Promoter methylation of yes-associated protein (YAP1) gene in polycystic ovary syndrome.

BACKGROUND: DNA methylation modification has been proved to influence the phenotype of polycystic ovary syndrome (PCOS). Genome-wide association studies (GWAS) demonstrate that yes-associated protein (YAP1) genetic sites are associated with PCOS. The study aims to detect the methylation status of YAP1 promoter in ovary granulosa cells (GCs) of PCOS patients and explore novel therapeutic targets for PCOS. METHODS: Randomized controlled trial was applied and a total of 72 women were included in the study, including 36 cases of PCOS patients and 36 cases of health controls. Ovary GCs were extracted from in vitro fertilization embryo transfer. Methylation status of YAP1 promoter was detected by bisulfite sequencing PCR (BSP). Protein and mRNA expression of YAP1 were measured by western blotting and real-time quantitate PCR. RESULTS: Overall methylation level of YAP1 promoter region from PCOS group was significantly lower than that from control group. CpG sites analysis revealed that 12 sites (-443, -431, -403, -371, -331, -120, -49, -5, +1, +9, +15, +22) were significantly hypomethylated in women with PCOS (P < 0.05). A significant upregulation of YAP1 mRNA and protein expression levels was observed. Testosterone concentration could alleviate the methylation status and demonstrate obvious dose-dependent relation. CONCLUSION: Our research achievements manifest that hypomethylation of YAP1 promoter promotes the YAP1 expression, which plays a key role in the pathogenesis and accelerate PCOS.

[Expression of R2 protein in gestational trophoblastic diseases].

OBJECTIVE: To study the expression of the small subunit ribonucleotide reductase (R2) in gestational trophoblastic diseases (GTD) and to assess its prognostic value. METHODS: The expression of R2 was detected with immunohistochemical method in 15 cases of normal villi, 38 cases of hydatidiform mole (HM), 42 cases of invasive moles (IM) and 18 cases of choriocarcinoma (CC). RESULTS: R2 expression in HM, IM and CC was significantly increased compared with that of normal villi (P=0.000). There were no significant differences in R2 protein expression among HM, IM and CC. Among 38 cases of HM, R2 expression in 8 cases with malignant transformation was significantly higher than in 30 cases of non-malignant transformation mole (P=0.02). Preoperative chemotherapy of gestational trophoblastic tumor including IM and CC did not influence the R2 expression. Compared with patients of stage I (WHO), the R2 protein in gestational trophoblastic tumor (GTT) patients of stage III or stage II was significantly increased (P=0.023 and P=0.038, respectively). The value of R2 in GTT patients with middle or high risk in WHO prognostic scoring system was higher than in the patients with low risk (P=0.018 and P=0.006, respectively). CONCLUSION: R2 expression in GTD is increased, which may be associated with the hyperplasia of trophoblasts, malignant transformation of hydatidiform mole and drug resistance of trophoblastic tumor.

[Effect of antisense oligodeoxynucleotide of small subunit component of human ribonucleotide reductase on human choriocarcinoma cell line in vitro].

OBJECTIVE: To study effect of the antisense oligodeoxynucleotide (ASODN) of small subunit of human ribonucleotide reductase (RRM2) mRNA on cell line of human choriocarcinoma in vitro. METHODS: Two 20-mer gapmer ASODNs with a full phosphorothioate backbone were artificially synthesized, which were complementary to nucleotides 626-645 (a coding region) and 1572-1591 (a 3'untranslated region) of RRM2, respectively. ASODNs were transfected into JAR cells through oligofectamine. The survival rate was assessed by methyl thiazolyl tetrazolium (MMT) assay, and RRM2 expression was detected by immunoblot and reverse transcriptase polymerase chain reaction (RT-PCR) methods. RESULTS: Antisense oligodeoxynucleotide one (ASODN1) targeting the coding region significantly inhibited growth of JAR cells in a dose- and time-dependent manner and downregulated RRM2 expression in a time-dependent manner. ASODN1 at 100 nmol/L could inhibit significantly cell growth (P = 0.000), and the effects of ASODN1 on JAR cell proliferation were enhanced with increase of ASODN1 concentration and reached the peak point at 400 nmol/L concentration (P = 0.000). Cell growth was significantly inhibited by 200 nmol/L of ASODN1 after 24 h of treatment (P = 0.000). The effect of ASODN1 was at the maximum at 48 h (P = 0.000), and began to decrease at 72 h of treatment. RRM2 expression started to reduce after ASOND1 treatment for 12 hours, and was obviously downregulated at 24 h of treatment, and decreased to the lowest level at 48 h (P < 0.05). RRM2 expression began to recover at 72 h of treatment. Antisense oligodeoxynucleotide two (ASODN2) corresponding to 3'untranslated region and control oligodeoxynucleotides had no significant effect on the proliferation and RRM2 expression, but the combination of ASODN2 and ASODN1 was more effective on reducing RRM2 expression and inhibiting cell proliferation than ASODN1 alone. CONCLUSIONS: ASODN1 of RRM2 can inhibit the proliferation of JAR cells and downregulate RRM2 transcription and translation in a selective and specific manner. The combination of multiple ASODNs targeting different regions of RRM2 mRNA would be an important approach to improve antisense effectiveness.

[Hemeoxygenase expression and activity in human placenta of pregnancy induced hypertension].

OBJECTIVE: To investigate the expression of heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2) in placenta tissue of pregnancy induced hypertension (PIH), and the relationship between HO protein expression and enzymatic activity. METHODS: Protein expression was analyzed qualitatively and semi-quantitatively by Western blotting in placental tissue of PIH (PIH group, n = 30) and normal late pregnant women (control group, n = 30). The levels of HO enzymatic activity in placental tissue were measured with the double wavelength scanning by spectrophotometer. RESULTS: (1) Western blotting analysis demonstrated that the relative protein levels of HO-1 and HO-2 in placental samples of control group were 0.7 +/- 0.4 and 0.8 +/- 0.4 respectively, there were no significant difference between HO-1 and HO-2 protein levels (P > 0.05). (2) The relative levels of HO-1 were 0.6 +/- 0.4 in PIH group, there was no significant difference from those in the control group (P > 0.05); the relative levels of HO-2 protein were 0.6 +/- 0.3 in PIH group, that were obviously lower than those in the control group (P < 0.01). The levels of HO enzymatic activity of PIH group were (0.3 +/- 0.2) nmol x mg(-1) x h(-1), significantly lower than that of the control group [(0.5 +/- 0.3) nmol x mg(-1) x h(-1)] (P < 0.01). The levels of HO activity correlated with HO-2 protein levels. CONCLUSIONS: The expression levels of HO-2 protein were decreased and HO enzymatic activity reduced in PIH group. HO may play a role in the pathophysiology of poor placental perfusion and tissue damage in placenta of PIH.

[Expression of RFC2 and PCNA in different gestational trophoblastic diseases].

BACKGROUND & OBJECTIVE: High expression of replication factor C (RFC) mRNA in hydatidiform mole and choriocarcinoma were detected previously with DNA microarray. Replication factor C subunit 2 (RFC2) was reported to be associated with DNA duplication, DNA repair, and the function of cellular checkpoint. The aim of current study was to investigate the expression levels of RFC2 and proliferating cell nuclear antigen (PCNA) in gestational trophoblastic diseases (GTD) and to explore the relationship of expressions of two proteins with GTD. METHODS: The expression of RFC2 and PCNA were detected with immunohistochemical method in 15 cases of normal villi, 38 cases of hydatidiform moles (HM), 42 cases of invasive moles (IM), and 18 cases of choriocarcinomas (CC). RESULTS: The expression of RFC2 and PCNA were significantly increased in HM, IM, and CC than in normal villi (P=0.000 for RFC2,P=0.004 for PCNA). There was no significant difference of the expression of RFC2 among HM, IM, and CC. The PCNA expression was significantly higher in CC than in HM (P=0.037). PCNA expression was significantly higher in the patients with malignantly transformed molar pregnancy than in the patients without malignantly transformed molar pregnancy (P=0.039). RFC2 expression in no preoperative chemotherapeutic group of gestational trophoblastic tumors (GTT) including IM and CC was significantly higher than that in the group with the chemotherapy of more than 3 cycles (P=0.028). Compared with patients at stageI, patients at stage III (WHO) had significantly increased expression of RFC2 (P=0.01). The level of RFC2 was higher in the patients with high risk in WHO prognostic scoring system than that in the patients with low risk (P=0.018). The levels of PCNA were significantly higher in the patients with high risk and middle risk than that in the patients with low risk (P=0.036 and P=0.048, respectively). The expression of PCNA protein was not associated with the preoperative chemotherapy and WHO stage of GTT. The RFC2 expression was positively correlated with the PCNA expression (P=0.000). CONCLUSION: The over-expression of RFC2 and PCNA in GTD may be associated with the malignant transformation of HM and the behavior of trophoblastic tumor.

[Changes in gene expression profiles of hydatidiform mole and choriocarcinoma as compared with trophoblast hyperplasia].

OBJECTIVE: To study the relationship of changes in gene expression profiles of hydatidiform mole and choriocarcinoma with hyperplasia of trophoblasts. METHODS: The differentially expressed genes were analyzed in two pairs of tissues of hydatidiform mole versus normal villi, and in two pairs of normal primary culture trophoblasts versus JAR cell line of chariocarcinoma, using cDNA microarray containing 4096 genes. To confirm the results of cDNA microarray analysis, expressions of some up-regulated genes related to DNA synthesis in normal villi, hydatidiform mole, and 2 choriocarcinoma cell lines (JAR and JEG-3) were examined by immunohistochemistry, immunoblotting and RT-PCR. RESULTS: A total of 89 genes were differentially expressed in all hydatidiform moles, accounting for 2.2% of the genes arrayed. Of the 89 genes, 24 were up-regulated and 65 were down-regulated. Compared with normal primary trophoblasts, there were 433 genes up-regulated and 380 genes down-regulated in JAR cell line. Forty six genes were up-regulated in both hydatidiform mole and choriocarcinoma, while 13 genes were down-regulated. Some genes associated with cell proliferative inhibition were significantly down-regulated, whereas those associated with cell proliferation, malignant transformation, metastasis and drug resistance were highly up-regulated. The expressions of thymidine kinase 1, the small subunit of ribonucleotide reductase (RRM2) were significantly increased in hydatidiform mole, JAR and JEG-3 cells. CONCLUSION: Abnormal expression of genes exists in hydatidiform mole and choriocarcinoma. Hyperplasia of trophoblasts may be related to over-expression of genes coding for synthetic enzymes.

[Preliminary study on gene expression profile of endometrial adenocarcinoma].

OBJECTIVE: To determine candidate genes of endometrial adenocarcinoma. METHODS: To compare the gene expression profile in 2 endometrial adenocarcinoma tissues and 2 normal endometria by HGEC-40s GeneChip probe including 4096 genes array. Expression differences between normal and malignant tissue groups were measured by GenePixPro3.0 software. RESULTS: 350 genes with a ratio below 0.5 and above 2.0 showed discrimination between normal and malignant groups. Thirty three genes with ratio above 3 were up-regulated, forty-four genes with ratio below 0.3 were down-regulated. CONCLUSION: The overexpression of oncogenes with their disturbed or constitutively activated signal transduction cascades alone or in combination with the mutation-induced silencing of tumor suppressor genes is associated with malignant transformation.

[Study of DNA microarray chip of associated genes of hydatidiform mole].

OBJECTIVE: To screen genes which may associate with the development and malignant transformation of hydatidiform mole. METHOD: The differentially expressed genes were analyzed between the tissues of two cases of hydatidiform mole and the normal placental tissues of two cases of pregnancy which had almost the same pregnant ages as hydatidiform mole, using cDNA chips containing 4,096 genes. RESULTS: There were total 89 genes significantly differently expressed in all hydatidiform moles, counting for 2.2% of total genes. Compared with normal villi, 24 genes in hydatidiform mole were up-regulated and 65 genes were down-regulated. Bioinformatical analysis of genes showed genes associated with cell proliferative inhibition, such as Ras GTPase activating protein, TGF-beta IIR alpha, BTG2 were significantly down-regulated, whereas genes associated with cell proliferation, malignant transformation and tumor metastasis as thymidine kinase, ribonucleotide reductase, glucose transport protein were highly up-regulated. CONCLUSION: cDNA chip technique is one kind of very effective methods in screening associated genes in hydatidiform mole.