mg letrozole, both the number of mature follicles and pregnancy rate were significantly higher than those in women having the half dose (P<0.05). The levels of FSH, LH, E2, and T on the third day of menstruation and on the day of HCG injection were similar between the two groups (P<0.05). CONCLUSION: Letrozole at the dose of 5.0 mg/day produces higher pregnancy rate and more mature follicles in fertile women with PCOS.

[Allogeneic leukocyte immunization combined with IVF-ET for treatment of infertility induced by recurrent spontaneous abortion].

OBJECTIVE: To investigate the effect of allogeneic leukocyte immunization combined with in vitro fertilization-embryo transfer (IVF-ET) for treatment of infertility induced by habitual abortion. METHODS: Allogeneic leukocyte immunization was performed in 9 patients with infertility induced by habitual abortion, with another 9 patients undergoing IVF-ET without habitual abortion as the control group. All the patients were treated with long GnRH-a protocols. The infertility patients with recurrent spontaneous abortion history were immunized with lymphocytes from the husband for before IVF-ET and after clinical pregnancy. RESULTS: The fertilization rates of the immunotherapy group and control group were 81.3% and 82.2%, respectively, showing no significant difference (P>0.05). Five patients in each group had clinical pregnancy, and a twin pregnancy occurred in the control group. The embryo implantation rates were also comparable between the two groups (22.7% vs 28.6%, P>0.05). All the fetuses resulted from IVF-ET developed normally and were healthily delivered. CONCLUSION: Allogeneic leukocyte immunotherapy along with IVF-ET is effective for treatment of infertility resulting from recurrent spontaneous abortion.

[Function of F10 gene, a novel hydatidiform mole-related gene: a preliminary study].

OBJECTIVE: To study the function of F10 gene, a novel hydaditiform mole-related gene. METHODS: A549 cell line was transfected with the F10 gene of forward or reverse sequence or with the empty vector, respectively. The cellular mRNA was extracted after 24 h of transfection to screen for the differentially expressed genes among the 3 transfected and the control cells using differential display-polymerase chain reaction (ddPCR). RESULTS: The bands representing differentially expressed genes were amplified from the cells, and the products were linked to T-Vector for sequence analysis. Several genes were screened by Blasting and their expressions were confirmed by fluorescent quantitative PCR. CONCLUSION: F10 gene is functionally related to cell proliferation and apoptosis.

[F10 gene knock-down mediated by RNA interference induces apoptosis of KLE cells].

OBJECTIVE: To study the effect of short double-stranded RNA (dsRNA) on F10 gene expression in KLE cells and the effect of F10 knock-down on KLE cell apoptosis. METHODS: The short dsRNA specifically targeting F10 gene prepared by in vitro transcription was transfected into KLE cells via lipofectamine 2000. The expression of F10 mRNA in the transfected KLE cells was detected by real-time quantitative RT-PCR, and the apoptosis of the cells was assayed by flow cytometry. RESULTS: Real-time quantitative RT-PCR demonstrated that transfection of the KLE cells with the short dsRNA induced effective knock-down of F10 gene, and transfection of the cells with 20 nmol/L dsRNA for 48 h decreased the expression of F10 mRNA by 83%. Compared with the control, the apoptosis index of the transfected KLE cells increased from 0.36% to 8.91%. CONCLUSION: F10 gene in KLE cells can be specifically knocked-down with dsRNA prepared by in vitro transcription, and the down-regulation of F10 gene induces apoptosis of KLE cells.